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2013 EAS Abstracts November 2013

2013 EAS Abstracts This volume contains the final abstracts for the oral and poster presentations which take place on Monday, November 18, through Wednesday, November 20, 2013. For each abstract provided, a complete mailing address for the presenting author is shown. Additional authors are indicated, however, their mailing addresses are not provided. Schedule and meeting room information for the technical sessions, as well as information concerning short courses, exhibitor workshops, and the exposition, are contained in the Final Program Book. More Information To obtain answers to EAS-related questions after the meeting:

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1 2013 EAS Abstracts November 2013

ABSTRACTS OF TECHNICAL PAPERS

1 Synchrotron X-Ray Spectroscopy - How 10 Orders of Magnitude 4 The Good, the Bad and the Ugly: Understanding the Roles of Makes Hard Things Easy Metals in Biology Using Synchrotron Radiation Stephen P. Cramer, University of California, Lawrence Berkeley Lab, Graham N. George, University of Saskatchewan, 114 Science Pl., Department of Chemistry & Physical Biosciences Division, Davis, CA Sasaktoon, SK S7N5E2, Canada 95616 One of the major strengths of X-ray absorption spectroscopy (XAS) is its ability to Over the past 40 years, the brightness of synchrotron radiation sources has in- probe chemical form in any sample without any chemical pre-treatment. The low creased by about 10 orders of magnitude. It is still improving today. In this talk I give concentrations of metals and metalloids in biological tissues have meant that in examples of experiments that were once heroic and that are now almost routine. the past XAS has been difficult to apply to these samples. Recent developments in I start with the EXAFS of MoFe nitrogenase, comparing early data with the latest synchrotron beam line and detector technology mean that XAS of intact biological analysis and the 1 Å crystal structure. By combining the EXAFS Debye-Waller factor samples is now achievable. XAS can reveal the molecular details of metals within with empirically calibrated molecular force-fields, we can obtain average distances living systems, how they interact with one another, how they confer beneficial prop- and rms disorder both with ~0.01 Å accuracy. I then discuss progress in soft X- erties, and how they act as poisons. As most tissues have spatial structure, there is ray spectroscopy and detectors, showing the potential going forward for arrays of great additional interest in visualizing the spatial location of the metal or metalloid hundreds of superconducting tunnel junctions to probe highly dilute systems. Next, as well as its chemical forms. The use of microfocus X-ray beams gives the oppor- I compare our earliest efforts at “high-resolution” X-ray fluorescence at 1 eV resolu- tunity of producing maps of elements and of specific chemical types of elements in tion with recent work on inelastic X-ray scattering a 1 meV resolution. I conclude dilute biological systems. These developments are discussed, drawing upon recent with recent progress in nuclear inelastic scattering. In 1995, Seto did the first ex- examples from our research on XAS of toxic elements in biological tissues. periment at 7 meV resolution on pure Fe metal. We are now looking at modes in metalloproteins at sub-millivolt resolution from samples with millimolar Fe. I end with 5 Analysis of Vesicle Contents with Electrochemistry and Mass some prognostication of what might be possible in the future in the current bright- Spectrometry ness trend continues. Andrew G. Ewing, Chalmers University and Gothenburg University, Kemivagen 10, Gothenburg 41296, Sweden 2 X-Ray Emission Spectroscopy - A Powerful Tool to Study Amperometry can be used to measure individual exocytosis events from secreting Biocatalysts cells. We have analyzed amperometric peaks corresponding to release at PC12 Uwe Bergmann, SLAC National Accelerator Laboratory, 2575 Sand Hill cells and found stable plateau currents during the decay of the peaks, indicating Rd., Menlo Park, CA 94025 closing of the vesicle after incomplete release of the vesicular content. From careful Much of the chemistry essential to life is governed by catalytic reactions that take analysis of these data, we have now proposed a process for most exocytosis events place at metal centers within biological systems. Among the various tools to study where the vesicle partially opens to release transmitter and then closes directly such metal centers X-rays are particularly powerful due to their sensitivity to the again, leaving the possibility for both a stable pre- and post-spike foot to be ob- geometric and electronic structure at an atomic level. X-ray emission spectroscopy served with amperometry. The experiments correlate well with those from the elec- (XES) is such a probe and with the advent of very powerful X-ray sources and novel trochemical cytometry method we have developed to count electroactive molecules X-ray spectrometers the technique has now been successfully applied to study the in individual synaptic vesicles in directly sampled populations from cells or brain extremely weak X-ray spectral signals from some of the most important biocata- tissue. With this method we can compare the total numbers of molecules to those lysts. We introduce the technique, present the state-of-the-art X-ray sources and released and only a fraction is in fact released both at cell models and in mammalian instrumentation, and discuss research examples. In particular we show how XES brains. We have combined these experiments with mass spectrometry imaging to has provided a breakthrough in the understanding of the atomic structure of the compare the concentration of transmitters across single vesicles. iron-molybdenum co-factor in nitrogenase, the enzyme used by some organisms to fix atmospheric nitrogen gas by breaking the N≡N triple bond, one of the strongest 6 Autophagy from an Individual Organelle Perspective bonds in nature. The other example focuses on a series of studies of the MnCaO Edgar A. Arriaga, University of Minnesota, 207 Pleasant St. SE, cluster in photosystem II, responsible for splitting water into molecular oxygen. Be- Minneapolis, MN 55455, Chad P. Satori sides using synchrotron X-ray sources for many years, we have recently started Autophagy is responsible for the degradation of damaged intracellular components. to perform studies at the Linac Coherent Light Source, the world’s first X-ray free Current measurements, such as those done by Western blotting, are insufficient to electron laser. We discuss our first results and show how this revolutionary machine understand autophagy and its roles in aging and disease. Capillary electrophoresis can be used to study the photosynthetic water splitting mechanism in real-time. coupled to laser induced fluorescence detection (CE-LIF) has been used previously to count and determine properties of individual organelles, but has never been used 3 Coherent and Inelastic Resonant X-Ray Scattering on autophagy organelles or for determination of temporal changes of such proper- Esen E. Alp, Argonne National Laboratory, Advanced Photon Source, ties. In this presentation we report on the use of this technique to investigate au- Bldg. 431-D004, Argonne, IL 60439 tophagy in L6 cells, a skeletal muscle model. Autophagy organelles were isolated Five decades ago, Nobel Prize was given to R. Mössbauer for the discovery of from L6 cells expressing GFP-LC3 and analyzed by CE-LIF for the determination of the eponymous effect. For the last three decades, many groups around the world the number of autophagy organelles, their individual GFP-LC3 fluorescence intensi- started research programs to make use of synchrotron radiation to replace the ra- ties, and their individual electrophoretic mobilities. Basal and rapamycin-driven au- dioactive parent isotope as a source. The early efforts were amply rewarded with the tophagy were compared under steady state and dynamic conditions. Under steady arrival of high-brightness third generation synchrotron radiation sources. Nuclear state conditions, there were differences in organelle number and electrophoretic resonant X-ray scattering of synchrotron radiation is now a useful tool for investigat- mobility distributions. Under dynamic conditions, the temporal trends of individual ing thermodynamic properties of organic and inorganic materials in various scien- organelle properties upon treatment with vinblastine were the opposite of the trends tific disciplines including condensed matter physics, materials science, biophysics, observed under basal autophagy. Individual organelle analysis by CE-LIF is a pow- mineral physics and bioinorganic chemistry and geochemistry. Interesting studies erful technology to investigate the complexity and temporal nature of autophagy, a of iron proteins and enzymes, and their model compounds like single crystal por- process that plays critical roles in response to drug treatments, aging, and disease. phyrins with different ligand substitutions provides important clues to the enzymatic reactions. Study of metallic and network forming glasses or nanoparticles provide 7 Microscale Separations Applied to Studies of Protein Aggregation new insights to vibrational properties of complex systems. Measurements of phonon Doug Gilman, Louisiana State University, Chemistry Department, 232 density of states under pressures exceeding two Mb, and at temperatures as high Chopin Hall, Baton Rouge, LA 70803, Suresh C. Regmi, Indu Kheterpal as 3000 K yields important clues to earth’s interior structure. This talk reviews the Methods to study aggregation of amyloid beta peptide based on capillary electro- recent trends from development point of view and tries to put some perspective phoresis combined with spectroscopic detection techniques are presented. Protein for various opportunities at the Advanced Photon Source with respect to optics, self-assembly plays important roles in normal biology, but aberrant protein aggrega- different isotopes and sample environments. This work is supported by U.S. Depart- tion is linked to a number of diseases including Alzheimer’s disease (amyloid beta ment of Energy, Office of Science, Basic Energy Sciences under contract DE-AC02- peptide), Parkinson’s disease (alpha synuclein) and prion diseases (prion proteins). 06CH11357. Unwanted protein aggregation is also a problem for pharmaceutical applications of proteins. Analysis of protein aggregation and protein aggregates is extremely

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challenging. One major challenge is the heterogeneity of a typical solution of an silica particle are described. TitanTM columns prepared with monodisperse 1.9-µm aggregating protein. A second obstacle is a lack of analytical standards for protein C18 silica can demonstrate more than 300,000 plates per meter for small molecules aggregates. Finally, aggregated structures that are functionally important are equili- and greatly enhance the performance of UHPLC instruments while keeping pres- brating with other structures, and the population of aggregates can be altered by sure lower than porous particles in the 1.7-1.8-µm range. the measurement technique. Capillary electrophoresis is well suited for analyzing protein aggregates because it is a relatively rapid and gentle separation technique 11 Superficially Porous Particles Offer a Complete Solution for LC that is can resolve species ranging from small ions to large particles. For amyloid Analyses beta peptide aggregation studies, we are combining capillary electrophoresis with Anne E. Mack, Agilent Technologies, 2850 Centerville Rd., Wilmington, several spectroscopic detection methods ranging from simple UV absorbance to la- DE 19808, William J. Long ser-induced fluorescence anisotropy measurements and multichannel fluorescence Over recent years, superficially porous particles have evolved to provide a compre- and light scattering detection of individual aggregates. hensive solution for liquid chromatography analyses. 2-3-µm superficially porous particles have become widely implemented because of their high sub-2-µm like ef- 8 Punctuated Microgradients for Bioanalysis ficiency, while generating lower back pressure, allowing for use on a broader range Mark A. Hayes, Arizona State University, PO Box 871604, Tempe, AZ of liquid chromatography (LC) instrumentation. Moreover, the recent releases of 85287 a broad range of phase chemistries have helped to overcome the limited method Early demonstrations of capillary- and microchip- based electrophoresis have shown development flexibility of these materials. This presentation reviews the benefits of extraordinary resolution using high fields, showing deuterated vs. hydrogenated and using superficially porous LC solutions compared to traditional totally porous particle sub-millisecond separations fully based on interactions of the analyte with the sol- solutions, including 1.8, 3.5 and 5-µm configurations, as well as easy method trans- vent/buffer and the applied field. These results were generally obtained in systems fer from these traditional LC columns via highly similar phase chemistries. Addition- operating in a linear mode - spreading and diluting a mixture out along a single axis. ally, the ample selection of superficially porous phase chemistries are discussed to This presentation focuses on a new approach enabled by the short length-scale of demonstrate easy method development via orthogonal selectivities, as well as other microdevices and strong local field gradients. Electrophoretic exclusion (EpE) and tips for getting the most out of superficially porous columns. gradient dielectrophoresis (gDEP), which use electric field gradients, are presented in detail here. Electrophoretic exclusion uses a constant flow field with an induced 12 Speed vs. Sensitivity: Compatibility of Ultra High-Performance electric field gradient, differentiating species based on electrophoretic mobility. Gra- Liquid Chromatography Coupled to Electrospray Ionization High- dient dielectrophoresis sets electrophoresis against dielectrophoresis. We have de- Resolution Mass Spectrometry (ESI-HRMS) veloped an underlying theoretical framework for both projects. It turns out that they Samantha M. Mahmoud, Kean University, 1000 Morris Ave., Union, NJ were largely equivalent in a non-obvious way: the core feature that generated some 07083, Dil Ramanathan unexpected results is the creation of punctuated microgradients separated by flat Advances in high-performance liquid chromatography (HPLC) have been made to field zones. This uniquely minimizes the effects of diffusion by dramatically increas- improve productivity and analysis speed with ultra high-performance liquid chroma- ing the local restoring forces. The most important outcome and the unifying feature tography (UHPLC) first pioneered by WatersTM. These advancements increased of this approach is that the models extend to any force which can be induced into speed, sensitivity and resolution of the chromatographic separation by reducing the dispersed microgradients in the presence of a static counter field. This concept has particle size of the column packing material to achieve narrower chromatographic been shown for two applications and points to improved metrics of time, dynamic peaks. This LC system was developed with enhanced ultra-violet-visible spectros- ranges (number of species and concentration), resolution, physical footprint, power, copy (UV-VIS) photodiode array detectors with an increase in data acquisition rate. cost, portability, range of targets, and system integration. Resulting UHPLC chromatograms contained peak widths of about 3-5 seconds and a total separation time 1/10 of HPLC. Though an efficient and cost effective tool, 9 Monolith Versus Core Shell - Which One is Better? these detectors are not sufficient enough to fully characterize unknowns, metab- Egidijus Machtejevas, EMD Millipore, Darmstadt 64293, Germany, Karin olites and impurities encountered during drug discovery and development. Mass Cabrera spectrometry (MS) as a detector is the most precise and accurate solution to resolve In contrast to conventional particle-packed columns, monolithic silica columns are this ambiguity in LC analysis. MS detectors detect based on the mass-to-charge made of a continuous piece of high- purity porous silica. High-permeability and ratio of the analyte of interest present in the sample, therefore the masses of interest porosity of the silica skeleton and the resulting low backpressure allow for more can be analyzed simply by knowing the mass of interest and unknowns and me- flexible flow rates compared to particulate columns, enable high-throughput and tabolites can be identified using the elemental compositions predicted by the mass robust analysis without loss of separation efficiency or peak capacity even for rela- measurement. Recent advancements in MS detectors have increased resolution tively “dirty” samples. Core shell materials are also made to deliver high efficien- to identify masses to +/-5 ppm mass accuracy. But can these MS detectors detect cies at lower back pressure comparing with totally diffusive particles. Chromolith® as fast as the UHPLC can separate? Optimizing mass spectrometry parameters of HighResolution monolithic silica high-pressure liquid chromatography columns pro- the LTQ-OrbitrapTM can effectively make UHPLC compatible by maintaining speed vide very similar selectivity like core shell particulate columns. However Chromolith® without losing sensitivity or mass accuracy. This is very important in pharmaceutical HighResolution columns possess a list of superior properties compared to core shell quantitative studies when determining the quantity of analytes in a sample of inter- materials such as lower back pressure, lower carry over effect, higher load-ability, est that may or may not be present at harmful enough concentrations to patients. the possibility to speed up analysis and longer column life time. We support our statements with numerous experimental data and applications. 13 The Impact of Acquity UPLC on Dissolution Method Development: A Case Study 10 Performance Advantages of Monodispersed Porous Particles in Dante Pillon, Celgene Corporation, 86 Morris Ave., Summit, NJ 07039, UHPLC Xiaoxuan (Jason) Shen, Yali Sun Richard Henry, Supelco Division of Sigma-Aldrich, 595 North Harrison An ultra-performance liquid chromatography (UPLC) method using a Waters Ac- Rd., Bellefonte, PA 16823, William H. Campbell, David S. Bell, Gaurang quity Classic UPLC system was developed for the assay of drug product dissolution Parmar, Wayne K. Way samples. It was found that a difference in sample assay was observed between the A careful investigation of columns prepared with monodisperse, Fused-core® silica results obtained with an Acquity Classic UPLC and those generated with an Acquity particles has previously shown that an important contribution to their surprising ef- H-Class UPLC. Based on this study, it is recommended that the application of the ficiency arises from a very low eddy diffusion (multipath) term in van Deemter plots. Acquity UPLC system to dissolution sample assay should be carefully evaluated The very narrow particle size distribution (PSD) of 6% relative standard deviation for prior to its selection. 2.7-µm core-type silica particles may simply create a more consistent column bed and uniform sample flow path than particles having broader distribution. Uniform 14 Determination of Carbohydrates Using a New Integrated Capillary column beds may also preserve initial high efficiency by providing more resistance High-Performance Ion Chromatography System with to voiding and channeling under variable flow and pressure conditions. Porous silica Electrochemical Detection made by usual processes exhibit a broad size range that requires careful classifica- Jun Chen, Thermo Fisher Scientific, 445 Lakeside Dr., Sunnyvale, CA tion before efficient, stable high-performance liquid chromatography (HPLC) or ul- 94085, Petr Jandik, Yan Liu, Chris Pohl, Michael Hvizd tra-HPLC columns can be prepared. A new process has been developed that avoids A new integrated capillary high-pressure ion chromatography (HPIC) system has waste and creates silica particles that are already monodisperse and ready for use been recently developed. This new capillary Reagent-Free™ (RFIC™) system uses in HPLC or UHPLC columns. The unique process allows monodisperse silica to be deionized water to electrolytically generate high purity eluents with well controlled prepared in a wide range of porosities and diameters. If small porous silica particles concentrations. The electrochemical detector is one of the detectors integrated into having a similar, narrow PSD can be made conveniently and economically, they the capillary HPIC system. It provides a highly sensitive and selective detection of may allow preparation of columns with performance advantages much like core- electrochemically active analytes separated using the capillary ion chromatography type particles. Specifications, performance and applications for the monodisperse system. We used the capillary HPIC system equipped with pulsed amperometric de- 3 2013 EAS Abstracts November 2013

tection (PAD). PAD is one of the electrochemical detection methods used for achiev- 18 Assessment of Nasal Drug Deposition Using Anatomical Models ing high reproducibility of response over long periods of time. We obtained excellent Samiran De, Next Breath, 1450 S Rolling Rd., Baltimore, MD 21227 results for the analysis of carbohydrates in protein hydrolyzates, beverages and in a Human nasal anatomy is primarily designed to allow exchange of air while pre- number of different food samples. Because of its low flow rates (5 – 10 μL/min), cap- venting foreign particles for entering upper airways. Since drug absorption can be illary HPIC with pulsed amperometric detection offers the convenience of extremely rapid in the nasal mucosa, intranasal drug delivery is currently targeted for allergic low eluent usage (1L of water lasts ca. 2 months) and waste. An additional advan- infections, vaccinations, pain management, osteoporosis, and Alzheimer’s disease. tage is in the very small injection volumes (0.4 μL) when analyzing limited samples. Intranasal drug delivery is highly dependent on the droplet size distribution and fluid dynamics of the aerosol. An in-vitro in-vivo correlation (IVIVC) between in-vitro tests 15 Oligonucleotide Analysis with Different Liquid Chromatography typically used to evaluate performance of nasal sprays and in-vivo outcomes has Methods not been established. Because of the importance of deposition and lack of IVIVC, Mirlinda Biba, Merck, PO Box 2000, Rahway, NJ 07065, Bing Mao, many researchers have turned to nasal casts and fluid dynamic (CFD) models to Christopher J. Welch, Joe P. Foley assess new devices and/or formulations may deposit. Often used in early develop- There has been a considerable recent interest in oligonucleotide therapeutics based ment, nasal cast studies have become easier to perform with increasing ease in the on synthetically prepared short oligonucleotides in the 20-25 base pair range. These creation of nasal casts from MRI and CT scans. With rapid prototyping techniques, oligonucleotides are readily prepared by chemical synthesis. However, the products nasal casts can be machined for use in a lab setting. These casts are typically typically contain a variety of closely related impurities that can be difficult to sepa- coated with a material to simulate the mucus layer and to prevent particle bounce. rate. Therefore, an important challenge in the field of oligonucleotide therapeutics The casts can provide both a qualitative and quantitative picture of the sites of drug is the development of analytical methods that provide good separation and analysis deposition. In addition, the casts can be incorporated with impaction based tech- of these complex mixtures. In these studies, short ribonucleic acid oligonucleotides niques to quantify the mass of drug exiting the nasal cavity. A review of different (21-mers) were analyzed using different liquid chromatographic separation modes, nasal cast models and intranasal drug delivery studies is presented. including strong anion-exchange and ion-pair reversed-phase liquid chromatogra- phy. A systematic approach was used to evaluate different conditions for each sepa- 19 Development and Validation of an HPLC-MS Method for Tobramycin ration technique in order to improve the separation and analysis of oligonucleotides. in Isolated Perfused Rat Lung (IPRL) Perfusate: Applicability to Pulmonary Kinetic Studies 16 Using Chaotropic Anions for Optimizing the Retention of Basic Min Li, Virginia Commonwealth University, 410 N 12th St., PO Box: Pharmaceutical Compounds in HPLC Method Development for 980533, Richmond, VA 23298 Complex Sample Matrix in OTC Drug Products A high-performance liquid chromatography-mass spectrometry (HPLC-MS) method Hugh V. Ta, McNeil, 7050 Camp Hill Rd., Fort Washington, PA 19034, was developed and validated to quantify tobramycin at low concentrations for its Gail Reed pulmonary disposition studies in a realistic ex-vivo model, IPRL. In these studies, Multi-active, over-the-counter drug products present the analytical chemists with determination of tobramycin’s concentration, in the circulating perfusate (modified significant challenges in developing robust, stability indicating methods. Resolution Krebs-Henseleit solution (KHS) with 4% w/v bovine serum albumin) in the pulmo- of degradation products, impurities, actives, and excipient components requires nary vasculature at different time following administration to the IPRL airways, is the good chromatographic resolving power. The use of chaotropic reagents in meth- prerequisite for the investigation of its pulmonary absorption. Tobramycin samples, od development has been shown to assist in developing high-performance liquid withdrawn from the circulating perfusate, were previously deproteined with 5% v/v chromatography (HPLC) methods with improved selectivity, retention, and robust- heptafluorobutyl acid, followed by the ion pairing solid phase extraction to remove ness for basic compounds. This presentation includes method development and the buffer salts. An isocratic elution, using the mobile phase consists acetonitrile and validation for multi-active over-the-counter drug products using chaotropic anions. 0.1% v/v trifluoroacetic acid (TFA) (30:70), was employed for the chromatographic Methodology was developed for the separation and quantitation of over 25 analytes, separation by a Hypersil Gold C18 column, where TFA was used as the ion pairing including actives, excipients, and related compounds. Various columns, buffers, and agent to obtain the retention of tobramycin. A single quadrupole mass spectrom- chaotropic counter-anions (H2PO4-, CF3COO-, BF4-, ClO4-, PF6-) as mobile phases eter in positive detection mode with electrospray ionization was used for the mass were evaluated during method development. The use of hexafluorophosphate chao- detection (m/z 467). This assay method was validated to be sensitive, accurate tropic reagent gave the best separation for all the related compounds, actives, and and reliable for the study of pulmonary disposition of tobramycin with lower limit of excipients. It is demonstrated that the use of the chaotropic reagents, along with the quantitation at 50ng/ml. The accuracy, precision and extraction recovery rates at the appropriate pH adjustments and mobile phase modifier, robust, stability indicating, concentration rang of 50-5000ng/mL were found to be 89.89-102.3%, 2.6-4.0% rela- transferrable methods were developed and validated per current industry standards. tive standard deviation, and 96.5-103.7%, respectively. This method was success- fully used for the study of tobramycin’s pulmonary disposition in the IPRL system. 17 Improving Cascade Impaction Return on Investment Adrian Goodey, Merck, 556 Morris Ave., Summit, NJ 07901, Buchilingam 20 Bringing Patients into the Lab Bupathi, Xiuhua Fang, Henry Tat, Kathleen Hogan, John Jekonski, Benoit Adamo, MannKind, One Casper St., Danbury, CT 06810, Chad Jennifer Loria, Maya George, Kuriakose Jacob, Antonio Macaluso, Smutney, Spencer Kinsey Ying Li Orally inhaled dry powder delivery technologies enable the delivery of therapeutics Aerodynamic particle size distribution (APSD) is perhaps the single most important locally to the lung or systemically through the lung. The delivery format includes attribute of a pharmaceutical aerosol. However, APSD testing by cascade impaction a dry powder formulation containing the active pharmaceutical ingredient and an (CI) is notoriously labor intensive, often accounting for more than 25% of the ana- inhalation device. The combination product is intended to allow patients an easy lytical labor on an orally inhaled drug product. Here we present two ongoing efforts self-administration of medicine. Anatomical and physiologic aspects of the patient to improve the return on investment from CI testing, one which aims to reduce the dominate this delivery route. Successful development of such inhaled products links investment, and one which targets increased returns. To address the magnitude dry powder formulations, particle engineering, and in-vitro/in-vivo system perfor- of the investment, we present an abbreviated impaction (AIM) strategy designed mance. All of these components contribute to overall product performance and must to drastically reduce labor while preserving the utility of the APSD data. As with all be driven by innovative development tools to reduce risk, expedite progress, and AIM systems, labor is reduced via the removal of impactor components. However, establish the critical momentum needed to drive development efforts forward. For careful selection of the remaining components, coupled with intelligent treatment of that purpose, MannKind has developed several new technologies and techniques the resulting data, allows determination of common parameters such as material that allow efficient development of inhaled drug products. This includes MIDAS, balance, fine particle dose, stage groupings and mass median aerodynamic diam- MannKind Inhalation Data Automated Simulator, a mechanical syringe pump de- eter. Meanwhile, there is also ample room to improve investment returns by adding signed to replicate patient inhalation efforts, and BLUHALE, a wireless microphone- relevance to the test. In particular, the static flow conditions typically employed for based technology adapted onto MannKind’s dry powder delivery systems to charac- CI teach us little about product performance in the hands of a patient. To this end, terize patient inhalation. Use of anatomically correct airway models and inhalation the conventional cascade impactor was modified to allow variable air flow through simulators is encouraged during development of candidate combination products. the test inhaler, while maintaining the required constant flow through the impactor. Quantification of delivered mass resulting from varied inhalation effort and inhaler As such, analysts can design custom inhalation profiles mimicking different patient in-use position can be made and provide more meaningful insight to product perfor- populations and inhalation maneuvers, thereby adding a new dimension to tradition- mance than traditional testing. al APSD data. The insight afforded by these studies enhances our understanding of inhalers, and helps guide the development of future products.

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21 Ultrafast GC for Residual Solvents in Drug Substance Scale-Up 25 Extending the Hydrocarbon Range above Naphthalene for Soil and Manufacturing Using a Low Thermal Mass GC Vapor and Air Samples Using Automated Thermal Desorption-Gas Yuwen Wang, Boehringer Ingelheim, 900 Ridgebury Rd., Ridgefield, CT Chromatography-Mass Spectrometry 06877 Lee D. Marotta, Perkin Elmer Instruments, 710 Bridgeport Ave., Shelton, Temperature programming in gas chromatography (GC) is considered the second CT 06484, Bill Hahn, Cindy Liang most important parameter to control the selectivity of a column. A new GC technolo- Efficient methods are needed for soil vapor intrusion of toxic compounds as con- gy to achieve ultrafast temperature programming with an unprecedented cool down cerns continue for the impact to human health. The sampling is performed at con- time has recently become available. This technology is referred to as low thermal taminated sites to investigate if potentially present sub-surface volatile contami- mass GC which represents a significant break-through reducing sample analysis nants have pathways to indoor air. Through rigorous investigation, a new sampling time. Conventional GC has been used for decades to determine residual solvents tube and method have been developed. In addition to excellent recoveries of all in drug substance and intermediates. It usually takes thirty to sixty minutes long for regulated target compounds specified in methods TO-15 and TO-17, these tubes a single GC run. This is not sufficient for in-process testing, which requires a quick and method allow for the recovery of compounds greater than naphthalene specifi- delivery of results to ensure the quality of the products. Our lab has developed fast cally covering the boiling point range from C3 to C24. This recovery was performed GC methods for in-process testing using a narrow bore column which reduces the in sites with high diesel and moisture content. The unregulated contaminants in GC run time to eight minutes. Due to the slow cool down time, the GC cycle time is the diesel were easily desorbed from the tube enabling quick turnaround time for still around fifteen minutes. A low thermal mass GC was evaluated in our lab for the re-sampling. In addition, polynuclear aromatic hydrocarbons to pyrene may be determination of residual solvents for drug substance scale-up and manufacture. added to the target analyte list. Since the compound range greater than naphtha- The GC run time was reduced to two minutes for eighteen commonly used process lene were added, breakthrough experiments were performed to ensure that lighter solvents and the total GC cycle time was reduced to three minutes. The method compounds, even the most volatile gases, remained on the tube during sampling. was validated in terms of specificity, repeatability, sensitivity, recovery and linearity. In this presentation, the following are discussed: 1) data from the above recovery Examples of process samples in drug substance scale up and manufacture are also and breakthrough experiments; 2) using thermal desorption and tube technology to presented. optimize water management; 3) analytical parameters such as precision, linearity and detection limits; 4) optimizing productivity by using tube technology; 5) using 22 Chiral Constituents and Enantiomeric Composition in the Essential these tubes for most air sampling. Oil Obtained from Artemisia Vulgaris L. Using Selectable 1-D/2-D Gas Chromatography/Mass Spectrometry 26 Petroleum Biomarker Analysis by Atmospheric Pressure Gas Jack D. Williams, Mercyhurst University, 501 East 38th St., Erie, PA Chromatography Tandem Mass Spectrometry (APGC-MS-MS) and 16546, Tianyue Xie, Michael A. Campbell, Michael C. Jaskolka Electron-Impact Ionization Tandem Mass Spectrometry (EI-MS-MS) Identification of the chiral constituents present in the essential oil obtained by hydro Chang S. Hsu, Florida State University, 2525 Pottsdamer St., distillation of the aerial parts of Artemisia vulgaris L. is reported for the first time. Tallahassee, FL 32310, Quan Shi, Douglas M. Stevens Analytes were detected using a selectable one-dimentional / two-dimentional gas Through numerous science evidence, petroleum is generally believed to be derived chromatography mass spectrometry system. Enantiomers were identified by heart from ancient living organisms buried in oxygen depleted environment under thermal cutting from a DB5-MS column to a Cyclodextrin-B chiral column. stress over geological time. A class of compounds that retain basic carbon skeleton of the original living organism is known as petroleum biomarkers. Their isomeric 23 Identification of Impurities and Recovery of Starting Material from structures and relative abundances reveal the history of geothermal conversion Industrial Distillation Bottom from y- Butyroactone Production of the living organisms into petroleum, through depositional environment, thermal Bala Balasanmugam, Ashland Inc., 1361 Alps Rd., Wayne, NJ 0470, stress by tectonic movement, compartmentation/mixing, and exposure to air, water Joycelyn Yapchulay, Maria Diss and bacteria. These geophysical and geochemical factors lead to changes in the bio- Unreacted residual chemicals and by-products are sometimes considered waste in marker profile as well as the bulk composition of oils in source rocks and reservoirs. a manufacturing environment. However, if these waste chemicals are identified they The oil/oil and oil/source rock correlation of biomarker profiles have been used in can be used to regenerate valuable pure starting materials. The cost savings from petroleum discovery, exploration and exploitation. Biomarkers are generally present this type of recovery are very significant for chemical industries. γ- Butyroactone is in crude oils and source rocks in relatively low abundances. This leads to the need produced from 1,4-butanediol in the presence of a catalyst. After the product is dis- for selective and sensitive analytical techniques for biomarker analysis. Steranes tilled, a fair amount of unreacted 1,4-butanediol along with other side products end and hopanes have been commonly used in the biomarker analysis due to their sta- up in the bottom of the distillation unit. Identification of these chemicals in the bottom bility and richness in geochemical information. In early days, 70-eV electron-impact residue is rather important if one wants to recover useful starting materials from it. ionization in combined gas chromatography-mass spectrometry (GC-EIMS) was This presentation discusses the identification of two major impurities present in the used for highly repeatable results for geochemical studies and correlations. Later, residual material using gas chromatography mass spectroscopy. Positive identifica- GC coupling with tandem mass spectrometry (MS-MS) was developed for specificity tion of these compounds was carried out by synthesis of the appropriate impurities among geochemically critical isomers. Recently, we have developed atmospheric and regenerating the mass spectra. pressure chemical ionization as GC ion source (APGC) which by collision-induced dissociation yields similar MS-MS results as 70-eV EI MS-MS. This presentation 24 Ionic Liquid Submerged Single Drop Microextraction and Static discusses further studies of APGC under different cell collision energy functions Headspace Single Drop Microextraction of Aromatic Hydrocarbons for the reproduction of the MS-MS results under EI. Furthermore, the comparison from Water of sensitivity and selectivity among different biomarker classes is also discussed. Ramkumar Dhandapani, Seton Hall University, 400 South Orange Ave., South Orange, NJ 07079, Nicholas H. Snow 27 An Approach to Quantifying Petroleum Ether in Active The interest of using ionic liquids as gas chromatographic stationary phase has Pharmaceutical Ingredients increased in recent years. The low volatility, tailor-able physico-chemical property Jana Stavova, Bristol-Myers Squibb, One Squibb Dr., New Brunswick, and high thermal stability of ionic liquids make them an ideal choice for gas chro- NJ 08903, Curtis Tinker, Michelle Kubin, William Fish matographic stationary phase. For the present research, commercially available Petroleum ether is used in the pharmaceutical industry during drug substance ionic liquid capillary columns were investigated for thermodynamics and kinetics of manufacturing as a less costly alternative to n-heptane. Petroleum ether is a distilla- the retention of alkanes and aromatic hydrocarbons. Van ’t Hoff plot was made and tion fraction of petroleum, consisting of C5-C7 alkane isomers, whose composition the thermodynamic parameters ΔG, ΔH, ΔS were determined. The thermodynamic varies by vendor and region. This multi-isomeric composition may contain class parameters of SLB-IL 61 were compared with a conventional polyethylene glycol 2 solvents, e.g., n-hexane, posing a health concern to patients. The International based stationary phase having same Rohrschneider-McReynolds constants. A plot Conference on Harmonization (ICH) guidelines for residual solvents limits the class of retention factor was made against claimed polarity of the ionic liquid columns. 2 solvents in pharmaceutical products due to their toxicity. The imposed limits for The plot revealed that change in the claimed polarity had no definite relationship class 2 solvents (2.9 mg/day for n-hexane) are lower than for less toxic class 3 sol- with the change in the distribution constant of analyte for these columns. This shows vents (50 mg/day for n-heptane). Even though petroleum ether may contain class 2 that Rohrschneider-McReynolds constants by themselves do not predict the actual solvents, it is not listed as a class 2 solvent in the ICH guidelines. In this presenta- polarity of these columns and the five probe molecules used for ascertaining the tion we discuss a recent project where petroleum ether was employed during drug McReynolds constant do not account for all the possible interactions. A Van Deem- substance manufacture. Results are demonstrated for the identification of individual ter plot was made for SLB-IL 100 using aromatic hydrocarbons as probe analyte petroleum ether components by gas chromatography (GC) flame ionization detec- and air as career gas. This kinetic study revealed the possibility of using ionic liquid tion and GC-mass spectrometry to determine class 2 solvent risk. Additionally, the stationary phase with air as career gas. The stability of ionic liquid stationary phases analytical testing strategy, which includes the use of a hexanes identification solu- in the presence of oxygen in air may significantly expand their application.

5 2013 EAS Abstracts November 2013

tion and quantitation using n-hexane, to mitigate class 2 solvent risk is described. 31 Supercritical Fluid Chromatography: Breaking out of the Finally, the key results from method development, validation and batch testing are Pharmaceutical and Chiral Paradigm shared. Terry Berger, SFC Solutions, Inc., 9435 Downing St., Englewood, FL 34224 28 Evaluation of Non-Conventional Polar Modifiers on Immobilized In 1989 pharmaceuticals accounted for less than 10% of supercritical fluid chroma- Chiral Stationary Phases with Supercritical Fluid Chromatography tography (SFC) published applications, including chiral separations, which account- (SFC) ed for less than 4% of the total. Today, pharmaceutical applications account for over Jimmy DaSilva, Merck, 126 E. Lincoln Ave., Rahway, NJ 07065 90% of SFC, and chiral, both at the analytical and semi-preparative scale, accounts As the diversity of drug intermediates and active pharmaceutical ingredients (APIs) for most of that. Many of the older applications employed pressure programming increase, solubility has become a big concern. These compounds are less and less and/or the flame ionization detection. The latest generation of SFC equipment offers soluble in the conventional solvents used in chiral supercritical fluid chromatography neither of these features. However, this doesn’t mean that many of those older ap- (SFC) like methanol, ethanol, 2-propanol, and acetonitrile and in carbon dioxide plication areas can’t be addressed by modern equipment. The low dispersion and itself. Chlorinated solvents have typically been used to aid in the dissolution of sam- high UV sensitivity of recent instruments should extend the applications of SFC back ples with poor solubility characteristics. Only a small percentage, usually 10-20% of into some of these neglected application areas. Examples of ultra high-performance chlorinated solvent in the diluent, can be used with standard coated chiral columns SFC, for the analysis of polymers, natural products, vitamins, food additives, and without washing the coating away. In some cases the chlorinated solvent has been residues in foods are presented. added, again at a very small percentage (< 5%), to the mobile phase to increase sol- ubility. However, the coated chiral stationary phases are not compatible with many 32 Unattended Method Development for Optimizing Selective solvents because the phase is only coated onto the surface of the silica rather than Supercritical Fluid Extraction immobilized onto the silica. Use of non-conventional polar modifiers can cause the John A. MacKay, Strategic Technology, Waters Corporation, Milford, MA coating to be washed away. In this presentation we discuss the systematic investi- 01757, Jeffery Wright gation of 11 non-conventional solvents and mixtures thereof with methanol using six Supercritical fluid extraction (SFE) is a powerful tool for selective isolation of key immobilized chiral stationary phase columns from chiral technologies and nine com- components in samples. The parameters of pressure and temperature over a spe- mercially available racemates. We demonstrate the utility of using non-conventional cific amount of time, impact the classes of compounds extracted from the sample of polar modifiers for adjusting retention times and to address solubility issues for both interest. By adding of co-solvent, such as ethanol, with the supercritical CO2, it pro- analytical and preparative supercritical fluid chromatography. vides even more fine tuning of an extraction. During this time we demonstrate how to optimize these three parameters for an extraction with unattended method devel- 29 Development of a Strategy to Transfer SFC Methods from Analytical opment. The power of programing static and dynamic extraction is also reviewed. to Preparative Scale Christopher J. Hudalla, Waters, 34 Maple St, Milford, MA 01757, Abhijit 33 Utility of NMR in the Development of Protein Drugs Tarafder, Kenneth J. Fountain Luciano Mueller, Bristol Myers Squibb, Route 206 and Provinceline

For separations using CO2 as the principal component of a mobile phase, analyte Rd., P.O. Box 4000, Princeton, NJ 08543, Brian O’Mara, Matt Conover, retention factors are influenced largely by the mobile phase density. Because of the James Tamura, Yongmi An, Nelly Aranibar, Sam Bonacorsi, John

compressibility of CO2 under standard operating conditions, the density can change Devincentis, Kai Cao, Brad Maxwell significantly with changes in pressure (under isothermal conditions) with retention New applications of protein nuclear magnetic resonance (NMR) in support of biolog- factors decreasing with increasing mobile phase density (pressure). In addition, the ics programs have emerged at Bristol-Myers Squibb. These new uses of NMR are selectivity and resolution of the analytes may be impacted as they respond differ- discussed. Protein NMR has been employed to monitor protein refolding kinetics of ently to the same changes in mobile phase density. This can be challenging when E. Coli expressed inclusion bodies. In addition, NMR was employed in optimizing attempting to transfer methods between different column configurations that involve refolding buffers of adnectin-based biologics. NMR also found its use as a quality changes in column length or stationary phase particle size, which in turn alters the control tool whereby subtle difference in biologics production processes could be pressure (density) profile for the separation. This is best exemplified when analytical delineated. Most recently, NMR was employed in analyzing mutants of anti ILx. scale separations developed using UltraPerformance Convergence Chromatogra- phy (UPC2), employing sub-2-µm stationary phases, are scaled up for preparative 34 Regulation of Protein Tyrosine Kinase Signaling in Bacteria supercritical fluid chromatography (SFC) conditions, employing 5-µm particle size Ranajeet Ghose, The City College of New York, 160 Convent Ave., New stationary phases. The difference in the density profiles across the column can lead York, NY 10031 to very different chromatography unless the scale-up procedure is guided by a sys- Bacterial tyrosine kinases (BY-kinases), and their cognate tyrosine phosphatases, tematic approach. Here we present a strategy for scaling SFC separations between form counteracting enzyme pairs that regulate diverse physiological processes in various column configurations. This is demonstrated for the transfer of methods both Gram-negative and Gram-positive species, most notably the biosynthesis and between analytical columns of different configurations, and more interestingly, for export of polysaccharides responsible for the formation of virulence-determining methods developed under analytical UPC2 conditions with subsequent scale up to capsules or biofilms. BY-kinases contain a cluster of tyrosine residues (YC) at their preparative SFC conditions. This ability enables the rapid screening of methods on C-terminus that is autophosphorylated and subsequently dephosphorylated by a the faster analytical scale, with the direct transfer of the final method to preparative tyrosine phosphatase. This phosphatase-mediated phosphorylation/ dephosphory- chromatography, resulting in significant savings in time and mobile phases. lation cycle in BY-kinases, rather than the quantitative phosphorylation state of the YC, is essential for polysaccharide production. Despite extensive genetic and bio- 30 Use of Supercritical Fluid Chromatography to Improve Efficiency chemical evidence of phosphatase-catalyzed YC-dephosphorylation in BY-kinases, of Medicinal Chemistry Purification the specific nature of their mutual interactions, and the regulatory mechanisms Larry Miller, Amgen, 360 Binney St., Cambridge, MA 02142 that govern dephosphorylation, are unknown for any BY-kinase/phosphatase pair. Over the past years supercritical fluid chromatography (SFC) has become an es- We have applied solution nuclear magnetic resonance (NMR) methodology to the tablished technology for analysis and purification in the pharmaceutical industry. canonical BY-kinase, Escherichia coli Wzc, and its corresponding tyrosine phos- With SFC a majority of the solvent in the mobile phase, usually greater than 60%, phatase, Wzb, to provide the first detailed insight into the regulatory interactions is supercritical CO2. The low viscosity and high diffusivity of the SFC mobile phase between a BY-kinase and its opposing phosphatase. allows higher flowrates relative to high-performance liquid chromatography, result- ing in shorter analysis times and increased throughput. For purification the higher 35 Molecular Dynamics Revealed: A 2H Solid-State NMR Investigation flowrate reduces time for purification and the incorporation of CO2 in the mobile Bernie O’Hare, Bruker Biospin, 19 Fortune Dr., Billerica, MA 01821 phase greatly reduces solvent requirements, making it a green technology. This pre- All 2H nuclear magnetic resonance (NMR) powder patterns are not created equal! In sentation discusses the utilization of SFC for medicinal chemistry scale purifications fact some powder patterns, based on their motional symmetry, are often mistaken to improve productivity and reduce costs. While preparative SFC has been utilized for isotropic NMR signals. This can lead to costly mistakes and misunderstandings. for 10+ years for chiral purifications, over the past few years its use has expanded 2H NMR is well suited for studying molecular motion for a number of reasons. Be- to achiral purification as well as library purifications. A number of examples from the cause of its sensitivity to motion, 2H NMR can be used to characterize motions with laboratories of Amgen are presented to demonstrate the improvements in purifica- frequencies ranging from ~ 1 s-1 < ν < 1013 s-1. During this talk, both low and high tion capacity and cost through the use of SFC. symmetry motion are discussed as well as 2H relaxation and powder pattern analy- sis. A thorough analysis of motions less than, equal to and greater than the 2H QCC interaction is given. Finally, examples of how one can use this information to answer questions in the materials and pharmaceutical science areas are also covered.

6 2013 EAS Abstracts November 2013

36 Anisotropic Interactions in MAS Solid-State NMR Spectroscopy: py measurements of the hyperfine couplings in the S2 state of the OEC of PSII. We Methodology Development and Applications in Protein Assemblies also conduct a detailed comparative study of the electronic and geometric structures Guangjin Hou, University of Delaware, Department of Chemistry, of the OEC with other natural and artificial hydroxo- and peroxo-splitting complexes. Newark, DE 19716, Alexander J. Vega, Sivakumar Paramasivam, Si This study is supported by the Photosynthetic Systems Program, Office of Basic Yan, Christopher L. Suiter, Huilan Zhang, Ago Samoson, In-Ja L. Byeon, Energy Sciences, United States Department of Energy (DE-FG02-07ER15903). Jinwoo Ahn, Angela M. Gronenborn, Tatyana Polenova Solid-state magic angle spinning nuclear magnetic resonance (NMR) spectroscopy 39 Reaction of Hydrazine with Cl-Terminated Si(111) Surfaces is rapidly emerging as the main technique for structural and dynamics studies of Fei Gao, University of Delaware, Department of Chemistry, Newark, DE large proteins and protein assemblies inaccessible to other methods. Dipolar inter- 19716, Andrew Teplyakov action and chemical shift anisotropy are the primary NMR observables that yield in- This work focuses on obtaining well-defined surface of silicon functionalized with formation about geometric and electronic structure as well as dynamics of biological hydrazine to produce an oxygen-free platform for further functionalization. Single and inorganic solids. However, most of the currently existing recoupling sequences crystalline Si(111) surface has been prepared using modified RCA procedure to work at slow or moderate MAS frequencies, and also the determination of the 1H produce a well-defined H-terminated Si(111) surface. Next, Cl-terminated Si(111) chemical shift anisotropy (CSA) tensors remains challenging due to strong 1H-1H ho- surface is prepared from H-terminated Si(111) surface using PCl5 in chloroben- monuclear dipolar interactions. Here, we introduce several families of rotor-synchro- zene solvent with trace amount of benzoyl peroxide as a reaction initiator under nized R-type symmetry pulse schemes, for dipolar and chemical shift anisotropy nitrogen atmosphere following previously established procedures. Hydrazine-func- (CSA) recoupling, and for spin diffusion experiments.[1-4] These sequences work at tionalized Si(111) sample is obtained from Cl-Si (111) surface with either anhydrous a wide range of MAS frequencies, including fast and very fast MAS conditions, and hydrazine at 30 °C or 1M hydrazine solution in tetrahydrofuran at 25 °C under Ar are readily incorporated in multidimensional experiments permitting site-resolved atmosphere. To confirm the presence of Si-N bonds following this procedure and measurements in uniformly, extensively, or sparsely isotopically enriched samples. to establish the structures of surface species produced, we followed the reaction These symmetry sequences have been examined experimentally and by numerical by Fourier-transform infrared spectroscopy, X-ray photoelectron spectroscopy, and simulations, and excellent performance has been demonstrated in peptides, pro- time-of-flight secondary ion mass spectrometry. Density functional theory (DFT) teins, and protein assemblies. calculations were performed to infer the mechanisms of surface reactions and to compare the predicted vibrational spectra and core-level energies with the results References: of experimental studies. [1] Hou G., Byeon I., Ahn J., et al., J. Am. Chem. Soc., 2011, 133, 18646. [2] Hou G., Byeon I., Gronenborn A., et al., J. Chem. Phys. 2012, 137, 134201. Towards Formation of Oxygen-Free Interface by Cyclocondensation [3] Hou G., Paramasivam S., Yan S., et al., J. Am. Chem. Soc., 2013, 135, 1358. 40 on H-Terminated Si(111) Surface [4] Hou G., Yan S., Sun S., et al., J. Am. Chem. Soc., 2011, 133, 3943. Yuexing Cui, University of Delaware, Academy St. and Lovett, 113 Lammot DuPont Lab, Newark, DE 19716, Fangyuan Tian, Andrew Molecular Insights into the Recognition of Cellular Membrane 37 Teplyakov Geometry Hydrogen terminated Si(111) surfaces (H-Si(111)) were modified using nitroben- Fang Tian, The Pennsylvania State College of Medicine, 500 University zene and nitrosobenzene to yield a well-defined interface based on Si-N bonding. Dr., Hershey, PA 17033 H-Si(111) is obtained from n-type Si(111) with RCA cleaning and etching procedure. Many essential cellular processes such as endocytosis, vesiculation, organelle H-Si(111) sample is heated in either neat nitrobenzene at 100 °C, or in nitrosoben- synthesis and cell division require transient membrane deformations. Recently, it zene solution in tetrahydrofuran at 65 °C under nitrogen atmosphere. Nitrobenzene was discovered that the activity of some proteins (e.g., human ArfGAP1) dramati- reaction on H-Si(111) follows cyclocondensation mechanism demonstrated previ- cally increases with the curvature of the membrane bilayer, and that membrane ously. Nitrosobenzene reaction was expected to follow similar set of reaction steps curvature could serve as a geometric cue for the subcellular localization of some to produce an oxygen-free interface as a result of reaction; however, this process proteins (e.g., SpoVM and DivIVA). In addition, the entry of some bacteria and virus is complicated by dimerization of this compound. The differences in surface reac- into human cells also requires alteration of membrane shapes. Membrane geom- tions of nitrobenzene and nitrosobenzene with H-Si(111) were followed by Fourier- etry is increasingly viewed as a critical component for creating microenvironments transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), for membrane fusion and fission, protein localization, trafficking and signaling. This and time-of-flight secondary ion mass spectrometry. Density functional theory stud- underscores the importance of understanding the molecular mechanisms respon- ies were used to establish surface reaction mechanisms and to verify experimental sible for the generation, recognition, maintenance, and regulation of membrane observables recorded by FTIR and XPS. architecture. SpoVM, a 26-residue peptide, was recently found to recognize and preferentially localize to the slightly curved outer surface of the forespore (diameter Probing the Orientation of 2,3-Dichloro-1,4-Naphthoquinone on of curvature, R, ~1 mm) during Bacillus subtilis spore development. [1] However, 41 Gold Nano-Rods and Ag Colloids little is known about how this is accomplished. SpoVM was predicted to exist as a Maraizu Ukaegbu, Howard University, 2400 Sixth St. NW, Washington straight, amphipathic -helix that shallowly inserts into the membrane surface. How- α DC 20059, Oladapo Bakare, Charles Hosten, Alberto Vivoni ever, using solution nuclear magnetic resonance (NMR), we have found that the The mode of interaction between 2,3-dichloro-1,4-naphthoquinone (DNQ) and Au SpoVM molecule adopts a loop-helix structure and that the helix deeply embedded nano-rods and Ag colloids was probed using surface enhanced Raman scattering into bicelles. This model has provided new structural insights into SpoVM function. (SERS) and, FT-Raman spectroscopy. Interpretation of the SERS data required a We are extending this study with new model membranes that are similar in curva- complete assignment of the vibrational modes of 2,3-dichloro-1,4-naphthoquinone ture and lipid composition to that of the B. subtilis forespore using solid-state NMR. and this was performed using B3LYP/6-31G (d) and potential energy distribution Reference: calculations. SERS spectroscopy was useful in determining the adsorption and ori- [1] Ramamurthi, K.S., Lecuyer, S., Stone, H.A. and Losick, R. (2009) Geometric cue entation of the monolayer as well as estimating its order. By analyzing the relative for protein localization in a bacterium. Science 323: 1354-1357. band intensities and using surface selection rules for SERS, the orientation of the adsorbed DNQ was determined. The red shift of the weak Raman band at 397 cm- 38 The Electronic Structure of Manganese Catalysts for Hydroxo- and 1in solution (solid; 384 cm-1) to 370 cm-1 in the SERS spectrum suggests that DNQ Peroxo-Splitting Reactions is adsorbed onto the nanosurfaces. The bands at 384 and 476 cm-1 were assigned K.V. Lakshmi, Rensselaer Polytechnic Institute, Department of to C-C=O and ring I/II bending vibrations respectively. The SERS intensity of the Chemistry, Troy, NY 12180, Sergey Milikisiyants, Christopher Coates, C=O stretch band (1670 cm-1) was reduced, while a strong C-C-Cl vibration was Ruchira Chatterjee, James Whittaker, Faisal Koua, Jian-Ren Shen observed at 243 cm-1 compare to the solution. This suggests that the adsorbate/ sur- The recent 1.9 Å resolution X-ray structure of Photosystem II (PSII) [Umena et al. face interaction is via one of the C=O. The ring, C-C=O bending modes at 476 and Nature, 2011,473, 55] provides a detailed molecular geometry of the oxygen-evolv- 370 cm-1 were enhanced by factors of 6 and 4.13 respectively in the SERS spec- ing complex (OEC) in the dark stable S1 state. Despite the remarkable breakthrough trum relative to the band at 1670 cm-1. This suggests that the adsorbed DNQ is ad- in the resolution achieved by the new X-ray crystal structure of the OEC in the S1 sorbed with a tilted orientation on the surface of the gold nano-rods and Ag colloids. state, its structure in the higher oxidation states remains largely unknown. Also, there is limited knowledge of the electronic structure of the tetra-nuclear manganese

calcium-oxo (Mn4Ca-oxo) cluster in the OEC of PSII. Weak magnetic interactions between the paramagnetic Mn4Ca-oxo cluster and surrounding magnetic nuclei are very sensitive to both the molecular geometry and electronic structure of the OEC. Two-dimensional hyperfine sublevel correlation (HYSCORE) spectroscopy is a powerful technique to resolve hyperfine interactions in multinuclear paramagnetic centers, such as, the OEC of PSII. In this study, we describe HYSCORE spectrosco-

7 2013 EAS Abstracts November 2013

42 Ultrafast Microscopy of Single CdSSe Nanowires 45 Terahertz Spectroscopy Advancement to a Broadband Source: Peter S. Eldridge, University of Delaware, Department of Chemistry and Pushing the Limits of Vibrational Spectroscopy and Time-of-Flight Biochemistry, 105 Lammot DuPont Laboratory, Newark, DE 19716, Jolie Analysis C. Blake, Lars Gundlach David A. Heaps, Advantest America, 508 Carnegie Center, Ste. 102, A substantial challenge in optical studies of nanostructured materials is the inherent Princeton, NJ 08540, Eiji Kato, Edward E. King, Richard McKay, Mark inhomogeneity of size, morphology and composition. The resulting distribution of Sullivan, Akiyoshi Irisawa, Motoki Imamura static properties and the dynamic response to external stimuli severely limits the in- A Cherenkov-type THz radiation emitter based on frequency conversion from a formation which may be reliably extracted from ensemble averaging measurements. nonlinear crystal overcomes the output limitations caused by strong self-absorption

We present ultrafast wide-field microscopy measurements utilizing the recently de- in photoconductive antennas (PCA). We report here the development of a LiNbO3 veloped femtosecond Kerr-gated microscope [1] on single CdSSe nanowires. II-VI Cherenkov source with an exceedingly narrow pulse width of 146 fs and ultra-broad- ternary semiconductor materials are of growing interest in the fields of photovoltaics band spectral range of 0.5 to 7 THz (~16.7 to 233 cm-1). The Cherenkov THz wave and optoelectronics. CdSxSe1−x nanoparticles can be prepared in a variety of dif- generation has efficiency improvements over the more commonly used PCA result- ferent shapes and dimensions and offer widely tunable optical properties.[2] How- ing in improvements in spectroscopic data collection. The Cherenkov THz pulse is ever, they are known to exhibit complex photodynamics and carrier recombination narrower than the PCA pulse allowing for analysis of thinner layers in time-of-flight kinetics. The ability to collect and spatially resolve the femtosecond carrier dynam- type measurements. A minimum of 10-µm of silicon is measured in a layered silicon ics of individual emitting objects enables insights which are impossible to obtain in insulator chip with the Cherenkov source. In comparison the PCA source was able an ensemble measurement. The improved resolution of our design for a femtosec- to measure only to a thickness of 30-µm. ond wide-field Kerr-gated microscope enabled us to study ultrafast time-resolved luminescence dynamics of different regions on a single CdSSe nanowire. At high 46 Sequential Electrochemical Reduction Analyses as a Tool for excitation powers several regions with high emission intensity were identified. In Surface Evaluation contrast to earlier measurements these hotspot are not restricted to the tips of the Peter Bratin, ECI Technology, 60 Gordon Dr., Totowa, NJ 07512, Michael wire. The dynamics of these hotspots was measured simultaneously under identi- Pavlov, Eugene Shalyt cal experimental conditions. The different spots on the nanowire showed distinct State of surface oxidation and modification has seen extensive studies; however temporal dynamics that can be related to structure and composition by comparison with the advancement of high-technology industries, the need has become more to SEM and TEM measurements. This allows identification of the prevailing param- important. Electron microscopy, auger and electron spectroscopy for chemical anal- eters that influence the carrier dynamics and lead to luminescence enhancement ysis (ESCA), X-ray photoelectron spectroscopy (XPS), focused ion beam (FIB) and in specific areas. various other methods and their modifications are very powerful and used quite often in the research environment, they are however quite expensive and time con- References: suming for more routine investigations and production environments. Sequential [1] L. Gundlach, P. Piotrowiak, Opt. Lett. 33, 992 (2008). electrochemical reduction analysis (SERA) was developed to study oxidation state [2] A. Pan, H.Yang, R. Liu R. Yu, B. Zou, Z. Wang, J. Am. Chem. Soc. 127, 15692 of the SnPb protective coatings to predict solderability of printed circuit boards (2005) (PCB). SERA has seen increasingly wider use for solderability of not only the SnPb 43 Merging Methods - Correlating Structure and Chemistry with Light coatings, but mainly the lead-free alternatives, such as electroplated and immersion Microscopy, SEM and XPS Imaging Sn, Ag, Au, Pd and Bi, OSP and Cu. Original method was expanded from study of Peter Eastman, The Dow Chemical Company, 727 Norristown Rd., the reducible species such as oxides and sulfides to oxidizable coatings and con- Spring House, PA 19477, Michaeleen Pacholski taminants, or even chemically oxidizable and reducible species. Use of the method We have recently been challenged with measuring the distribution of an olefin poly- was expanded from the original area of solderability, to evaluation of surfaces in mer on a fibrous cellulose sheet. A combination of analytical techniques was em- semiconductor processing, oxidation and protection of thin wires (15µm) for wire- ployed to obtain both chemical and spatial information. Stitched composite scanning bonding, epoxy bonding and environmental applications, such as air quality and electron microscope (SEM) images with relatively low magnification but with high corrosion. SERA equipment is significantly simpler than Auger or ESCA equipment, pixel resolution were used to obtain a wide field of view with high spatial resolution. however it allows for virtually limitless possibilities when it comes to study the sur- For chemical imaging, a number of methods were investigated and X-ray photo- faces. It provides complimentary information to these techniques, with simpler set- electron spectroscopy (XPS) imaging was selected as a technique capable of ob- up and much higher throughput. This presentation focuses on novel approaches in taining chemical distributions over the desired fields of view (1 mm-3 mm). Among electrochemical analysis of various surfaces as used in semiconductor and printed the challenges met and solved were understanding the distribution of carbon-rich circuit manufacturing, as well as examples from environmental studies. chemistries on non-uniform organic substrates and accurate registry of the SEM Environmental Analysis for Cultural Heritage images with the XPS images. This proved difficult because of differences in scale, 47 John Scott, New York Conservation Foundation, 261 Fifth Ave., Rm. rotation and image contrast as well as the issue that many of the traditional registry 2000, New York, NY 10016 methods, such as marking with ink or gluing markers to the surface, are ill-suited No abstract submitted by author. to use on absorbent cellulose. The method that proved successful was to align relatively low magnification light microscope images from a stereoscope with optical ATP Testing to Determine if Cultural Artifacts are Contaminated images captured directly in the XPS instrument. These images were then used as a 48 with Active Biological Materials “bridge” to align the higher magnification SEM and XPS images. With this method, Jessica Keister, Conservation Center for Art & Historic Artifacts, 264 deposited polymer and chemical information were correlated with high spatial ac- South 23rd St., Philadelphia, PA 19103, Jessica Silverman curacy. Composite images showing the chemical information as colored overlays on In late October 2012, Hurricane Sandy caused major flooding to huge areas of the SEM images were generated to clearly display the correlation. coastal New York and New Jersey. Storm flooding also caused extensive damage to materials held by archives, galleries, and other cultural institutions throughout the Polarized Raman Spectra of Single Isolated Strained Fibers of 44 Mid-Atlantic. The hurricane-damaged archives of a New York City fine art gallery Cellulose were subsequently brought to the Conservation Center for Art & Historic Artifacts Fran Adar, HORIBA Scientific, 3880 Park Ave., Edison, NJ 08820, Craig (CCAHA) in Philadelphia, Pennsylvania for salvage. Reports reached Philadelphia Clemons, Umesh Agarwal of heavy metal, organic, and biological contaminants present within the hurricane Polarized Raman spectra of single isolated fibers of cellulose derived were recorded flood waters. Safety precautions were taken during the initial salvage, but these on the Aramis with the 633nm HeNe laser excitation with the goal of monitoring reports raised questions not only in further salvage, but for use of the archive mate- frequency shifts and/or changes in polarization ratios with strain. In the case of rials once returned to the gallery. To this end, water samples were collected during frequency shifts the changes are due to changes in interatomic spacing or bond subsequent treatment of the photographic materials in the collection. They were angles. In the case of changes in polarization ratios, the changes are due to re- analyzed using the industrial water ATP Test, which detects actively growing micro- orientation of functional groups with respect to the fiber axis. For example, the di- organisms by tagging the adenosine triphosphate (ATP) present within all bacteria, rections of the OH and CH bonds can change significantly when the molecule is mold, fungi, and algae. The ATP test is a firefly luciferase assay, producing digital under strain, and this type of change would affect the polarization ratios. These output roughly quantitative to the biological mass present within the sample. This observations are discussed within the current model for crystalline cellulose with paper outlines the salvage operation and safety precautions taken during the sal- suggestions as to how strain in supported in this material. vage operation, as well as the ATP testing and implications for future use of the archival materials.

8 2013 EAS Abstracts November 2013

49 The Influence of Select Pigments and Aging on the Detection of 52 Effect of Orientation on the Dichroism of Dyed Fibers Proteinaceous Materials with ELISA Dale K. Purcell, Chaminade University of Honolulu, 3140 Waialae Ave., Natalya Atlasevich, Metropolitan Museum of Art, 1000 Fifth Ave., New Honolulu, HI 96816, John A. Reffner, Thomas A. Kubic, Fran Adar York, NY 10028, Brian Baade, Caroline Tokarski, John Loike, Julie Textile fibers, both synthetic and natural, tend to exhibit molecular orientation. This Arslanoglu orientation is easily documented by recording polarization-dependent Raman spec- The accurate identification of protein sources in paint binders and glues significantly tra. However, a more widely used measurement of fibers, especially for forensic contributes to the preservation and technical study of artworks. Recently, enzyme- applications, is that of their absorption spectra. The application of absorption mea- linked immunosorbent assay (ELISA) has emerged as a promising technique for surements for forensics is for comparing trace evidence with source material. These protein analysis in art because it can detect low abundant proteins and gums that measurements exhibit unexpected challenges. Due to the inhomogeneous distribu- are present in complex mixtures.[1-2] However, several experiments also suggest that tion of the dyes, the absolute values for absorbance may not offer a characteristic pigments and aging may interfere with the ELISA response.[3-4] Thus, it is important value. In addition, differences in orientation of one fiber compared to another may to fully characterize the technique before it can be adapted for practical applications produce different spectra, even though the fiber and the colorant are identical. How- in cultural heritage institutions. In these experiments, a series of replica paints are ever, the measurement of the dichroism of the absorbance can produce meaningful prepared and artificially aged to study their impact on ELISA. Preliminary results[3] results, because the dichroism is a ratio measurement, and is less sensitive to varia- suggest that certain pigments such as chalk and verdigris reduce protein detection tions in dye concentrations. We show non-polarized spectra of a fiber as a function by ELISA to a greater degree than pigments such as lead white. Furthermore, pig- of angle, demonstrating the sensitivity to misorientation, and then the dichroism of ment interference is protein specific, where ovalbumin detection in whole egg is the same fiber. more susceptible to pigment interference than borax casein, when both systems are mixed with chalk. Proteomic techniques will also be used to characterize pigment 53 Luminol for Blood Detection - Should It Be an Instrumental and protein interactions on the molecular level, with a long term goal to identify Method? stable markers for proteinaceous material detection in artworks. John Allison, The College of New Jersey, PO Box 7718, Ewing, NJ 08628, Nicole Renkel, Samantha Nowak References: The luminescence that results when luminol interacts with blood, bleach, or any [1] Arslanoglu, J., et al. Journal of Bioscience, 2010, 35, 3-10. number of other chemical compounds is well known in the Forensic Community. [2] Cartechini, L., et al. Acc. Chem. Res. 2010, 43, 867-876. Often, little documentation accompanies the “experiment,” except for perhaps pho- [3] Lee, H. Y., et al. manuscript in preparation. tographs of the blue light that is emitted. We propose that it would be beneficial in an [4] Duce, C., et al .Anal. Bioanal. Chem., 2012, 492, 2183-2193. investigation to collect spectroscopic data. If the intensity of the light vs. time could 50 Determining the Kinetics of Paper Degradation in Reduced Oxygen be collected, these data may be useful in suggesting whether the catalyst is indeed Environments blood, or bleach, or perhaps some transition metal salt. If the actual visible spectrum Ami L. Pass, University of Lincoln, Brayford Pool, Lincoln LN6 7TS, could be collected, the reaction could be confirmed. We have observed that in some United Kingdom, Belinda J. Colston, Barry Knight cases, if the amount of blood is relatively high, emission spectra can be skewed, Oxidative degradation is one of the main mechanisms of paper decay resulting in because blood can absorb some of the light that is being emitted. When this occurs, physical and chemical changes. As such, it is of major concern for the preservation the spectrum of the blood can be extracted from the experiment. With experimental of our paper-based heritage. Although it is known that the rate of oxidation is slowed data accompanying the observations made, the reliability of the investigator’s ob- under anoxic environments, these conditions are expensive to achieve and main- servations could be improved. Development of such a device, to use with luminol, is tain for whole collections. This research, in collaboration with the British Library, discussed and demonstrated. has evaluated the effect of various reduced-oxygen atmospheres (1–20%) on the Rapid Quantification of DOSS Oil Dispersant in White Pelican Bill kinetics of oxidative degradation, and has shown that significant rate reductions 54 Knobs Inhabiting the Shores of the Gulf of Mexico Using can be achieved. Oxidative reactions in artificially aged paper have been remotely QuEChERS Extraction and UPLC-MS/MS Analysis monitored under a range of environments using transition metal redox reactions as Aliaksandr V. Yeudakimau, University of Connecticut, 3107 Horsebarn colorimetric indicators. Samples of Iron (III) chloride-doped Whatman paper were Hill Rd., Building 4, Annex U-4210, Storrs, CT 06269, Anthony V. heat-sealed in food grade barrier film bags, with volatile organic compound (VOC) Provatas, Brianna M. Galos, Christopher R. Perkins, James D. Stuart absorbent carbon cloths added. The package was flushed with a mixture of oxygen In order to contain the oil spill that occurred in the Gulf of Mexico (GoM) in 2010, and nitrogen (1–20% oxygen) and exposed to accelerated ageing conditions for up millions of gallons of Corexit 9500 oil dispersant was applied, resulting in the mas- to 105 days. The colorimetric change was measured through the barrier film and sive application of chemicals with unknown consequences to the environment and monitored every 100h (approx.). As part of the design of their new high density stor- wildlife. Dioctyl sulfosuccinate sodium salt (DOSS) is the main component of Corexit age facility, the British Library has utilized OxyReduct® technology - a reduced oxy- 9500 and has been shown to affect the endocrine system, resulting in modulations gen fire-suppression system (14.8% oxygen). The research has shown that even at to hormone balance and embryonic development in animals. White pelicans winter these oxygen concentrations, the rate of paper degradation is significantly reduced. in the GoM and were a species known to have been impacted by the spill. While on These results have implications for future design of large-scale storage for paper- the wintering areas both sexes grow knobs on the top of their bills that fall off after based collections, indicating that fire-suppressant technologies like OxyReduct® their eggs are laid, acting as an accumulator and integrator of certain contaminants may also be used as a preventative tool. that the pelican was exposed to on the wintering grounds. DOSS was extracted from 51 Spectroscopic Document Analysis - In Pursuit of a “Forensic bill knobs using QuEChERS (quick, easy, cheap, effective, rugged, and safe), which Scanner” proved to be an effective extraction technique and is commonly used in isolation John Allison, The College of New Jersey, PO Box 7718, Ewing, NJ of organic contaminants from a variety of fruits and vegetables. The QuEChERS 08628, Kelly Wisnewski extraction technique significantly shortened the sample preparation time compared Perhaps the most substantial investigation into a document was that of the “Archi- to other traditional techniques. This method provided data quality comparable to medes Palimpsest”, a thirteenth century prayer book made from an older docu- other extraction techniques that require significantly more time to complete. The ment – by erasing the text and reusing the parchment. The erased text, written by method detection limit for the analysis of DOSS was 2.03 ng/mL based on 7 repli- Archimedes, was spectroscopically recovered. This led to the idea that, if one could cates spiked with a concentration of 10 ng/mL and quantified on ultra-performance scan a document using specific wavelengths of light, from the ultraviolet through liquid chromatography tandem mass spectrometry (UPLC-MS/MS). For the accu- the infrared, multiple images could be generated, and using “image math,” perhaps racy study the average recovery was 97% based on 4 replicates spiked with 100.0 questions about the questioned document could be answered. This presentation ng/mL and %RSD of 4.1%. discusses instruments and methods that allow such work to be done, their strengths Automating the Fractionation of Extractable Petroleum and weaknesses. Also presented is the initial concept of a forensic document scan- 55 Hydrocarbons with a Scaled Down Silica-Gel Cartridge ner, and early investigations into the approach – focusing first on distinguishing be- Brian LaBrecque, Horizon Technology, 16 Northwestern Dr., Salem, NH tween pen inks on paper. The challenge is a substantial one - to generate light with 03079, Alicia J. Cannon, William R. Jones a narrow bandwidth, from the UV through the visible, that can be used to illuminate To assess the toxicological health risk of extractable petroleum hydrocarbons (EPH) a document, and to collect images using a black and white camera, which is capable found in contaminated water and soil from leaking underground storage tanks or of responding to UV, visible, and infrared light. Finally, a powerful program for image surface spills it is important to know how much of the petroleum compounds are manipulation is discussed. aliphatic and aromatic. The aliphatic compounds (C9-C36) pose less of a health risk as compared to the polyaromatic hydrocarbons (PAHs) in the C11-C22 range. The 2004 method developed by the Massachusetts Dept. of Environmental Protection

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fractionates the extracts into the two hydrocarbon classes using manual solid phase chromatographic (HPTLC) method for the simultaneous determination of five drugs extraction (SPE) with 5 g of silica gel packed into a 20 mL cartridge. Aliphatic com- prescribed for the treatment of BPH. These drugs include the α-adrenergic blockers; pounds are eluted in the first fraction with hexane while the aromatics are collected alfuzosin (ALF), terazosin (TER), prazosin (PRZ), and doxazosin (DOX) in addition in the second fraction with methylene chloride. The fractions are concentrated down to the 5 α-reductase inhibitor; finasteride (FIN). The cited drugs were separated on to 1 mL and analyzed separately by gas chromatography flame ionization detection. TLC-silica plates using a mobile phase composed of methylene chloride: n-hexane: The manual fractionation process is prone to poor recovery and reproducibility if methanol (8.8: 0.3: 0.9, by volume). Densitometric analysis was carried out at 254 proper technique and care of the cartridge is not followed. Automation of the frac- nm for the α-blockers while FIN was measured at 220 nm. The five drugs were tionation step reduces operator error and variation while reducing time and con- detected at Rf values of 0.26 ± 0.02, 0.36± 0.02, 0.45± 0.02, 0.59± 0.02, 0.69± sumption of solvent waste. A 6 mL cartridge filled with 2.5 g of silica gel was devel- 0.02, respectively. The developed method was validated according to the Interna- oped to interface with the automated solid phase extraction instrument. To eliminate tional Conference on Harmonization (ICH) recommendations regarding; linearity, hydrocarbon extractables from the polypropylene cartridge a Teflon coated variety ranges, accuracy, precision, selectivity and limits of detection and quantification. was selected. In this presentation the optimization of the instrument method on this The proposed method showed good linearity (r > 0.9990) in the ranges; 30-350, custom silica-gel cartridge is reviewed along with the percentage recovery and the 30-350, 20-200, 30-350, 200-2000 ng/spot for the cited drugs, respectively. The quality of the fractionation on standards and real samples. applicability of the proposed method was verified through the analysis of laboratory- prepared mixtures and percentage recoveries between 98.27 and 101.97 % were 56 Double Lab Productivity Overnight: Extract PAHs and PCBs obtained. Commercial tablets were also analyzed by the developed methodology Simultaneously from Tissue and Soil Samples Using a New with no interference detected from the co-formulated excipients. The high sensitivity, Accelerated Solvent Extraction (ASE) Procedure simplicity and selectivity of the proposed method suggest its applicability for routine Greg Malek, Thermo Fisher Scientific, 1228 Titan Way, Sunnyvale, CA quality-control analysis purposes of any of the titled drugs in their pharmaceutical 94085, Peter Bodsky, Art W. Fitchett, Michael Hvizd preparations. Laboratories that are involved in food safety or environmental analysis are often dealing with many types of matrices and broad ranges of compounds. In some cas- 59 In-Situ Monitoring of Form Change as a Function of Relative es, certain solvents are used for specific analyte types, and more than one extrac- Humidity in the Solid-State by VTI-Raman tion of the same sample may be required to obtain the levels of all of the contami- Candice Choi, Bristol-Myers Squibb, One Squibb Dr., New Brunswick nants present. For example, a dichloromethane/acetone mixture is often used for NJ 08903, Duohai Pan, Denette Murphy, Anisha Patel, Roxana Schlam, polycyclic aromatic hydrocarbons (PAHs) while hexane/acetone mixtures are often Shawn Yin, Sruthi Janakiraman used for printed circuit boards (PCBs). In many cases, it would be advantageous to Understanding changes that occur as a function of water activity has become an have a single extraction procedure for both of these compound classes. Accelerated important aspect of physical characterization of the active pharmaceutical ingredi- solvent extraction (ASE) is accepted under United States Environmental Protection ent (API) in all stages of pharmaceutical product development due to the recent Agency Method 3545A for the extraction of a broad spectrum of compound classes. increase in number of compounds forming both stoichiometric and nonstoichiomet- ASE uses elevated pressures and temperatures to effect rapid extractions with ric solvates and/or hydrates. Currently, moisture sorption isotherms are acquired to small solvent volumes. We will present the results of our work to develop a single better understand the relationship between water activity and moisture content at a extraction procedure for the determination of PAHs and PCBs in soil and animal given temperature for an API form/material in the solid-state. However, the addition- tissue samples. Analysis of the PAHs was done by gas chromatography (GC) mass al complexity of API form/phase change that can occur during a sorption analysis spectrometry, and GC with electron capture was used for the PCBs. In-cell clean- can make the interpretation of the moisture sorption isotherm difficult. Integration of up procedures were used to provide interference free extracts of the animal tissue moisture sorption isotherm and Raman spectroscopy provides better understanding samples. We demonstrate the impact of adsorbent and extraction temperature on of these changes that may be occurring to the API as a function of equilibrated water analyte recovery and amount of co-extractables present in the final extract. content and/or %RH in the solid-state. This poster illustrates, via three examples, applications of VTI/Raman in the pharmaceutical field. 57 Comparison of Methods for the Determination of Asbestos Soil Surface Contamination; Pretoria, South Africa 60 The Correlation Between Actuation Profile and Dose Content Frank Ehrenfeld, International Asbestos Testing Laboratories, 9000 Uniformity: Design and Implementation of a Non-Invasive Meter Commerce Pkwy, Mount Laurel, NJ 08054, Raymond Kennedy, Kristen Dose Inhaler Actuation Profiler Goedde, Thomas Barkley Jagruti A. Patel, Merck, 556 Morris Ave., Summit, NJ 07901, Francisco This study concerns a global engineering and consulting company from the United Alberto, Paul Bjorndal, Justin Pennington Kingdom that is managing an investigation of asbestos surface contamination over Metered dose inhalers (MDIs) are a widely used dosage form for pulmonary deliv- several hundred square kilometers outside of Pretoria, South Africa. The site was ery of anti-asthmatic drugs for the treatment of respiratory diseases. Dose content affected by early mining (primarily amphibole asbestos) operations that existed from uniformity (DCU) testing is required to measure the performance of pressurized 1893 until 2001. The investigation is to be submitted to a South African govern- metered dose inhalers by determining the amount of drug delivered through the ment transportation agency that is collecting environmental assessments as part of actuator from the unit. In this test, a dose is actuated into a sample collection tube a nationwide inventory of similar sites. International Asbestos Testing Laboratories under a vacuum flow rate of 28.3 L/min. The sample is extracted and quantified us- was contracted to analyze a few hundred such samples taken from surfaces in the ing chromatography. In this research study, the traditional DCU test was performed initial test site. A random selection of samples, representing different soil types and with a modification in the actuator to incorporate a linear displacement transducer surface conditions, as well as laboratory spikes, were homogenized, split, and ana- and a single axis accelerometer. The modifications were designed to have no im- lyzed under the client’s suggested screening method (United States Environmental pact on how the analyst holds or doses the MDI device. The data was collected Protection Agency 600 R93-116), the California Air Resource Board (CARB) 435 using an in-house designed Labview application and processed within Matlab. The method, as well as the American Society for Testing and Materials (ASTM) D7521 data system records the shaking and actuation profile and can be configured to soil method. A total of 56 samples in the several matrix categories were analyzed display the results real-time to the analyst. The doses were subsequently extracted by the three methods for a total of over 400 data points. The analytical data detail and analyzed to provide DCU recovery values for each unique collection profile. The the precision of the methods as they relate to the matrices involved and yield sig- system provides a unique opportunity to view how changes in shaking and actuation nificant observations about both the advantages and the limitations of the selected directly impact the delivered dose without fundamentally altering the MDI device. methods. Key observations include shortcomings of the CARB 435 method when The resulting data allows for improvement in training of analysts, and can be used to compared to the ASTM D7521 method. These findings confirm the validity of ASTM minimize the variability in critical collection steps in performing DCU test. D7521 for asbestos testing of soil. 61 AIM (Abbreviated Impaction) for Success: Streamlining Inhaler 58 Development, Validation and Application of a New HPTLC Method Characterization for the Simultaneous Determination of Alfuzosin, Terazosin, Buchilingam Bupathi, Merck, 556 Morris Ave., Summit, NJ 07901, Prazosin Doxazosin and Finasteride in Pharmaceutical Xiuhua Fang, Kathleen Hogan, Kuriakose Jacob, John Jekonski, Formulations Jennifer Loria, Adrian Goodey, Ying Li Mona M. Khamis, University of Alexandria, Pharmaceutical Chemistry Aerodynamic particle size distribution (APSD) is perhaps the single most important Department, Alexandria 21521, Egypt, Hoda G. Daabees, Mohamed S. attribute of an inhaled drug product. APSD measurement by cascade impaction is Mahrous, Magdi M. Abdel-Khalek, Tarek S. Belal extremely labor intensive, often accounting for more than 25% of the analytical labor Benign prostate hyperplasia (BPH) is one of the most common chronic diseases on a drug product. Although no suitable surrogate for inertial impaction has yet been affecting men and it increases in both incidence and prevalence with age. This developed, abbreviated impactor (AIM) systems are tentatively emerging as an al- work presents a simple, sensitive and fast generic high-performance thin layer ternative to traditional cascade impaction. However, to date, no abbreviated impac-

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tor technique has been demonstrated which provides a significant reduction in labor to cause the change in variable interactions. For the current study, an eight stage without a concomitant reduction in the utility of the data. As such, AIM apparatuses cascade impactor equipped with a pre-separator and glass throat was used to frac- are typically relegated to screening studies. This poster demonstrates an innova- tionate particles into nine different groups based on size. Nine potential laboratory tive approach to abbreviated impaction which dramatically reduces labor require- errors were chosen as a test case. A design of experiment program was then used ments without compromising the utility of the data. As with all AIM systems, labor to randomize experiments and identify which lab errors caused statistically signifi- is reduced via the removal of impactor components. However, careful selection of cant changes in the data. Six of the parameters assessed had a p-value less than the remaining components, coupled with intelligent treatment of the resulting data, 0.05. New data sets were then created for each of the significant parameters to allows determination of all the parameters used in product evaluation and regulatory design a PCA model. Each separate lab error defined a discrete class and each size filing documents, including material balance, fine particle dose, stage groupings and group defined a variable. A single model was created in which five classes could mass median aerodynamic diameter. The abbreviated impaction technique that was be distinctly identified. The results of the study conclude that PCA analysis can be described has shown a good agreement with conventional cascade impaction all successfully utilized to determine the impact of potential laboratory errors related to the while reducing labor and solvent usage by half. This novel approach allows for a Andersen Cascade Impaction. noteworthy reduction in the burden of labor for testing inhalation products. 65 ELS Detectors: A Simplified Approach to Mathematic Modeling of 62 Relating Surface Wettability Measured by IGC to Dissolution Dry Powder Inhaler Blend Uniformity Properties of Dry Powder Inhaler Formulations John Lena, Merck, 556 Morris Ave., Summit, NJ 07901, Justin Daniel J. Burnett, Surface Measurement Systems, 2125 28th Street Pennington SW, Suite 1, Allentown, PA 18103, Armando R. Garcia, Majid Naderi, Content uniformity analysis is conducted on dry powder inhaler products to ensure Manaswini Acharya consistency of blending operations at the unit dose. Previously, work was completed To deliver drug substances to the lung via the inhalation route, it is common to using a high-throughput mathematical approach for content uniformity analysis of reduce the primary particle size of the active ingredient. These high-energy pro- agglomerate based dry powder inhalers using a mass spectrometer. Mass spec- cesses often change the physicochemical properties of the active pharmaceutical trometry (MS) was used for quantitation due to the lactose carrier molecule lacking a ingredient (API), which can ultimately alter final product performance. This study in- UV chromophore. While the method was effective, MS detection requires expensive vestigates the surface energy and wettability of busesonide powders by inverse gas instrumentation and methods tend to be difficult to validate for routine good manu- chromatography (IGC). These results are compared to dissolution behavior using facturing practice (GMP) testing. A Waters Acquity evaporative light scattering de- a next generation impactor (NGI) dissolution method. Crystalline budesonide was tector (ELSD) was selected as an alternative to mass spectrometry due to straight- used as received. Micronized budesonide (MBD) was created using an air jet mill. forward integration into ultra high-performance liquid chromatography (uHPLC) Spray-dried budesonide (SBD) was created using a 1% w/v solution of budesonide systems and the ability to detect molecules that lack a UV chromophore. The ELSD in high-performance liquid chromatography grade ethanol at an inlet temperature was used to quantify the amount of lactose in dry powder inhaler blend samples. 84 °C. The surface wettability of these samples was measured by IGC and the dis- ELSDs are robust and not influenced by variations in mobile phase content because solution behavior was determined using the NGI dissolution method (stage 4). IGC the liquid portion is evaporated before measurement, thus giving a true measure- surface energy heterogeneity results indicated the dispersive surface energy values ment of the mass of analyte present. The chromophore containing active pharma- (γD) for the SBD and MBD samples were similar. However, the acid-base surface ceutical ingredients were quantified using a photodiode array detector. Uniformity energy component (γAB) was significantly higher for the SBD material, indicating samples containing lactose and APIs at varying levels were measured using both the SBD sample has a higher degree of wettability (γAB/γTotal). For dissolution stud- the aforementioned uHPLC-MS method and the ELSD/UV method to assess their ies, stage 4 of the NGI was optimized for a constant 44 μg deposition for both MBD equivalency. Both sets of data were successfully processed and gave equivalent and SBD. A faster dissolution rate was observed for the spray-dried budesonide results. By expanding the mathematical approach to uniformity to simplified detec- formulation than the micronized formulation. An increase in surface wettability of tors that can readily be analyzed on a validated data system in a GMP environment, SBD, compared to MBD correlated to faster dissolution rates as measured using there is a path forward to validation of the approach for use in routine testing. the NGI dissolution method. 66 Design of Experiments (DoE) and Response Surface Methodology 63 Automated Analysis of In-vitro Nasal Spray Deposition Patterns to (RSM) to Improve the Robustness of a Particle Size Method Guide Formulation Development Shasad Sharif, Bristol-Myers Squibb, One Squibb Dr., New Brunswick, Xiuhua Fang, Merck, 556 Morris Ave., Summit, NJ 07901, Elaine Pu, NJ 08902, Mario Hubert, Beth Sarsfield Adrian Goodey, Kuriakose Jacob, Julianne Berry Solid-state powder properties of active pharmaceutical ingredients (APIs) play a In-vitro characterization of nasal sprays is critical for understanding and predict- critical role in pharmaceutical development. Specifically, particle size of APIs can ing product performance. Typically, dose delivery testing is supplemented by plume impact bioavailability and dissolution of the solid dosage form. Hence, there is a geometry and spray pattern testing to assess product performance. However, these need to control the targeted particle size during API manufacturing. Further, it is tests do not directly probe deposition of the drug product, which relates not only also critical to quickly develop robust and accurate analytical particle size methods to safety and efficacy, but also to patient comfort. Recently, a method has been to reduce the analytical error, provide meaningful information to formulators, and demonstrated which allows direct visualization of spray deposition within a cast of a avoid potential regulatory issues. Design of experiments and statistical analysis, es- human nasal system. Upon delivery of the drug to the model nose, presence of the pecially response surface methodology, can be used to improve method robustness drug is indicated by a water-sensitive dye coating the interior of the nasal cast. In the and accuracy. This approach also reduces testing and cycle time during method current work, automated image analysis techniques are applied to the established development. The overall objective of the development process is to design a ro- nasal cast method, in order to aid development of an aqueous nasal spray formula- bust particle size method for an API across the different stages of development that tion. This approach allows quantitation of changes in regional deposition (anterior is applicable to the different morphologies of the API. In this work, DoE and RSM vs. posterior or nasopharyngeal) over time, after discharge of drug to the nasal were used to design a method to measure the primary particle size of an agglom- cast. As such, both the initial deposition pattern and the tendency of the formulation erated API. The statistical approach began with a screening experiment using a to migrate within the nasal cast are captured. Indeed, the impact of changing the fractional-factorial design, 2(III)^3-1, which identified two significant factors. Further formulation is readily observed in the nasal cast. In particular, increasing formula- a 2^2 factorial design with a center point was used to identify possible curvature tion viscosity is demonstrated to favor deposition in the anterior region, and also (non-linearity) to adjust the RSM modeling. This was followed by a face-centered to prevent formulation running down into the throat. The application of automated central composite design that was suitable for applying RSM to identify the optimum image analysis to this direct-visualization method offers a powerful tool for the rapid method conditions. In conclusion, by using statistical analysis, the robustness in evaluation of nasal sprays. The method complements existing in-vitro performance the particle size method development could be improved and testing was reduced. tests, providing crucial insight to guide formulation development. 67 A Quality-by-Design (QbD) Based LC-CAD Method for Oleic Acid in 64 A PCA Model of Atypical Andersen Cascade Data Metered-Dose Inhalers (MDI) Lauren Seabrooks, Merck, 181 Passaic Ave., Summit, NJ 07901 Honggen Zhang, Merck, 556 Morris Ave., Summit, NJ 07901, Zhenyu Andersen cascade impaction is a popular technique used to measure the aerody- Wang namic particle distribution of medical inhalers. It is a size fractionation technique Oleic acid is common excipient in pharmaceutical industry. Currently, quantitation which quantifies the mass of particles belonging to a certain size group. Principal of oleic acid is typically carried out using time-consuming sample methylations prior component analysis (PCA) was used to model atypical Andersen cascade results to gas chromatography method. The purpose of this abstract is to describe a newly to assist in identifying the root cause of out of specification data. PCA is a useful developed QbD based liquid chromatography - charged aerosol detection (LC-CAD) tool for understanding how samples relate to one another. Samples with a similar method for direct quantitation of oleic acid and related fatty acids. Incorporating variance in data group together and can be classified based upon factors known QbD principles, a systematic screen approach was used to evaluate columns, pH

11 2013 EAS Abstracts November 2013

ranges and organic modifiers. Separations of oleic acid and eight related fatty acids 71 Pesticides in My Beverage - Screening (and Subsequent were carried out using a Waters 2695 LC system equipped with an ESA Corona Quantification) of Pesticides in Beverages Originating from CAD detector. A C18 column (Halo), 2.7-µm, 75 x 3.0 mm was used. A gradient Leaves, Grapes, Grasses, and the Hydrologic Cycle (Tea, Wine, elution (50% to 100% organic) over 30 minutes was used. Software (DryLab) was Milk, and Water) Using Automated SPE used during method development to assist separation optimization. The method Peter Bodsky, Thermo Fisher Scientific, 1228 Titan Way, Sunnyvale, CA was characterized in specificity, linearity, accuracy, and precision. There was no 94085, Jennifer Peterson interference from sample matrix. Good recoveries (98.5% - 102.5%) were observed Solid phase extraction (SPE) is a very efficient technique for concentrating contami- over a linear range of 40 µg/mL-120 µg/mL (r > 1.0000). The method is reliable, nants from beverages and other aqueous samples. SPE offers many benefits when reproducible, and robust. compared with traditional liquid-liquid extraction. Minimal solvent usage and contact with solvents saves on disposable costs and operator health concerns. Automated 68 Design of Experiment Studies of the Robustness of a Chiral Method SPE instruments also have the advantages of reducing operator interaction and Using a Modeling Software streamlining the sample preparation portion of analysis. The AutoTrace 280 is an Hui (Viviane) Zhao, Bristol-Myers Squibb, One Squibb Dr., New automated SPE instrument that can process up to 6 samples in parallel. In this pre- Brunswick, NJ 08903, Xin (Amy) Bu, John Castoro sentation we present data demonstrating the use of the AutoTrace 280 for isolation A majority of new drug substances under development are chiral molecules with of pesticides from beverage samples using SPE prior to analysis by gas chromatog- one or multiple chiral centers. The active ingredients and their enantiomers usually raphy mass spectrometry. Beverage samples that have been investigated include possess very different biological activities in terms of pharmacological effects or drinking water, tea, wine, and milk. With properly optimized conditions, we have toxicity. Therefore, the control and determination of the enantiomeric composition obtained quantitative recovery of target pesticide compounds at ppb levels. of chiral drug substances is critical to ensure the quality and safety of the drug. In this case, the initial enantiomeric impurity method was developed as an isocratic 72 Development of an In-Vitro Assay for Assessing the Anti- reversed phase high-pressure liquid chromatography method using Chiralcel OJ- Inflammatory Drug Effects Based on Human Airway Epithelium RH column with on-line mixing of aqueous trifluoroacetic acid buffer and Acetonitrile. Antonio Macaluso, Merck, 556 Morris Ave., Summit, NJ 07901, Ivy When the method was transferred to another laboratory, the method’s robustness Hamill, Jason Ehrick, Song Huang, Ludovic Wiszniewski, Samuel became a concern due to retention time shifts. Subsequently, there was significant Constant difference of retention time observed between pre-mixing and on-line mixing of the Major respiratory diseases such as asthma and chronic obstructive pulmonary mobile phases. A detailed evaluation of the chromatographic parameters indicated disease (COPD) are inflammatory in nature. By expressing a large panel of cy- that multiple factors, such as buffer concentration, ratio of organics in the mobile tokines and chemokines, the airway epithelia play an essential and central role in phase and the column temperature, all influence the retention, the separation and these inflammatory responses. Therefore, the airway epithelium is an ideal target the peak shape of the main compound and its enantiomer. To better understand the of anti-inflammatory drugs. Based on a long shelf-life three-dimensional in-vitro cell method robustness, a design of experiment (DoE) study was conducted using the model of the human airway epithelium (MucilAir), which is fully differentiated and liquid chromatography modeling software to explore the concurrent effects of the closely mimicking the morphology and function of the native tissues. A test method multiple chromatographic conditions on the chiral separation. The results from the was developed to assess efficacy for two classes of reference anti-inflammatory DoE study enabled the method to be optimized with satisfactory robustness. drugs: the steroids (e.g., Budesonide, Fluticasone and Mometasone) and the non- steroidal anti-inflammatory drug (NSAID) (e.g., Piroxicam and Ibuprofen). In order 69 Quality-by-Design (QbD): A Life Cycle Approach for Analytical to simulate the chronic inflammation, the epithelia were treated with tumor necrosis Method Development factor-α, or lipopolysaccharide, or 10% fail back syndrome every day for 5 days. Prasad Panzade, Aditya Birla Science and Technology, Plot No. 1 & 1- The anti-inflammatory drugs were then added into the culture medium at three dif- A/1, MIDC Taloja, Taloja 410208, India ferent concentrations: 10, 100 and 200 μM. IL-8 released by the airway epithelial In 2002, the Food and Drug Administration introduced a major initiative, recogniz- cells was used as an inflammation endpoint for assessing the efficacy of the drugs. ing that regulations of pharmaceutical manufacturing and product quality needed The steroids significantly reduced the IL-8 release at all three experimental con- to be enhanced and modernized. In 2003, the International Conference on Har- centrations, but Fluticasone and Mometasone appeared to be more potent than monization (ICH) workshop participants proposed a new version of pharmaceutical Budesonide. They each showed 4-fold inhibition at 10 μM, however, to achieve an development- “The Desired State.” The basic concepts of QbD as expressed in equivalent level of inhibition 100 μM Budesonide was needed. In conclusion, we ICH Q8 guidance are described in this presentation. Adoption of quality-by-design have leveraged MucilAir to develop a reliable and convenient tool for screening, (QbD) concepts in pharmaceutical development and manufacture is becoming in- validating, and ranking the potency of anti-inflammatory drug candidates, including creasingly well-established. QbD concepts are aimed at improving the robustness steroids and NSAIDs. of manufacturing processes based upon adopting a systematic and scientific ap- proach to development and implementing a control strategy based on the enhanced 73 Quantifying the Effect of Powder Caking by Powder Rheometry process understanding this provides. Many pharmaceutical companies have also Mike Delancy, Freeman Technology, 17 Wakefield Dr., Medford, NJ recognized that QbD concepts can be used to improve the reliability of analytical 08055, Tim Freeman, Brian Armstrong, Katrina Brockbank methods. In this presentation we describe how traditional approaches to analytical Significant numbers of materials in the food, chemical and pharmaceutical indus- method transfer and validation also may benefit from alignment with QbD concepts, tries are supplied as relatively free-flowing powders, intended to be suitable for the and propose a three-stage concept to ensure that methods are suitable for their manufacturing process or final application. For logistical reasons these materials intended purpose throughout the analytical lifecycle: method design, method quali- often require storage for extended periods, increasing the potential to gain strength fication and continued method verification. due to particle/particle interactions. This is referred to as ‘caking’ and can significant- ly limit the ability of a powder to pass uninterrupted through the process train as well 70 Scientific Instrument Regulation and Qualification as detrimentally impacting product quality. Caking occurs through several mecha- Alex White, Anton Paar, 10215 Timber Ridge Dr., Ashland, VA 23229 nisms – typically, mechanical, chemical and thermal – with the absorption/migration Qualification of equipment is a must in most industries today, as the scope of and of water the most influential. Limiting caking can be achieved by managing envi- number regulations continues to increase. Companies who choose to complete the ronmental conditions, by adjusting the time at which the material is stationary or by often rigorous qualification of their own instruments must commit huge amounts of modifying the product formulation. Regardless of the mechanism, determining the time and money into the process. Anton Paar offers a range of instruments which precise conditions which minimize the potential for caking requires a comprehensive provide complete conformity and traceability, and can further support companies understanding of the changes in flow properties that occur as a consequence of with an instrument-specific qualification and validation package. Our most compre- such caking. Powder rheometry can assist by measuring a range of powder char- hensive package uses a risk-based approach and complies with all relevant phar- acteristics, including dynamic, shear and bulk properties, which can quantify the maceutical regulations: GMP, GAMP 5, 21 CFR Part 11, USP <1058>. We now offer progression of caking – for example, as a function of time, humidity and/or con- less comprehensive qualification packages for those companies who are not re- solidation stress. This poster presents case studies evaluating the flow properties quired to comply with the entire scope of regulations listed above. With these pack- of three different powder systems affected by chemical, moisture and temperature ages, companies can efficiently achieve and maintain the qualified status of their based caking mechanisms. It shows how the propensity to cake can be quantified instruments. If preferred, a certified Anton Paar representative can assist throughout with respect to the powders’ flow properties and how this can assist with adapting the whole process, saving you money and time during the implementation process the processing environment to limit caking and retain optimal processability. and shortening the time until the instrument can be used in the production process.

12 2013 EAS Abstracts November 2013

74 The Characterization of Granulation Wet Masses Using Powder compound separation. We developed two novel HPLC-UV methods to separate car- Rheometry boplatin and decitabine, both of which are very polar molecules. We tested several Mike Delancy, Freeman Technology, 17 Wakefield Dr., Medford, NJ kinds of C18, C8, polar C18 columns, and found that they hardly retained carbo- 08055, Tim Freeman, Brian Armstrong platin and decitabine without a strong buffer. We then explored the use of Reliasil The wet granulation of powders is a widely used unit operation in many process Polychrome column, 5-µm, 50 x 4.6 mm. For carboplatin, it was found to retain on industries to primarily increase the particle size but also has the benefits of main- the column with water as mobile phase, with a retention time of 2.3 minute; or using taining content uniformity, reducing segregation and improving the flowability and 5% acetonitrile as mobile phase, the retention time was 1.4 min. For decitabine, compaction properties of a formulation. Simply measuring variables such as particle the isocratic mobile phase is 5% acetonitrile in water, and the retention time is 1.8 size distribution does not provide sufficient information to predict downstream pro- minute. Testosterone and epitestosterone isomers can also be well separated by cessing behavior, which may be influenced by properties such as permeability, com- using same Reliasil PFPP column. The mobile phase is 40% acetonitrile with 0.1% pressibility and flowability. The results of a series of laboratory based experiments formic acid which gives a better peak shape and mass spectrometer signals. The are presented where simple granulation formulations were created and the physical resolution of 4 was found between Testosterone and Epitestosterone. characteristics (permeability, flowability, compressibility and shear properties) of the wet masses were evaluated, using a powder rheometer immediately following the 78 Statistical Comparison of Unique Chemically Modified granulation process, with respect to the proportion of water added. The data shows Carbohydrate Based Chiral Stationary Phases to Improve Chiral that there was a significant and distinct change in the flowability energy at the point Separations of granule formation in all cases. Permeability was also found to increase linearly Matthew Przybyciel, ES Industries, 701 South Rt 73, West Berlin, NJ up to the granulation formation point, after which a deviation from a straight line was 08091 observed. These trends in permeability and flow energy are known to be influenced The chromatographic separation of chiral compounds is an important tool in the by large and stiff particles, thus the results correspond to the traditional concept of search for new pharmaceutical entities. Both high-performance liquid chromatog- granule formation. Using these physical characteristics of granulated wet masses, raphy and supercritical fluid chromatography separations of chiral chemicals are it is possible to derive relationships between the process parameters and the tablet important tools for analytical determination and preparative isolation of enantiomeric quality to achieve a model for determining scaling parameters from the laboratory mixtures. Existing chiral stationary phases can separate many chiral mixtures. Many bench to full scale granulators. of these chiral stationary phases are based on chemically modified carbohydrates. However, even with the existing chemically modified carbohydrates stationary phas- 75 Stability Studies of a Novel Silica-Based Diol-Bonded 3-µm Particle es there are still many enantiomeric mixtures that are difficult to separate limiting the Size 30nm Pore Size-Exclusion Chromatography Analytical ability to characterize and purify chemical mixtures containing chiral compounds. Column Especially Designed for the Separation of Aggregates In this study we are chemically modifying carbohydrates, such as cellulose and Justin Steve, Tosoh Bioscience, 3604 Horizon Dr., King of Prussia, PA amylose, with functional groups that have not been routinely employed. Chemical 19406, Atis Chakrabarti modifications of the carbohydrates include halogenated, aromatic and hetero-aro- TSKgel UltraSW Aggregate 7.8 mm ID × 30 cm analytical columns are novel col- matic functional groups. We present information on the chiral separation character- umns packed with 30 nm pore size, 3-μm silica particles specially designed for the istics and overall separation capabilities for these chemically modified carbohydrate separation of aggregates of monoclonal antibody monomer. Larger pore size with based chiral stationary phases. the estimated exclusion limit of ~4 x 10^6 Da provides improved separation and quantitation of protein aggregates and oligomers, particularly for the separation of 79 Thermodynamics and Multivariate Analysis of HPLC Stationary the monoclonal antibody aggregates. For safe bio therapeutic application separa- and Mobile Phase Behavior tion of the pure monomer needs to be very well resolved from its dimer and higher Wilmer R. Barndt, Ursinus College, 601 E. Main St., Collegeville, PA order aggregates. Similarly for quality control and regulatory purpose the separa- 19426, Eric J. Williamsen tion of antibody fragments is also very essential since the species other than the High-performance liquid chromatography (HPLC) is a common analytical technique monomer might induce toxic side effects to the body if not removed. Analytical size that relies on separation caused by physical interactions between the analyte and exclusion chromatography (SEC) columns are costly, so a stable column yielding stationary phase, analyte and mobile phase, and the mobile phase with the station- a high degree of reproducibility of retention time, peak symmetry and column ef- ary phase. Even with its popularity, some aspects of the mechanism of these inter- ficiency over a large number of injections is very important to the analyst. Losses actions remain unclear. Using a phenyl stationary phase and 10% methanol/90% of resolution, peak broadening or significant tailing that may affect quantitation are water mobile phase, retention of approximately 40 aromatic analytes including the symptom column failure. Most important warning that the column may be on various functionalities such as alcohol, cyano, halogen, and nitro were measured at the verge of failing can be predicted from gradual increase in backpressure. Reten- temperatures ranging from 5.0 to 65.0 ˚C. In addition to these results, trends in this tion time shift can sometimes be related to a loss of packing material or stationary data set are compared to results obtained with other mobile phase compositions phase. Here we report the results of a column lifetime study of a new silica based (various organic/water compositions and replacement of methanol with acetonitrile) TSKgel UltraSW Aggregate columns. and fluorophenyl stationary phases. Because of the variety of analyte functionality, retention patterns are emphasized using multivariate analysis techniques such as 76 Separation Improvements with 2-D Liquid Chromatography principal component and cluster analysis methods. The thermodynamics of reten- William J. Long, Agilent Technologies, 2850 Centerville Rd., Wilmington, tion are obtained through van ’t Hoff analysis. Linear solvation energy relationships DE 19808, Anne Mack are used to determine the relative importance of various physical interactions that The separation of complex samples can be improved by using two-dimensional liq- occur during retention. Through these results a better understanding of the interac- uid chromatography (2-D LC) with two different types of columns. The main advan- tions that underlie retention in HPLC can be obtained. tage of 2-D LC compared to conventional single dimension chromatography with a single column is the increase in peak capacity or resolution. But this is maximized 80 Characterization and Resolution of the Deteriorative Effects of only as long as the two column selectivities are completely orthogonal. However in Hydroxypropyl Methylcellulose on UHPLC Column Performance the real world this othogonality is often only partial due to available column choices Karthik Jayaraman, Bristol-Myers Squibb, One Squibb Dr., New or difficulties in solvent/mobile phase compatibility. In this work, a range of superfi- Brunswick, NJ 08901, Hutton Chapman, Thomas Hooker, Frank cially porous columns and conditions are evaluated to determine the best available Tomasella othogonality. These include the use of reversed-phase and hydrophilic interaction Recently, an ultra high-performance liquid chromatography (UHPLC) method using liquid chromatography column choices. These are then used to separate complex sub-2-µm particles was developed for the analysis of potency and impurities for pharmaceutical and environmental mixtures using a 2-D LC solution. modified release tablet formulations that contain hydroxy propyl methyl cellulose (HPMC) as a major excipient. The tablet extraction procedure involves 40 minutes 77 HPLC Separations of Highly Polar Compounds and Isomers Using of sonication and 20 minutes of mechanical shaking. In addition, the procedure in- Reliasil Polychrom Column volves centrifugation and two steps of filtration to remove any insoluble components Xuejun Zang, Orochem Technologies, 340 Shuman Blvd., Naperville, IL or particulates. A guard column was used as a final attempt to remove any insoluble 60563, Dipali Parikh, Anil Oroskar, Asha Oroskar material and to preserve the analytical column. Irrespective of all these precaution- There is increasing demand for separation of highly polar or isomeric compounds ary steps, the life time of the guard and the analytical column was rather low, typical- in reversed phase conditions, especially using less buffer, acid or base to increase ly less than 60 injections. The main focus of this study was to determine the source the lifetime of columns and the sensitivity of the assay. The PFPP (Pentafluorophe- of the column degradation, devise methodology to reduce column deterioration and nyl Propyl) high-performance liquid chromatography (HPLC) column has multiple optimize method robustness. Through a series of experiments, it was demonstrated binding mechanisms, including hydrogen bonding, dipole-dipole, aromatic and hy- that HPMC gets retained on the column and has a deteriorative effect on the sta- drophobic, and it has been used to solve some challenging highly polar or isomeric tionary phase which was irreversible with both aqueous and/or organic washing.

13 2013 EAS Abstracts November 2013

The effect of separation temperature and stationary phase particle size on HPMC by optimizing bonded density of C18 phase. This feature provides superior reten- retention and the solubility of HPMC in organic/aqueous mixtures were also studied. tion and resolution of hydrophilic compounds and compatibility with 100% aqueous Based on the findings, a multifaceted approach was designed to enhance method mobile phase. Additionally, YMC-Triart Diol-HILIC provides alternative separation robustness which involved: 1) selection of a diluent with higher organic composition of hydrophilic compounds that are not retained on C18 phase. In this poster, we to extract HPMC as part of the sample preparation, 2) selection of a lower column introduce characteristics of these new hybrid columns through some examples of temperature to minimize HPMC retention and 3) selection of a larger particle size hydrophilic compounds analysis. We also show the guide for selecting column and/ stationary phase to maximize column life time. or developing analytical method on separation of hydrophilic compounds. 81 Particle Size Considerations of Superficially Porous Particles 84 Increased Throughput and Purity of Combinatorial Libraries Stephanie A. Schuster, Advanced Materials Technology, 3521 Silverside Utilizing a Targeted Gradient Profile Based on Method Scouting Rd., Ste. 1-K, Quillen Building, Wilmington, DE 19810, Joseph J. Software DeStefano, Robert S. Bichlmeir, William L. Johnson Todd M. Anderson, Shimadzu Scientific, 1404 Zook Rd., Atglen, PA Columns of fused-core, superficially porous particles demonstrate higher efficiency 19310 than columns packed with totally porous particles of similar size. This result is likely The purpose of this poster is to describe a technique where the analyst can obtain a because of superior eddy and longitudinal diffusion properties (smaller van Deemter higher purity and better resolution using information from the preliminary analytical A and B terms) observed for core-shell particles resulting from exceptionally nar- screening of these samples prior to purification with Shimadzu’s Method Scouting row particle size distributions and higher density contributing to an ability to form software. By utilizing the retention time of the compound from preliminary runs, an homogeneous packed beds. The achievement of efficiencies comparable to those optimal set of conditions may be obtained that allows for a greater success rate of obtained using columns of sub-2-micron totally porous particles without the need for separation. Compared to the standard 5 to 95% elution gradient, a narrow, short ultra high-pressure instrumentation is a distinct advantage of fused-core columns. gradient profile can be determined. Along with a better separation, the chromato- Fused-core particles with a wide range of uniform particle sizes were synthesized to graphic time can be shortened, saving the analyst both instrument time and solvent allow the preparation of stable, efficient packed columns for this study. These col- consumption, while allowing for purification with a single injection. umns were used to measure the effects of particle size on chromatographic perfor- mance and pressure requirements. This report describes the effect of particle size 85 Determination of Organic Acids in Electroless Nickel, Silver, and on several factors of separation importance, including plate heights, reduced plate Gold Plating Solutions Using High Performance Liquid heights, pressure, and ease of use. Depending on the intended application, certain Chromatography particle sizes are recommended for use over others. The performance of larger Peter P. Yeh, MacDermid, 245 Freight St., Waterbury, CT 06702, Shelby fused-core particles exceeds even the high expectations observed for smaller su- Hale perficially porous particles, likely because larger particles form more homogeneous Organic acids, such as lactic, malic, citric, succinic, maleic, and fumaric acids, are packed beds than smaller particles. The utility of larger and smaller superficially often used in the plating bath solutions to serve a variety of functions including metal porous particles are compared and evaluated for high-speed and high-resolution chelation and pH buffering. Maintaining the proper organic acid level in the process applications. solution is important to ensure the optimum plating performance. Analytical methods have been developed for determining the organic acid concentrations in the plat- 82 Efficient and Rapid Method Development with Hybrid Silica Based ing bath solutions. In these analytical procedures, organic acids are determined Material by Utilizing Particle Size, Temperature and Bonded via high performance liquid chromatography (HPLC) using a reversed-phase C18 Chemistry column for separation in conjunction with an acidic mobile phase solution contain- Ernest J. Sobkow, YMC Co., 941 Marcon Blvd, Ste. 201, Allentown, PA ing methanol or acetonitrile for elution. Sample preparation often involves simply 18109, Noriko Shoji, Takatomo Takai, Takashi Sato, Naohiro Kuriyama diluting the process solution with de-ionized water and injecting it directly into the Recently, the requirement for enhancing efficiency of analysis in a laboratory is liquid chromatograph. Filtration of the sample solution to remove particulates is rec- increasing. Ultra high-performance liquid chromatography (UHPLC) that uses the ommended in order to prevent clogging of the column frits. For difficult samples highly efficient columns packed with sub-2-µm particles, has been proposed and containing interfering components, sample pre-treatment using solid phase extrac- widely used for the analyses in the various fields. Although UHPLC method offers tion (SPE) technique to remove the interference may be necessary. The organic shorter analysis time, higher resolution and higher productivity than conventional acids are separated, eluted, and are detected using a spectrophotometric detec- LC method, it often requires a special LC instrument due to high back pressure pro- tor set at a selected wavelength. Quantitation of the organic acid concentrations duced by sub-2-µm particles and the increased linear velocity. There are also cases is accomplished by peak height or peak area measurement against those of the where even the smaller particles and longer column length cannot achieve an ac- standard solutions of known concentration. In this paper, HPLC analysis procedures ceptable separation because selectivity is not sufficient to gain resolution between developed for determining organic acids in the electroless nickel, electroless silver, target analytes or between analyte and sample matrix. Selectivity has a greater and electroless gold plating bath solutions are described, and the accuracy and impact on resolution than column efficiency and is changed by mobile phase as well precision of the analytical results obtained are discussed. as stationary phase in reversed-phase (RP) LC. Column temperature also plays an important role in controlling selectivity and there is the additional advantage that 86 Evaporative Light Scattering Detection - Improved Detector Design increasing temperature reduces column pressure. We have developed a new type for the Detection of Semi-Volatile Compounds of hybrid silica based column named “YMC-Triart series” to make a LC method de- Graham M. Cleaver, Agilent Technologies, Stokewood Rd., Craven velopment faster and more efficient. Triart column is based on multilayered organic/ Arms SY7 8NR, United Kingdom, Stephen Bullock inorganic hybrid particles with 5-µm, 3-µm and 1.9-µm diameters, and available in Evaporative light scattering detection (ELSD) is a powerful tool for detecting any C18, C8, Phenyl, PFP bonded phase for RPLC. In this poster, we show some ex- sample that is less volatile than the mobile phase and has become widely used in ample cases of developing the methods of the challenging separation and reducing the field of high-performance liquid chromatography (HPLC) since its introduction analysis time by using these new hybrid columns. in the late 1970’s. For non-UV absorbing compounds, ELSD is a primary choice since the principle of detection does not rely on the optical properties of the solute. 83 Efficient Method Development for Polar Compounds Analysis on Even if a compound does contain UV active components, ELSD can offer benefits Hybrid Silica Based Particle Column in terms of uniformity of response for non-volatile solutes since the effect of extinc- Ernest J. Sobkow, YMC Co., 941 Marcon Blvd, Suite 201, Allentown, PA tion coefficient on UV response is not a feature in ELSD. The detector also offers 18109, Noriko Shoji, Takatomo Takai, Takashi Sato, Naohiro Kuriyama significant benefits as it can operate over very rapid changes in eluent composition In the field of chromatography, there has been much attention given to hybrid mate- and temperature with no effect on the baseline stability, and can offer very high rials that have two aspects of inorganic and organic characteristics: high efficiency, sensitivity, courtesy of a new blue laser light source and advanced detection optics. and high mechanical and chemical stability. Recently, we have developed a new One disadvantage that has been cited for some designs in the past, has been that type of hybrid silica based ultra high-performance liquid chromatography (UHPLC) high operating temperatures are required to fully evaporate the eluent can cause and HPLC column named “YMC-Triart series”. YMC-Triart series is based on mul- loss of semi-volatile analytes. An improvement in this respect would be a key benefit tilayered organic/inorganic hybrid particles with 5-µm, 3-µm and 1.9-µm diameters, in the analysis of small molecules, e.g. drug candidates. This presentation describes and available in C18, C8, Phenyl, PFP and Diol-HILIC chemistries. Nowadays, sev- the design and operation of the Agilent 1290 Infinity Evaporative Light Scattering eral hybrid based columns are marketed. However, sufficient retention and separa- Detector, which incorporates design improvements that allows ambient temperature tion, or reproducibility on analysis of hydrophilic compounds is not given by such operation; so that losses of semi-volatile sample components are minimized. By products. To solve this problem, we have lined up various phases that are applicable preserving sample integrity, the detector offers maximum sensitivity for a wide range to analyses of hydrophilic compounds. YMC-Triart C18 is designed to retain both of HPLC and ultra HPLC applications. moderate hydrogen bonding capacity and hydrophobicity on the surface of particle

14 2013 EAS Abstracts November 2013

87 Preparative Isolation of Active Components from Natural Products difficult than it is in liquid chromatography. Since supercritical fluids are highly com- Using Low-Pressure Gradient Preparative High Performance pressible, their density varies significantly along the column and so does the local Liquid Chromatography volumetric flow rate. In most cases, the volumetric flow rate set in the instrument Kenichiro Tanaka, Shimadzu Scientific Instruments, 7102 Riverwood software is not the actual average volumetric flow rate through the column but the Dr., Columbia, MD 21045, William Hedgepeth, Lincoln Grimes, Tsutomu flow rate in some location. In this poster, we explain how it is possible to calculate Watanabe, Takaei Kitagawa, Yosuke Iwata under various experimental conditions an average value of the volumetric flow rate Natural products contain a variety of active components. Many medicines have that properly takes into account the variations of the local mobile phase density their origin in natural products and it is said that they account for 50% of the total along a column operated in supercritical fluid chromatography. Giving estimates medicines. Preparative high-performance liquid chromatography (HPLC) is used to of this average value requires knowledge of some critical properties of the system obtain active components from complex mixture such as natural products. In this such as the mass flow rate of the supercritical fluid and the variations of its tempera- poster, we show preparative separation from rosemary. Rosemaries have several ture and pressure along the column. Knowing these data permits the determination effects such as odor eliminating, antibacterial activity, and antioxidant activity, so it of valid estimates of the volumetric flow rate with a high accuracy. has been used for a meat dish in Europe for a long time. Rosemaries contain rose- maric acid, carnosic acid, carnosol, and etc., as their active components. Rosmaric 91 High Performance Ion Mobility Spectrometry-Liquid acid has the ability to control allergic symptoms. Carnosic acid and carnosol exert Chromatography: An Integrated Tool for 2-D Separation a revitalizing influence on biological defense mechanism and enhance detoxifica- Carol Moraff, Excellims Corporation, 20 Main St., Acton, MA 01720, tion. It is also reported that carnosic acid has the ability to improve memory perfor- Ching Wu, Clinton Krueger, Anthony Midey mance and to facilitate creation of nerve growth promoting substances which has A novel integration of high-performance ion mobility spectrometry (HPIMS) with liq- a significant role in maintaining nerve cells. We used newly released low-pressure uid chromatography (LC) proved to be a powerful tool for chemical analysis. HPLC- gradient preparative HPLC system in this study. This system enables combination HPIMS combines two orthogonal techniques with entirely different separation mech- of four mobile phases, so it is useful for method development. Shim-pack PREP- anisms to create a two-dimensional (2-D) separation. In an integrated HPLC-HPIMS ODS was used as a column for preparative separation and water, methanol and 2% system, a liquid sample elutes from the LC column after being separated based on formic acid water solution were used as mobile phases. As a result, rosemaric acid, polarity. It then flows through a sample line to an electrospray ionization needle, carnosic acid, and carnosol were successfully separated from other components. where it ionizes and enters the ion mobility drift tube as a collection of gas-phase molecular ions. These ions are pulled through the tube by an applied electric field, 88 Exploring Low and High pH Eluents Using a New Silica-Based and separate based on their size and shape due to collisions with a drift gas flowing Extended pH Range C18 Column for Analysis of Amphetamines in the opposite direction. A Faraday plate detector at the end of the tube creates a and Opiates from Urine by LC-MS-MS signal that is plotted as a 2-D spectrum, with LC elution time on the Y-axis and IMS Alan P. McKeown, Advanced Chromatography Technologies, One Berry drift time on the X-axis. 2-D plots were collected in this manner using a standard St., Aberdeen AB25 1HF, United Kingdom, Geoffrey Faden C18 column, and these plots showed separation of compounds that were not fully Selectivity is one of the most important principles in chromatography and many separable by either the HPLC or HPIMS method alone. method factors including eluent pH can be highly influential. Whilst eluent pH can be powerful for changing the elution order of charged species in mixtures, many 92 Studying Stability and Degradation Pattern for Sunscreens standard high-performance liquid chromatography (HPLC) – ultra HPLC silica- Containing TiO2 Nanoparticles and Other Sunscreen Agents Under based phases have limited stability and robustness under alkaline conditions which UV Light Exposure Through Universal LC-ESI-MS is a significant limitation for the method developer and column use on screening Michael M. Puppolo, Temple University, 1901 N. 13th St., Philadelphia, systems. Advances in bonding and endcapping technology during stationary phase PA 19132, Siddharth Shah manufacture have allowed the production of a new wide pH range stable silica- Sunscreens have become increasingly popular consumer products. Public health based phase: ACE SuperC18. This highly versatile phase can be used from pH 1.5 demands that these sunscreens provide protection against increasing levels of UVA – 11.5 giving maximum flexibility without compromising performance for the method and UVB. In the United States, sunscreens are regulated by the Food and Drug developer and/or screening system setup. The ACE SuperC18 phase is demon- Administration (FDA) as over-the-counter drugs. The FDA has responded to recent strated to be highly robust across the pH range with guaranteed reproducibility col- public concerns over sunscreen by increasing inspectional surveillance. The United umn to column and batch to batch. This poster explores the impact on elution order, States Pharmacopeia contains no monographs for any of the combination cream or retention and selectivity values for a range of analytes with low and high pH eluents. lotion products. Inspections have found inadequate firm oversight of the drug ingre- In addition, the application of this phase for high eluent pH LC tandem mass spec- dients in these products and ineffective or no analytical testing methods to analyze trometry (MS-MS) analysis (low ppb detection levels) of amphetamines and opiates these combination products. Measurement capability is imperative for reinforcing extracted from urine are demonstrated. safety assessments, ensuring quality control of consumer products and evaluating toxicity concerns. We have developed a universal liquid chromatography electro- 89 Exploring the Power 0f Chromatographic Selectivity for Polar and spray ionization mass spectrometry method to analyze common sunscreen ingredi- Non-Polar Analytes with a Unique HPLC-UHPLC Polar Embedded ents for quantification of most commonly used organic sunscreen agents allowed in Stationary Phase US market by the FDA and we are studying stability and degradation pattern kinetics Alan P. McKeown, Advanced Chromatography Technologies, One Berry for most commonly used sunscreen agents in the presence of TiO2 nanoparticles to St., Aberdeen AB25 1HF, United Kingdom, Geoffrey Faden study their long term effectiveness under UV light exposure. Selectivity is the most powerful term in the resolution equation. Understanding which parameters affect selectivity is helpful for rapid method development. In this 93 Identification of Purified Flavonoids (Proanthocyanidins and poster, we investigate the influence of stationary phase on selectivity with a novel, Flavonols) from American Cranberry polar embedded stationary phase: ACE C18-Amide. This new stationary phase has Yifei Wang, Rutgers University, Department of Plant Biology and been specifically designed to minimize phase bleed, be stable at low and inter- Pathology, 59 Dudley Rd., New Brunswick NJ 08901, Ajay P. Singh, mediate eluent pH values and have enhanced hydrophobicity whilst maintaining Nicholi Vorsa polar retention and phenolic resolution properties. The impact of organic modifier American cranberry (Vaccinium macrocarpon) has long been recognized as one of choice and eluent pH on selectivity values was also determined and highlighted the the leading sources of bioactive phytochemicals with important implications to hu- versatility of the C18-Amide phase. A variety of polar analyte separations including man health. Cranberry proanthocyanidins (PACs), with specific A-type linkage pre- catecholamines, resorcinols, vanillins, wine acids and steroids were developed and sented, have been shown to exhibit unique anti-bacterial adherence and anti-tumor compared under the same conditions to a standard C18 phase to highlight retention, bioactivities.[1,2] Cranberry flavonols, conjugated with different sugar moieties, have elution order and selectivity differences. shown anti-inflammation and anti-tumor properties among various cell models.[3] Identification of cranberry flavan-3-ols has been problematic with regular high-per- 90 Determination of the Average Volumetric Flow Rate in Supercritical formance liquid chromatography (HPLC) method since large molecular weight (MW) Fluid Chromatography polymers, such as PACs with high degree of polymerization (DP), invariably yield Peter Vajda, University of Tennessee, 413 Buehler Hall, Knoxville, TN wide overlapped peaks with poor separation. With flavonols, such as quercetin gly- 37996, Georges Guiochon cosides of cranberry, their similar UV-Visible spectra and retention times make them In all applications of gas, liquid, and supercritical fluid chromatography (SFC), the difficult to be identified. With Dionex® Ultimate 3000 UPLC system coupled with determination of the exact volumetric flow rate of the mobile phase is critical. The Applied Biosystems API 3000TM triple quad liquid chromatography tandem mass accurate knowledge of this flow rate is particularly important to understand retention spectrometry (LC-MS-MS) mass spectrometer, the MW of the array of cranberry mechanisms and to plan analytical or preparative scale separations. The properties PACs and flavonols has been precisely identified. Under full scan MS Q1 negative of the supercritical fluids used as the mobile phases in SFC make this task more ion mode with atmospheric pressure chemical ionization (APCI), DPs of PACs have

15 2013 EAS Abstracts November 2013

been confirmed by comparing the total MW with MW of single building block mono- 97 GC-MS Study of the Composition of Saturated Hydrocarbons in mer. Number of probable A-type linkage has been calculated from MW reduction High-Boiling Fractions of Georgian Oils caused by losing hydrogen upon linkage formation. Structure of cranberry flavonols, Elza N. Topuria, Melikishvili Institute of Physical & Organic Chemistry, including the different conjugated sugar moieties, has been analyzed by MS/MS Djikia Street 5, Tbilisi 0186, Georgia, Natela T. Khetsuriani, Kirill V. product ion scan with APCI. Data from both Q1 and Q3 has been precisely matched Tretyakov, Carlos A. Gonzalez, Anzor I. Mikaia with compound structure and fragmentation pattern, difference between Q1 and Q3 Structure determination for over 100 mono- , di-, tri- and tetracyclic hydrocarbons can be used to determine the specific sugar moiety. and their alkyl-substituted homologs in high boiling point fractions of Georgian oils References: have been completed. It was achieved with the preliminary use of special separa- [1] Foo, L. Y., Lu, Y., Howell, A. B., & Vorsa, N. (2000). A-Type Proanthocyanidin tion techniques for the extraction of alkanes and cyclaoalkanes, followed by gas Trimers from Cranberry that Inhibit Adherence of Uropathogenic P-Fimbriated chromatography mass spectrometry (GC-MS) analysis. The special separation Escherichia c oli. Journal of natural products, 63(9), 1225-1228. techniques included distillation, adsorption chromatography, a multi-stage thermal [2] Singh, A. P., Singh, R. K., Kim, K. K., Satyan, K. S., Nussbaum, R., Torres, M., ... diffusion and extractive crystallization with thiocarbamide. The complex GC-MS & Vorsa, N. (2009). Cranberry proanthocyanidins are cytotoxic to human cancer data files were analyzed by the automated mass spectral deconvolution and identifi- cells and sensitize platinum-resistant ovarian cancer cells to paraplatin. Phyto- cation system (AMDIS). Complex study of mass spectral data and evaluation of gas therapy Research, 23(8), 1066-1074. chromatography retention index values led to the differentiation of region-isomers. [3] Neto, C. C. (2007). Cranberry and its phytochemicals: a review of in-vitro anti- Common and specific fragmentation pathways of tri- and tetra-cycloalkanes under cancer studies. The Journal of nutrition, 137(1), 186S-193S. electron ionization are discussed. 94 Selectivity of Chloride Ion Attachment in Negative-Ion Atmospheric- 98 GC-MS Study of Polycyclic Aromatic Hydrocarbons in High-Boiling Pressure Chemical Ionization LC-MS with Applications to Fractions of Norio Oil Pharmaceutical Development Natela T. Khetsuriani, Melikishvili Institute of Physical & Organic Keith B. McKellop, Boehringer Ingelheim, 900 Ridgebury Rd., Ridgefield, Chemistry, Djikia Street 5, Tbilisi 0186 Georgia, Elza N. Topuria, Nino G. CT 06877, Scott Pennino, Shirley A. Rodriguez, Daniel Norwood Todua, Carlos A. Gonzalez, Anzor I. Mikaia Gas phase chloride ion attachment negative ion atmospheric-pressure chemical Gas chromatography mass spectrometry (GC-MS) study of a vacuum gas oil frac- ionization (APCI) liquid chromatography mass spectrometry (LC-MS) has been ap- tion with boiling point 340-590 ˚C has been studied, which is about 28% of the Norio plied to the analysis of drug molecules, such as nitroglycerin, which are difficult to oil (Republic of Georgia). Eight portions of this gas oil were obtained by a fractionat- ionize by more conventional LC-MS methods. [M+Cl]- ions are formed by non-cova- ing column distillation. Further separation of these fractions was carried out on a lent gas phase interactions of analyte molecules with Cl- derived from the addition 300 cm x 5 cm silica gel chromatography column with the use of petroleum ether of chlorinated reagents (e.g., chloroacetonitrile) to the high-performance LC mobile as an eluent under nitrogen gas pressure. The remaining organic compounds were phase. This presentation describes a systematic approach to evaluate the selec- extracted with hot benzene. As a result 876 petroleum ether and 78 benzene elu- tivity of chloride attachment ionization to a diverse group of compounds including ates were obtained. GC-MS was used for the structure elucidation of components of hydroxylated (sugars), phenols, sulfur and phosphorus containing, and aliphatic vs. these eluates. The GC-MS data files were analyzed by the automated mass spec- non-aliphatic structures using a standard LC-MS platform and ionization conditions. tral deconvolution and identification system (AMDIS). Fragmentation under elec- The affinity of these compounds to form [M+Cl]- anions is summarized and experi- tron ionization conditions and gas chromatography retention index data of some mental considerations are presented. General trends which describe the selectivity aromatic hydrocarbons and their hetero-analogs are discussed. Emphasis is made of the chloride ion attachment process are considered. on the Isomers of mono- and poly-alkylated Indenes, tetralines, dinapthylbenzens, naphthalines, acenaphthylenes, fluorenes, phenanthrenes, naphtofluorenes and 95 Novel SFC Additives and Modifiers for Separating Drug Candidates -phenanthrenes, pyrenes and chrysenes. in Discovery Chemistry Peng Li, Bristol-Myers Squibb, Route 206 & Province line Rd., Princeton, 99 Withdrawn by the author. NJ 08543, Dawn Sun, Yip Shiuhang, Wu Dauh-Rurng, Arvind Mathur, Joel Barrish Supercritical fluid chromatography (SFC), a powerful preparative separation tech- Characterizing Copolymer Compositional Heterogeneity by GPC- nique, has been successfully used in drug discovery of pharmaceutical industry. 100 FTIR Combined System SFC utilizes carbon dioxide (CO ) as the mobile phase with a co-solvent (e.g., al- 2 Tracy Phillpott, Spectra Analysis Instruments, 411 University Ridge, Ste. cohols) resulting in faster equilibration, lower pressure drop, reduced solvent con- 245, Greenville, SC 29601, William Carson sumption, and greater ease of solvent removal at the end of the process. For chiral Fourier transform infrared (FTIR) spectroscopy is a powerful technique for determin- separations, SFC has demonstrated to be the best technique, since it reduces hy- ing structural and compositional information of copolymers. Hyphenating gel perme- drocarbon usage and improves productivity. Furthermore, even for achiral purifica- ation chromatography (GPC) with a FTIR detector through an automated solvent tions, SFC has gained popularity in our drug discovery process. The authors have removing interface allows compositional and structural characterization across the successfully separated polar compounds by SFC with novel additives and modifiers. whole molecular weight distribution of a complex copolymer. A GPC-IR hyphenated This presentation discusses chiral and achiral SFC separations/purifications of a system was used to characterize various copolymer samples of styrene-butadiene polar drug candidate and its analogue successfully carried out with water and am- rubber (SBR), styrene ethylene butylene styrene (SEBS), Copovidone polyvinyl- monium hydroxide as additives. pyrrolidone-vinyl acetate (PVP/VA), polyethersulfone, poly(lactic-co-glycolic acid (PLGA) and acrylate-styrene copolymers for compositional variations across mo- Analytical Challenges in Residual Chlorobutanol Analysis in the 96 lecular weight (MW) distributions. Significant amounts of compositional drifts were Presence of Trace Amount of THF observed for SBR, PVP/VA samples, whereas smaller levels of compositional drifts Vera Leshchinskaya, Bristol-Myers Squibb, One Squibb Dr., New for SEBS and no compositional drifts were observed for PLGA and polyethersul- Brunswick, NJ 08903, Yande Huang, Yun Ye fone copolymers. GPC-IR was also used to investigate a complicated copolymer Chlorobutanol is an alkylating agent and therefore considered to be a genotoxic system based on polymethyl methacrylate-butyl acrylate (PMMA-BA) (~6:1 ratio) impurity. It has the potential to be a by-product if the reaction conditions contain tet- main backbone but incorporated with small amounts of methacrylic acid, styrene rahydrofuran (TFH) as the solvent in the presence of concentrated HCl. An analyti- and Diacetone acrylamide (DAAM) comonomers in order to improve various co- cal method capable to determine 20 ppm of chlorobutanol as well as residual THF polymer end-use properties. Different patterns of compositional heterogeneity of needed to be developed. Analytical challenges included presence of highly reactive those copolymers with the 3-5 comonomers were found for a structure-property matrix, absence of high purity standard (highest purity of commercially available correlation study. GPC-IR hyphenated system is well suited to characterize copoly- reagent is ~85%), and suspected thermal instability of chlorobutanol. In addition, mer compositional drift across MWD, supplier-to-supplier / lot-to-lot variations and residual THF analysis also presented a challenge since chlorobutanol may contain polymer compositional changes from a thermal processing. trace of THF either as residual starting material or as a result of the reagent deg- radation. The challenge was solved by a combination of gas chromatography (GC) and quantitative nuclear magnetic resonance techniques. A simple and sensitive GC method was developed within a short period of time.

16 2013 EAS Abstracts November 2013

101 Extraction of 22 Pain Management Drugs from Human Urine Using known amounts of estrogens were analyzed by this automated approach and by Supported Liquid Extraction (ISOLUTE® SLE+) in 96-Well Plate conventional techniques. The automated approach achieves a sample throughput Prior to LC-MS-MS Analysis of 8 samples per hour on a one-channel system. Throughput can be doubled or Victor E. Vandell, Biotage, 10430 Harris Oaks Blvd., Charlotte, NC quadrupled by multiplexing across two- or four-channel systems, respectively. 28262, Brad Nolt The screening of various prescribed pain medications in patient urine samples has 105 Evaluation of Centroid and Profile Mode Data Collection Methods, grown significantly in the United States. As a result, clinical testing laboratories have for High-Resolution Accurate Mass Spectrometry (HRMS) Based moved to develop fast, reliable quantitative and qualitative liquid chromatography Integrated Qualitative and Quantitative Analysis tandem mass spectrometry (LC-MS-MS) methods. Clinical labs prefer to use a sin- Eldho Raju, Kean University, 1000 Morris Ave., Union, NJ 07083 gle extraction technique for speed and cost effectiveness. A fast and simple sample Many scientists acquire high resolution mass spectrometry (HRMS) data in centroid preparation method was developed using supported liquid extraction, specifically mode preferred to profile mode. On contrast of a recent conflict in mass spectrom- ISOLUTE SLE+ 96 well plates to extract 22 pain management drugs. This poster etry community regarding these data acquisition modes in HRMS, we decided to demonstrates a rapid and reliable supported liquid extraction assay for the simulta- investigate on effectiveness of centroid versus profile data acquisition mode. We neous extraction of pain management drugs from urine prior to LC-MS-MS analysis. investigated, using four pharmaceuticals and their metabolites, on the effectiveness of centroid and profile mode data acquisitions for HRMS experiments using an LTQ- 102 Use of Sorbent Coated Metal Probes with Direct Analysis in Real Orbitrap mass spectrometer. We also studied different mass resolving power, mass Time (DART) Ionization for Rapid Analysis of Herbal Supplements accuracy, mass extraction window (MEW) and peak smoothening algorithms neces- Joseph LaPointe, Ionsense Inc., 999 Broadway, Saugus, MA 01906, sary to obtain accurate m/z measurement in both centroid and profile modes. The Brian Musselman, Robert Goguen entire study was performed by analyzing the effect of internal calibration. A mass Determination of composition and quality of herbal supplements are time consuming resolving power of 15K FWHM was optimized for m/z measurements with sufficient due to the nature of the active components and complexity of the sample matrix. mass accuracy in HRMS studies. An optimum MEW with a mass tolerance of 10 Published methods often incorporate solid phase microextraction (SPME) of supple- ppm and Gaussian peak smoothening algorithm with 7 data points, were found suf- ment extracts followed by either gas chromatography mass spectrometry (GC-MS) ficient for the generation of an extracted ion chromatogram. Centroid data found or liquid chromatography (LC) MS analysis. More recently, flexible and rugged sor- to show better signal response and preferred for industrial research. Profile data bent coated wire solid phase extraction devices have been produced for use in with reasonable signal response with an option of manual enhancing is preferred simplifying sample prep. We utilized these wires for both sample extraction and as a in academic and independent researches. While using liquid chromatography tan- support for direct surface desorption ionization facilitating rapid analysis. Compari- dem mass spectrometry (LC-MS-MS), the fragments of drugs showed equal signal son of the results using this new direct analysis from sortbent coated wire method responses in both centroid and profile modes. The profile mode data showed high with analysis of liquid samples from syringe-based and column-based solid phase percent abundance for precursor ions upon high-performance LC-MS-MS. Profile extraction devices using bare wire screens are shown. mode data acquisition with a resolution of 15K FWHM and MEW of 10 ppm in LC- MS-MS was preferred for structural determination of smaller molecules. 103 Extraction of Caffeine from Tea and Water Using QuEChERS with Gas Chromatography-Mass Spectrometry Detection 106 Rapid Separation of Anthocyanins in Bilberry and Cranberry Michelle L. Schmidt, Seton Hall University, 400 South Orange Ave., Extract using Core Shell Columns South Orange, NJ 07728, Nicholas H. Snow Peter Bodsky, Thermo Fisher Scientific, 1228 Titan Way, Sunnyvale, CA Caffeine and its extraction from tea and water have been used as a model system 94085, Pranathi Perati, Art W. Fitchett, Michael G. Hvizd in many applications including organic chemistry teaching, environmental and phar- Anthocyanins are powerful antioxidants that are responsible for the red, orange, and maceutical analysis. The extraction of caffeine from tea and water is interesting blue coloration in fruits and flowers. Major anthocyanin sources include blueberries, because it provides the opportunity to investigate fundamental chemistry param- pomegranates, and cranberries. The consumption of cranberries and bilberries has eters such as extraction conditions and partition coefficients and how they affect increased globally due to the reported health benefits. Anthocyanin profiles have extraction recoveries, for a very well-known model analyte. Tea provides a model shown to be critical in the authentication of fruit juices containing these antioxidants. complex matrix, similar to both food and plants, which can result in overlapping High-performance liquid chromatography (HPLC) columns with fully porous 2-μm peaks, providing, in turn, modeling for matrix interferences. Partition coefficients and and solid core-porous shell particles, both deliver separation efficiencies higher than extraction conditions for the sample preparation method, QuEChERS (quick, easy, traditional packing materials, i.e., fully porous particles of 3-5-μm nominal diameter. cheap, effective, rugged, and safe) were studied in this work. The extraction of caf- The fully porous 2.2-μm particles are designed to increase peak efficiency, but at the feine from tea and water using QuEChERS combines a liquid-liquid microextraction expense of increased system pressure requirements. Core-shell particles improve with a dispersive solid phase extraction (d-SPE) clean-up step to remove matrix the mass transfer kinetics by restricting intraparticle diffusion to the thin porous shell interferences, providing a clean sample for gas chromatography mass spectrom- while maintaining the hydraulic permeability associated with the total particle diam- etry (GC-MS) analysis with the use of limited solvent. QuEChERS has been used eter. This study compares the separation of anthocyanins in cranberry and bilberry for the extraction of pesticides and other analytes from many matrices; however, extracts using a fully porous 2-μm and a core-shell column. The fully porous 2-μm the fundamental aspects of this method have never been fully investigated. In this particle is designed for fast separations and the core-shell column uses a solid core study the partition coefficients and optimum extraction conditions for caffeine were developed to generate high speed, high resolution separations without excessive determined using several solvent systems including acetonitrile, acetone, and ethyl backpressure. The study concluded that the fully porous column can be used to acetate. The depletion study used to determine the partition coefficients through the quantify anthocyanins in both cranberry and bilberry extracts, but the core-shell col- calculation of extraction ratios is discussed along with the resulting partition coef- umn provided a qualitative analysis of bilberry extract (more complex sample) and ficients and percent recoveries from each extraction. a quantitative analysis of cranberry extract with a 20 minutes time saving. Method precision was evaluated by analyzing three independent preparations of cranberry 104 Measuring Estrogens by Automated On-Line Extraction, extract over three days. The intraday and between-day peak area relative standard Derivatization and Cleanup Coupled to LC-MS-MS deviation ranged from 1.01%-1.31%, and 1.26%-1.87% respectively, suggesting Heather L. Heilman, Thermo Fisher Scientific, 130 Dean St., Apt. 8, good method precision. West Chester, PA 19382, Joseph M. Di Bussolo The amount of estrogens such as estrone (E1), 17β-estradiol (E2) and estriol (E3) 107 Ramp Up the Pressure, Turn Up the Heat: Secondary Effects in in various fluids can be measured by liquid chromatography mass spectrometry Chromatographic Method Scaling (LC-MS) methods utilizing electrospray or atmospheric-pressure ionization to in- David Lloyd, Bristol-Myers Squibb, One Squibb Dr., New Brunswick, NJ duce negative ions. Unfortunately, the efficiency of such ionization is poor and limits 08903, Holly M. Shackman, Wei Wang, Xujin Lu, Zhen Wu, Samantha the sensitivity to approximately 5 pg/mL. Derivatization with dansyl chloride provides Mahmoud, Hannah Lo estrogens with a positive charge, thereby allowing quantitation below 0.1 pg/mL. Very high-pressure liquid chromatography (VHPLC) has become commonplace in However, prior to dansylation, the estrogens must be extracted using liquid-liquid industrial laboratories. Nevertheless, with a huge installed base of regular LC equip- or disposable solid phase techniques, which are labor-intensive and expensive. We ment, ideally our methods should be usable in both regular- and very-high pressure have devised a modification to a two-dimensional LC system designed for reusable environments. In order to translate method conditions between HPLC and VHPLC, on-line solid phase extraction to automate the initial extraction, derivatization and geometric scaling [1] is implemented in VHPLC instrument software. This is often final cleanup steps prior to the final high-performance LC separation and detection quite effective, but surprises do occur, e.g., changes in selectivity or occasionally by tandem mass spectrometry. To demonstrate its utility, urine samples spiked with gross differences between predicted and observed retention. Given that significant

17 2013 EAS Abstracts November 2013

frictional heating occurs in VHPLC, high pressure is an inherent feature of the tech- and cytosolic/Nuclear receptors is discussed together with the key parameters to be nique, and both temperature and pressure affect chromatographic retention, why considered for the development of a reliable bioaffinity tool. is geometric scaling so often successful? We show results from successful and References: unsuccessful scaling of pharmaceutical impurities methods, and demonstrate the [1] E. Calleri et al. J. Pharm. Biomed. Anal. 54 (2011) 911-925. utility of resolution maps generated during method development to ensure robust- [2] C. Temporini et al. Anal. Biochem. 384 (2009) 123-129. ness against frictional-heating induced temperature changes. In addition, a system- [3] C. Temporini et al. Anal. Bioanal. Chem. 405 (2013) 837-845. atic study of retention of model compounds is presented where either pressure, [4] E. Calleri et al. J. Chromatogr. A 1232 (2012) 84-92. temperature or both were altered. While increasing temperature alone caused a [5] C. Temporini et al. J. Chromatogr. A 1284 (2013) 36-43. reduction in retention, increasing pressure alone increased retention. However, in a normal VHPLC setup when the flow rates were increased (analogous to method 111 When Someone Says “Everybody Knows That ...” Stop Listening! scaling) and thus both temperature and pressure changed, little change in retention Adventures in Basic and Translational Research was noted, apparently due to the self-cancelling nature of the effects of temperature Irving Wainer, National Institute on Aging/NIH, 251 Bayview Blvd., and pressure. Baltimore, MD 21224 Reference: This presentation explores the potential benefits derived from listening to data [1] U. D. Neue, D. McCabe, V. Ramesh, H. Pappa, J. DeMuth, Pharmacopeial Fo- instead of “experts.” While this is a recurring theme in my research, only two is- rum 35 (2009) 1622. sues are addressed. The first is molecular chiral recognition and will address the statement “Everyone knows that chiral recognition is based upon a three-point in- 108 Chiral Bioanalysis and Metabolite Monitoring in the Era of teraction.” The presentation explores the origins of this theory and propose that Enantiomerically Pure Drugs molecular chiral recognition is a multi-step process flowing from the tethering of Anne-Françoise Aubry, Bristol-Myers Squibb, MS: K12-04, Rte. 206 and the analytes to the chiral selector to conformational adjustments within the diaste- Provinceline Rd., Princeton, NJ 08540, Michael Furlong, Jian Wang, reomeric complexes which leads to the expression of stereochemcial differences. Daria Wentzel, Song Zhao, Moucun Yuan, William R. Mylott Jr. The second addresses the statement “Everyone knows that the clinical effects of Since the 1980’s, the trend in drug discovery has been the synthesis of enantiomeri- (R,S)-ketamine are due to the inhibition of the NMDA receptor by (R,S)-ketamine cally pure drugs. In the last 10 years alone, chiral drugs comprised about 70% of and its active metabolite (R,S)-norketamine.” The presentation discusses our re- new drug approvals worldwide, of which 95% were marketed as pure enantiomers. cent studies of the use of low dose (R,S)-ketamine in the treatment of depression Several of these drugs were the result of a chiral-switch strategy, in which a pure and neuropathic pain. The data indicates that the observed clinical response is due enantiomer of a previously approved racemate is developed with the expectation of to the selective and potent inhibition of the alpha7-subtype of the nicotinic ace- superior activity and/or diminished side effects. As a result of the near-elimination tylcholine receptor by the (R,S)-ketamine metabolites (R,S)-dehydronorketamine of racemates in new drugs, the need for chiral evaluation of pharmacokinetics has and (2S,6S;2R,6R)-hydroxynorketamine, which results in an indirect attenuation of been reduced but not eliminated. Case studies will illustrate situations in drug de- NMDA receptor activity. The potential use of these compounds in the treatment of velopment when a stereoselective bioanalytical assay may be needed, including depression and neuropathic pain is discussed. in-vivo racemization, where the drug undergoes a chiral inversion, stereoselective Abstracts 112-200 drug-drug interactions between a development drug and a racemic drug, and meta- bolic chiral inversion that impacts the drug safety or activity profile of the drug. The 112 Deep UV Resonance Raman Spectroscopy of Biopharmaceuticals examples involving metabolites are discussed in the context of the Metabolites in Sergey Arzhantsev, United States Food and Drug Administration, 1114 Safety Testing (MIST) guideline and the tiered approach to validation. Market St., Room 1002, Saint Louis, MO 63101, Liwei Yuan, Vincent Vilker, John Kauffman 109 High-Performance Affinity Microcolumns: Recent Developments in The number of protein based therapeutics entering the US market is increasing Clinical Testing, Pharmaceutical Analysis and Biointeraction rapidly. To assure the safety and quality of therapeutic protein products, it is impor- Studies tant to develop rapid analytical methods to screen for degradation of protein drug David Hage, University of Nebraska, Chemistry Dept., 704 Hamilton products, which is one of the major concerns of the biopharmaceutical industry. Hall, Lincoln, NE 68588 Some degradation pathways result in changes in the secondary structure of pro- Biologically-related binding agents such as antibodies and serum transport pro- teins. Deep ultraviolet resonance Raman (DUVRR) spectroscopy is a good alterna- teins have long been of interest for the selective isolation and analysis of chemi- tive to techniques such as to nuclear magnetic resonance, infrared, ultraviolet and cals in complex mixtures. The selectivity and moderate-to-strong binding of these CD for determination of secondary structure of the protein. The resonance excita- agents have also made these ligands of great interest for use in affinity-based high- tion provides high selectivity and sensitivity to secondary structure compared to performance liquid chromatography methods and, more recently, in microaffinity non-resonance Raman spectroscopy and other vibrational spectroscopies. DUVRR separations. This presentation discusses recent developments in the creation of spectra are dominated by protein vibrational modes that are coupled to the amide pharmaceutical and clinical applications of high-performance affinity microcolumns. electronic state, including C-O and N-H vibrations that are extremely sensitive to This includes an examination of how such columns can be made and used in chro- secondary structure. The main advantages of DUVRR spectroscopy are minimum matographic-based immunoassays, ultrafast immunoextraction, and biosensors. sample preparation and the possibility to analyze formulated product without any The use of affinity microcolumns for studying the thermodynamics and kinetics of separation step. This presentation describes the chemometrics analysis of DUVRR biological interactions will also be considered, as well as the possible extension of spectra of proteins with known secondary structures that enables us to predict this approach to the high-throughput screening of drug-protein binding. The benefits secondary structure of a protein with an unknown structure. Application of DUVRR and current challenges in using affinity microcolumns are also discussed. spectroscopy for study of forced degraded protein samples is presented. Mixture analysis for determination of the protein contribution to the resonance Raman spec- 110 Biochromatography: An Endless Frontier tra of formulated protein products is discussed. Gabriella Massolini, University of Pavia, via Taramelli 12, Pavia 27100, Italy 113 Characterization and Spectroscopic Applications in Pulmonary The drive to bring innovative drugs to market faster, without compromising quality Delivery and Advanced Dry Powder Inhalers and safety, induced need for new experimental techniques and methodologies. It is, Heidi M. Mansour, University of Arizona-Tucson, Office 434/Lab 435, thus, crucial to determine biological activity, drug-target interactions and physico- 1703 E Mabel St., Tucson, AZ 85721 chemical properties of the new potential drugs. The development of rapid meth- Targeted pulmonary delivery as inhalable multifunctional microparticulate/nanopar- odologies enabling to obtain this information is a key aspect in the drug discovery ticulate dry powder aerosols offer unique advantages to address unmet medical process. In the wide scenario of available assays, biochromatography has emerged needs in the treatment and prevention of pulmonary diseases. Incorporation of as a useful and promising label-free technique. This method relies on the immo- nanotechnology with model drugs representing different therapeutic classes was bilization of a biological target on a chromatographic support and the developed optimized for aerosol delivery as a therapeutic dry powder nanotechnology platform. stationary phase can be used for determine all the parameters useful in drug dis- A systematic quality-by-design (QbD) approach in the solid-state was used including covery process to characterize the binding of new potential drugs to the target of comprehensive physicochemical characterization, confocal Raman microspectros- interest.[1] For several years, our group has been involved in the development of copy (chemical imaging), Fourier transform infrared spectroscopy, design of experi- different biochromatographic systems for binding studies. This presentation reports ments, performance modeling, and prediction. The physicochemical properties and an overview of the strategies followed and the realization methods used to develop spray drying conditions were correlated and modeled with dry powder aerosol dis- biochromatographic stationary phases based on membrane receptors (GPR-17 and persion performance profiles in the solid-state. A2A) and nuclear receptors (PPAR alpha and gamma). [2-5] In particular, the power of frontal affinity chromatography mass spectrometry approach using membrane

18 2013 EAS Abstracts November 2013

114 Application of In-Line Raman for Real-time Measurement of Carbon ize for APCI and APPI. An alternative method for ionization is metallation, which is Source in Mammalian Cell Culture Fermentation the process of forming metal cations in solution which can then be analyzed using Louis Obando, Merck, 770 Sumneytown Pike, West Point, PA 19486, ESI. This paper demonstrates how didodecylpolysulfides can be analyzed using ESI Daniel C. Johnson with Ag(I) complexation. Using a dilute AgNO3 solution as a post column reagent, Merck has adopted quality-by-design (QbD) principles outlined in the International we characterized the alkyl and Sx groups in the didodecylpolysulfide. C22, C23, Conference on Harmonization guidelines for the development many of its products. and C24 alkyl polysulfides and S4-12 chains were detected. The formation of [M + This work demonstrates the strong link between QbD and real-time, in-line Raman Ag]+ and [2M + Ag]+ ions for S4-5 and [M + Ag]+ ions for S6-12 was identified. We spectroscopy for the quantification of the carbon source for mammalian cell culture also studied how the fragmentor voltage affected the ions observed and performed during fermentation. This presentation will address model and method develop- collision induced fragmentation to understand the fragmentation pathways of the ment, the multivariate method and the logistics of implementation in an industrial silver complexes. environment. The benefits of this type of process analytical technology (are also presented. 118 Rapid Disulfide-Containing Protein Structure Analysis Using Combined Electrochemistry and Mass Spectrometry 115 Challenges and Issues of Spectroscopy and Chemometric Hao Chen, Ohio University, Chemistry Department, Athens, OH 45701 Applications in Protein Therapeutics Development and Redox-active disulfide bonds are one of the most common protein post-translational Manufacturing modifications but the presence of disulfide linkages increases the complexity for Seongkyu Yoon, University of Massachusetts-Lowell, One University protein structure determination by mass spectrometry (MS). This paper presents Ave., Lowell, MA 01854, Nicholas Trunfio, HaeWoo Lee a rapid and convenient method for structure analysis of disulfide bond-containing Spectroscopic techniques have proven to be a powerful tool for the development proteins, employing desorption electrospray ionization-mass spectrometry (DESI- and manufacturing of mammalian cell cultures that produce therapeutic proteins. MS) coupled with online pepsin digestion and online electrochemical reduction of Since these techniques can provide fast, non-invasive methods to obtain biochemi- disulfide bonds. Peptides are generated in high digestion yield as its precursor pro- cal information about biopharmaceutical processes, they offer significant advan- tein in acidic aqueous solution flows through a pepsin column, which can undergo tages over traditional off-line analyses in the areas of raw material characterization, direct electrolysis. The electrolytic behaviors of peptides, as online monitored by bioprocess monitoring and product quantification. An important challenge is that bio- DESI-MS, not only tells the presence/absence of disulfide bonds in the peptides, but pharmaceuticals require several complex raw materials whose compositional vari- also provides information to relate disulfide bond-containing peptide precursors to ability is high and whose influence on cell culture is poorly understood. Application their corresponding reduced products. Furthermore, selective electrolysis simply us- of spectroscopic techniques like near-infrared, Raman, fluorescence and X-ray fluo- ing different reduction potentials can be adopted to generate either partially or fully rescence spectroscopy combined with chemometric tools like ensemble partial least reduced peptides to assist disulfide bond mapping. In addition, it turns out that DESI squares can provide fast, non-destructive ways to evaluate raw material quality as is suitable for ionizing peptides in water without organic solvent additives that would it relates to cell culture performance. Viable cell density (VCD) is one of the most not be compatible with the use of pepsin column. The feasibility of this method was crucial parameters when monitoring the current status of cell growth and protein demonstrated using insulin, a protein carrying three pairs of disulfide-bonds as an production. Dielectric spectroscopy is a reliable tool for the real-time monitoring of example, in which all disulfide bond linkages and most of the protein sequence VCD. However, a critical drawback of dielectric spectroscopy is that its accuracy fre- were successfully determined. Strikingly, this method shortens the sample diges- quently deteriorates during the late-growth phases. By using locally-weighted partial tion, reduction and MS detection from hours to 7 min, which could be of high value least squares as the multivariate regression technique, process modeling capabili- in high-throughput proteomics research. ties were improved, especially in the late-growth phases. The real-time monitoring of chromatographic protein purification processes is important. However, a majority 119 Process Mass Spectrometers - Now a PAT Tool for Cell Culture of techniques still rely on the use of a single ultraviolet (UV) absorbance, or the Todd B. Colin, Thermo Fisher Scientific, 1410 Gillingham Ln., Sugar utilization of expensive and time-consuming instruments. Therefore, the study was Land, TX 77478, Peter J. Traynor conducted to show that chemometric models based on multi-wavelength UV spectra Mass spectrometers have been used to provide online PAT for industrial fermenta- have sufficient accuracy for estimating protein concentrations. tions for 30 years or more. They continue to provide valuable insight regarding the trajectory of batch and fed-batch processes. Their use has been somewhat limited 116 Analysis of Tetracycline Transformation and Degradation Using in cell culture processes, however. The distinction between microbial fermentation Vetiver Grass by Ultra High-Performance Liquid Chromatography/ and cell culture is important from a process control perspective since hardy bacteria High-Resolution Mass Spectrometry need less protection from process variation than do mammalian cells which require Chinmayi Parikh, Kean University, STEM Bldg., 1000 Morris Ave., tight control of temperature, pH and the sheer forces produced by agitation and Union, NJ 07083, Dil Ramanathan, Aparupa Sengupta, Rupali Datta aeration. Control of dissolved carbon dioxide is also important in cell culture control. Tetracycline, a well-known antibiotic group, has been widely used in treatment Low and intermittent sparge rates provide challenges for mass spectrometers when of bacterial infection of plants, aquaculture and animal growth. However, it is an 5-10 Liter bench-top bioreactors are used for process development. This paper dis- emerging environmental and human health concerns due to a large amount of pres- cusses the challenges and introduces a new mass spectrometer specifically de- ence in water and soil systems via manure, slurry, biomedical waste and sewage. signed to accommodate low sample flows without compromising the high precision In this study, a sensitive method was developed to trace tetracycline transformation provided by magnetic sector technology. and degradation by using ultra high-performance liquid chromatography – high res- olution mass spectrometry (UHPLC-HRMS) combined with solid phase extraction 120 Evaluation of Centroid and Profile Mode Data Collection Methods (SPE). Vetiver grass (root and shoot) samples were grown in various concentration for High-Resolution Accurate Mass Spectrometry, (HRMS) Based of tetracycline such as 0ppm, 5ppm, 35ppm, 75ppm in hydroponic media. After 30 Integrated Qualitative and Quantitative Analysis days, these grass samples were analyzed in UHPLC-HRMS system. In UHPLC Eldho Raju, Kean University, Department of Chemistry, Union, NJ system, C18 column was used at 30 °C column temperature along with 0.2 mL/ 07083, Dil Ramanathan min flow rate and 20uL injection volume. Furthermore, sixteen minutes gradient Recently a global conflict has aroused in the mass spectrometry community regard- program was developed using both water and acetonitrile (ACN) with 0.1% formic ing the data acquisition modes in high resolution mass spectrometry (HRMS). Many acid as aqueous (A) and organic (B) mobile phases respectively for separation. scientists acquire HRMS data in centroid mode data files, in spite of profile mode. In Then, tandem mass spectrometry was used for the detection of tetracycline and this conflicting contrast, we decided to investigate on effectiveness of centroid ver- their degradative products. sus profile data acquisition mode. We investigated, using four pharmaceuticals and their metabolites, on the effectiveness of centroid and profile mode data acquisi- 117 Electrospray Ionization Mass Spectrometry of tion methods for HRMS experiments using an LTQ-Orbitrap mass spectrometer. We Didodecylpolysulfides Using Silver(I) Complexation also studied different mass resolving power, mass accuracy, and mass extraction Christie Bowden, Arkema, 900 First Ave., King of Prussia, PA 19406, window (MEW) necessary to obtain accurate m/z measurement in both centroid and Sriraj Srinivasan, Gary S. Smith profile data acquisition modes. The effect of different peak smoothening algorithms Analyzing didodecylpolysulfides using traditional atmospheric pressure ionization was also studied. The entire study was performed by analyzing the effect of internal techniques can be challenging for numerous reasons. With electrospray ionization calibration. A mass resolving power of 15K FWHM with an optimum MEW 10 ppm (ESI), ionization occurs in solution; therefore, requiring analytes to be polar so that and Gaussian peak smoothening algorithm with 7 data points, were optimized for protonation or deprotonation can occur. With atmospheric pressure chemical ioniza- m/z measurements with sufficient mass accuracy in HRMS studies. Centroid data tion (APCI) and atmospheric pressure photoionization (APPI), ionization occurs in found to show better signal response, which is preferred for industrial research. Pro- the gas phase therefore requiring analytes to have some volatility. Unfortunately, file data with reasonable signal response with an option of manual processing would didodecylpolysulfides are neutral, non-polar molecules, making ESI difficult. In addi- be preferred in academic and independed research. The most and least intense tion, didodecylpolysulfides have high boiling points, making them difficult to volatil- fragments of all drugs upon MS/MS, showed equal signal responses in both centroid 19 2013 EAS Abstracts November 2013

and profile modes. In contrast profile mode data showed comparatively high percent enhanced chromatography is superior to gas chromatography for high-boiling-com- abundance for precursor ions upon HPLC-MS/MS, which is preferred for structural pounds analysis and offers more resolution than high-performance liquid chroma- determination of smaller molecules. tography. FAMEs analysis by supercritical fluid chromatography (SFC) has been attempted earlier with some success. We have investigated the benefits brought to 121 Elucidation of Polysaccharide Structures, Containing Charged the analysis of FAMEs and FAMEs containing materials by the recent progress in Polysulfated/Polycarboxylated Moieties, Using Exact-Mass ESI SFC instrumentation. Negative Ionization MSMS Data and the MASSPEC Algorithm Marshall M. Siegel, MS Mass Spec Consultants, 39-24 Grant St., Fair 124 Overloaded Chiral Separations on an Analytical and Preparatory Lawn, NJ 07410, Gary Walker, Lingyun Li, Robert J. Linhardt (with Collection) Scale in SFC Arixtra (C31H53N3O49S8, M = 1506.95134 Da) is a pentasaccharide containing John Whelan II, Waters, 159 Lukens Dr., New Castle, DE 19720, John 8 sulfo and 2 carboxyl groups.dP7 (C44H71N3O57S7, M = 1777.07943 Da) is a Baugher septasaccharide containing 7 sulfo and 3 carboxyl groups. These compounds pro- One of the advantages of using supercritical fluid chromatography (SFC) for analyti- duce very complex spectra in the electrospray ionization (ESI) negative ionization cal and prep scale separations is the ability to quickly separate chiral compounds tandem mass spectrometry (MS-MS) mode that is challenging to correlate with the with low solvent use and faster dry down times. In the past many chiral presenta- structures. The MASSPEC algorithm, derived from Graph Theory and combinator- tions have been done, but typically with only a baseline separation being used as ics, was used to correlate the fragment ions observed in the ESI-MS-MS spectra an example in the area of SFC. Contained in this study we demonstrate overloaded with corresponding substructures of the proposed structures. All ionizable protons separations that yield two peaks or more peaks. The body of work includes effects were replaced with sodium during the electrospray process using a carrier solvent of slight co-solvent/modifier changes with regards to retention time correlated to the containing NaOH. Exact mass data were acquired (Delta = 3 ppm) with an Orbi- degree of overloading with comparison to recovery yield. trap mass spectrometer in the ESI-MS-MS negative ionization mode for the [M- 10H+7Na]3- parent ions at m/z 552.601 for Arixtra and m/z 642.6423 for dP7. The 125 Targeted Isolation of Impurities in Pharmaceutical Compounds Arixtra and dP7 structures were each re-expressed as sets of bonded and floating Using SFC (Strategy Development for Pharmaceutical Impurity superatom substructures. Expected fragmentation rearrangements were expressed Isolation) in the organization of the bonded super atom structures and through the use of Mikey Rego, Averica Discovery Services, One Innovation Dr., Biotech 3, floating super atoms. The MASSPEC algorithm automatically computed the masses Worcester, MA 01605, Paul Lefebvre, John Tipping, Jeff Kiplinger of all possible substructures with up to four bonds removed and compares the pre- Chromatographic isolation of impurities can be a painstaking process for the analyti- dicted masses to the observed masses. The MASSPEC scoring algorithm incorpo- cal laboratory. Generally a suite of analytical instrumentation and multiple chromato- rates MS fragmentation processes and degree of fit of the predicted and observed graphic methods are required. Additionally, traditional preparative high-performance masses. The most probable fragment ion assignments were the highest scoring liquid chromatography (HPLC) approaches result in large (often aqueous) fraction predicted substructures. This structure elucidation work is virtually impossible to be volumes, jeopardizing the stability of the collected fractions. Supercritical fluid chro- successfully achieved by hand calculations since only with computer based algo- matography (SFC) is one tool that can help eliminate some of these problems. In rithms are the calculations exhaustive. our laboratory, we systematically approach this process using an approach we term “targeted isolation” which maximizes the benefits of SFC. In comparison with re- 122 Chromatographic Method Development Strategies for Convergence versed-phase and normal phase HPLC, SFC offers rapid method development and Chromatography high efficiency of preparative separations through increased loading capacity, faster Paula Hong, Waters, 34 Maple St., Milford, MA 01757, Michael D. linear solvent velocity, and drastically reduced fraction dry down time. The efficiency Jones, Patricia McConville gained allows us to use stepwise protocols to, such as impurity enrichment by main Convergence chromatography is emerging as a chromatographic technique that peak extraction, that increase process productivity. Targeted isolation is a problem- provides alternative selectivity to traditional reversed phase high-performance liquid solving process in which we strategically design economical ways to isolate specific chromatography while reducing solvent consumption. Using supercritical carbon peaks of interest. Targeted isolation, and the benefits of SFC leveraged in the isola- dioxide, this normal phase technique is based on the numerous modes of inter- tion process, are described using several case studies. action between the stationary phase and the solute, including hydrogen bonding, hydrophobic interactions, dipolar interactions and solute partitioning. These interac- 126 Improved Chiral SFC Screening for Separation of Complex tions can be manipulated by a number of variables including sample diluent, column Mixtures chemistries, organic modifiers and additives, and physical parameters. Combining Erik L. Regalado, Merck, 126E. Lincoln Ave., PO Box 2000, Rahway, NJ and optimizing the impact of these factors can be a complex and time-consuming 07065, Wes Schafer, Tilak Chandrasekaran, Zainab Pirzada, Chaowei method development process. In order to streamline this process, we are develop- Zhang, Xiaoyi Gong, Mirlinda Biba, Christopher J. Welch ing a systematic approach to achiral convergence chromatography method devel- In this study we describe the evaluation of a recently developed supercritical fluid opment. Initial steps will evaluate the properties of the analyte of interest, including chromatography (SFC) instrument for automated chiral SFC method development. hydrophobicity and functional groups. Based on these chemical properties, column, The greatly improved gradient dwell volume and liquid flow control of the new instru- organic modifiers and additives will be screened for their impact on peak shape and ment in combination with the use of shorter columns containing smaller stationary selectivity. In addition, physical factors such as pressure and temperature will also phase particles affords chiral SFC method development that is faster and more be examined for maximizing efficiencies and throughput. This process, when com- universal than previous systems. By using this instrument, we develop new methods bined with sub-2-µm column chemistries will allow for a streamlined approach to to isolate and identify complex isomeric species. convergence chromatography method development for a wide range of compounds. 127 Comparison of Two-Dimensional 1H-19F NMR Scalar Coupling 123 Novel Supercritical Carbon Dioxide Enhanced Chromatography Experiments in Small Molecules Method for the Comprehensive Analysis of Fatty Acid Methyl Alexander Marchione, DuPont, PO Box 80328, Wilmington DE 19880, Esters (FAMEs) and FAMEs Containing Materials Elizabeth F. McCord, Rebecca J. Dooley François J. Huby, The Dow Chemical Company, 727 Norristown Rd., PO Two-dimensional (2-D) nuclear magnetic resonance (NMR) experiments correlating Box 0904, Spring House, PA 19477, Robert M. Campbell 1H and 19F via scalar coupling are often very valuable tools in the structural analysis FAMEs (fatty acid methyl esters produced from vegetable oils and animal fats by of polyfluorinated organic compounds. Nevertheless, the extant literature on these trans-esterification with methanol), are used as fuels or fuel additives in vehicles experiments is scarce, despite their utility and high sensitivity. A comprehensive (biodiesel), in machines and power generators, in cosmetics and pharmaceuticals, comparison of gradient coherence-selected 1H-19F 2-D scalar coupling experiments, as lubricants, as industrial solvents and as carriers for fertilizers and pesticides. including hetero-COSY (a.k.a. FUCOUP), HETCOR, HMQC, and HSQC, is pre- They are also used in the food and agriculture industry because they are biode- sented here on polyfluorinated and monofluorinated analytes. The J selectivity of gradable and eco-friendly. New uses are being developed as their availability and the latter experiments is discussed, as well as the benefits of adiabatic pi pulses in cost permit it. FAMEs are generally analyzed by gas chromatography, most of the 19F, and the factors influencing the choice of observe nucleus. time using the ASTM D6584, 7371 and 7398 methods or similar. Unfortunately gas chromatographic analysis of these relatively high boiling compounds is plagued by 128 Spectroscopic and Computational Analyses of Polymorphic many problems, especially when the samples contain significant amounts of impuri- Organic Materials ties such as residual fatty acids, glycerol, water and partially converted glycerides. Sean T. Holmes, University of Delaware, 102 Brown Laboratory, Newark, Moreover the derivatization reagents used to volatilize the more polar compounds DE 19716, Fahri Alkan, Cecil Dybowski and the elevated temperatures required for eluting the highest boiling fractions de- Two forms of 1,10-phenanthroline, which differ in the number of unique molecules in grade the gas chromatography columns very rapidly. Supercritical carbon dioxide the asymmetric unit and by the presence of water, were analyzed by C-13 nuclear magnetic resonance spectroscopy. Assignments of the isotropic spectral peaks are

20 2013 EAS Abstracts November 2013

based on dipolar-dephasing experiments in which the signal intensity is inversely provement of the analysis method is necessary to detect relatively small but relevant proportional to the strength of the C-H dipolar coupling constant. The symmetries of changes in dynamics, such as those between wild-type and mutant proteins. Using the two forms were investigated through infrared spectroscopy and were found to our newly optimized method for analyzing NMR relaxation data, we recently charac- be consistent with X-ray diffraction data from the literature. The experimental C-13 terized backbone dynamics of a multi-drug-resistant variant of HIV-1 protease with chemical shifts were correlated with calculated chemical shieldings obtained from a combination of mutations at the edge of the active site, within the active site, and density functional theory (DFT) calculations on molecular clusters with a diameter in the flaps (L10I, G48V, I54V, V82A), and compared the results with those for the of over three nanometers. We discuss the importance of a realistic input geom- wild-type protease. etry in these types of calculations; specifically, the cluster size and the placement of protons (which cannot be resolved sufficiently by single crystal X-ray diffraction 132 Exploring Amyloid Structure by Quenched Hydrogen Exchange data) are shown to affect the overall accuracy. Results show that the two forms of Solution NMR 1,10-phenanthroline differ significantly. Differences in chemical shift between the Andrei T. Alexandrescu, University of Connecticut, 91 N Eagleville Rd., two forms are rationalized using a model in which two equivalent nuclei can be split U-3125, Storrs, CT 06269 by differences in the local field induced by a neighboring molecule in the crystal Amyloids are protein aggregates with a fibrillar cross-beta-sheet structure. Over lattice. 30 amyloidogenic proteins are involved in human diseases including Alzheimer’s, Parkinson’s, and HIV infectivity. Because amyloids are large and complex they are 129 Characterization of Polymer Electrolyte Membranes by Gel-State extremely challenging for structural studies. Solid-state nuclear magnetic resonance MAS NMR (NMR) has afforded detailed structural information but the insolubility of fibrils pre- Haiming Liu, Arkema, 900 First Ave., King of Prussia, PA 19406, Dave cludes direct studies by solution NMR. To this end, we have developed a quenched Mountz, Tao Zhang, Wensheng He hydrogen exchange (qHX) method to study amyloid fibrils indirectly by solution Polymer electrolyte membranes (PEM) play essential functions of proton transport NMR. In the qHX experiment, HX is initiated by suspending amyloid fibrils in D2O. and separation of reactants in fuel cells. Their proton conductivity and thermal stabil- After varying periods of time, HX is quenched by flash-freezing. Partially exchanged ity are the key properties for their functions, which to a large extent are determined fibrils are lyophilized and dissolved in a strongly denaturing solvent such as 95% di- by their chemical composition and structure. PEM characterization is usually chal- methyl sulfoxide (DMSO), which dissociates most types of amyloid fibrils to unfolded lenging, due to insolubility, complex composition or formulation, and interference monomers. Because DMSO is an aprotic solvent, HX from the denatured state oc- from water, acid, and counterion. In this study, a comprehensive approach to PEM curs on timescales of hours compared to minutes or seconds in H2O, allowing the sample preparation is developed, and variable temperature (VT) gel-state magic detection of amide protons trapped in the fibril. The experiment has been used to angle spinning (MAS) nuclear magnetic resonance (NMR) is applied, to overcome study artificial amyloids formed by the bacterial protein CspA where residues exhibit challenges. High resolution 1H and 13C MAS NMR spectra of PEM are successfully uniformly strong protection and exchange occurs through dissociation of monomers obtained, which allows in-depth compositional analysis. For Arkema polyeletrolyte- from the fibrils. In the case of IAPP, an amyloid involved in type 2 diabetes, protec- fluoropolymer blends (APEM), key components including poly(vinylidine fluoride) tion identifies residues in hydrogen-bonded beta-sheet structure and differences in (PVDF), polyelectrolytes, and tetraalkyl ammonium are well resolved and quanti- protection result from the packing of beta-strands in the protofibrils. Additional ap- fied by NMR, which enables determination of polyelectrolyte-to-PVDF ratio and the plications of the qHX experiment include determining if fibrils form in an all-or-none capacity and degree of protonation. The method can be applied to study PEM after process or through intermediates, and mapping the binding sites for ligands and fuel cell testing, which could help better probe or understand possible chemical inhibitors of fibrils. degradation of PEM. 133 Two-Dimensional IR and Two-Dimensional SFG Spectroscopies: 130 Investigating the Molecular Framework and Assembly of Synthetic New Techniques for the Analytical Sciences and Fungal Melanins by Solid-State NMR Martin Zanni, University of Wisconsin-Madison, 1101 University Ave., Subhasish Chatterjee, CUNY Institute for Macromolecular Madison, WI 53706 Assemblies,160 Convent Ave., MR 1209, New York, NY 10031, Sindy Infrared spectroscopy and vibrational sum frequency generation (VSFG) spectros- Tan, Hsin Wang, Ruth E. Stark, Rafael Prados-Rosales, Susana Frases, copies are two of the most widely used analytical tools for characterizing surfaces Arturo Casadevall, Boris Itin and solutions. In this talk, I introduce multi-dimensional variants of these spectros- Melanins constitute an important class of ubiquitous natural pigments that play im- copies. These two-dimensional (2-D) techniques allow vibrational modes to be cor- perative roles in protection against sunlight, camouflage, free radical scavenging, related with one another, which provide information on binding, solvent exposure, and microbial virulence. Nonetheless, structural elucidation of these enigmatic pig- vibrational lifetimes and many other variables that provide information about mo- ments remains a challenging task because of their insolubility and heterogeneous lecular identity and structure. New pulse shaping technologies make it quite simple architecture. High-resolution solid-state nuclear magnetic resonance (NMR) spec- to collect 2-D infrared or 2-D sum-frequency generation spectra. I focus on 2-D SFG troscopy offers a unique tool to investigate melanin structure, and melanogenesis spectroscopy as it is applied to interfaces, and present recent results on a Pt-CO in Cryptococcus neoformans (CN) requiring exogenous obligatory catecholamine electrochemical cell and a peptide bound to a gold monolayer. In both of these sys- precursors such as L-dopa provides an unparalleled means to probe melanin bio- tems, 2-D SFG provides structural information not previously obtainable. synthesis and assembly in fungal cells. One-dimensional (1-D) solid-state 13C NMR and electron paramagnetic resonance demonstrated the structural differences 134 Photoexcited Excitons in Single Walled Carbon Nanotubes among CN melanins produced by a range of catecholamine precursors and in cell- Todd Krauss, University of Rochester, Department of Chemistry, 120 free media. Broad aromatic resonances in both fungal and synthetic melanins sug- Trustee Rd., Rochester, NY 14627, Michael Odoi, Julie Smyder, Lisa gested a heterogeneous disordered aromatic domain in these pigments, whereas Nogaj, Andrea Lee, Xiaoyong Wang differences among the aliphatic moieties indicated incorporation of glucose-derived Semiconducting single-walled carbon nanotubes (SWNTs) display remarkably cellular materials into the fungal pigments. 13C and 15N solid-state NMR, includ- stable and size-tunable photoluminescence at near-infrared wavelengths, making ing 2-D through-space and through-bond correlation spectra of pigments produced them fundamentally interesting and technologically relevant materials. However, with exogenously 13C-and 15N-enriched L-dopa and/or [U-13C6]-glucose supplied to individual SWNTs typically have a photoluminescence (PL) quantum yield (QY) of the growth media, provided information on the molecular framework formed by the about a few percent. It is currently not understood whether the poor QY of SWNTs pigments and in CN melanization. The molecular architecture of natural melanin is an intrinsic property or the result of extrinsic quenching mechanisms indicative revealed by this solid-state NMR study can guide the design of new drugs against of non-optimal sample quality, such as structural defects or dopants. We discuss microbial virulence and novel therapeutics for melanoma tumors. several experiments designed to elucidate the nature of the PL emission in SWNTs. Upon addition of reducing agents to an aqueous solution of SWNTs, an enormous 131 NMR Relaxation Method to Detect Small Dynamical Differences in enhancement of the QY for individual SWNTs is observed accompanied by dramatic Proteins change in the single nanotube fluorescence dynamics. Correlated measurements Rieko Ishima, University of Pittsburgh, 3501 Fifth Ave., Pittsburgh, PA of fluorescence and topography show only ~11% of all SWNTs were found to be 15213 emissive, while approximately 36% of the SWNTs displayed weak luminescence, Nuclear magnetic resonance (NMR) relaxation provides site-specific information in which PL spectra were not observed for corresponding bright pixels in the PL im- on protein internal dynamics associated with configurational energetics and con- age. The unexpectedly high QY values indicate that SWNTs are intrinsically bright formational equilibrium. Since NMR techniques to study structure and dynamics emitters, and that current understanding of the optical properties of SWNTs has of biomolecules have tremendously improved in the last decade, experiments that most likely been obtained from defective SWNTs having their PL partially quenched. previously did not yield sufficiently sensitive data can now provide new insights into Further, the low ensemble QYs for carbon nanotubes can be largely attributed to a biomolecular mechanisms. Although a set of 15N longitudinal relaxation rate (R1), small percentage of bright SWNTs in the ensemble, and not to a large population of 15N transverse relaxation rate (R2), and the heteronuclear 15N-{1H} NOE experi- poorly emitting SWNTs. ments are routinely applied to characterize protein backbone dynamics, further im- 21 2013 EAS Abstracts November 2013

135 Quadrupole Enhanced Raman Scattering of Spiky Gold Nanoshells 139 Environment Monitoring in Developing Countries and Remote Zahra Fakhraai, University of Pennsylvania, Department of Chemistry, Assessment of Cultural Materials Through Nano-scale Weight 231 South 34th St., Philadelphia, PA 19104, Simon P. Hastings, Zhaoxia Monitoring Qian, So-Jung Park, Nader Engheta, Pattanawit Swanglap, Ying Fang, Henoc Agbota, University College, Gower St., London WC1E 6BT, Stephan Link United Kingdom Recent studies have shown the importance of dark non-radiant plasmonic modes This presentation reports on the Accessible Heritage project which aimed at devel- in the Raman enhancement efficiency of plasmonic nanostructures. Dark modes oping an environment monitoring tool suitable for heritage sites, especially in devel- are important in the generation of localized hotspots that strongly interact with Ra- oping countries. The monitoring system is designed to allow seamless integration man active molecules. However it has been extremely challenging to design such of any number of small electronic environmental sensors. Since low-cost sensors hotspots in nanostructures through simple synthesis routs. Furthermore, the mol- for individual pollutants are not yet commercially available, a dosimeter array is ecule of the interest needs to be located inside such hot-spots for maximum Raman used. The array consists of piezoelectric quartz crystal microbalances coated with efficiency. In this presentation I demonstrate that spiky nanoparticles have a tun- different metals, each differently sensitive to pollutants. The prototype can measure able, global electrical quadrupole that efficiently and reproducibly enhances Raman temperature and relative humidity and monitor nanometric changes in the weight scattering. The imperfections in the nanoparticle structure help broaden the quadru- of different materials. The measurements can be accessed ‘live’ from the project pole and allow for the enhancement of both the excitation and Stokes modes. The website: www.ucl.ac.uk/accessible-heritage. The system has been deployed at optimum excitation frequency for best Raman enhancement factor can be predicted Apsley House, an English Heritage property in London and at the Royal Palaces using finite difference time domain (FDTD) simulations. These nanoparticles are of Abomey, a Unesco World Heritage Site in Benin, West Africa. The project ad- produced using a simple synthesis technique and are ideal for applications such as ditionally involved 25 heritage sites in developing and emerging economies. The biosensing, plasmonic nanoantennaes and photovoltaics. goal of this study was to understand the issues surrounding environmental monitor- ing, and air pollution monitoring in particular, in these regions. The responses show 136 Using Ultrafast Pump-Probe Microscopy to Image Carrier Migration that financial reasons are not the only reason why environmental data is lacking and Carrier Recombination in Si and ZnO Nanowires from developing regions. Politics and lack of expertise also play major roles. The John Papanikolas, University of North Carolina - Chapel Hill, Department presentation also reports on an environmental monitoring campaign that has been of Chemistry, Chapel Hill, NC 27599 conducted and which involved heritage sites in 12 countries in Africa, Middle East, We have used femtosecond transient absorption microscopy to investigate the Latin America and Asia and six European countries. The measurements, which in- charge carrier dynamics in needle-shaped ZnO rods and Si nanowires. The struc- clude sulphur dioxide, nitrogen dioxide and ozone, are presented and analyzed. tures, which are 10-30 microns in length and 200-1800 nm in diameter, are excited by a femtosecond pump pulse that is focused to a diffraction limited spot size by a 140 Evaluating the use of Agricultural Moisture Probes in the Historic microscope objective, creating a localized region of photoexcited carriers a spe- Built Environment cific point in the structure. The carrier dynamics are probe by a second laser pulse Nicole Fielding, University of Lincoln, Brayford Pool, Lincoln LN6 7TS, that is delayed in time, and whose position relative to the pump pulse is controlled. United Kingdom, Belinda J. Colston, Adrian M. Goodman The pump-probe microscope has a lateral resolution of 350 nm and a time resolu- Much of the deterioration affecting historic buildings is a direct result of moisture tion better than 500 fs. Experiments performed at different locations within the ZnO penetration in porous building materials. This damage can be wide-ranging and structures reveal dramatically different dynamical signatures, with the ends of the severe – from “puddle staining” and mold, to decay fungi and deterioration due rod exhibiting faster electron-hole recombination than the interior. We also directly to the crystallization of soluble salts. The relationship between masonry moisture observed spatial evolution of the charge carrier cloud produced by photoexcitation content and the extent of the decay is complex, and monitoring moisture move- of Si nanowires. ment, particularly within mass masonry structures is problematic. If; however, ef- fective conservation strategies are to be developed and adopted, it is imperative 137 Evaluating Exposure During Cleaning and Conservation of Metallic that a full understanding of the causes and mechanisms behind moisture-driven Lead deterioration is achieved, and the development of an effective method for monitor- Shaundree T. Davis, Princeton University, 262 Alexander St., Princeton, ing masonry moisture movement key. Current research has been evaluating the NJ 08540 potential of utilizing technology, normally used in agriculture, for the historic built en- Since 2003, Princeton University has housed a 40-foot by 25-foot sculpture created vironment. Methodologies for establishing calibration protocols for the Delta-T PR2 by Frank Gehry. The sculpture, made from lead sheeting that is supported by wood capacitance-type soil moisture probe (Delta-T Devices Ltd) is discussed for a range framing, was recently cleaned and encapsulated. These activities raised concerns of historic masonry materials, and an assessment given of its suitability for use in regarding the potential exposure of bystanders and those conducting the work to historic masonry structures. A three-year case-study at North Foreland Lighthouse lead dust during both the cleaning and conservation process. This talk reviews the (a grade II listed building in Kent, England), carried out in parallel with the probe cleaning and encapsulation method selection process, as well as how exposure was evaluation and calibration, provided invaluable ‘real’ data for validating the use of controlled and monitored. the probe in historic contexts. The impact of moisture ingress into the thick masonry walls of North Foreland’s tower, and its subsequent impact on the internal environ- 138 Long Term Environmental Monitoring: Museum Size Strategies and ment and observed salt-induced material decay, was evaluated, and has been used Wooden Objects Hygro-Mechanical Reactivity to Temperature and to inform an effective conservation strategy for the lighthouse. Relative Humidity Fluctuations Paolo Dionisi-Vici, Metropolitan Museum of Art, 1000 Fifth Ave., New 141 Supercritical Fluid Chromatography (SFC), a Widely Applicable York, NY 10028, Masahiko Tsukada, Lucretia Kargère, Hendrik F. Technique Used in Industries from Pharmaceutical to Food Hamann, Sergio A. Ramirez Bermudez, Levente Klein, Vanessa Lopez, Jennifer Van Anda, Agilent Technologies, 330 Locust Grove Rd., West Alejandro G. Schrott, Marco Leona Chester, PA 19382, Terry A. Berger Museums worldwide are currently engaged in a lively debate on gallery climate The use of SFC in a variety of applications and industries has increased with the conditions, namely acceptable levels of relative humidity and temperature and ac- advent of second and third generation instruments. SFC is widely accepted for ceptable fluctuation ranges. Due to the complexity of the building, this issue is very pharmaceutical applications, particularly chiral separations and subsequent puri- relevant to The Cloisters. The Metropolitan Museum is undertaking a pilot study fication of the enantiomers. Monitoring of reactions to determine the formation of with IBM T. J. Watson Center involving state-of-the-art smart building technologies, product or the conditions needed for optimal product formation are routinely per- which provide real-time three dimensional models of temperature, humidity, dew formed with SFC by the pharmaceutical medicinal chemistry group. In the food and point distributions, and air pollutants in a number of galleries. The study provided nutraceutical industries, SFC is used for the analysis of caffeine in beverages and some encouraging results about the effectiveness of the platform in highlighting the characterization of nutraceuticals from various naturally occurring matrices. The potential anomalies in thermal stratification and out-of-target humidity values in any petroleum industry has two American Society for Testing and Materials methods that area. The sensors mesh network installed will become a useful tool to improve the are currently used for fuel analysis. The SFC separation of polynuclear aromatic interaction among conservators, curators and engineers. A future phase deals with compounds and pesticides has enabled SFC to provide an orthogonal and faster the possibility of installing sensors on some objects (real ones or dummies), made of separation of these components for the environmental industry. Finally the chemical organic hygroscopic materials, in order to understand their reactivity to the climate industry has employed SFC in the separation of complex mixtures. fluctuations around them. In this frame a testing campaign on wooden dummies in controlled climate chambers at the DSR of the Museum are being carried out, in order to evaluate the preliminary parameters to safely move to the next step in the exhibiting area.

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142 Keeping Our Food Safe: Detection of Veterinary Drug Residues of the endogenous metabolites we observe in the circulating blood is presented. Marilyn J. Schneider, United States Department of Agriculture, 600 Here the endogenous metabolites in numerous different organs (liver, pancreas, Mermaid Ln., Wyndmoor, PA 19038 kidney, etc.) are determined as well as in plasma and urine, and the metabolic pat- A great number of people and several different agencies are responsible for helping terns compared.[1] to keep the U. S. food supply safe. One aspect of this involves monitoring for vet- References: erinary drug residues in food. The residue chemistry research group at the United [1] Trygg, J., Holmes, E., and Lundstedt, T. 2007. Chemometrics in metabonomics. States Department of Agriculture (USDA) Agricultural Research Service Eastern Journal of proteome research 6:469-479. Regional Research Center supports our sister agency, the USDA Food Safety In- spection Service, and others, by providing increasingly effective and efficient meth- 146 Multivariate Approach to Reveal Biomarker Signatures for Disease ods to facilitate the monitoring process. Specific examples illustrating the historical Classification development of such methods, from single analyte chromatographic methods, to Tarja Rajalahti Kvalheim, Haukeland University Hospital, Department of multiresidue methods, to multiclass, multiresidue methods are described. Neurology, Bergen N-5021, Norway Clinical laboratories are facing fundamental changes when it comes to methodol- 143 Analytical Development in Over-the-Counter Drugs – Straddling ogy and instrumentation. Views of health and disease are influenced by the pres- Pharmaceutical and Consumer Products ent post-genomic era, driven by huge advances in bioanalytical technologies and Mary Selman, The Mentholatum Company, 111 Rock Rd., Horsham, PA bioinformatics. The study of human genome has been followed by the study of hu- 19044 man proteome and metabolome, providing us complementary information for the The over-the-counter (OTC) drug business is a hybrid of pharmaceuticals and per- analysis and understanding of complex pathological processes. These approaches, sonal care products. The incessant need to appeal to the demanding consumer with the so-called “omics” sciences, have given a novel insight into human biology. Bio- features above and beyond efficacy creates ever-more-complicated formulations in markers are tests or measurements that provide information about the biological multiple package configurations. Without the need for individual Food and Drug Ad- condition of the subject being tested. In addition, they have an ability to discriminate ministration (FDA) preapproval, products are conceived and readied for market on between different biological states. In most cases multiple biomarkers are needed a very short timescale, leaving little room for method development and refinement. to diagnose the disease. This talk presents a general approach where full spectral Many products are related to earlier formulations, and so methods are often derived profiling (e.g., using mass spectrometry or nuclear magnetic resonance) and mul- from existing methods. Compendial (e.g., United States Pharmacopeia) methods tivariate data analysis are combined to reveal biomarker signatures from complex may be used if validated for the particular product, and those for some authorized body fluids. The instrumental profiles are modeled by latent variables using partial OTC drug ingredients are antiquated and fall short of current expectations. Adopting least squares regression. Target projection is subsequently performed to condense FDA’s risk-based approach, we use chromatography as an essential tool in helping the features related to each y-variable onto a single latent variable. The ratio be- to understand our products and processes while also meeting regulatory obliga- tween the variance explained on the target projected latent variable and the residual tions. Case studies from analytical development at a small manufacturer of topical variance, i.e., selectivity ratio (SR), provides a ranking of the explanatory variables products illustrates the ongoing challenge to satisfy ever-more-stringent regulatory according to their ability to predict the investigated y-variable. Biomarker candidates requirements in a fast-paced and ever-changing market. are thus easily identified. A probability measure is calculated for each explanatory variable and can be used to assess the reliability of each biomarker candidate. 144 Systematic Investigation of Factors that Affect Precision in HPLC Mary Ellen McNally, DuPont Crop Protection, Stine Haskell Research 147 The OPLS Approach for Multi-Block Data Integration Center, 1090 Elkton Rd., Newark, DE 19711, Steven Hansen, Karyn Johan Trygg, Umeå University, Computational Life Science Cluster, Usher Department of Chemistry, Umeå 901 87, Sweden In an effort to establish the best possible value when measuring the assay of an ac- Partial least squares (PLS) projections to latent structures has been a workhorse tive ingredient (AI) precision measurement comparisons have been conducted. In in chemometrics for more than three decades. Fifteen years ago, several variants this study, a group of agricultural product AI’s were used to thoroughly compare the of the PLS method, denoted orthogonal signal correction (OSC) and orthogonal internal standard versus the external standard method of analytical calibration. In an projections to latent structures (OPLS) methods were introduced. The driving force external standard calibration, the absolute analyte response is plotted against the behind that development was that the model interpretation of our projection-based analyte concentration or amount in order to create the calibration curve. In the inter- latent variable models (including PLS) was not straightforward because of its con- nal standard method, a carefully chosen compound that is not the analyte is added founding of different systematic variations in the model. The OPLS method sepa- to every standard and sample to give the same final concentration. In an internal rates the systematic variation in X into two parts, one that is correlated (predictive) standard calibration the ratio of the analyte response to the internal standard (IS) to Y and one that is uncorrelated (orthogonal) to Y. This has consistently shown to response is plotted against the ratio of the analyte amount to the IS amount. This facilitate model interpretation and make it more straightforward. Now, OPLS has calibration curve is then applied to the results obtained for the samples so that the been developed into a suite of different methods for different purposes such as required values can be calculated. The internal standard method can be used to im- OPLS-DA for discriminant analysis, K-OPLS for non-linear modeling, O2PLS for prove the precision and accuracy of results when there are volume errors involved two block data integration and OnPLS for multi-block data integration and path mod- in the method that are difficult to control. Examples of the types of errors that can eling. I present several examples and applications on the success of orthogonal be minimized by the use of an internal standard are those due to: 1) evaporation of based methods, including recent developments in multi-block analysis, the OnPLS solvents, 2) injection errors, and 3) complex sample preparation involving transfers, methods. Further the talk covers theoretical and algorithmic similarities to published extractions and dilutions. In addition to the chosen method of calibration, we have alternatives to OPLS with focus on the post-PLS transformations. In addition, I try found that there are other factors that influence the precision of an assay measure- to give a future outlook on the exciting possibilities that lie ahead of us, and what I ment of a technical substance. These have been studied in a systematic series of believe chemometrics need to focus on in this area to have maximum impact. experiments. The results of these experiments are presented. 148 Latent Variables - What are they? 145 Endogenous Metabolic Profiling as a Fundament in Personalized Svante Wold, Umeå University, Department of Chemistry, Umeå 901 Theranostics 87, Sweden Torbjörn Lundtstedt, Uppsala University, Chemistry Department, Models in latent variables (LV’s) have a long and honorable history in applied math- Uppsala S-75123, Sweden, Katrin Lundstedt-Enkel, Thomas Moritz, ematics from Cauchy (1829) to Kvalheim (2013). These models run under many Frida Troell, Johan Trygg, Kate Bennett names, i.e., factor analysis, principal components analysis (PCA), singular vector Metabolomics is an established tool in research for: 1) diagnosis, i.e., classification; decomposition (SVD), and bilinear modeling (BLM). Very similar approaches, spe- 2) identification of biomarkers in relation to e.g., diseases; 3) dynamic studies i.e., to cial for a particular field of science include extra-thermodynamic relationships (ETR) identify effects from e.g., medical treatment, changes in food intake, environmental in physical organic chemistry. LV’s can be defined operationally, e.g., as optimal or genetical changes to a living species such as human, animal, or plant. In this pre- summaries of a set of measured variables, or, philosophically as inherent proper- sentation the use of metabolomics as a tool in diagnostics, biomarker identification, ties of a system that can be only indirectly observed; and this without mutilating the drug development and theranostics are highlighted. In the first part, the differences data by variable selection. LV models are able to well approximate the information in biochemical profile between healthy volunteers and persons with the diagnosis in a data matrix, provided some assumptions – similarity and continuity -- about the rheumatoid arthritis (RA) are shown; and identification of novel biochemical path- data are fulfilled. In addition, since LV’s are weighted averages of the measured ways for understanding the underlying factors of the disease is discussed. In the variables, they also work well with many and collinear variables, even much more next part a comparison to different RA animal models are made, in order to identify numerous than the number of observations, N. The LV structure in these models the most relevant for describing the disease in humans. The most appropriate model is expressed as a sequence of products of vector pairs (ta pa’). Here the “scores” can then be used for evaluation of novel treatments of RA that can be drugs, but (ta) and the ”loadings” (pa), express the information about the observations and also changes in diet or lifestyle. In the last part, an attempt to understand the origin 23 2013 EAS Abstracts November 2013

the variables, respectively. Plots of these vectors facilitate the interpretation of the sults indicate that kinetic rather than transport processes are limiting in most cases, models and provide a strong link to the chemistry/biology/etc., underlying the data. with the desorption rate most critical. This rate depends especially strongly on the Theoretically, LV’s can be derived in several, apparently different, ways. The most strength of adsorption but also to a smaller extent on the extent of protein loading. common of these are discussed and illustrated by a few chemical examples. These results suggest that the multilayer partitioning of protein into the polymer extenders, while increasing the sorption capacity, may be detrimental in that it limits 149 Interpretation and Measures of Variable Importance in Multivariate the desorption and efflux of proteins from the adsorbent. Data by Latent Variable Modeling Olav Martin Kvalheim, University of Bergen, Chemistry Department, 153 Understanding Retention in Reversed-Phase Liquid Realfagbygget - Allégt. 41, Bergen N-5007, Norway Chromatography: Insights from Molecular Simulation Interpretation and reduction of multicomponent profiles from complex samples rep- J. Ilja Siepmann, University of Minnesota, Department of Chemistry, resent a challenging area. Target projection (TP) [1] and orthogonal projections to 207 Pleasant St. SE, Minneapolis, MN 55455 Mark R. Schure, Jake latent structures (OPLS)[2] reduce the y-related information in a latent variable re- L. Rafferty gression (LVR) model to a single y-related latent variable. Although TP and OPLS Chromatographic separations involve a delicate interplay of enthalpic and entropic provide enhanced possibilities for the interpretation, they do not solve the interpre- factors arising from interactions of the analyte molecules with the stationary and tation problem. One reason is that TP or OPLS loadings may be dominated by x- mobile phases, where the former can be altered by the presence of solvent (mobile- variables with large co-variances but small correlations to the y-variables. Rajalahti phase) molecules. Particle-based simulations allow one to probe chromatographic et al.[3] introduced the so-called selectivity ratio (SR) plot to solve the problem. This systems on the molecular level and to provide detailed information on structural measure is scale independent, plots like a spectrum or a chromatogram, and is also characteristics and retention mechanisms. This talk highlights recent applications useful for variable selection [4] since it provides a ranking of all x-variables accord- of configurational-bias Monte Carlo simulations in the Gibbs ensemble to under- ing to their importance for the y-variables. The concept and use of SR and related stand the retention mechanisms of small flexible analytes and of shape-constrained plots for interpretation and variable selection are illustrated for different applications. polycyclic aromatic hydrocarbons in reversed-phase liquid chromatography (RPLC). References: Accurate molecular mechanics force fields are used for these systems and enable [1] Kvalheim O.M. and Karstang T.V. (1989) Interpretation of latent-variable regres- direct comparison to experimental retention data. The simulations elucidate the in- sion models, Chemometrics and Intell. Lab. Syst., 7, 39-51. fluence of mobile-phase composition, embedded polar groups, and grafting density [2] Trygg J. and Wold S. (2002) Orthogonal projections to latent structures (O-PLS), for alkyl-silane based stationary phases. J. Chemometrics, 16, 119-128 ASAP (Accelerated Stability Assessment Program): Opportunities [3] Rajalahti T., Arneberg R, Berven F.S., Myhr K.-M., Ulvik R.J., Kvalheim O.M. 154 and Challenges (2009) Biomarker discovery in mass spectral profiles by means of selectivity ratio Fenghe Qiu, Boehringer Ingelheim, 900 Ridgebury Rd., Ridgefield, CT plot, Chemometrics & Intell. Lab. Syst., 95, 35-48. 06877, Madhuri Jerfy, Lisa Hernandez, Zeena Williams, Prince Korah [4] Bleie O., Westerhuis J.A., Arneberg R., Rajalahti T., Smilde A.K. and Kvalheim ASAP, developed by Dr. Kenneth Waterman, has been a popular topic in pharma- O.M., Variable selection based on selectivity ratios from latent variable regres- ceutical stability testing in recent years. The basic principles of the ASAP approach sion models with continuous response variables, manuscript. include the use of the humidity modified arrhenius equation and “Isoconversion.” In 150 Orthogonal Separations an ideal world, an appropriately executed ASAP study can provide enhanced under- Mark R. Schure, Kroungold Analytical, Inc., 1299 Butler Pike, Blue Bell, standing of the critical stability attributes (e.g., T/RH) and more accurate and faster PA 19422, Joe M. Davis expiry predictions of a pharmaceutical product. However, in reality, not all drugs The term “orthogonality” usually refers to the statistical concept of spreading peaks behave the same. Sometimes, as the saying goes, “The more you know, the more across a two-dimensional chromatogram. However, this concept can be generalized you don’t know”. This presentation aims to provide a balanced picture of the ASAP to a one-dimensional chromatogram (spreading peaks across a line chromatogram), approach through real world case studies. The advantage of this approach is fully and three-dimensional chromatograms (spreading the peaks across a volume demonstrated in some cases, while in other cases, challenges or even confusions or three dimensions), etc. For very complex separations, for example separating exist. Based on authors’ first hand experiences, best practices for drug substance hydrocarbon mixtures like diesel fuel, there is just too much saturation in a one- and drug product applications are recommended. dimensional chromatogram and one is forced to use multidimensional techniques. The Material Mass Balance Investigation of API in Orlistat Drug This talk focuses on a quick survey of orthogonality metrics and then focuses on a 155 Product Stored at Accelerated Condition: Where is the Missing more rigorous definition of orthogonality where both local effects of peak spacing Orlistat? and spatially-averaged properties are considered. It appears that separation sci- Zbigniew Marcinow, GlaxoSmithKline, 1500 Littleton Rd., Parsippany, entists understand the concept of separation orthogonality but have not universally NJ 07054 accepted any particular separation metric or definition. We think current metrics Orlistat, also known as Tetrahydrolipstatin (THL), is the active pharmaceutical in- and definitions neglect the importance of the peak distribution within the separation gredient (API) in weight control products. It reduces the absorption of dietary fat hull, relative to the hull size. In fact, both are important. We attempt some rigor to by preventing digestion of triglycerides (lipase inhibitor). The results of the infor- a definition by connecting metric properties to certain optimization criteria that con- mal stability study at accelerated conditions (40 oC/75%RH) in Orlistat drug prod- sider both attributes. We also mention the connection of this problem to other fields uct showed a significant decrease in assay over a relatively short time period, but like range ecology and the distribution of stars and interstellar mass in the galaxy. the amount of impurities did not account for the amount of lost Orlistat. For that 151 Adventures in Two-Dimensional Liquid Chromatography reason 40 ºC/75%RH storage conditions are not considered appropriate for expiry Peter W. Carr, University of Minnesota, Department of Chemistry, 207 determination of Orlistat tablets. Contrary under shelf life time condition this formu- Pleasant St. SE, Minneapolis, MN 55455 lation undergoes transformation only to secondary degradation products detected No abstract submitted by the author. by analytical method; therefore, there is no deviation from 100% mass balance. These studies provided mass balance justification based on the interaction of the 152 Resolution of Transport and Kinetic Limitations in Protein API primary degradation product either with excipients or itself (self-condensation) Exchange in Polymer-Functionalized Adsorbents resulting in the formation of products not possible to detect by analytical method. Abraham M. Lenhoff, University of Delaware, Department of Chemical This rationale was supported by designing a series of experiments and confirmed and Biomolecular Engineering, Newark, DE 19716 by liquid chromatography (LC) mass spectrometry and LC nuclear magnetic reso- The addition of polymer extenders has been found to enhance the performance nance analysis. of ion exchangers in preparative protein separations by such measures as both static and dynamic binding capacity. Despite the constriction of the pore space, 156 The Impact of Extractable Materials from Plastic Sample even protein transport rates during loading are seen to be extremely rapid in these Preparation Devices on Formulation Analysis materials. On the other hand, elution in these materials is often found to be slower Jigna Patel, Bristol-Myers Squibb, One Squibb Dr., New Brunswick, NJ than in conventional ion exchangers, and displacement of one protein by another 08901, William Fish, Anne Kelly may be similarly retarded. The mechanisms responsible for these pronounced dif- Plastic devices such as syringes, transfer pipettes, vial caps, filters, etc., are widely ferences in transport rates are incompletely understood, and this presentation de- used to prepare samples for many analytical techniques. Components from these scribes efforts to probe the underlying mechanisms. A key tool is confocal micros- devices can be extracted when the surface comes into contact with the pharmaceu- copy, which is used here to perform real-time observations of fluorescence recovery tical product or product-related solutions. The significance of these contaminants is after photobleaching (FRAP) to investigate the limiting steps in exchange of labeled increased with low dosage unit strength, such as a 0.25 mg/unit, since impurities are and unlabeled protein molecules sorbed on the chromatographic particles. The re- typically reported relative to the active ingredient. This results in additional labora-

24 2013 EAS Abstracts November 2013

tory investigations around unknown impurities or degradants. In this presentation graphic band broadening significantly reduced method sensitivity; 4) the analyte we describe recent efforts to understand, identify and control the source of plastic is very hydrophobic and does not dissolve in polar solvents such as acetonitrile material contaminants for a low dosage unit product. These studies include inves- and alcohol. This presentation describes the development of a simple reversed- tigation of the effect of diluent and sample component, amount of extractable from phase HPLC method that can quantitate the total amount of polymeric degradants different type of devices (e.g., syringe, filter, centrifuge tube, etc.) and from different with quantitation limits of ~0.05% w/w. The method utilizes a simple derivatization brand and batches of device. procedure to increase aqueous solubility of the polymeric analyte by removing its hydrophobic function group. The HPLC conditions were optimized using a 250 x 157 A Complete Solution for Sample Prep Technique for New USP<232> 4.6 mm Waters XSelect CSH column maintained at 30 °C with a mobile phase of and <233> water-acetonitrile-trifluoroacetic acid (20:80:0.1 v/v/v). The feasibility of other HPLC Arshad Kokardekar, Milestone Inc., 25 Controls Dr., Shelton, CT 06484, approaches including size exclusion chromatography and normal phase chroma- David Gunn tography were also investigated and found to be less suitable for this particular The control of inorganic impurities has always been a critical issue to the pharma- application. Validation data for this method in terms of precision, linearity, accuracy ceutical industry. These impurities can be harmful to human health as well as reduce and sensitivity are also presented. drug stability and shelf life. United States Pharmacopeia (USP) <231> is a current test method for measuring the content of metallic impurities by colored sulfide pre- 160 New Aspects in Drug Development Using (U)HPLC Methods and cipitates. This antiquated method suffers as a qualitative method with limitations Robustness Evaluation According to Quality-by-Design Principles including poor-sensitivity, loss of volatile elements to go along with the time and with the Help of Modeling Techniques labor demands. USP is proposing three new chapters, effective May 1st 2014 - Im- Imre L. Molnar, Molnar-Institute, Schneegloeckchenstr.47, Berlin 10407, purities Limits<232>, Methods<233>, Dietary Supplements Metal Limits <2232> to Germany, Hans-Jürgen Rieger, Alexander H. Schmidt overcome these issues with the current method. The new method <233> describes The disaster with the active pharmaceutical ingredient (API) Thalidomid was ruining two analytical techniques, inductively coupled plasma optical emission spectrometry the life of thousands of childen and their parents. Since then the pharmaceutical (ICP-OES) and ICP-mass spectrometry, both using closed vessel digestion as the industry did a great deal to improve the quality of drugs, introducing modern high- sample prep technique. Closed vessel microwave digestion, one of the standard performance liquid chromatography (HPLC) started by Csaba Horváth in 1967 and techniques for digestion over the past 25 years has recently seen substantial devel- with the understanding the theory of reversed-phase chromatography in 1976. We opments, like improved vessel design, precise temperature and pressure control. have nevertheless still much to do. From ca. 100,000 known human diseases we Current instruments are safe, easy to operate and can digest variety of sample could develop until today drugs for only ca. 20,000 diseases. For ca. 80,000 dis- types. A new and unique alternative to closed vessel digestion is single reaction eases there is still no treatment available. Many people suffer everyday world wide, chamber (SRC)technology. With SRC, all samples are placed inside the digestion due to lack of drugs, which could not be developed for several reasons. Therefore vessel and digested simultaneously in the Teflon lined stainless steel chamber, as it we have to increase the speed of the process of developing new drugs. One of can go to very high temperature and pressure – up to 300 ºC, 200bar. This presenta- the reasons is the high cost of discovering new API’s. Another hindrance is the im- tion includes both-conventional microwave and SRC, along with their critical feature mense costs involved with the bureaucracy in submissions and necessary changes enhancements in sample prep optimization of a variety of pharmaceutical products of the pharmacopeial processes. Quality-by-design (QbD) is the way out of this. for trace metals analysis as a sample-prep solution for USP<233>. Primarily we have to reduce the time needed for the development and the quality control of a drug. The HPLC method is part of the drug product. We can observe a 158 A System to Perform Autocalibration, Automated Sample Dilution, decrease in analysis time using ultra-PLC instead of HPLC by a factor of 3-5. We and Auto-Spiking for Analyzing Pharmaceuticals by ICP and ICP- recently improved a pharmacopeial method from 160 minutes to only 3 minutes by MS using a modeling software in combination with UPLC instrumentation. The second Kyle Uhlmeyer, Elemental Scientific, 1500 N 24th St., Omaha, NE step is to reduce the bureaucracy involved with analytical methods. We need faster 68110, Paul Field, Dan Wiederin processes to be able to help those waiting for faster help. This presentation shows Trace metal impurities in pharmaceutical products are either the direct result of practical case studies of resolution modeling for fast and reliable separations in the catalysts added during drug formulation or contamination during production. Based pharmaceutical industry. on the toxicity of elemental impurities the United States Pharmacopeia (USP) has set values for a permissible daily exposure (PDE) of a suite of trace elements and 161 DART-MS Collision Induced Dissociation for Structural Analysis of defines a target (J) value as a function of PDE (mg/day) and a drugs daily dose (g/ Designer Drugs day). USP <233> outlines specific protocols for the determination of toxic metals by Jason Shepard, University at Albany, Department of Chemistry, 1400 tandem inductively coupled plasma mass spectrometry (ICP-ICP-MS) and defines Washington Ave., Albany, NY 12222, Rabi Musah, Ashton Lesiak, Marek sample preparation, standardization, and spike recovery based on the J values. A Domin, Robert Cody, John Dane system utilizing four independent syringes combined with unique software to assist Designer drugs have been tied to large spikes in emergency room visits, overdoses, in the automated analysis of USP samples is presented. The system automatically and deaths. Identifying these substances is difficult because these compounds are: calculates flow rates for carrier, diluent, and internal standard. The syringes push 1) novel or unknown; 2) closely structurally related, and 3) not testing positive in each solution, mixing them in a specialized valve before delivering the final solution routine drug screens. The rapidity with which they appear and change on the mar- to the instrument. In this way, the system automatically dilutes solutions to build ket is problematic. Direct analysis in real-time mass spectrometry (DART-MS) is a linear calibration curves from a single stock standard (r2 > 0.999s) and precisely/ technique utilizing an atmospheric pressure ion source that vaporizes and ionizes accurately dilute samples. This study illustrates the practical benefits of an ICP-MS liquids or desorb and ionizes molecules from solid surfaces in open air under ambi- coupled to an inline, auto dilution and auto calibration sample delivery system to de- ent conditions. Small molecule drugs, like “white powder” cathinone mixtures or termine a group of toxicologically-relevant elements in various pharmaceutical prod- cannabinoids mixed on plant material, were tested by DART-MS. The ionization ucts. This system easily surpasses the validation criteria of USP<233>,1) stability < process results in [M+H]+ ions for the drugs and the high mass accuracy of the ± 5% (USP limit ± 20%), 2) repeatability < ± 3% (USP limit ± 20%),3) ruggedness < time-of-flight mass analyzer establishes the presence of the active component(s). ± 4% (USP limit ± 25%) and 4) accuracy < ± 10% (USP limit ± 20%). Additionally, DART-MS in-source collision induced dissociation (CID) induces frag- mentation of the [M+H]+ ions when the electrode voltage at the inlet orifice to the 159 Determination of Polymeric Impurities in Pharmaceutical Products mass spectrometer is increased. CID analysis employs the utility of fragmentation by Reversed-Phase Chromatography patterns for distinguishing among closely related structures. Closely related com- Zongyun Huang, Bristol-Myers Squibb, One Squibb Dr., New Brunswick, pounds fragmented in a similar fashion, but inherent structural differences resulted NJ 08901, Yan Zha, Yue Hu, Zuguang Tian, Robert Perrone, Robert in unique fragments that served as a means to identify each substance. This method Francis, Joan Ruan circumvents time-consuming sample extraction, derivatization, chromatographic Studying the chemical stability of drug substances and formulated drug products and other sample preparative steps required by more traditional methods. The high- is a critical requirement of the pharmaceutical development process. Recently, we throughput of DART-MS enables designer drugs to be detected more quickly, reduc- encountered an atypical degradation pathway where a small molecule drug under- ing the time necessary to triage analytical evidence. went polymerization when exposed to ambient light and mild heat (e.g., 40 °C). This degradation pathway yielded large, polymeric molecules with estimated molecular weights > 1 million Dalton. Quantitating levels of these polymeric degradants by high-performance liquid chromatography (HPLC) is particularly challenging at trace levels in the sample matrix due to: 1) the analyte is a mixture of species of different molecular mass; 2) the analyte is typically detected as a broad band of peaks in chromatography, which could coelute with peaks from sample matrix; 3) chromato-

25 2013 EAS Abstracts November 2013

162 A Study of Positional Isomers Using Linear Ion Trap LC-MS-MS and 167 Surface Enhanced Raman Scattering: New Pathways for Rapid, Q-TOF LC-MS Parallel and Low-Level Analyte Detection Ada Kong, DEA Northeast Laboratory, 99 Tenth Ave., Suite 721, New Jennifer H. Granger, Nano Institute of Utah, 36 South Wasatch Dr., Salt York, NY 10011 Lake City, UT 84112, Marc D. Porter Designer drugs are becoming increasingly more common in forensic laboratories The detection of biological agents diagnostic of human health stands as an ever- across the country. Many of these compounds have multiple isomers making iden- present need. Whether it be detecting the early stages of cancer or infectious dis- tification challenging. The majority of forensic laboratories utilize retention time eases in setting beyond the clinical laboratory, the demand for platforms for the to differentiate positional isomers. Although this is a valid technique, a reference rapid, parallel and low-level biomarker measurements has never been greater. This standard for each isomer is needed for comparison. These reference materials are presentation focuses on the use of surface-enhanced Raman scattering (SERS) expensive and difficult to obtain. The purpose of this project was to investigate an ef- as a readout tool for immunoassays. As a diagnostic tool, we have found that this ficient and inexpensive approach to determine isomeric position. Two non-traditional platform can be effectively applied to the multiplexed determination of biomarkers in instruments were utilized, the linear ion trap liquid chromatography tandem mass complex sample matrices at femtomolar concentrations and lower. This presenta- spectrometry (LC-MS-MS) and quadrupole time-of-flight (Q-TOF) LC-MS. Although tion discusses the creation, performance assessments, and challenges in using this these instruments are not commonly found in forensic laboratories, they are increas- platform as an integrated system in measurements of biomarkers central to pancre- ing in popularity due to their characterization and accurate mass capabilities. This atic cancer, human immunodeficiency virus, and tuberculosis in both spiked human presentation presents data utilizing both instruments for a variety of designer drugs. specimens and in disease-positive patient samples. 163 Forensic Applications of Comprehensive GC with TOF/MS 168 An Atypical Query about the Subjectivity of “Objective” Frank Dorman, Pennsylvania State University, 107 Whitmore Spectroscopy-Based Cancer Recognition Laboratories, University Park, PA 16802 Ishan Barman, Massachusetts Institute of Technology, 77 Massachusetts No abstract submitted by the author. Ave., Rm 6-016, Cambridge, MA 02139 Label-free molecular sensing methods, such as vibrational spectroscopy, provide 164 Development of a Surface-Enhanced Raman Spectroscopy Method objective biomarkers for diagnoses, and have the potential to permit disease de- for the Detection of Benzodiazepines in Urine tection prior to morphologic manifestations. Indeed, spectroscopic imaging, which Erika L. Doctor, Florida International University, 11200 SW 8th St., combines the molecular basis of spectroscopy with the imaging capabilities of mi- Miami, FL 33199, Bruce McCord croscopy, may represent a novel approach to automated cancer identification and, Benzodiazepines are among the most prescribed compounds and are commonly thus, to accurate, reproducible and economical diagnoses without necessitating present in many toxicological screens. Currently these compounds are predomi- human interpretation. Nevertheless, in developing a test for the detection of any nantly analyzed using immunoassay techniques; however more specific screening disease state, it is important to assess the precision of the measurements upon methods are needed. This project shows the applicability of surface-enhanced Ra- which the diagnosis is based, as is it a key factor in determining the repeatability, man spectroscopy (SERS) as a method for the analysis and detection of benzodi- reproducibility and reliability of the diagnosis. This is an issue of concern for all azepines. The procedure involves mixing urine extracts with gold nanoparticles and optical spectroscopy techniques in development for tissue-based disease diagnosis aggregating agents for trace detection of these compounds and their metabolites. that has been little explored to date. Importantly, all methods under development The optimization of various parameters of this technique as well as its application are validated against “gold standard” histopathological diagnosis, which inherently to screening urine samples is discussed. Eleven different benzodiazepines and is a subjective measure and may vary significantly from one observer to another. metabolites were examined. Previous work by this laboratory has shown that for In this talk, I discuss the challenges faced and the possible solution approaches for benzodiazepines an aggregate solution made of MgCl2 prepared at a concentra- developing a truly “objective” algorithm for spectroscopy-based cancer recognition. tion of 1.67 M provided the highest signal intensity at the lowest drug concentration and thus was used in this study. A supported liquid extraction method specific for 169 Tailor-Made Transporter Assays for Stage-Specific Drug Discovery benzodiazepines was used. The method investigated was found to allow for the and Development detection of a wide variety of benzodiazepines and their metabolites. The presence Imad Hanna, Novartis, One Health Plaza, East Hanover, NJ 07936 of individualizing spectral peaks provides a high degree of specificity for sample de- The investigation of the potential consequences of new drugs interacting with drug termination. The technique is sensitive with a limit of detection of 2 ng/mL and linear transporters and drug metabolizing enzymes is carried out at various stages of drug over several orders of magnitude for the drugs chosen. This method has shown discovery and development. The bulk of this work is usually conducted by dedicated the applicability of SERS for the detection of trace quantities of benzodiazepines in research groups that perform both in-vitro and in-vivo studies and provide guidance toxicological samples as well as the optimization of the technique over a wide range on selecting new drug candidates. Recently the role played by drug transporters on of compounds. influencing the intracellular concentrations of certain drugs, or drug classes, has emerged as the main driver for both drug efficacy and toxicity. In particular there is 165 Semi-Conductor Compatible Plasmonic Materials: The Next Wave considerable interest in the field to understand factors that may impact the intracel- of Optical Sensing Technology lular drug concentrations in the liver, as it is the main organ for drug disposition and Stefan Franzen, North Carolina State University, 116 Cox Hall / Campus efficacy. These investigations include an understanding of how new drug candidates Box 8201, 363D Partners III, Raleigh, NC 27695 may act as inhibitors of uptake and efflux endogenous substances such as bilirubin, No abstract submitted by the author. bilirubin metabolites, and bile salts. The inhibition of either process can result in changes in systemic, and more importantly intracellular, concentrations of poten- 166 Modeling Spectroscopic Imaging Optical Paths for New Biomedical tially toxic compounds. In some cases, the inhibition of transport or metabolism of Sensing Opportunities endogenous compound can serve as a biomarker for the inhibition of either process. Rohit Bhargava, University of Illinois at Urbana-Champaign, 405 N. Moreover, the extent by which these changes occur would additionally be depen- Mathews Ave., Urbana, IL 61801, Thomas van Dijk, Rohith Reddy, Paul dent on the genetic makeup of individual human subjects. S Carney The path traversed by light as it interacts with matter critically determines the op- 170 Leveraging High-Throughput Mass Spectrometry-Based tical response recorded from any sample in analytical measurements. The mod- Transporter Assays to Drive Decision-Making and Enhance ADMET eling of these traversal and light-sample interactions is possible using a modular Knowledge approach that includes optical components from the source to the detector as well Andrew Wagner, Bristol-Myers Squibb, 5 Research Pkwy, Wallingford, as interactions with the sample. Here, we first present a general framework we CT 06492, Wilson Shou, Matt G. Soars have developed using both an electromagnetic theory and a scalar wave theory Over the last decade, it has become clear that transporters play a pivotal role in approach for infrared spectroscopy and imaging. We demonstrate that many of the the absorption, distribution, metabolism, and excretion - toxicity (ADMET) profile of commonly observed spectral features can be explained by considering the effects many drugs. At Bristol-Myers Squibb (BMS) we have developed a panel of relatively of the sample and optics. Electromagnetic theory is also applied to simulate sur- high-throughput (HT) transporter inhibition assays to screen for potential transport- face enhanced spectroscopy from single particles. We show that measurements er liabilities earlier in drug discovery in order to inform lead chemotype selection. involving light interactions with many such particles can be modeled using an ef- These 384-well format assays are conducted in freshly-plated cryopreserved cell fective medium theory. The results, validated with experiments, show a non-linear lines and/or vesicular systems expressing a transporter of interest and utilize clini- dependence on concentration and provide surprising clues for maximizing signal in cally relevant probe substrates to assess compound inhibition of transporter activity. surface-enhanced Raman spectroscopic measurements. Together, the results dem- While many of these assays employ microplate-based scintillation counting to as- onstrate that the interplay of scattering and absorption is crucial to understanding sess probe substrate transport, we have found that HT mass-spectrometry-based the recorded signal from spectroscopic experiments. The implications of this inter- detection methodologies offer a flexible and robust solution for assessing uptake play for biomedical sensing are discussed using illustrative examples. 26 2013 EAS Abstracts November 2013

of clinically relevant probe substrates. In an effort to reduce the cost and waste NMR-active isotopic labeled residue for a variety of different peptides and proteins streams associated with using radiolabelled drug substrates, we have converted which ultimately can be used to determine the difference between an alpha-helical several radiometric transporter inhibition assays to 384-well assays utilizing either and beta-sheet secondary structure. DEER can be used to measure distances be- solid-phase extraction-mass spectrometry (SPE-MS-MS) or liquid chromatography tween 2 spin labels out to about 70 Å. We have shown a huge improvement is sen- MS-MS to detect probe substrate uptake. Validation with reference compounds sitivity with DEER measurements at Q-band when compared to X-band. demonstrates that these assays are robust, reproducible, and generate data con- sistent with the literature. Implementing this HT-assay panel has facilitated informed 174 Structural Basis for Disruption by Model Amyloid Peptides from clinical candidate selection at BMS, and has also helped to establish a foundation Solid State NMR upon which we can build a better understanding of the role transporters play in Simon Sharpe, The Hospital for Sick Children, 555 University Ave., ADMET biology. Toronto, ON M5G1X8, Canada Protein misfolding in amyloid diseases leads to the accumulation of multiple types 171 High-Throughput LC-MS-MS Based Permeability and Transporter of beta-sheet rich assemblies, including amyloid fibrils and a host of smaller oligo- Assays in Early Drug Discovery meric entities. Current evidence suggests that non-fibrillar aggregates represent the Hui Zhang, Pfizer, Eastern Point Rd., Groton, CT 06340, John P. cytotoxic entities in amyloidosis, and that these are responsible for the cell death Umland, Carrie Funk, Michael West, Matthew D. Troutman that results in the degenerative pathology of these diseases. Defining the molecular Permeability is an important fundamental drug property as it directly impacts the basis for cytotoxicity is essential for a complete understanding of amyloidosis, and drug absorption/disposition and oral bioavailability. Besides the predominant mech- for identifying potential therapeutic targets. One potential mechanism for the cyto- anism as of passive diffusion, drugs can also be carried in and out through cell toxicity of amyloid oligomers is a direct disruption of the membrane, leading to loss membrane by uptake and efflux transporters. Accurate measurements of passive of cell integrity and apoptosis. We have used a solid-state nuclear magnetic reso- permeability and active transport via major transporters for drug candidates are criti- nance (NMR) based biophysical approach to determine the high-resolution struc- cal parts of the drug discovery process. In-vitro permeability and transporter screens tures of fibrils and non-fibrillar assemblies formed by several neurotoxic peptides are available during early drug discovery stages to address these needs including derived from the human prion protein. We find structural features that correlate with PAMPA, vesicle assays, as well as various cell based assays. Different cell based their ability to interact with model membranes, and have used imaging and NMR assays are developed in house to assess the passive permeability, active uptake, methods to determine the molecular mechanism for membrane disruption by these and active efflux respectively. All assays are in 96-well based high throughput plat- peptides. Specifically, we find that the mode of action depends on both the pep- forms and are highly automated. End points of assays are analyzed by liquid chro- tide sequence, and the membrane composition – such that a single toxic amyloid matography tandem mass spectrometry (LC-MS-MS) based bioanalytical systems oligomer may exhibit multiple mechanisms for membrane disruption and induction which provide high sensitivity, selectivity and throughput. Our system is capable of of cell death. delivering 15 sec/sample analysis speed routinely and it is also capable to analyze cassetted samples when test compounds are cassette-dosed ( e.g., 4-in-1 dosed 175 Mechanism of Transient Sequential Domain Interactions in for passive permeability assay). The fast turnaround of high quality permeability Nonribosomal Peptide Synthetases Revealed by NMR and transporter assay results enabled the effective compound profiling and specific Dominique Frueh, Johns Hopkins School of Medicine, 725 N. Wolfe St., absorption rate optimization. Baltimore, MD 21205 Non-ribosomal peptide synthetases (NRPSs) are microbial enzymatic systems 172 A Generic Mass Spectrometry-Based Methodology for Sensitive that synthesize important natural products, often with pharmaceutical applications and Fast Analysis of Non-Radiolabeled Substrates or Test (antibiotics, antitumor agents or immunosuppressants). NRPSs use a series of do- Compounds in Transporter Study mains, organized in contiguous modules, to covalently load substrates and con- Ming Yao, Bristol-Myers Squibb, Rte. 206 & Province Line Rd., dense them in an assembly line fashion. These catalytic steps require a series of Princeton, NJ 08543, Hong Shen, William G. Humphreys, Mingshe Zhu transient, sequential domain/domain and domain/substrate interactions, which are Transporters play a key role in drug disposition in humans. Inhibition of transporters currently poorly understood. We have used nuclear magnetic resonance (NMR) to by a co-administered drug could lead to increased toxicity or reduced effectiveness probe and characterize chemical and protein substrates binding sites for several of a victim drug that is a substrate of the transporter(s). Transporter uptake is mainly NRPS domains. With a combination of biophysical methods and NMR, we show that accomplished using radiolabeled compounds. However, the use of a radiolabel re- chemical substrates play a role in regulating the timing of sequential domain inter- quires significant cost and time of radio synthesis and manual operation in analysis. actions. Novel NMR methods were developed to monitor domain dynamics during The key objective of this study was to develop and validate a robust and sensitive these interactions. Understanding NRPS domain communication is a prerequisite liquid chromatography mass spectrometry (LC-MS) methodology for transporter to understand the biosynthesis of many natural products and offers prospect for substrate and inhibition assays. Cells were incubated with non-labeled compounds, reprogramming NRPS assembly lines. then washed, lysed with a special buffer and precipitated with acetonitrile contain- ing an internal standard. After processing using a liquid handler, samples in 96-well 176 Stochastic Dipolar Recoupling NMR Spectroscopy for the plates were analyzed by LC/MS. IC50 values of OATP1B1 inhibition by rifampin Structural Studies of Biological Solids were determined using non-radiolabeled atovastatin, pravastatin and rosuvastatin Wei Qiang, National Institutes of Health, Rm. 406, Bldg. 5, 9000 as substrates, consistent to data generated using radiolabeled substrates and re- Rockville Pk., Bethesda, MD 20892, Robert Tycko ported in the literature. In addition, dabigatran as a model compound was tested Deposition of beta amyloid (Abeta) fibrils in human brain is considered as one of for substrate analysis, which showed that the LC-MS based method worked well in the crucial clinic hallmarks for the identification of Alzheimer’s diseases (AD). Over OCT, OAT, MATE uptake experiments. The results from the study demonstrate sev- decades, the Abeta fibrils are utilized as targets for the design of either inhibitors eral advantages of the LC-MS-based methodology, including high sensitivity, fast that have therapeutic potential to AD or diagnosis reagents for the disease. In order analytical time and capability of direct determination of protein levels in the lysates. to get a rationale design of inhibitors or diagnostic markers, there has to be a better In addition, the use the filtration approach not only eliminated the interference of understanding of the atomic resolution structures of Abeta fibrils, especially the fibril protein and lysing detergents to column and MS source, but also enabled automa- structures inside of the amyloid plaques from human brain. In this presentation, I tion processing. As a consequence, the LC-MS-based approach has replaced radio first describe the structural purification and identification of a novel full-length anti- detection method in transporter substrate and inhibition assays in our lab. parallel Abeta fibril formed by the familial Iowa mutant (Asp23 to Asn23 mutation). As the same primary protein sequence can form either parallel or antiparallel fibrils, 173 EPR and NMR Studies of Membrane Proteins I then describe our attempt to design the structural-specific inhibitors which modify Gary A. Lorigan, Miami University, 701 E. High St., Oxford, OH 45056 the direct ion of fibrillation. This serves as an example of rationale inhibitor design Currently, limited structural and dynamic information on proteins embedded in- based on known high resolution structures of Abeta fibrils. Finally, I talk about our side a membrane exist, because they are difficulty to crystalize. New biophysical/ most recent studies on the correlation between the high resolution structures of the structural biology methods are needed to probe these systems in a lipid bilayer. brain-extracted Abeta fibrils from AD patients and their clinic histories. This work The Lorigan lab is applying unique hybrid solid-state nuclear magnetic resonance was motivated by the fact that the neurotoxicity of different Abeta fibril structures (NMR) and spin-label electron paramagnetic resonance (EPR) spectroscopic tech- may be distinct. In the future, we hope to combine the information from all these niques to study membrane proteins. Magnetic resonance spectroscopic data of works to achieve structural-specific inhibitors that can reduce the formation of cer- 15N-, 2H-labeled and/or spin-labeled membrane proteins incorporated into vesicles, tain highly-toxic Abeta fibrils inside of human brains. lipodisc nanoparticles, and bicelles are presented. State-of-the-art magnetic reso- nance techniques such as solid-state NMR, electron spin echo envelope modulation (ESEEM) spectroscopy, and double electron-electron resonance (DEER) spectros- copy are used. The ESEEM technique can determine short to medium range dis- tances (out to about 8 Å) between a site-specific nitroxide spin label and a nearby 27 2013 EAS Abstracts November 2013

177 Withdrawn by the author. 182 Heterogeneous Reactions on Ice and Metal Oxide Surfaces Studied In-Situ by X-Ray Photoelectron Spectroscopy Hendrik Bluhm, Lawrence Berkeley National Laboratory, One Cyclotron 178 Application of NMR Crystallography to Drug Development Rd., Berkeley, CA 94720 Heather Frericks Schmidt, Pfizer, 445 Eastern Point Rd., Groton, CT Metal oxide and ice surfaces act as heterogeneous catalysts in the environment 06339, Yuriy A. Abramov, Linda L. Lohr and atmosphere. Molecular level investigations of processes at the metal oxide and The bonding information present in a single crystal structure of a new pharmaceuti- ice interface with the surrounding gas atmosphere is thus of great importance for cal candidate provides vast amounts of information to aid in its development. In a basic understanding of heterogeneous processes in the environment. However, addition to reporting on chemical structure, the crystal interactions can be used quantitative investigations of these interfaces under relevant humidities and trace to identify the most stable polymorph, understand the solid form landscape, pre- gas concentration are difficult since many of the surface sensitive spectroscopies dict chemical/physical stability and predict physical properties in manufacturing. In are limited to vacuum conditions. In this talk we discuss how ambient pressure X-ray cases where a suitable single crystal is not available for structure determination by photoelectron spectroscopy can be used to quantitatively determine hydroxylation single crystal X-ray diffraction alternative methods must be explored. Solid-state and molecular water adsorption on metal oxide surfaces, as well as the influence of nuclear magnetic resonance (NMR) can provide crystallographic information and the adsorption of small hydrocarbons and nitrogen dioxide on ice surfaces on the in some cases determine the crystal structure from a powdered sample. I present premelting transition. a couple of applications of this emerging field, termed NMR crystallography, to the development of pharmaceutical candidates at Pfizer. NMR crystallography methods 183 Diverse Spectroscopy on Modern and Traditional Materials at the have been routinely used to confirm salt/co-crystal formation and identify hydro- Library of Congress gen bonds. In one example, chemical shift calculations and analysis were used to Fenella G. France, Library of Congress, 101 Independence Ave SE, identify the hydrogen bonding in a metastable solid form to derisk the polymorph Washington D.C. 20540, Lynn Brostoff, Eric Breitung landscape. For another drug candidate, proton-proton correlations, combined with The Library of Congress (LC) has been expanding non-invasive research tech- chemical shift calculations proved the formation of a co-crystal and identified the niques to better understand degradation mechanisms for preservation of traditional counterion location within the crystal lattice, in addition to other crystal packing in- and modern materials. The Library holds more than 750,000 magnetic tapes. Many teractions. are degrading rapidly, and a method to triage tapes before mounting for digitization is needed to speed the digitization process and limit damage to one of a kind collec- 179 Scenes from the Edge: Atmospheric Chemistry of Environmental tion items. Research into the use of Fourier transform infrared (FTIR) spectroscopy Interfaces and optical microscopy for rapidly identifying degraded tapes, enabled the separa- V. Faye McNeil, Columbia University, 500 W. 120th St., Rm. 816, New tion of degraded from non-degraded, but needed increased accuracy to become a York, NY 10027 useful collection assessment tool. By utilizing statistical spectral analysis and image Processes at environmental interfaces such as aerosol and ice particle surfaces analysis in addition to FTIR, this has enabled the development of a comprehensive profoundly influence atmospheric chemistry and climate. In the first part of my talk, method for distinguishing degraded from non-degraded tapes. One area of expand- I present an overview of my group’s recent experimental and theoretical work on ing research at LC is assessing the long-term effects of treatments on collection organic material at the gas-aerosol interface and its impacts on aerosol surface ten- materials. The on-going technical study and conservation of an important 1513 sion and cloud condensation nuclei ability. In the second part, I discuss our work on hand-colored, printed edition of Ptolemy’s Geographia at LC has posed interesting trace gas-ice interactions and the effects of impurities on the state of the ice surface. challenges, especially in terms of the treatment of select maps that are in poor con- dition due to the presence of verdigris pigment on a poor quality paper, as well as a 180 Interactions of Oxalic Acid on Ice later restoration treatment. Treatment was necessary to stabilize these seven maps, Liang Chu, Wadsworth Center - University at Albany, PO Box 509, and a combination of quantitative X-ray fluorescence (XRF) trace element analysis Albany, NY 12201 before, during and after treatment of several maps, spectral imaging, and Raman Understanding the interactions between oxalic acid and ice is important to reveal spectroscopy has served as a key aid in the treatment decision-making process. the nature of the heterogeneous reactions occurring in the polar boundary layer. The reflection-absorption infrared spectroscopy and temperature programmed de- 184 Scientific and Computational Imaging: Research & Conservation sorption (TPD) were used to study the interfacial chemistry of oxalic acid with ice of Smithsonian Collections on a polycrystalline Cu surface at 155−283 K. The study shows that the interaction E. Keats Webb, Smithsonian Museum Conservation Institute, 4210 between ice and oxalic acid does not lead to the ice-induced deprotonation of oxalic Silver Hill Rd., Suitland, MD 20746, Melvin Wachowiak acid. However, the H-bonding interaction between ice and oxalic acid can lead to A variety of scientific and computational imaging techniques are used as non-de- the formation of a metastable oxalic acid-ice complex in some cases during the TPD structive examination and analysis tools in research and conservation of cultural process. Desorption of H2O from the oxalic acid covered H2O/Cu surface is sug- heritage objects. Documentation methods using a range of cameras, lighting, and gested to involve the diffusion of H2O molecules through the top oxalic acid layer. In setups, aid and inform the condition, treatment and overall understanding of an the oxalic acid + ice coadsorption system, ice desorption is followed by a rearrange- object. This presentation describes scientific and computational imaging techniques ment of oxalic acid to form an oxalic acid adlayer on Cu. Uptake coefficient of oxalic including reflectance transformation imaging (RTI), high dynamic range imaging acid on ice was determined to be ~10-3 to 10-4, suggesting relatively weak interaction (HDRI), multispectral imaging, gigapixel imaging and axial specular illumination, between oxalic acid and ice as well. and their applications to specific projects involving the Smithsonian Collections. 181 Photolysis of Pollutants at Ice Surfaces in the Presence of 185 Structure from Motion/Photogrammetry and the Long-term, Environmental Contaminants Archival Survival of 3D Information Tara F. Kahan, Syracuse University, 1-014 Center for Science and Mark Mudge, Cultural Heritage Imaging, 2325 3rd St., Suite 323, San Technology, Syracuse, NY 13244 Francisco, CA 94107 Ice presents a complex reaction environment; reaction kinetics there can differ sig- Photogrammetry refers to the practice of deriving three-dimensional (3-D) measure- nificantly from those in aqueous solution. In order to predict the role of ice in the ments from photographs. Recent technological advances in digital cameras, com- transformation of aromatic pollutants and other atmospheric species, we need to puter processors, and computational techniques, such as sub-pixel image match- know kinetics and mechanisms of important processes there. While a number of ing, make photogrammetry a portable and powerful technique. It yields extremely laboratory studies have measured reaction kinetics in ice, reported rate constants dense and accurate 3-D surface data. It can be generated using a sequence of pho- can vary by over an order of magnitude due to different techniques being sensitive tos and captured with standard digital photography equipment, in a relatively short to different regions of ice (i.e., liquid veins and pockets within ice compared to the period of time. Photogrammetry is one example of a larger set of digital technologies disordered region at the air-ice interface). I discuss the reasons for these discrep- called “computational photography”. Based on digital photography, computational ancies as well as ways to resolve them. I also discuss recent work that examines photography tools extract relevant information from the individual photographic im- the role of environmental contaminants such as halide salts and organic matter in ages of an image sequence. The extracted information is fed to a computer algo- determining reactivity at ice surfaces. rithm, which generates new information not present in any of the original individual photographs. One of the great virtues of computational photography is that all the information required to generate the final result is contained in the original photo- graphic image sequence. As the long-term archiving of digital photographs is well understood, there are significant benefits to the use of computational photography methods in cases were long-term archival survival desired. In the case of 3-D im- aging, and where archival methodologies are not well established, image-based

28 2013 EAS Abstracts November 2013

structure from motion/photogrammetry empirical acquisition technologies can offer its urea degradant. The two derivatization reactions are simple, fast and quantita- an enhanced chance that important cultural heritage and scientific data will remain tive. The two LC-MS-MS methods were qualified and both have a reporting limit of available to future scholars. 0.1 ng/mL with good precision (RSD < 6%, N = 6, S/N > 350). The HOPO method has a linear range of 0.1 – 25 ng/mL; EDAC has a linear range of 0.1 – 100 ng/mL. 186 Computed Tomography meets Archaeology - Virtual Excavation Both methods have excellent recoveries in each of three different API matrices. In and Reconstruction addition, a selected reaction monitoring based confirmatory methodology was de- Nicole Ebinger-Rist, Landesamt für Denkmalpflege Baden-Württemberg, veloped to verify the identity of HOPO when present at ≥ 0.6 ppm. Berlinerstrasse 12, Esslingen am Neckar – 73728, Germany The archeological heritage preservation authority Ba-Wue has used computed X- 189 Enabling Rapid, Sensitive Peptide Quantitation Through ray tomography (XCT) routinely since 2009. This non-destructive method is par- Automation and Nano/LC/MS ticularly advantageous in the case of excavated blocks containing finds of different Weixun Wang, Merck, 51 Central Square Park, Metuchen, NJ 08840 materials. Some astonishing results have been achieved with it over the past years, No abstract submitted by the author. especially on composites of metal and organic materials. One of the exigencies of daily routine is that objects have to be processed by a specified deadline. Not 190 Data Independent High Resolution Mass Spectrometry Methods to only does the preparation of archeological finds for evaluation have to be carried Integrate Drug Metabolism and Pharmacokinetics Studies out within an ever shorter time, the volume of finds is also increasing year by year. Ragu Ramanathan, QPS, 110 Executive Drive, Suite 7, Newark, DE This made it necessary to look for alternative, yet also highly precise processing 19711 methods that could be used alongside conventional approaches. Due to finds hav- A work flow involving high-resolution mass spectrometry (HRMS) and ultrahigh- ing lain the ground, especially in the case of burials, their materials are often found pressure liquid chromatography (UHPLC) has been proposed as an option for compressed in layers only a few centimeters thick. Separating individual layers of streamlining drug metabolism and pharmacokinetic studies. The streamlined work- finds often leads to destruction of their context. XCT is well suited for documenting flow involves simultaneous gathering of qualitative and quantitative (qual/quant) individual find layers because it permits their virtual separation. To be able to visual- information from in-vitro and in-vivo samples. When acquiring qual/quant data, the ize organic structures it is necessary to have a knowledge of the specific character- scan rate of the mass spectrometer is important because the number of points used istics of the material in question, since they cannot be identified otherwise. When to define a chromatographic peak is important for accurate quantitation of drugs objects are so poorly preserved from having lain in the ground that they can hardly and their metabolites. Often the scan rate has to be optimized when acquiring data be removed from the surrounding earth XCT often provides the only way to obtain a with higher mass resolving power or complex scan functions to include metabolite three-dimensional image of their appearance. The lecture deals with the possibilities identification data. In this presentation, HRMS-based qual/quant experimental op- of and limits to the innovative methods of X-ray computed tomography and shows tions, including data independent options, that are available to improve sensitivity, different results. signal-to-noise ratio, and selectivity are discussed using recent in-vitro and in-vivo applications. 187 Measuring Dysregulation of Kynurenine Metabolites in Mouse Models of Inflammation Associated Depression 191 Atomic Emission Beacons for Long-Distance Chemical Signaling David Budac, Lundbeck, 215 College Rd., Paramus, NJ 07652 Christopher N. LaFratta, Bard College, PO Box 5000, Annandale Metabolism of tryptophan to serotonin has been studied intensely due to the impor- on Hudson, NY 12504, Ian Pelse, Jose Luis Ffalla, Yi Liu, Manuel A. tance of serotonin in neuroscience. A less understood set of tryptophan metabolites, Palacios, David R. Walt, George M. Whitesides kynurenines, are also implicated in neurological and psychiatric disorders. For ex- In an effort to exploit chemistry for information science, we have constructed a sys- ample, increased levels of quinolinic acid, a kynurenine metabolite, is found in the tem to send a message powered by a combustion reaction. Our system uses the brain of Huntington disease patients and increased levels of kynurenic acid, also a thermal excitation of alkali metals to transmit an encoded signal over long distances. kynurenine metabolite, and is observed in the brain of schizophrenia patients during A message is transmitted by burning a methanol-soaked cotton string embedded post-mortem studies. Liquid chromatography tandem mass spectrometry (LC-MS- with combinations of high, low, or zero levels of potassium, rubidium, and/or cesium MS) techniques provide an important tool for exploring the involvement of kynuren- ions. By measuring the intensities at the characteristic emission wavelengths of ines in neurological and psychiatric disorders. We have developed a LC-MS-MS each metal in the near-infrared, 19 unique signals can be distinguished. We have method and sample preparation techniques which allow us to monitor changes in detected these signals from 1 km away for nearly ten minutes. Potential applications tryptophan metabolites in a wide range of sample types including: plasma, brain and of this platform include covert messaging or remote self-powered environmental liver tissue, cell cultures, enzyme assays and micro dialysis samples. The last of sensors. This work, which seeks to encode and transmit information using chemistry these is the most demanding with respect to limits of quantitation, with analyte levels instead of electronics, is part of the new field of “infochemistry”. in the low pg/mL concentration range. Analysis of brain sections weighing as little as 5 mg also requires attention to detail to allow measurement of the kynurenines in 192 Mass Spectrometry in RIC ROC Applications - Part 01 in Theory specific brain regions. This presentation outlines the sample preparation required to and Practice achieve the desired measurement of tryptophan metabolites in the various sample Srivas R. Srinivas, Anadams, 2850 Webb Ave., Bronx, NY 10468 types along with a description of the LC-MS-MS methodology. Examples of the role A critical review of literature reveals that physical methods are employed in the that this technique has played in investigating mouse models of inflammation-asso- characterization of organic/inorganic compounds. Application of ultraviolet, infra- ciated depression are presented. In addition collaborative efforts with academia are red, nuclear magnetic resonance and X-ray spectroscopy has made it possible to presented which highlight the importance of this analytical technique. resolve very complex problems. Contra distinctively, mass spectrometry as a tool in experiments has yet been neglected. The mass spectrometer characterizes the 188 A Chemical Derivatization Approach for the Quantification of molecular species of the sample, rather than bulk properties as in the case of ab- Genotoxic Impurities HOPO and EDAC-HCl at Sub-ppm Level by sorption spectroscopy. An important aspect of mass spectrometry is the amount of LC-MS-MS sample (milli-moles) to obtain a usable mass spectrum. Anomalies in library search Wei Ding, Bristol-Myers Squibb, One Squibb Dr., New Brunswick, NJ results of data base are also covered. These aspects are reported in another com- 08903, Yande Huang, Jeff Dai, Jonathan Marshall, Scott Miller munication. 2-Hydroxypyridine N-oxide (HOPO) and 1-ethyl-3-[3-dimethylaminopropyl] carbodi- imide (EDAC) hydrochloride are reagents used to form amide bonds in the syn- 193 Mass Spectrometry In RIC ROC Applications - Part 02 theses of active pharmaceutical ingredients (APIs). Recently it was confirmed that Pharmaceutical Analysis HOPO and EDAC have positive responses in Ames assays classifying them as Srivas R. Srinivas, Anadams, 2850 Webb Ave., Bronx, NY 10468 genotoxic impurities and as such must be controlled to the threshold of toxicological The aim of this work was to identify and quantify the quality of the United States concern. Direct detection for trace amounts of HOPO was unsuccessful with atmo- Pharmacopeia (USP) Standard Pharmaceuticals, viz., Hydralazine,UV max 260 nm, spheric pressure ionization liquid chromatography tandem mass spectrometry (LC- and Hydroxozine UV max 230 nm by the application of mass spectrometry. Further, MS-MS) and direct determination of EDAC was challenging due to its propensity to the USP method involves the use of high-performance liquid chromatography meth- hydrolyze to the corresponding urea under some solution conditions. To overcome od for separation and UV detector for identification and quantification. Thin layer these analytical challenges, chemical derivatization for HOPO and EDAC in multiple chromatography (TLC) may also be used, but it should be noted that these sub- APIs at sub-ppm level using LC-MS-MS was explored. The derivatization reagent stances are highly light sensitive and prolonged exposure might lead to inaccurate dansyl chloride was applied to convert HOPO into the corresponding dansyl-de- results due to the formation of undesirable degradatives. On the other hand, mass rivative which significantly improved chromatographic performance and enhanced spectrometry reveals not only structural and sub structural units but also enables the sensitivity by four orders of magnitude. For EDAC, sodium hydrogen sulfide was analyst to ascertain the quality of the product. Fallacies in the library search results successfully used to convert EDAC into its thiourea derivative to differentiate it from of a large data base are also covered.

29 2013 EAS Abstracts November 2013

194 On the Fermentation of Cabbage by NMR 198 Label-Free Imaging and Characterization of Cancer Cell Responses Kenith M. Conover, Princeton University, Frick Chemistry Laboratory, to Polymethoxyflavones Using Raman Microscopy Princeton, NJ 08544 Hua Zhang, University of Massachusetts-Amherst, 247 Chenoweth Civilizations throughout the world have fermented vegetables for thousands of Laboratory, 100 Holdsworth Way, Amherst, MA 01002, Hang Xiao, Lili He years. Not only does fermenting allow for the preservation of vegetables, it also Polymethoxyflavones (PMFs) are almost exclusively found in the citrus genus. They increases the nutritional value of them. The majority of vegetables are fermented have shown great anticancer potentials. However, their exact mode of action against by lactic acid bacteria (LAB). When fresh vegetables are salted and submerged in cancer cells remains unknown. Raman spectroscopy is molecular vibrational and the resultant brine, an anaerobic environment emerges favoring the proliferation of can be used to noninvasively measure chemical signatures of cells without chemical LAB. Sauerkraut is an example of this fermentation process. A holistic approach was labels. The objective of this study is to develop a label-free imaging method to char- taken to investigate the chemical change of cabbage when fermented by LAB. The acterize cancer cell responses to PMFs using Raman microscopy. Two colon cancer brine was subjected to 1H and 13C nuclear magnetic resonance (NMR) analysis on cells, namely HT29 and HCT116, were cultured and compared. HT29 were treated a system equipped with a cryoprobe for improved sensitivity. Databases and NMR with nobiletin (NBT) and 5-hydroxy-3,7,8,3’,4’-pentamethoxyflavone (5HPMF) for 48 prediction software were implemented to assist in the analysis of the brine mixture. hours. The cells were either fixed using formaldehyde or directly measured in media Findings from the analysis are compared with previously published results. under the Raman microscope. The spectral data were analyzed using TQ analyst. The results show that Raman mapping method could image a single cell based on 195 The Determination of Silicon Concentration on Hair Tresses Using this chemical Raman signature without adding chemical labels. Measuring live cells FTIR was more beneficial than the fixed cells for clearer nucleic acid peaks. Two types of Alex J. Augatis, Ashland Specialty Ingredients, 1361 Alps Rd., Wayne, cancer cells used in this study showed different spectral patterns illustrated in the NJ 07470, Raymond Bret Clark, Chi-san Wu, Osama Musa, Eugene W. principle component analysis plot. After treatment with PMFs, the Raman peaks of Oldham, James E. Brady lipid at 1301 cm-1, proteins at 1005 cm-1 and nucleic acids at 680 cm-1 changed The deposition of silicone on hair enables the hair to exhibit numerous performance differently. NBT induced more peak intensity changes than 5HPMF, indicating the attributes desirable in the personal care markets. As such, it is desirable to measure different sensitivity of cell responses to different PMFs. In conclusion, we demon- the amount of silicone present on hair as it is related to performance. In addition, it strated the capacity using Raman microscopy for label-free imaging and character- is also crucial to monitor the level of silicone after hair is exposed to typical external ization of cancer cell responses to two PMFs. This method could be extended to forces (washing, rinsing, heat treatment, etc.). Therefore, identification of an ana- study other cancer cells and anticancer compounds. lytical technique that can determine silicone at low levels is desirable. In this study, a quantitative method is presented using Fourier transform infrared spectroscopy 199 Elemental Analysis of Ancient Pottery by Inductively Coupled (FTIR) to monitor silicone deposition on hair down to ppm levels. Monitoring the in- Plasma-Optical Emission Spectroscopy (ICP-OES) to Better tensity of the Si-CH3 stretching absorbance at 1260 cm-1, a linear calibration curve Understand Provenance was established with R2 = 0.9969. From this curve, silicone extracted from hair was Erika Portero, Drew University, 36 Madison Ave., Madison, NJ 07940, measured and reported. Further, this study also demonstrates the validity of this Astrid Castillo, Molly W. Crowther, Jon Kettenring, Carl Savage spectroscopic method by a correlation to values obtained chromatographically and At least thirty samples from shards of Herodian oil lamps (first-century, Bethsaida by inductively coupled plasma - atomic emission spectroscopy. area) have been obtained. In order to better understand the sample provenance, multi-element analysis is performed using inductively coupled plasma-optical emis- 196 Spectroelectrochemical Study of the Redox Activity of 2, sion spectrometry (ICP-OES). The samples are broken down to elemental constitu- 3-Dichloro-1, 4-Naphthoquinone ents and dissolved using strong acids and microwave digestion at high temperature Maraizu Ukaegbu, Howard University, 2400 Sixth St. NW, Washington and pressure. Several major component elements (Al, Ca, Fe, K, Mg, Na) and minor DC 20059, Oladapo Bakare, Charles Hosten component elements (Ba, Cd, Co, Cr, Cu, Mn, Ni, Pb, Sr, Zn) are simultaneously 2, 3-dichloro-1, 4-naphthoquinone (DNQ) is known as anti-microbial agent and elec- determined using ICP-OES and external standard calibration. Principal component tron acceptor. The electrochemistry of 2, 3-dichloro-1, 4-naphthoquinone on a gold analysis (PCA) and other statistical methods will be employed to determine pat- electrode was studied using cyclic voltammetry, bulk electrolysis and in-situ ultravio- terns, clusters, or outliers in the chemical composition that may ultimately reveal let spectroelectrochemistry. The cyclic voltammetry data show two cathodic peaks common or separate origins among the samples. This addresses the question of at - 0.73 V and - 1.38 V, and anodic peak at – 0.66 V and – 1.31 V referenced to Ag/ whether there was a cultural connection between Jerusalem and the Galilee in the Ag+ reference electrode. The ratio of cathodic and anodic potential of 1.10 and 1.06 first century CE. for peak I and II suggest that the electron transfer is reversible. The peak separation of 59 mV suggests one electron transfer process. For DNQ, it is 70 and 74 mV for 200 Trace Metal Screening of Barium Sulfate Raw Ores by ICP-OES peak I and II respectively. From bulk electrolysis studies, it was concluded that the Gregory W. White, Bracco Research USA, 305 College Rd. East, reductive process involves two single electron transfers. In-situ ultraviolet spectro- Princeton, NJ 08540, Nishit Gandhi, Thomas Katona electrochemistry was used to monitor the formation of the reductive intermediate. Inductively coupled plasma-optical emission spectrometry (ICP-OES) was used to The wavelength at 473 nm is a characteristics absorption peak for the formed inter- screen barium sulfate raw ores for trace metals. A concentrated sulfuric acid diges- mediate. From the electrochemical and spectroelectrochemical data a mechanism tion at 300 °C for four hours was employed to ensure solubilization of the barium sul- for the electrochemical reduction of DNQ was developed. fate and any metals including zirconium. Multiple lots of barium sulfate raw ore from three different vendor sources were semi-quantitatively screened for 55 elements by 197 Combined Headspace Karl Fischer and NIR System Makes NIR ICP-OES using an axial view instrument with a high performance Echelle spectrom- Modeling of Lyophilized Products Quicker, Safer and More Reliable eter and collision induced dissociation detector, providing greater speed, accuracy George E. Porter III, Metrohm USA, 6555 Pelican Creek Circle, and sensitivity (~ 10 – 100 fold) than previous radial view techniques. Variations in Riverview, FL 33578, Hari Narayanan, Denise Root trace metal levels were observed between ore suppliers with up to 14 elements (Al, Near-infrared (NIR) spectroscopy has been used for decades as a rapid and cost Ca, Cu, Er, Fe, K, Mg, Mn, Na, P, Si, Ti, Zn and Zr) repetitively detected above the effective tool for determining the water content of lyophilized drug products while method limits of detection in some or all of the samples. Results for all 14 elements during production and even phases of research and development. NIR has no sam- were well below current International Conference on Harmonization (ICH), United ple prep and is fully compatible with glass vials making it the perfect candidate for States Pharmacopeia, and European Medicines Agency safety levels assuming a rapid screening of mass produced freeze dryer vials. The most common drawback maximum 10 g daily dose. General method validation to determine instrument limits of NIR testing is creating a new quantitation model when the matrix or packaging of detection included determinations of specificity (55 elements, 80 wavelengths), goes through a change via a primary analysis technique such as headspace Karl limit of detections (LODs) ( ≤ 0.25 µg/mL), precision (CVs ≤ 5.0%), and robustness Fischer titration. This process usually involves creating a large number of samples, (LODs consistently achievable). Validation of a quantitative method for the determi- collecting NIR spectra and then analyzing by Karl Fischer (KF) for water content. nation of trace amounts of Zr in barium sulfate included determinations of specific- We show that by mounting a NIR probe on the turntable of a KF oven we are able ity (spectral scans of spiked and unspiked barium sulfate), LOD (0.01 µg Zr/mL), to streamline this process. By integrating NIR and headspace KF, there is immense minimum quantifiable limit (0.05 µg Zr/mL), linearity (r = 0.99999), precision (CVs ≤ time savings, higher productivity and more reliable results than the former method. 5.3%), accuracy (62.1 – 74.3 %), range (0.05 – 0.6 µg Zr/mL), and robustness (LOD consistently achievable). All validation parameters were established in compliance with current ICH guidelines.

30 2013 EAS Abstracts November 2013

201 FT-Raman Mapping of Multi-Component Solid Dosage Forms degradation behavior of a polymeric additive (hydrogenated styrene-butadiene co- Ronald L. Rubinovitz, Thermo Fisher Scientific, 4410 Lottsford Vista polymer) used in a motor oil formulation under an accelerated ageing test at 170C. Rd., Lanham, MD 20706 The oligmeric degradants were detected and eluted after the additive but before The benefits of Fourier transform (FT)-Raman spectroscopy to provide material the low-MW base oil. Longer ageing times led to more degradants via oxidative characterization across a broad range of applications are well known. These include cleavage as evidenced by the increased oxygen functional groups: ether(1000-1200 straightforward sample handling, since samples can be quickly and easily measured cm-1), oxirane(806 cm-1) and carbonyl(1700-1750 cm-1). Other polymeric additive and the frequencies of interest are compatible with glass. Further, the spectra are was partially oxidized as evidenced by the oxygen incorporation in the ether region typically characterized by sharp features, similar but complimentary to the common- (1000-1200 cm-1) and into the oxirane ring (806 cm-1), and appeared at the similar ly used mid-infrared (IR) absorption spectroscopy. In addition, the laser frequency SEC elution time without any polymer chain breakdown. (1064 nm) of FT-Raman avoids the fluorescence often observed when visible laser excitation is used. This study presents data measured with a newly developed FT- 205 Coupling Raman Spectroscopy and Gravimetric Vapor Sorption Raman unit that is utilized in an FTIR spectrometer sample compartment. Measure- Methods to Monitor Vapor-Induced Phase Transitions ments collected of the interior of solid dosage forms through the use of a computer Daniel J. Burnett, Surface Measurement Systems, 2125 28th St. controlled sample stage are analyzed, providing composition as well as spatial dis- SW, Ste. 1, Allentown, PA 18103, Armando R. Garcia, Majid Naderi, tribution information. Dosage forms are presented that include various component Manaswini Acharya levels to illustrate the mapping and data extraction capabilities that are possible. As Raman spectroscopy is a widely used physical characterization technique. The shown in these results, although the simplest method of creating spectral maps is combination of Raman spectroscopy and dynamic vapor sorption (DVS) allows for a through the representation of a unique peak height across the area measured, this more complete understanding of vapor-solid interactions for pharmaceutical materi- method may not always yield all the possible information from the collected spectra. als. This paper investigates the in-situ monitoring of a vapor-induced transforma- To that end, analysis techniques beyond mapping of direct spectral features, such tions of pharmaceutical ingredients using a combined Raman spectroscopy-vapor as principal component analysis and multivariate curve resolution are presented sorption technique. Spray-dried lactose was used to investigate moisture-induced and contrasted. The results shown illustrate the potential for applications including amorphous to crystalline transformation. Mannitol was used to investigate polymor- monitoring manufacturing processes and counterfeit product screening. phic changes. Mannitol is known to exist in at least three polymorphic forms: alpha, beta, and delta. The beta form is considered to be the most stable. Humidity and 202 PMMA/PLA Blends Compositional Analysis. ATR-FTIR Coupled organic vapors are known to convert the less stable delta form to the beta form. Sal- with Chemometrics Methods butamol sulphate (SS) was used to illustrate effects of processing route. Samples Dana Garcia, Arkema, 900 First Ave., King of Prussia, PA 19406, Susan were studied after micronization (MSS), spray-drying (SDSS), and re-crystallization Pirollo, Florence Mehlmann, Thomas Richards (CSS). Gravimetric and spectroscopic results on spray-dried lactose showed clear Plexiglas® RnewTM PMMA/PLA blends are bio-based high-performance thermo- evidence of moisture-induced crystallization above 60% RH at 25 °C. Ethanol vapor plastics with superior performance, suitable for a number of applications requiring at 95% P/Po and 45 °C was sufficient to convert the less stable δ form of D-mannitol optical transparency, chemical resistance, and easy processing, while striving to to the more stable β form. SDSS showed evidence of crystallization above 70% meet sustainable solutions. In order to tailor their performance, the composition RH in both the Raman spectra and gravimetric data. However, the MSS did not of the blends has to be carefully controlled. Attenuated total reflectance Fourier show any evidence of crystallization. The combination of gravimetric vapor sorption transform infrared (ATR-FTIR), coupled with chemometrics techniques, is ideally with Raman spectroscopy allowed for the monitoring of the vapor-induced trans- suited to this type of analysis. FTIR techniques overcome the limitations of clas- formations in real-time. The unique combination of these two techniques allows for sical separation methods, which are not always well suited for polymer analysis, increased understanding of vapor-induced structural changes of pharmaceutical due to high molecular weights and lack of solvent specificity. Further coupling with ingredients. chemometrics addresses spectral complexity. The methodology has the advantage of requiring minimal to no sample preparation and can easily be implemented in a 206 Modifying Gold Nanoparticles for Analysis of Lipophilic Food plant quality control laboratory. In this poster presentation we show the development Components and implementation of a robust method for at-line product analysis. Plexiglas as well Michael Driver, University of Massachusetts, 228 Chenoweth Laboratory, as Rnew, are a registered trademark of Arkema. 100 Holdsworth Way, Amherst, MA 01003, Yue Li, Lili He Surface enhanced Raman spectroscopy (SERS) is a sensitive method for molecular 203 FT-NIR Applications in Tablet API Analysis and Tablet Coating characterization. However, little study has applied this technique in lipophilic food Process Monitoring components. Gold nanoparticles (AuNPs) prepared by citrate reduction method Herman He, Thermo Fisher Scientific, 4410 Lottsford Vista Rd., are suspended in water, not mixable with lipophilic compounds. The objective of Lanham, MD 20706 this study was to modify AuNPs to be hydrophobic so that they can be compatible The majority of commercially available pharmaceutical formulations are tablets and with lipophilic molecules for SERS measurement. The experiment modified 50 nm capsules designed to be taken orally. Fourier transformed near-infrared (FT-NIR) AuNPs with octanethiol in various concentrations in hexane. Dark field microscopic analysis is a powerful technique to measure these materials. The advantages of images and Raman spectra and images were taken to measure the surface modifi- using both diffuse reflectance measurement and transmission measurement on the cation and characterize the modified AuNPs. Additionally, the modified AuNPs were Thermo Scientific Antaris™ FT-NIR analyzer are discussed in this presentation. This mixed with canola oil to analyze their solubility as well as the Raman signals of presentation also shows an example of process analytical technology application the resulting mixture. The results showed that after mixed with octanethiol, AuNPs to monitor tablet spray coating process. Because of the fast measurement speed lost the characteristic pink color from the colloidal gold. The modified AuNPs were of the FT-NIR analyzer, now process operators can have the ability to estimate the concentrated at the interfacial layer of water and hexane, which demonstrated that coating time, and to fine tune process parameters in order to optimize the coating the successful modification with the octanethiol. The Raman spectra and principle process and to achieve better consistency in product quality. component analysis showed that the spectra characters of modified AuNPs were in between those of unmodified and octanethiol spectra. When mixing the interfacial 204 SEC-IR Coupled Technology to Characterize Polymer Grafting and layer, which contained modified AuNPs, with canola oil, a water-in-oil emulsion was Polymer Degradation formed. Analysis showed that the AuNPs were at the surface of the droplets which Ming Zhou, Spectra Analysis Instruments, 257 Simarano Dr., indicates the amphiphilic property of modified AuNPs. The modified AuNPs can be Marlborough, MA 01752, Tom Kearney used for interfacial characterization in emulsions. Infrared (IR) spectroscopy is the premier analytical tool for determining the composi- tion and structure of polymers. The coupling of IR spectroscopy with size exclusion 207 SERS Characterization of Hydrophobic Compounds by Silver chromatography (SEC) through an automated solvent-removing interface provides Dendrites a map of changing composition across the molecular weight distributions. The time- Yue Li, University of Massachusetts-Amherst, 248 Amherst Rd., Apt. J3, ordered IR spectra can be used to understand and monitor polymer modifications Sunderland, MA 01375, Michael Driver, Eric Decker, Lili He such as grafting reactions and to characterize the mechanism of polymer degra- Surface enhanced Raman spectroscopy (SERS) has been exploited widely for sen- dation. SEC-IR was used to characterize a polymer grafting reaction of poly(vinyl sitive molecular characterization and has been used extensively as a signal trans- alcohol-co-vinyl acetate) copolymer (A,1739 cm-1 band) with an epoxy-terminated duction mechanism in biological and chemical sensing. However, little application siloxane oligomer (B, 803 cm-1 band) under a specific catalyst condition. The band has been explored in measuring hydrophobic compounds, e.g., edible oils. In this chromatogram based on B-specific 803 cm-1 band gave a new polymer peak in the study, gold colloids (50 and 100 nm) and silver dendrites (60 nm) were used as higher molecular weight (MW) region than polymer A, confirming that silicone B was SERS substrate for measuring canola oil with different concentrations of 0.01%- 5% grafted onto the polymer A chain. The IR spectra of the grafted product in this high in hexane. Three microliter of oil was directly deposited onto the gold and silver MW region have all the IR features of both starting materials with band ratios 803 substrate and dried for Raman measurement. Large variation of signals and signal cm-1/1739 cm-1 indicating the grafting levels. SEC-IR was also used to study the enhancements were observed for the oil spectra on gold nanoparticles. The SERS 31 2013 EAS Abstracts November 2013

spectra showed much better consistency and enhancement when silver dendrites 211 Effect of a Fluorophore on Bovine Serum Albumin Using Molecular were used as the substrates. The major bands of canola oil in the normal Raman Imprinting Process spectra were all enhanced. Interestingly, a new peak at 1616 cm-1 was observed in Anne E. Okafor, St John’s University, 8000 Utopia Parkway, Jamaica, the SERS spectra which was absent in the normal Raman spectra. The overall peak NY 11439, Enju Wang, Neil Jespersen, Viviana Scocco, Mustafa Sadoqi intensity firstly rose with the increasing oil concentrations and reached the highest This paper discusses the effect of a fluorophore-dimethylaminonitrostilbene (DMA- value at 1% oil concentration. While further increasing the oil concentrations to 5% NS) tethered to a triethoxysilane coupling agent creating a dye known as DMSCA on reduced the peak intensity. The relative analytical SERS enhancement factor was bovine serum albumin (BSA). The fluorescence spectra of BSA excited at a wave- calculated to be 580, by comparing the peak intensity of SERS spectra of 0.01% length of 280nm is used to evaluate the fluorescence emission spectra of BSA. canola oil and normal Raman spectra of 5% canola oil. In conclusion, this study The shift plus/minus of the wavelength (nm) revealed the conformation of the BSA. demonstrated the capacity of using silver dendrites for SERS characterization of Fourier transform infrared is also used to evaluate the three homologous domains hydrophobic compounds. of BSA-Amide I,II,III. The imprinted BSA topography is monitored with atomic force microscopy. Stability and reproducibility of the imprinted BSA is also evaluated. This 208 Use of Surface Dissolution Imaging to Determine the Effect of study helps in tailoring, designing and fabricating a sensor for drug delivery and Counter Ions on Dissolution Rate of Several Standard Drugs intraction (to mention a few) with BSA. Jun Yin, Sirius Analytical, 100 Cummings Center, Beverly, MA 01915, Jon Mole, Rebeca Ruiz, Karl J. Box, John Comer 212 SARA – A Software Environment for the Analysis of Relaxation Purpose Counter ions in dissolution media can have a significant impact on dis- Data Acquired with Accordion Spectroscopy solution rate of ionisable drugs. For those drugs, it is critical to select appropriate Bradley J. Harden, Johns Hopkins University, 725 N. Wolfe St., counter ions and their concentrations in dissolution media for the dissolution method Hunterian 701, Baltimore, MD 21231, Dominique P. Frueh development. Here a fast method is described to determine counter ion effect on We present SARA (Software for Accordion Relaxation Analysis), an interactive and the dissolution rate of papaverine, aripiprazole and chlorprothixene. Method UV im- user-friendly MATLAB software environment designed for analyzing relaxation data aging was performed using a Sirius SDI dissolution imaging system. Compacts of obtained with accordion spectroscopy. Accordion spectroscopy can be used to mea- the drugs were inserted into the flow-through cartridge. Direct measurements and sure nuclear magnetic resonance (NMR) relaxation rates in a fraction of the time visualisation in real-time of dissolution events directly at or near the surface of the required by traditional methods, yet data analysis can be intimidating and unified product, and downstream from the compact, were recorded. The study compared software packages are not available to assist investigators. Hence, the technique the intrinsic dissolution and mass released of papaverine HCl, aripiprazole and has not achieved widespread use within the NMR community. SARA offers users chlorprothixene HCl in an experiment with different starting media: de-ionised water a selection of analysis protocols spanning those presented in the literature thus and 0.01M HCl containing different concentrations of chloride counter-ion. KCl con- far, with modifications permitting a more general application to crowded spectra tent varied between 0% and 2.5%. Experiments were compared at flow-rates of 0.2 such as those of proteins. We discuss the advantages and limitations of each fit- mL/min and 0.8 mL/min. The temperature was maintained at 37°C and 2 to 5 mg ting method and suggest a protocol combining the strengths of each procedure of powder was compressed into the sample cup at pressure of 60 cN m. A 280 nm to achieve optimal results. In the end, SARA provides an environment for facile UV filter was used for this study. Experiments ran for up to 30 min. Conclusion The extraction of relaxation rates and should promote routine application of accordion study confirmed that counter ions affect the dissolution process and can suppress relaxation spectroscopy. the dissolution of the drug. This is especially important when dealing with the high dose formulations of the same drug, when the dissolution profiles start to deviate 213 Dual-optimized 1JCC-Edited 1,n-ADEQUATE: A Novel NMR from the solubility profiles. Structure Elucidation Technique Mikhail Reibarkh, Merck, 126 E. Lincoln Ave., PO Box 2000, Rahway, NJ 209 On the Interpretability of O-PLS Filtered Models 07065, Gary E. Martin, Mary Senior, R. Thomas Williamson, Wolfgang Barry M. Wise, Eigenvector Research, 3905 West Eaglerock Dr., Bermel Wenatchee, WA 98801, Jeremy M. Shaver Establishing the carbon skeleton of a molecule greatly facilitates the process of Orthogonal projections to latent structures (O-PLS), introduced originally by Trygg structure elucidation. Recent advances in the family of ADEQUATE experiments and Wold in 2002[1], is a patented algorithm that has received much attention for its have demonstrated their potential in this regard with 1,1-ADEQUATE, which pro- perceived ability to simplify model interpretation. Since its introduction, it has been vides direct 13C-13C correlation via 1JCC, and 1,n-ADEQUATE, which yields both shown by Ergon[2] and Kemsley and Tapp[3] that results identical to the original O- long-range (typically 3JCC) and direct 1JCC correlation. However, direct and long- PLS formulation can be obtained by post-processing conventional PLS models in range correlations are indistinguishable in 1,n-ADEQUATE spectra so one would a non-patented way. This demonstrated, unequivocally, that O-PLS models have need to collect both spectra in order to establish a carbon skeleton of a molecule. A predictive properties identical to their non-rotated versions. The authors did not, modified pulse sequence was developed in 2012 that semi-selectively invert 1JCC however, consider the interpretability of the models at length. In this poster we ex- correlations in 1,n-ADEQUATE spectra. This change provided a significant improve- plore the issue of interpretability of O-PLS models by applying the method to care- ment, allowing 1JCC and nJCC correlations to be differentiated in the same spec- fully constructed simple systems and well characterized data. trum. However, the experiment required careful matching of the amplitude transfer 1 References: function with JCC coupling constants in order to achieve near uniform inversion, 1 [1] J. Trygg and S. Wold, “Orthogonal Projections to Latent Structures (O-PLS),” J. and even then some JCC correlations could still have positive intensity due to the Chemo, 16, 119-128, 2002. oscillatory nature of the transfer function. Both shortcomings significantly limit the [2] R. Ergon, “PLS post-processing by similarity transformation (PLS+ST): a simple practicality and broad application of the method. We now report a new, dual-opti- 1 alternative to OPLS,” J. Chemo, 19, 1-4, 2005. mized JCC-edited 1,n-ADEQUATE experiment, that provides uniform inversion of 1 [3] E.K. Kemsley and H.S. Tapp, “OPLS filtered data can be obtained directly from JCC correlations across the range of 29-82 Hz. Unlike the original method, the dual 1 non-orthogonalized PLS1,” J. Chemo, 23, 263-264, 2009. optimization experiment does not require fine-tuning for the molecule’s JCC cou- pling constant values. Even more usefully, the dual-optimized version provides up 210 Classification Using Fused Data: Selectivity Enhancement and to two-fold improvement in signal-to-noise for some long-range correlations. Using Simplification Using a Hierarchical Approach modern, cryogenically-cooled probes, the experiment can be successfully applied Michael Dotlich, Eli Lilly, One Lilly Corporate Center, Indianapolis, IN to samples as small as 1 mg. Besides strychnine, we provide examples of applica- 46285, Bob Roginski, Jeremy Shaver tions of the method to known challenging molecules such as staurosporine and Fusion of multivariate data sets can aid in meeting modeling objectives that would coniothyrione. not be possible with the sets individually. In an effort to provide a rapid classifica- tion procedure to differentiate between solid pharmaceutical ingredients, Raman 214 Dynamic NMR Characterization of the Fluxional Rearrangement at spectroscopy was combined with reflectance color (RGB) measurements. In order an Eight-Coordinate Metal Center to successfully provide discrimination between each of the classes, a hierarchical Heather Siebert, Monmouth University, 400 Cedar Ave., West Long approach was successful where a “brute force” strategy fell short. Along the way, Branch, NJ 07764, Harpreet Sodhi, Gregory A. Moehring characteristics of the data were brought to light which greatly simplified the hierar- Eight-coordinate rhenium(V) polyhydride centers adopt a dodecahedral coordina- chical model by significantly flattening it. tion geometry with anionic ligands occupying A coordination sites and neutral li- gands occupying the less crowded B coordination sites. Such complexes undergo a pseudorotational rearrangement that changes the stereochemical relationships of all eight atoms bound to the rhenium center. Measurements of 1H and 31P at a variety of different temperatures on several different systems supported by only monodentate ligands have established a free energy of activation for such pseudo-

32 2013 EAS Abstracts November 2013

rotational rearrangements very near to 10 kcal/mol. More recent work on systems mable and can be reconfigured for a different set of molecules if the analysis needs that include a single bidentate ligand such as an ortho-metalated aldimine ligand of the end user change. indicates that the free energy of activation for pseudorotational rearrangements of these systems is considerably higher. This work describes the dynamic NMR deter- 218 Analysis of Produced Water Samples: A Comparison of Techniques mination of the activation energies for the pseudoratational rearrangement of eight- Helen M. Boylan, Westminster College, 319 S. Market St., New coordinate rhenium(V) polyhydride systems supported by a single bidentate ligand. Wilmington, PA 16172, Danielle Murtagh, Tyler Umstead At Westminster College, a small, undergraduate institution, we make good use of 215 Progress Towards a Mid-Infrared Noninvasive In-Vivo Glucose collaborations to extend our access to instrumentation and real-world samples from Sensor the natural gas industry. Over the past year, we have been collaborating with Pro- Sabbir Liakat, Princeton University, F310 G4 Equad, Olden St., ChemTech International, Inc., to improve a treatment process for produced water, Princeton, NJ 08544, Kevin A. Bors, Laura Xu, Claire F. Gmachl, Jessica in which sellable products are recovered from the water. As part of this collabora- Doyle tion, we must support the work through good analytical data. Trustworthy analyti- The realization of a clinically accurate, noninvasive, in-vivo glucose sensor would cal data is difficult to obtain with produced water due to the high concentrations of improve the quality of life of the more than 343 million diagnosed diabetics in the total dissolved solids (>100,000 ppm), chlorides (>50,000 ppm), sodium (>20,000 world by eliminating the necessity of drawing blood to monitor blood glucose levels. ppm), and calcium (>7,000 ppm), as well as many other substances. Our work has We have made significant progress towards such a sensor by utilizing fundamental focused primarily on barium and strontium, two signature components of produced vibrational resonances of the glucose molecule between 1000-1200 cm-1, which water from Marcellus Shale. At Westminster, we analyze for barium and strontium in yield stronger absorption cross sections than overtone absorption or Raman scat- the water samples using flame atomic absorption spectroscopy (FAAS) and in solid tering. Previously, we showed that backscattered mid-infrared light can be detected samples (precipitates from the treatment process) using laser-induced breakdown from the skin’s dermis which is important given that the glucose content in dermal spectroscopy (LIBS). In order to confirm the in-house analyses, water samples and interstitial fluid correlates well to blood glucose. Furthermore, we demonstrated that filtrates were analyzed with inductively coupled plasma-atomic emission spectros- physiological glucose concentrations could be detected to clinical accuracy in-vitro. copy (ICP-AES) at a neighboring university. We also verified our LIBS analyses Here we report recent results involving the prediction of glucose to near clinical using a different LIBS system with higher resolution and a broader spectral range. accuracy using human in-vivo spectra. In-vivo spectra of backscattered light from Based on our research, we have developed strategies to overcome the calcium and human forearms recorded between 1060 and 1090 cm-1 (a prominent glucose ab- ionization interferences experienced with our in-house FAAS analysis. We also view sorption feature centers around 1075 cm-1) show differences in absorption depth chemometrics as a tool to improve our in-house LIBS testing. and width between low (recorded on an empty stomach) and high (after jellybean consumption) concentrations of glucose. Next, we obtained datasets for multiple 219 Measurement of Molecular Structure with Low Frequency Raman subjects and quantified the concentrations their spectra represented using a com- Spectroscopy mercially available glucose monitor and conducted quantitative analysis using par- James T. Carriere, Ondax, 850 E. Duarte Rd., Monrovia, CA 91016, tial least squares regression. Using the optimal calibration set, clinically accurate Randy A. Heyler, Frank Havermeyer predictions were achieved approximately 75% of the time. We are currently working Raman and Terahertz spectroscopy are both widely used for their ability to safely on improving both our optical setup and data acquisition in order to build on these and remotely identify unknown materials. Traditional Raman spectroscopy typically encouraging results. This work is supported by the Wendy and Eric Schmidt founda- measures molecular energy transitions in the 200-5000 cm-1 region correspond- tion and MIRTHE NSF-ERC. ing to sub-molecular stretching or bending transitions, while Terahertz spectroscopy measures molecular energy transitions in the 1-200 cm-1 region (30GHz-6THz) that 216 Cell Culture Media Identification with Handheld 1064 Nanometer correspond to low energy libration modes or vibrational modes of the entire molecule Raman as well as lattice modes. Alternatively, low-frequency or “THz-Raman” spectroscopy, Claire Dentinger, Rigaku Raman Technologies, 1043 Serpentine Ln., which covers the ~5 cm-1 to 200 cm-1 (150GHz-6 THz) regions and beyond, of- Unit H, Pleasanton, CA 94566, Mark Mabry, Alyssa Knightly fers a powerful, compact and economical alternative to probe these low energy The quality of cell culture media used for the growth of cells plays an important transitions. These low energy transitions can be several times larger than typical role in the quality and efficiency of many biopharmaceutical production processes. fingerprint transitions with diverse enough signals to become a second fingerprint A non-destructive, reliable, efficient and cost effective method to analyze complex region. Combining the information from both the low frequency and “fingerprint” re- cell culture media is extremely important to ensure high quality process control. gions of the spectrum can be quite powerful in identifying polymorphic materials that Handheld Raman spectroscopy can meet this need at the point of use. It is non- have very similar chemical structure but different physical structure. This is critically destructive, fast, requires little to no sample preparation and provides highly detailed important in the manufacturing process of many active pharmaceutical ingredients chemical information about a variety of samples. Many pharmaceutical companies (APIs) where bioavailability will be affected by the form of the material. We present have adopted Raman spectroscopy as an effective and efficient technique for raw results from a new approach for extending the range of Raman spectroscopy into material identification, and other uses. The majority of handheld Raman spectrom- the Terahertz regime using an ultra-narrow-band volume holographic grating based eters in the field today use 785 nm lasers. Fluorescence from cell culture media is a notch filter system. An integrated, compact Raman system is demonstrated utilizing common problem associated with Raman analyzers using 785 nm (or lower) excita- a single stage spectrometer to show both Stokes and anti-Stokes measurements tion wavelengths. Present fluorescence can significantly interfere with successful down to <10cm-1 on various compounds containing aromatic ring structures and analysis by Raman spectroscopy. Fluorescence can be reduced or eliminated via polymorphic API compounds. the purposeful selection of a Raman spectrometer’s excitation laser’s wavelength. A number of different cell culture media are analyzed with Rigaku Raman FirstGuard 220 Development of a Sensitive Analytical Method to Characterize Spectrometers using both 785 nm and 1064 nm laser excitations. The 785 nm spec- Polymer and API Release from Solid Dispersion Formulations - tra show significant fluorescence, making it difficult to obtain specific information Linking Bioavailability to In-Vitro Dissolution about the sample. In contrast, the 1064 nm spectra show clear Raman peaks that Jagdish A. Prajapati, Merck, 556 Morris Ave., Summit, NJ 07901, Zheng can be used to reliably distinguish even closely related media. Zhao, Joshua Hoerner Solid dispersion formulations can enhance drug bioavailability by significantly in- 217 Reprogrammable Multi-Species Gas Monitors Based on Fourier creasing the solubility and dissolution of Biopharmaceutics Classification System II Transform Millimeter-Wave Spectroscopy and IV compounds. The polymers used in solid dispersion formulations often play Justin L. Neill, BrightSpec, 770 Harris St., Ste. 104B, Charlottesville, a critical role in drug release. While traditional analytical techniques rely simply on VA 22911, Matthew T. Muckle, Robin L. Pulliam, Brent J. Harris, Brooks quantifying API release rates only, probing the relationship between polymer re- H. Pate lease and active pharmaceutical ingredient (API) release may provide a more com- BrightSpec is introducing a versatile multi-species gas sensor based on the prin- prehensive understanding of formulation bioperformance. Two major challenges ciples of Fourier Transform millimeter-wave (FT-mmW) spectroscopy. This instru- in quantifying (or measuring) polymer release are the lack of chromophores on ment can quantify trace levels of volatile organic compounds in a complex mixture most polymers and low method sensitivity. Here we present the development of without the need for chromatographic separation. A linear response is demonstrated an innovative method to solve these challenges. Reversed-phase chromatography over at least four orders of magnitude, and no instrument calibration is required. coupling UV detection with a universal detector such as evaporative light scatter- Because of the ultrahigh resolution and specificity of the submillimeter molecular ing (ELS) or charged aerosol detector (CAD) was developed as a versatile, sensi- spectrum, the probability for erroneous detections is extremely low even at trace tive, efficient, and general procedure for polymer quantitation. The reverse-phase levels or in complex mixtures. A single instrument can be programmed to monitor chromatographic method uses either a polymer PLRP-S column or a polymer X several volatiles simultaneously with ppb sensitivity, with typical analysis times of a column, both of which are inherently hydrophobic and contain no bonded phase, al- few seconds to minutes. Additionally, BrightSpec spectrometers are fully reprogram- lowing highly reproducible separations. The on-line use of multiple detection modes

33 2013 EAS Abstracts November 2013

provides simultaneous synergistic information on the active and polymer release 224 Super Saturation and Precipitation Behavior of Ionizable Drugs in in both conventional and biorelevant dissolution media. This information has the the Presence of Cellulose Polymers potential to be used to better understand in-vitro in-vivo relationships and to help Jon Mole, Sirius Analytical, 100 Cummings Ctr. 231C, Beverly, MA drive development of better and more robust formulations. In addition the specificity 01915, Karl J. Box, Robert Taylor, John Comer, Rebeca Ruiz of the polymer separation could be applied to determine and quantitate the polymer The aim of this study was to evaluate the supersaturation and precipitation behavior used in marketed products. of poorly soluble ionizable compounds in the presence of cellulose polymers. The CheqSol solubility method was used to monitor the crystallization tendency during 221 Development of a Discriminatory Dissolution Method for a BCS precipitation of the free acid or base form of the compound. CheqSol uses prin- Class II Tablet Formulation Containing a Basic Active Ingredient ciples of mass and charge balance to determine the solution concentration of the Himanshu Gandhi, Bristol-Myers Squibb, One Squibb Dr., New neutral species. Seven compounds were studied: piroxicam, metoclopramide, rosi- Brunswick, NJ 08903, Peter Tattersall, Amy Bu, Huang Zongyun glitazone, paliperidone, dipyridamole, aripiprazole and tolbutamide. Measurements A dissolution method was required for an amine salt of a Biopharmaceutics Clas- were made in the presence of hydroxypropylcellulose and hydroxypropylmethylcel- sification System (BCS) class II active pharmaceutical ingredient (API) that is more lulose to investigate the influence on solubility and precipitation behavior. At 1:1 soluble at low pHs. Dissolution methods developed for monitoring previous clinical cellulose:drug ratio, rosiglitazone precipitated in an amorphous form (solubility formulations (dissolution media of 0.1N HCl) provided rapid dissolution and inad- 110-µm), which was more soluble than the crystalline form found without cellulose equate discriminating capability for the commercial formulation. Media pHs ranging (5.5-µm). The amorphous form endured for approx. 30 mins. The solubility of dipyri- from pH 4 to pH 5 provided a promising dissolution profile; however, small changes damole, tolbutamide, aripiprazole and paliperidone only marginally increased with in media pH provided significant changes to the dissolution profile. As a result, con- cellulose, and crystallization could not be prevented. A high solubility form of meto- cerns regarding potential method robustness at these intermediate pHs prompted clopramide was achieved with cellulose and endured for longer time periods with in- the choice of a pH 6.8 media. However, lack of sink conditions necessitated the creasing cellulose content. A slow conversion to a low solubility was then observed. use of a surfactant. Experiments investigating several different surfactants revealed Peak supersaturation levels of piroxicam were proportional to polymer content; Klu- that several surfactants we had previously utilized for other projects did not pro- cel achieved a 5-fold increase at 1:1 cellulose:drug ratio and a 4-fold increase at vide appropriate dissolution profiles. Fortunately the use of 0.75% Brij-35 surfac- 1:4. Pharmacoat also performed well with a 3-fold increase at 1:1 cellulose:drug tant provided the required method robustness and discriminatory capability. In this ratio and a 2-fold increase at 1:4. Klucel and Pharmacoat cellulose polymers were presentation, we describe our approach to method development studies for this proven to be efficient in increasing the extent and/or duration of supersaturation of product, highlighting the challenges we encountered with media pH and surfactant several of the studied compounds. selection. We also briefly discuss experiments performed to justify other method pa- rameters such as agitation speed, apparatus type, and media ionic strength. Finally, 225 Withdrawn by the author. we demonstrate the capability of the method to discriminate between changes in composition, manufacturing process, and stability for the commercial tablet formula- tion. It is worth noting that these results have been reviewed by the Food and Drug 226 Spectrofluorimetric Determination of Two Pharmaceutical Mixtures Administration, and the method was deemed acceptable for use in our long-term Containing the Antihypertensive Drug Amlodipine Besylate stability studies for the product. Together with either Candesartan Cilexetil or Telmisartan Mona M. Khamis, University of Alexandria, Pharmaceutical Chemistry 222 Monitoring the Hydrolysis of Aspirin During the Dissolution Testing Department, Alexandria 21521, Egypt, Hoda G. Daabees, Mohamed S. of Aspirin by In-Situ IR Spectroscopy Mahrous, Magdi M. Abdel-Khalek, Tarek S. Belal Vrushali Bhawtankar, Seton Hall University, 2216 Village Dr., Avenel, Combination regimens serve as an effective treatment strategy to provide adequate NJ 07001 control of hypertension that is not satisfactorily controlled using monotherapy. Am- Dissolution test is the qualitative study for measuring the performance of a drug lodipine (AML), a dihydropyridine calcium channel blocker, is available in fixed product. In the past few years, the importance of the dissolution test has increased. dose combination tablets with angiotensin II receptor blockers as; candesartan ci- Using in-situ attenuated total reflection Fourier transform infrared spectroscopy lexetil (CAN) or telmisartan (TEL). This work describes a direct, simple, sensitive (ATR/FTIR) spectroscopy we developed a methodology of analyzing and monitoring and reliable spectrofluorimetric method for the analysis of AML/CAN and AML/TEL dissolutions of pharmaceutical active pharmaceutical ingredients. The accuracy of combinations without prior separation. The method involves measurement of the this technique was found to be ± 3% relative to high-performance liquid chromatog- native fluorescence of AML at excitation and emission wavelengths 367 and 452 raphy and ultraviolet. In fact, we also performed the hydrolysis of aspirin and study- nm, respectively in water without any interference from either drug. On the other ing the chemistry behind the dissolution by react infrared (IR). We are monitoring the hand, the intrinsic fluorescence of CAN was measured at excitation and emission hydrolysis while doing the dissolution of aspirin by in-situ IR spectroscopy. We are wavelengths; 265 and 392 nm, respectively in ethanol, while TEL was measured performing this study by using different body pHs, such as simulated gastric fluid, at 366 nm in 0.05 M sodium hydroxide solution using 294 nm as excitation wave- sodium acetate buffer and potassium phosphate buffer. With this we will study the length. The proposed spectrofluorimetric procedure was validated with respect to kinetics of this dissolution and hydrolysis. linearity, ranges, precision, accuracy, selectivity and detection and quantification limits. Regression analysis showed good correlation between relative fluorescence 223 The Effect of In-Situ pH Change on a Biphasic Dissolution Model in intensity and concentration over the ranges 0.1-1.4, 0.025-0.25 and 0.0025-0.05 μg/ the Presence of Simulated Intestinal Fluids mL for AML, CAN and TEL, respectively. The limits of detection were 0.0338, 0.0063 Jon Mole, Sirius Analytical, 100 Cummings Ctr. 231C, Beverly, MA and 0.0007 μg/mL for AML, CAN and TEL, respectively. The proposed method was 01915, Karl J. Box, Robert Taylor, John Comer successfully applied for the assay of both fixed dose combinations AML/CAN and A “biphasic dissolution model” has been developed to mimic the absorption of drugs AML/TEL in laboratory prepared tablets with good recoveries. No interference was under gastrointestinal pH conditions. This has been further developed to include observed from common pharmaceutical additives. The results were favorably com- simulated intestinal fluids (SIF) to understand their effect on the uptake of orally pared with those obtained by reported methods for the assay of these drug mixtures. dosed drugs. A compact of dipyridamole (approx 5 mg), was introduced into a stirred 45 mL chamber at pH2 to represent stomach conditions. Dissolution was recorded 227 Development and Validation of an Oven Karl Fischer Titration for for 30 minutes using a fibre-optic probe and UV spectrometer. A quantity of SIF was Testing Water Content in Thiohydrazide Derivative Pharmaceutical then added, followed by 20 mL of nonanol, during automated addition of potassium Active Ingredient hydroxide, to change the pH to pH4. This was thought to mimic the gastric empty- Chumang Zhao, Synta Pharmaceuticals, 45 Hartwell Ave., Lexington, ing process and enable partitioning of the drug into a pseudo lipid layer. This was MA 02421, Jinghua Lu, Jianhua Shen, Suresh Babu monitored by a second fibre-optic probe. Further pH adjustments were set to mimic Karl Fischer titration (KFT) is the most commonly used analytical testing technique transition through the intestinal tract. Under standard GI dissolution conditions (in- in pharmaceutical industry to determine water content in active pharmaceutical in- situ pH change, but no SIF or lipid), dipyridamole precipitated in the final pH period gredients (API), drug products and excipients. In most cases, water content in API (pH 7.4). The addition of SIF prevented precipitation at pH 7.4, as did the addition of must be determined to assess the assay on an anhydrous basis. Water content, lipid, which resulted in a final partitioned mass of 3 mg in the lipid. As expected, the in drug products, especially in sterile drug products, also needs to be determined, addition of both SIF and lipid prevented precipitation. However, the final partitioned because presence of residual water could promote drug product degradation and/ mass was down to 2 mg. The addition of SIF helped to maintain a solubilized state or microbial growth, which has a direct impact on the stability and shelf-life of drug of the drug. However the SIF also hindered the uptake of the drug into the pseudo product, and overall safety of patients. A case study is presented on determination lipid layer. This indicated that natural bile components may further inhibit the bio- of water content in Compound E, a thiohydrazide derivative API, and the associated availability of some drugs. challenges that needed to overcome. Direct Coulometric KFT (cKFT) for the API was found to yield inconsistent, highly variable water content results among differ- ent batches of the API and among different stability time points of the same batch 34 2013 EAS Abstracts November 2013

of API. Keeping the fundamental principle of KFT by Bunsen reaction, a hypothesis PIVKAII. AFP, PIVKAII and PIVKA+AFP of sensitivity are 78.6%, 84.7, 83.7%. We was developed for the potential root cause, and the underlying mechanism was conclude that the specifics are 83.9%, 88.1%, 93.5%. Combining the information elucidated by high-performance liquid chromatography mass spectrometry study. from AFP and PIVKAII increases the sensitivity and specificity over AFP alone and Thus, an optimized oven-cKFT method had to be developed based on the solid could be useful in early diagnosis of HCC. phase characterization and thermo studies of the API. The oven-cKFT method was finally developed, validated and implemented to generate consistent and accurate 231 Comparison Between Theoretical and Experimental Retention water content data. Times of Prostanoids and Sex Hormones Guoxiu Wei, Temple University, 1901 N. 13th St., Philadelphia, PA 228 Approaching USP Method 233 - Results of Sample Preparation and 19122, Susan Jansen-Varnum Analysis of Several Pharmaceutical Sample Types Prostaglandins (PGs) and sex hormones and their metabolites are very important Jason Keith, CEM Corporation, 3100 Smith Farm Rd., Matthews, NC biomarkers in many diseases, including hypertension, inflammatory conditions, and 28106, Daniel Iversen, Elaine Hasty, Tina Restivo, Ivana Mrvalj breast cancer. The prevalence of some of these diseases varies between the gen- The current United States Pharmacopeia (USP) Method 231 is to be replaced with ders. Female sex hormones have been suggested to play a protective role against USP Methods 232 and 233. Conformance to USP Methods 232 and 233 will be re- hypertension in women. PGs, which are inflammation mediators, are also involved quired on May 1, 2014 as part of the General Notices revision that appears in USP in the pathogenesis of disease. By separating and quantifying the various PGs and 37–NF 32. During the process of developing the new methods, several changes sex hormones from urine or tissue samples, it is possible to explore the balance were made to the sample preparation and sample analysis procedures, as well between the effects of inflammation mediators and the self-protection effects of as the limits of accuracy and precision. The official USP Method 233 requires that sex hormones in disease sufferers. Our studies are aimed at building a theoretical samples are completely dissolved prior to analysis. The sample can be directly dis- model to predict the retention times of prostanoids and sex hormones with com- solved in water or organic solvent or the sample can be dissolved by closed vessel putational software. The theoretical results are compared with the experimental microwave digestion. The dissolved samples are then analyzed with an inductively results from the separation of prostanoids and sex hormones in a C18 symmetry coupled plasma (ICP) optical emission spectrometry or ICP mass spectrometry column with high-performance liquid chromatography. We examine the effect of the (MS). A study was completed to compare accuracy and precision of several phar- chemical structures of prostanoids and sex hormones on their retention times and maceutical sample types. The samples were spiked with a part-per-billion solution polarities. Our results help predict separation measurements before performing real of volatile and non-volatile metals and were prepared by closed vessel microwave experiments, which will help to save time, decrease chemical consumption, and digestion. All samples were analyzed with an ICP-MS to compare accuracy and lower research costs. precision. The spike recovery results are discussed. 232 An LC-MS-MS Method for Quantitation of Hydrocodone and 229 Pressure Decay Pneumatic Integrity Testing of Medical Device Hydromorphone in Rat Plasma Tubing Components Yuexin Su, Teva Pharmaceuticals, 145 Brandywine Pkwy., West Peter J. Oehlberg, Bracco Research USA, 305 College Road East, Chester, PA 19380, Steve Gorman Princeton, NJ 08540, Irene K. Ropiak, Julius P. Zodda Hydrocodone is a semisynthetic opioid analgesic and antitussive that is widely Leak integrity is a critically important parameter to both the supplier and end-user of used for relief of moderate to moderately severe pain. Teva Pharmaceuticals is de- tubing components. A method for the pneumatic integrity testing of tubing compo- veloping a new extended-release formulation for various pain indications. A liquid nents was developed and validated using a CE certified instrument. The test method chromatography-tandem mass spectrometric (LC-MS-MS) analytical method was utilizes pressure decay with fixed time (60 second held at @ 50 psi) with acceptance developed and validated to support the safety studies of hydrocodone in rats. The criteria of ≤ ∆0.2 psig for each component. Due to the strict tolerance of the pres- rat plasma samples (0.1 mL) were prepared using a 96-well SLE+ plate to extract sure decay test, a method was established to consistently apply the tubing luer to the analytes from the matrix. Stable isotope-labeled analogues of the analytes the tester to alleviate false positive results. The tubing components, intended for were used as internal standards (IS). Samples were reconstituted and analyzed use in medical devices, were manufactured and tested per guidelines in Interna- by LC-MS-MS in positive mode. The method was thoroughly characterized during tional Standards Organization (ISO) 594/1 and ISO 8536, respectively. A torque the validation. The results show the linear range of the method was 0.300-30.0 ng/ limiting driver was adapted to fit the luer and apply the connection to the tester at mL for both hydrocodone and hydromorphone with a correlation coefficient > 0.99. 15 in/lb. The parts (n=90) affixed to the pneumatic integrity tester using the torque The method validation was conducted using quality control samples prepared at 4 driver technique demonstrated > 50% improvement in the pressure decay results concentrations covering each calibration range. The overall intra-day and inter-day over multiple-operator hand tightening. A system suitability test for the pneumatic assay precision (coefficient of variation) was <15% of nominal values and assay integrity tester was developed and validated to ensure proper functionality of the in- bias values were within 15%. The absolute recoveries were 93.0% and 90.4% for strument prior to initiating testing. Two leak rate standards (0.058 and 0.174 scc/m), hydrocodone and hydromorphone, respectively. The overall normalized matrix ef- manufactured by an A2LA accredited and ISO 17025 certified lab with calibration fect factors were 1.02 for both analytes. The analytes are stable in rat plasma for traceable to National Institute of Standards and Technology, were implemented to at least 25 days at -86 ºC. The rat plasma extracts are stable for 39 hours in an bracket the test parameters used during integrity testing. Unlike dye testing, the autosampler at 5 ºC, while the working solutions are stable for 9 day at -20 ºC. The method provides a non-destructive, rapid, sensitive and accurate means of detect- conclusion is this method has been shown to be accurate and reproducible for the ing pinhole tubing leaks or bonding failures in tubing components making it appli- concurrent quantitation of hydrocodone and hydromorphone in rat plasma. cable as a manufacturing in-process, or release test. 233 Rapid Determination of Polysorbate 80 Content in Therapeutic 230 Association Study of Serum Biomarkers (Alpha-fetoprotein and Monoclonal Antibody Products by a Sensitive Fluorescence-Based PIVKA-II) for Prognosis of Hepatocellular Carcinoma Method Mi-Ran Kim, Asan Medical Center, 88 Olympic-ro 43-Gil, Songpa-gu, Songyan Zheng, Bristol-Myers Squibb, One Squibb Dr., New Brunswick, Seoul 138-736, Korea NJ 08903, Pedro Smith, Monica Adams Hepatocellular carcinoma(HCC) is a major health problem worldwide, with more A sensitive and effective method has been developed for the rapid determination than 500,000 cases diagnosed annually. The increasing incidence of HCC is com- of polysorbate 80 content in therapeutic monoclonal antibody (mAb) products of pounded by the fact that tumors are diagnosed at a late stage when curative treat- the IgG1 subtype. The method is based on the detection of the fluorescence emis- ments are not possible. Therefore, surveillance of the at risk population could aid sion of 4,4`-dianilino-1,1`-binaphthyl-5,5`-disulfonic acid dipotassium salt (Bis-ANS) earlier detection of the disease and decrease the mortality rate. Present success is which is enhanced in the presence of polysorbate 80. Two approaches have been limited by the lack of sensitive biomarkers. The most widely used serum HCC mark- co-developed to determine the amount of polysorbate 80 in unknown samples; one er is alpha-fetoprotein (AFP), while PIVKA-II has been proposed as new biomark- method calls for removal of the mAb and the other for simply diluting the samples ers. We showed that the combination of AFP and PIVKAII is more valuable than AFP prior to measurements. In the presence or absence of mAb, linearity of the polysor- alone. This research has been adopted as standard surveillance in risk population bate 80 curve was comparable. The limits of detection and quantitation calculated of HCC. Serum PIVKAII and AFP levels were collected in 354 patients with liver from the calibration curve generated in the absence of mAb-A were 1.5 ppm and 4.5 disease and 196 patients with HCC. All of the HCC patients were diagnosed by fine ppm, respectively. Given the comparable linearity between the two conditions (i.e., needle biopsy under the guidance of ultrasonography and surgery. The serum AFP with or without mAb), the limit of detection and quantitation is assumed to be similar. was measured using routine automated method in Chemiluminescent Microparticle The diluting sample method is not only simple in terms of sample preparation but it is Immunoassay (ARCHITECT i2000SR, Abbott). The serum PIVKAII was measured also particularly suitable for analyzing the level of polysorbate 80 contained in highly by the sensitivity enzyme immunoassay (LUMIPULSE G1200, Japan).The cutoff concentrated mAb products. To this end, the method was utilized to determine the value for the AFP and PIVKAII was set at 20 ng/ml, 40 mAU/ml according to the concentrations of polysorbate 80 at 75, 200, and 250 ppm in a mAb product of 250 manufacturer’s instruction. No significant correlation was found between AFP and mg/ml. On the other hand, when the availability of mAb for analytical purposes is

35 Call for Papers ANNOUNCING NH March 1–April 15, 2014 2 2014 Eastern Analytical Symposium & Exposition HO EAS seeks contributed abstracts in Garden State Exhibit Center | Somerset, NJ these and other analytical fields: November 17–19, 2014  Bioanalysis  Ligand Binding Assays  Size Exclusion O Chromatography HO  Capillary  LC/MS, GC/MS Electrophoresis  Solid State Analysis HO  Microchemistry  Chemometrics  Space Analytics  Microscopy  Conservation Science  Supercritical Fluid  Nanoparticles HO O Chromatography  Environmental Analysis  Near-Infrared (NIR)  Surface Science  ESR Spectroscopy Spectroscopy  Very High-Pressure LC/ O  Food Analysis  NMR Spectroscopy The Art and ScienceHO of Analysis Ultra High-Pressure LC  Forensic Analysis  Pharmaceutical Cl  Vibrational Analysis O  Gas Chromatography Spectroscopy  Process Analytical  HPLC  Three-day technical program Science  ICP/MS O O Cl  Protein Analysis  Immunochemistry  State-of-the-art exposition featuring analytical  Quality-by-design  Industrial Hygiene equipment and services H  Quality/Regulatory/ N N O  Ion ChromatographyOH Compliance  Extensive selectionHN of short Hcourses and  IR Spectroscopy  Raman Spectroscopy professional development CO workshops H Laboratory Automation  Sample Preparation 2H N  Laboratory O Science Education  NManagement Employment bureau, and more O H  Sensors EAS.org  Laboratory O Miniaturization N O NHMe HO H OH NH Contact us by email at OH 2 [email protected] or call (732) 449-2280 in early 2014 for submission information.

13348-EAS_11x17_MagSpread_PREP.indd 1 8/29/13 3:15 PM Call for Papers ANNOUNCING NH March 1–April 15, 2014 2 2014 Eastern Analytical Symposium & Exposition HO EAS seeks contributed abstracts in Garden State Exhibit Center | Somerset, NJ these and other analytical fields: November 17–19, 2014  Bioanalysis  Ligand Binding Assays  Size Exclusion O Chromatography HO  Capillary  LC/MS, GC/MS Electrophoresis  Solid State Analysis HO  Microchemistry  Chemometrics  Space Analytics  Microscopy  Conservation Science  Supercritical Fluid  Nanoparticles HO O Chromatography  Environmental Analysis  Near-Infrared (NIR)  Surface Science  ESR Spectroscopy Spectroscopy  Very High-Pressure LC/ O  Food Analysis  NMR Spectroscopy The Art and ScienceHO of Analysis Ultra High-Pressure LC  Forensic Analysis  Pharmaceutical Cl  Vibrational Analysis O  Gas Chromatography Spectroscopy  Process Analytical  HPLC  Three-day technical program Science  ICP/MS O O Cl  Protein Analysis  Immunochemistry  State-of-the-art exposition featuring analytical  Quality-by-design  Industrial Hygiene equipment and services H  Quality/Regulatory/ N N O  Ion ChromatographyOH Compliance  Extensive selectionHN of short Hcourses and  IR Spectroscopy  Raman Spectroscopy professional development CO workshops H Laboratory Automation  Sample Preparation 2H N  Laboratory O Science Education  NManagement Employment bureau, and more O H  Sensors EAS.org  Laboratory O Miniaturization N O NHMe HO H OH NH Contact us by email at OH 2 [email protected] or call (732) 449-2280 in early 2014 for submission information.

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EAS-2013_Ad.indd 1 9/12/13 12:54 PM 2013 EAS Abstracts November 2013

limited, the method of removal of the mAb may be practically applied to samples chloride salt. This rapid and effective determination is a key element in assessing a containing mAb by first removing the mAb from the analyte and utilize polysorbate salt’s moisture content and suitability for use during biologics manufacturing. Cali- 80 standards prepared in a protein-free matrix such as PBS. bration standards (0.1 – 0.8% wt/wt) with different moisture levels were prepared us- ing water and different saturated salt solutions (sodium chloride, potassium chloride, 234 A Novel Approach to Greener and Healthier Medical Chemicals and potassium nitrate) Two different pre-process treatments were used: 1) S. Golay Purifications with 1st derivative, 9-point smoothing, 2nd polynomial fit; 2) baseline offset based Matthew Przybyciel, ES Industries, 701 South Rt. 73, West Berlin, NJ on the validation results, the best model was chosen to determine the water levels 08091, Dave Kohler, Joe Stefkovic, Xenosep, Rolf Schlake, Martin in sodium chloride salt. Fetner Dichloromethane (DCM) is both the lifeblood and main chemical waste of many 238 Screening Herbal Supplements Using Statistical Modeling to Find pharmaceutical medicinal chemistry laboratories employing flash chromatography. and Identify Adulterants and Contaminants Using Direct Analysis The solvating power of DCM for many organic compounds when combined with its in Real-Time (DART)-MS low boiling point make it an ideal non-polar solvent for this application. However, Robert Goguen, IonSense, 999 Broadway, Ste. 404, Saugus, MA DCM is not without its health risks to the scientist or specialized solvent waste con- 01906, Brian Musselman, Joseph LaPointe cerns. This paper discusses a uniquely novel approach utilizing carbon dioxide to The Food and Drug Administration Food Safety Modernization Act includes provi- completely eliminate and/or at the very least, greatly reduce the amount of DCM and sions for monitoring herbal supplement composition, mandating testing to ensure other non-polar solvents currently employed by pharmaceutical medicinal chemistry the presence and quality of active herbal ingredients while monitoring for poten- laboratories worldwide. tial contaminants and adulterants. Due to the active nature of substances involved and potential variability in formulation, batch-to-batch monitoring is desirable. Direct 235 Improving Peptide Mapping of a Therapeutic Monoclonal Antibody analysis in real-time mass spectroscopy (DART-MS) enables a fast, inexpensive (mAb) and Considerations for Enhancing Analysis analysis and measurement of the substance of interest. A program designed to com- James Martosella, Agilent Technologies, 2850 Centerville Rd., plete automated statistical analysis and comparison of samples against a custom Wilmington, DE 19808, Alex Zhu, Ning Tang model to identify the presence of the substance of interest, as well as the absence Peptide mapping by reversed-phase liquid chromatography (RPLC) is a common of contaminants and/or adulterants is described. A set of commercial supplements analytical technique for characterizing biologic therapeutic proteins and the combi- (Black Cohosh) from capsules was prepared for analysis. To simulate contaminants, nation of RPLC with electrospray ionization mass spectrometry (ESI-MS) provides pesticide was added at several different concentration levels. The samples were a powerful approach for developing effective peptide mapping protocols in the then analyzed using DART-MS. Analyses of different aliquots of sample at increas- biopharmaceutical industry. However, developing efficient peptide mapping separa- ing desorption gas temperatures. Resulting spectra were collected, and used to cre- tions requires the integration of key analysis parameters such as gradient length, ate a statistical model of spectra for both the supplement and supplement containing time and shape, LC eluent composition and MS optimization. In this study, peptide known contaminants. Subsequent tests were run to determine the effectiveness of mapping parameters of an IgG(1) therapeutic monoclonal antibody (mAb) were in- the model. vestigated using a novel superficially porous peptide mapping column coupled to an Agilent 6530 quadrupole time-of-flight. The unique resolving power of the peptide 239 Withdrawn by the author. column to maximize resolution and efficiency in combination with optimized LC-MS operating conditions provided efficient mapping and sensitive identification of pep- tides resulting in high sequence coverage. Additionally, optimization of the IgG(1) 240 Overcoming Challenges in Particle Size Measurement by Laser peptide gradients and LC-MS parameters is explored for achieving rapid and repro- Diffraction Technique ducible mAb peptide maps. Kalyan Potluri, Almac, 20 Seagoe Industrial Estate, Co Armagh BT63 5QD, United Kingdom, Noel Hamill, Linda McCausland, Catherine 236 Rapid Counter Ion/Residual Ion Analysis using LC-MS McClorey Holly M. Shackman, Bristol-Myers Squibb, One Squibb Dr., New The objective is to develop and validate a single method for particle size distribution Brunswick, NJ 08903, Mark S. Bolgar, Xujin Lu assessment for both API and drug product for a drug with particle size distribution For small ion determinations, common choices for analysis are ion chromatography as a critical quality attribute. The wet dispersion method development used focused (IC), gas chromatography, titrations, and charged aerosol detection. However liquid on instrument set-up parameters and conditions which could affect the accuracy, chromatography coupled to mass spectrometry (LC-MS) is an excellent and under- precision and robustness. The results showed the suitability of laser diffraction for utilized technique for this type of assay. The single quadrupole mass spectrometer particle size measurement was verified by successful correlation to cross sectional is ideal for small molecule analysis as it is easily qualified for GxP measurements, area by microscopy. Sample preparation, dispersant selection, sonication time and can be extremely sensitive, specific, couples easily to LC for rapid analysis, and is sampling techniques were all found to affect the quality and precision (% of relative capable of similar reproducibility and linearity as its more commonly used counter- standard deviation) of the data. A scientific approach for selection of these param- part, IC. First, a method to quantify the counter ion TRIS was developed and validat- eters was used, as opposed to merely focusing on a reduction in statistical variance. ed using hydrophilic interaction liquid chromatography (HILIC) MS. HILIC produced The method was successfully validated in compliance to International Conference the most Gaussian peak shape while retaining TRIS on column for separation from on Harmonization guidelines Q2 (R1) and United States Pharmacopeia <429>. In- other sample components. By employing isocratic elution at a flow rate of 1.5 mL/ creasing numbers of active pharmaceutical ingredients (API) are larger lipophilic min (split prior to entering the mass spectrometer) with single ion monitoring (SIM) molecules with poor solubility characteristics. Therefore, the impact of particle size for detection, a rapid and sensitive method was created. The method gave excel- on bioavailability is increasingly important and has become a critical quality attribute lent repeatability and good linearity, with relative standard deviation =0.3% and R2 for many new drugs. Pitfalls in development of laser diffraction methods include: 1) = 0.998457. Second, a novel method to determine the amount of NaCl present lack of benchmarking of laser diffraction data to image analysis; 2) poor develop- in sample was developed and validated using reversed-phase LC-MS. By utilizing ment of product specific methods or an absence of re-validation at late phase; 3) isocratic elution on a short (5.0 cm) column coupled to polarity switching with SIM unrealistic specifications; 4) lack of robustness; 5) issues with method transfer from channels, sodium and chloride were completely separated and detected in less than sieving to laser diffraction or from obsolete to modern laser diffraction systems. one minute. The method achieved good linearity (R2 > 0.99) and repeatability (<3%) for both ions in addition to being extremely rapid while using conventional LC. 241 Statistical Evaluation using Design of Experiments of Total Organic Carbon (TOC) Analysis to Combine Cleaning Agent Methods with 237 Rapid Determination of Water Content in Sodium Chloride Salt Drug Product Methods Using Portable NIR Spectrometer Peter A. Walsh, Bristol-Myers Squibb, One Squibb Dr., New Brunswick, Ravi Kalyanaraman, Bristol-Myers Squibb, One Squibb Dr., New NJ 08903, Nikunj Vasoya, Mariann Neverovitch, Antonio Fernandez Brunswick, NJ 08903, Sibylle Herzer, Judith Punzalan, Abdelhady Total organic carbon (TOC), a non-discriminatory technique, is currently used to Elkhouga, Nisha Patel support the majority of cleaning verification submissions. However, we still employ Accurate buffer preparation is a simple yet quintessential step towards developing a method-specific, legacy approach of taking two separate swab samples from the robust manufacturing processes. Many of the salts used in preparing buffers for pu- same piece of equipment; one to test for drug product residue, and another to test rification of biologics are inherently hygroscopic. The accuracy of buffer preparation for detergent residue. Since TOC is a non-specific technique (and thus will detect becomes more questionable over time as the solid is exposed to more and more and measure all organic carbon in a sample) these two analyses can theoretically moisture. While ingress of moisture into salts is easily recognized by an increasing be combined into one. Design of experiment (DoE) was used to evaluate combining propensity to form clumps, it is difficult to measure them rapidly. The handheld near- TOC methods for drug product and detergent. Since drug products are analyzed via infrared (NIR) spectrometer (microPhazir from Thermo) is a rapid and non-destruc- TOC using different instrument parameter settings (diluent volume, diluent pH, acid, tive tool that is used to determine the water content (0.1% - 0.8% wt/wt) in sodium and oxidizer flow rate) than the detergents, DoEs were performed to assess the 39 2013 EAS Abstracts November 2013

impact these different parameter settings have on the detection of detergents. Ex- 246 Filter Filtration Suitability for Sample Preparation in Drug Assay perimental data indicated that the three parameters tested had no significant effect Testing on the detection of detergents and hence separate methods for drug product and William Long, Agilent Technologies, 2850 Centerville Rd., Wilmington, detergents are not required. This result allows us to reduce the number of swabs, DE 19808, Limian Zhao vials and reagents used and the turnaround rate for equipment significantly while Various filters were tested thoroughly for sample preparation in drug assay testing. still maintaining existing scientific rigor. The potential impact on drug binding through filtration was evaluated based on the analyte recovery. Five representative drugs were selected for this study, covering a 242 High Sensitivity Glycan Profiling in Antibody Drugs Using the RF- wide variety of chemical physical properties and drug binding propensity. All of tests 20Axs Fluorescence Detector were performed following United States Pharmacopoeia methods. The results dem- Kenichiro Tanaka, Shimadzu Scientific Instruments, 7102 Riverwood onstrate that, with appropriate selection of filtration membrane based on sample Dr., Columbia, MD 21045, William Hedgepeth, Ayako Nomura, Tadayuki medium and target analytes, filters filtration can provide excellent recovery in a wide Yamaguchi, Yuki Sato, Kenichiro Todoroki variety of compound structures and chemistries. Glycans in antibody drugs may play roles in the antigenicity, pharmacokinetics and high-order structural stability of drugs, which could adversely affect drug safety and 247 Automated Extraction of Phthalates in Consumer Products for effectiveness. It is therefore necessary to investigate which glycans are present in Analysis by GC-MS antibody pharmaceuticals. Non-uniformity of glycans due to the instability of culture Fredrick D. Foster, Gerstel, 701 Digital Dr., Ste. J, Linthicum, MD 21090, conditions used for antibody drugs is a concern which requires rigorous manage- John R. Stuff, Jacqueline A. Whitecavage, Edward A. Pfannkoch ment of the production process. However, while there is currently no glycan test The United States Consumer Product Safety Commission’s (CPSC) Test Method method specified in the United States Pharmacopoeia, there is wide demand for CPSC-CH-C1001-09.3, is used by testing laboratories for the analysis of phthalate such an assessment method. Here, using the core-shell column AerisTM PEPTIDE content in children’s toys and child care articles covered by the standard set forth in XB-C18 and the Nexera X2 ultra high-performance liquid chromatograph, we intro- the Consumer Product Safety Improvement Act Section 108. The CPSC determined duce an example of the analysis of antibody drug glycans. For detection, the world- that an appropriate combination of methods of extraction and analysis is sufficient leading sensitivity fluorescence detector RF-20AXS was used. to determine the concentration of the six regulated phthalates in most consumer products. The general manual approach is to dissolve the sample completely in 243 Withdrawn by the author. tetrahydrofuran, precipitate any PVC polymer with hexane, filter and then dilute the solution with cyclohexane, and analyze by gas chromatography-mass spectrometry (GC-MS). A single robotic X-Y-Z coordinate autosampler commonly used for sample 244 Overcoming USP Chapter <233> Challenges: Introducing a Novel introduction in GC or high-performance liquid chromatography (HPLC) can be used Microwave Combustion Sample Digestion Technique for Complex to perform a wide variety of sample preparation techniques using a single instru- Pharmaceutical Matrices ment and controlling software. Among the autosampler capabilities controlled by Reynhardt Klopper, Anton Paar USA, 10215 Timber Ridge Dr., Ashland, Maestro software are filtration and centrifugation, which can be used to generate VA 23005 clear filtrate from a solvent extraction of polymer. The sampler can be configured as For more than 100 years, the United States Pharmacopeia (USP) General Chapter part of a GC or LC system or can be configured as a benchtop workstation. In this <231> was the standard method for the determination of metal impurities present work, we demonstrate an automated extraction for phthalates in consumer products in pharmaceutical products and raw materials. Improvements in analytical equip- based on CPSC method CPSC-CH-C1001-09.3 coupled directly to the GC-MS to ment and techniques; however, have elucidated several known drawbacks of this streamline the entire extraction and analysis process as well as avoid exposure to technique. USP recently approved General Chapters <232> and <233> to replace potentially hazardous materials by laboratory personnel. General Chapter <231>. Although the closed-vessel sample preparation techniques proposed in Chapter <233> offer improved accuracy, sensitivity, and elemental 248 From Helium to Hydrogen: Case Study in GC-MS Analysis of VOCs specificity, the question of the broad applicability over a wide range of organic com- and SVOCs in Water pounds used in the pharmaceutical industry remains. For example, many modern Alexander Semyonov, Thermo Fisher Scientific, 2215 Grand Avenue drugs have enteric coatings that consist of complex synthetic polymers or bio-poly- Pkwy., Austin, TX 78728, Jessie Butler mers. Using the most aggressive acid mixtures in high-temperature and high-pres- The global helium shortage has caused more and more laboratories to migrate to sure environments, complete digestion of these complex components is not certain. hydrogen as a carrier gas. This transition is easy for the gas chromatography (GC) Incomplete sample digestions can lead to analyte under-recoveries and high blank methods utilizing flame ionization (FID), thermal conductivity (TCD) and electron values. In this study we present the efficacy and applicability of a novel microwave- capture (ECD) and other non-mass-selective detectors. However, for the GC- mass induced oxygen combustion sample digestion technique coupled with inductively spectrometry (MS) methods, especially complex and regulated ones, migration to coupled plasma mass spectrometry analysis for pharmaceutical products. A variety hydrogen carrier gas presents significant challenges. In addition to the changes of over-the-counter aspirin tablets containing complex enteric coatings were tested in chromatographic conditions of the run due to the physical property differences according to USP <232> and <233>. Employing the microwave-induced oxygen of hydrogen, its chemical reactivity brings about chemical reactions in the mass combustion digestion technique and complete decomposition of sample matrices, spectrometer’s ion source that do not occur with helium. The analysis of volatile and independent of their structure and functional groups, resulted in superior analyte semi-volatile organic compounds in drinking and waste water by Environmental Pro- recovery data and detection limits when compared to legacy sample preparation tection Agency Methods 8270, 524, and 525 involves identification and quantitation techniques and other closed-vessel digestion techniques. of more than 120 analytes of varying chemical structure, polarity and volatility. The diversity of the analytes in these methods as well as the target bromofluorobenzene 245 Quantitative Application of Cyclic Voltammetry to Superoxide or decafluorotriphenylphosphine tuning requirements presents particular challenges Radical Scavenging by Antioxidants when migrating from helium to hydrogen carrier. We present and compare data of Stuart L. Belli, Vassar College, PO Box 111, Poughkeepsie, NY 12604, fast GC-MS analyses of volatile organic compounds (VOCs) and semi-volatile or- Francesco Caruso, Casey Bartow-McKenney, Charles Caldwell, Grace ganic compounds performed on TRACE 1310 GC coupled to ISQ single quadrupole Lee, Miriam Rossi mass spectrometer utilizing helium and hydrogen carrier gases. Key modifications Cyclic voltammetry can be used to measure the radical-scavenging effectiveness to both GC and MS set up in hardware and methods necessary for successful mi- of antioxidant compounds, such as flavonoids, by generating the superoxide radical gration to hydrogen carrier gas are discussed and effect of each parameter on the ion, O2- in a solution containing the compounds to be studied. The forward sweep data quality is demonstrated. The final, optimized methods fully migrated to hydro- of cyclic voltammetry generates the superoxide radical ion from dissolved molecular gen carrier gas with improved peak shape, resolution, run time, and sensitivity are oxygen and then subsequently, the reverse sweep oxidizes any remaining super- presented. oxide anion back to molecular oxygen. The oxidation peak height is proportional to the amount of superoxide left in the diffusion layer of the electrode and so is a 249 Enhancing Efficiency of Generic Residual Solvent Methods in API measure of the amount of the superoxide radical ion remaining a short time after James Girotti, Genentech, 1 DNA Way, South San Francisco, CA 94080, its generation. The superoxide radical ion is stable over the time of the experiment C.J. Venkatramani, Larry Wigman, Nik Chetwyn in an anhydrous aprotic solvent, such as dimethyl sulfoxide (DMSO), containing Pharmaceutical industries around the world strive hard to ensure patient safety by no reactive species and when a compound is added to this solution, the loss of delivering drug substance and drug products of high quality on an on-going basis. superoxide between generation and measurement can be related to the compounds Several analytical tests are performed as per agency guidance to assess purity and radical scavenging properties. In this study we report the calibration of the method quality of these. Analysis by gas chromatography is one test performed to monitor/ using standardized KO2 solutions in DMSO and its application to the investigation limit residual solvents in active pharmaceutical ingredients (API). Multiple solvents of antioxidant properties of several flavonoids. are used in manufacturing of API and their levels must follow International Con- ference on Harmonization (ICH) guidelines. Typically, generic gas chromatography 40 2013 EAS Abstracts November 2013

methods are used to simultaneously quantify relevant process related solvents. with the change in the distribution constant of analyte for these columns. This shows These methods are robust, but usually take about 30 minutes for one sample injec- that Rohrschneider-McReynolds constants by themselves do not predict the actual tion. Analytical testing for residual solvent involves system suitability, external stan- polarity of these columns and the five probe molecules used for ascertaining the dards, diluent blanks followed by sample injections. This can easily lead to 4 hours McReynolds constant do not account for all the possible interactions. A Van Deem- of analysis time for a sample. Often, manufacturing of API concludes with drying ter plot was made for SLB-IL 100 using aromatic hydrocarbons as probe analyte to lower residual solvent levels to comply with ICH limits. This can involve multiple and air as career gas. This kinetic study revealed the possibility of using ionic liquid rounds of sampling, testing and further drying. By significantly decreasing analysis stationary phase with air as career gas. The stability of ionic liquid stationary phases time, manufacturing can proceed without delay. To speed up analysis time the col- in the presence of oxygen in air may significantly expand their application. umn internal diameter and length were evaluated and optimized to gain efficiency. This produced comparable methods using shorter columns of smaller internal diam- 253 Analysis of Synthetic Controlled Substances; Cannabinoids, eter with run-times less than 10 minutes. This reduced analysis time from 4 hours to Cathinones, Phenethylamines and Related Isomers by GC-IR less than 90 minutes. A limitation of this approach is lowered sample capacity which Thomas M. Kearney, Spectra Analysis Instruments, 257 Simarano Dr., was addressed using multiple capillary columns in parallel without impacting speed Marlborough, MA 01752, Bill Carson and or sensitivity. The poster covers aspects of method optimization/efficiency gain Controlled substance labs have seen an increase in designer or synthetic drug sub- and regulatory guidelines. missions. The complexity of the submissions has grown as “manufacturers” try to avoid existing laws by making slight modifications to the parent structure. These 250 Headspace Single-Drop Microextration for the Detection of molecular modifications make the unequivocal identification of these substances Jasmine Flowers Essential Oil Content difficult as the isomers can be indistinguishable on gas chromatography mass Sevgi Kocaoba, Yildiz Technical University, Faculty of Art and Science, spectrometry (GC-MS). Solid-phase transmission spectra obtained from the GC- Department of Chemistry, Davutpasa Cad. No:127, Davutpasa-Istanbul, infrared (IR) analysis of synthetic cannabinoids, cathinones and phenethylamines 34210 Turkey, Ozge Erdogmus is presented. JWH-018 and its isomers as well as the emerging threats AM-2201, Several tropical flowers have distinctive fragrances which are very appealing to use XLR-11 and UR-144 are discussed in detail. Cathinones 4-FMC, 4-MMC and related in perfumery, cosmetics and spas. However, to obtain a ‘natural fragrance’ from positional isomers are compared and contrasted. Finally phenethylamines such as the flower is a challenge as the scent could change during the extraction process. phentermine and methamphetamine are reviewed. The infrared spectra from solid- The aim of the study is to find the suitable procedure for extraction of volatile oils phase GC-IR have sufficient resolution to differentiate the positional isomers, dia- from Turkish jasmine fragrant flowers. In this study, Jasminium Grandiflorum that is stereomers and analogs of these compounds. grown in the Istanbul region of Turkey, were collected in different months. The oils of the flowers were separately extracted using solvent extraction and head-space 254 Withdrawn by the author. solid-phase microextraction (HS-SPME). The volatile constituents were analyzed by gas chromatography mass spectrometry (GC-MS). The major components iden- tified by use of solvent extraction method were benzyl acetate (21.31%), phytol 255 Analysis of Caffeine in Coffee and Tea Products Using (10.27%), benzyl benzoate (7.75%), izo-phytol (6.93%), linalool (5.7%), methyl ChromatoProbe and GC-MS linoleate (3.16%), cis-jasmon (2.19%) and methyl palmitate (1.39%). The results Vivian Xianyu Chen, Bruker Daltonics, 40 Manning Rd., Billerica, MA were compared with (HS-SPME) method in May, July and September months and 01821 maximum benzyl acetate values (35.16%) were measured in July. Coffee and tea have become more and more popular around the world because of their pleasant taste and aroma. The popularity is also partially due to the caffeine 251 Analysis of Steroids in Water Using SPME with GC-MS-MS and content. While the percentage of caffeine content in coffee seeds themselves dimin- GCxGC-TOFMS ishes with increased roast level, the caffeine content in final products also depends Shilpi Chopra, Seton Hall University, Chemistry Department, 400 South on the brewing technique. Coffee and tea are also occasionally decaffeinated to Orange Ave., South Orange, NJ 07079, Nicholas H. Snow meet different needs of consumer. Commercial ground-coffee from the same brand Recently, interest in the analysis of steroids in water has expanded because ste- with three different roast levels (light, medium and dark) as well as the decaffeinated roids have been detected in trace (part per trillion and lower) quantities and are one was analyzed. Commercial decaffeinated and black tea from a major brand was suspected of having adverse effects on aquatic life. The techniques that have been also analyzed for comparison. A Bruker gas chromatography mass spectrometry employed for this analysis include liquid chromatography mass spectrometry (LC- (GC-MS) system with ChromatoProbe was used to detect caffeine and some other MS), UV spectrometry and traditional gas chromatography (GC)-MS. The problem major aroma compounds in coffee and tea products, eliminating the need for sample with traditional one-dimensional GC-MS is co-elution of steroids with each other preparation or extraction procedure. and with matrix components in often complex mixtures. In this work steroids were extracted from water using solid-phase microextraction (SPME) with polydimethyl 256 Nitrogen Carrier Gas for GC-MS siloxane-divinyl benzene (PDMS-DVB) coated fiber, was coupled to GC-MS-MS Adam J. Patki, PerkinElmer, 710 Bridgeport Ave., Shelton, CT 06484, and GCXGC-time-of-flight (TOF)MS as two approaches to solving the problem of Andrew Tipler, Ruben Garnica co-elution. Traditional GC coupled to a triple quadrupole mass spectrometer (GC- Helium is the most common gas chromatography-mass spectrometry (GC/MS) MS-MS) still showed co-elution, which was overcome using the high selectivity of carrier gas because of its chromatographic efficiency, inertness, safety, low mass, MS-MS. SPME-GC-MS-MS also provided excellent quantitation and part-per-qua- and familiarity. However, in the last few years there have been high and rapidly drillion detection limits of detection using selected reaction monitoring. In contrast, increasing costs (especially outside the USA), supply disruptions, shortages, and GCXGC-TOFMS overcame co-elution problems through orthogonal separation us- local unavailability forcing laboratories to find alternatives. Hydrogen is the most ing two columns. Analytical figures of merit (precision, calibration, reproducibility, common alternative, but has safety and inertness issues. This paper discusses the limits of detection and quantitation) were discussed for both techniques. GC-MS-MS advantages and disadvantages of nitrogen as an alternative GC-MS carrier gas, and GCxGC-TOFMS provide complimentary information, as GCxGC-TOFMS dem- and present some operating guidelines. High purity nitrogen can also be much less onstrates comprehensive separation and GC-MS-MS provides highly selective and expensive than equivalent purity helium, providing a potential cost savings. Helium sensitive detection. and nitrogen carrier gases are compared for a variety of analyses and compounds. The differences in the required GC and MS method conditions are investigated 252 Thermodynamic and Kinetic Performance of Ionic Liquids as Gas by comparing the chromatographic figures-of-merit, detection limits, and dynamic Chromatographic Stationary Phases ranges. Constant velocity carrier gas control is used to closely match the original Ramkumar Dhandapani, Seton Hall University, Chemistry Department, helium retention times and chromatographic resolutions. Optimal MS tuning condi- 400 South Orange Ave., South Orange, NJ 07079, Nicholas H. Snow tions for nitrogen differ from helium, requiring slight modifications to the parameters The interest of using ionic liquids as gas chromatographic stationary phase has for optimal performance. Nitrogen was found to give a lower signal-to-noise ratio increased in recent years. The low volatility, tailorable physico-chemical property than helium. Matching gas velocity allows replacement of helium by nitrogen carrier and high thermal stability of ionic liquids make them an ideal choice for gas chro- gas for many applications, with minimal change in chromatography. matographic stationary phase. For the present research, commercially available ionic liquid capillary columns were investigated for thermodynamics and kinetics of 257 A Design of Experiment (DoE) Approach to Develop and Optimize a the retention of alkanes and aromatic hydrocarbons. Van ’t Hoff plot was made and GC Method for Residual Pivalic Acid in Amoxicillin the thermodynamic parameters ΔG, ΔH, ΔS were determined. The thermodynamic Richard Desmangles, Corealis Pharma, 200 Boul. Armand-Frappier, parameters of SLB-IL 61 were compared with a conventional polyethylene glycol Laval PQ H7V4A6, Canada, Marie Di Maso, Yves Mouget based stationary phase having same Rohrschneider-McReynolds constants. A plot Pivalic acid is an intermediate used in the synthesis of various active pharmaceuti- of retention factor was made against claimed polarity of the ionic liquid columns. cal ingredient (API) such as Ampicillin, Amoxicillin and Cephalosporins. Monitoring The plot revealed that change in the claimed polarity had no definite relationship of residual pivalic acid is required to ensure the quality of the API when used as raw 41 2013 EAS Abstracts November 2013

material for the production of clinical or commercial pharmaceutical dosage forms. The extraction performance of the headspace system is further enhanced through The preferred technique for analysis of residual solvents is by gas chromatography the inclusion of adsorbent trap technology which enables all or most of the sample (GC), where methods are available for analysis of several residual solvents. Small vapor from the vial to be collected, concentrated and transferred to the GC column. organic acids, such as pivalic acid, are prone to interaction either with active sites This HS trap sampling system can improve detection limits by a factor of up to x100 in the GC injector or with the liquid phase of GC capillary columns and thus method and also, very importantly, deliver higher amounts of each compound to the olfac- development becomes more challenging. In determining residual Pivalic acid in tory port to aid their detection by the operator. Selecting an appropriate trapping Amoxicillin trihydrate raw material a significant decrease of the signal was observed material is vital to correctly sample the vapor and here we present several perfumes in spiked solutions following multiple sample injections. Investigations showed that that are sampled with different trapping materials. The consumer experience is de- this was due to matrix interactions in the GC injector. Derivatization of the acid scribed using an olfactometry and correct chemical identification is achieved with analyte is a choice solution to circumvent this problem. Fisher esterification is a mass spectrometry. favored option. The reaction and recovery of the derivatized analyte are influenced by temperature, time of reaction as well as by the concentration of the reagents. In 261 Structure Sensitivity in Adsorption and Desorption of Hydrogen on this presentation a DoE approach adopted to investigate the key parameters simul- Iridium Surfaces. taneously with a minimal number of experiments is described. The results obtained Wenhua Chen, Rutgers University, Department of Physics and from the DoE experiments were used to finalize the optimal parameters for a robust Astronomy, Piscataway, NJ 08854, Robert A. Bartynski, Payam analytical method. Kaghazchi, Timo Jacob Clean planar Ir(210) and clean faceted Ir(210), consisting of nanoscale {311} and 258 Analysis of Oxygenated Compounds in Jet Fuel Using Solid-Phase (110) facets, have been fabricated to investigate adsorption and desorption of hy- Extraction and Comprehensive 2-Dimensional Gas drogen. Temperature programmed desorption (TPD) spectra show that only one Chromatography H2 peak is seen from planar Ir(210) at all coverages whereas a single H2 peak is Raymond Yoder, Virent, Inc., 3571 Anderson St., Madison, WI 53704, observed at around 440 K at fractional monolayer (ML) coverage and an additional Rory McAloon, Mark Heininger, Jim Dudek H2 peak appears at around 360 K at 1 ML coverage on faceted Ir(210), implying Trace hetero-atomic species, particularly oxygenates, can affect jet fuel thermal sta- structure sensitivity in recombination and desorption of hydrogen on faceted Ir(210) bility and increase deposit formation.[1] Studies have shown that deposit formation versus planar Ir(210). Densityfunctional-theory calculations revealed the origin of correlated with the amount of polar species present in the fuel. When the polar the two TPD peaks of H2 from faceted Ir(210): the peak at higher temperature from species were scrubbed from a fuel, deposit formation decreased. Analysis of polar desorption of H2 on {311} facets while the peak at lower temperature from desorp- species in a complex jet fuel matrix is challenging. Various approaches have been tion of H2 on (110) facets. taken to extract the polar species from the fuel matrix prior to analysis. Extracts are normally analyzed using gas chromatography (GC) with several detection methods 262 Tuning Re(11-21) Surface Morphology by Kinetic Control of including mass spectrometric (MS) detection. This poster summarizes the develop- Adsorbate ment of a method to analyze polar species, particularly oxygenates, in an assort- Hao Wang, Rutgers University, Department of Physics and Astronomy, ment of jet fuel samples. The fuel is passed through a silica solid-phase extraction Piscataway, NJ 08854, Grant Junno, Wenhua Chen, Robert A. Bartynski, cartridge, rinsed with hexane to remove the hydrocarbon matrix, and eluted with Xiaofang Yang, Bruce E. Koel, Payam Kaghazchi, Timo Jacob methanol. An internal standard is added for quantitative purposes and the extract is The surface morphology of Re(11-21) tailored on the nanometer scale by kinetic analyzed using comprehensive two-dimensional gas chromatography (GC x GC). control of adsorbate has been investigated using low energy electron diffraction, The improved separation capability of GC x GC, coupled with time of flight mass scanning tunneling microscopy, Auger electron spectroscopy, and density func- spectrometry, allows for improved identification of the types of polar species present tional theory in combination with the ab initio atomistic thermodynamics approach. in jet fuel. Depending on adsorbate species at elevated temperatures, the initially planar re(11-21) surface becomes fully faceted and the faceted surface consists of either Reference: two-sided ridges formed by (13-42) and (31-42) facets (nitrogen) or three-sided pyr- [1] S. Zabarnick, Ind. Eng. Chem. Res. 33 (1994) 1348-1354. amids formed by (01-11), (10-11) and (11-20) facets (carbon). In addition, we have 259 Influence of Alkyl Spacer on Properties of L-Phenylalanine Ester observed N-induced (2x1) reconstruction of Re(11-21), which serves as a precursor Chiral Ionic Liquids state for N-induced faceting of Re(11-21). Calculations provide quantitative informa- Lydia R. Morris, St. John Fisher College, 3690 East Ave., Rochester, NY tion on the geometries and energetics of adsorbate (N or C) on the substrate and 14618, Nicole J. Savage, Irene Kimaru facets as well as the stability of the substrate and facets without and with adsorbate The goal of this project was to evaluate the influence of the alkyl spacer on physical at different coverages, which enables us to obtain an atomistic understanding of properties and chiral recognition ability of chiral ionic liquids (CILs), determine their facet formation. The N- or C-covered faceted Re(11-21) surfaces are nanoscale potential use as chiral solvents, and as stationary phases in gas chromatography model catalysts and catalyst supports. (GC). Four different CILs were synthesized by ion exchange between L-phenylala- Analysis of Surface Chemistry and Optical Properties in Post nine based esters as the cation and bis-(perfluoroethylsulfonyl) imide as the anion. 263 Synthesis Treated CdSe Quantum Dots The alkyl spacer on the cations was varied in order to test the influence of the Daniel J. Asunskis, Black Hills State University, 1200 University St., alkyl spacer on properties of the resulting CILs. The CILs were found to be pure by Spearfish, SD 57799, Samantha R. Brush, Wick D. Carr nuclear magnetic resonance (H1 and C13). They were found to be thermally stable Cadmium Selenide (CdSe) is a group II-VI semiconductor that has great potential up to 288.10 ºC via thermal gravimetric analysis. Their glass transition ranged from as a photovoltaic, photodiode, and nonlinear optical material. Surfactant-capped -49.47 oC to -29.51 ºC and melting points from 85.33 ºC -112.63 ºC. Variations were CdSe were synthesized using colloidal methods where a selenium precursor was in- observed in the physical properties of the CILs as the alkyl spacer changed. The jected into a high temperature vessel containing a solvent, cadmium, and surfactant fluorescence spectra of the neat CILs shifted to longer wavelengths as the excita- molecules. Ultraviolet-visible absorption, fluorescence, and synchrotron X-ray pho- tion wavelength was increased. The CILs demonstrated chiral discrimination for the toelectron spectroscopy were used to analyze changes in optical properties while pure enantiomers of 2,2,2-trifluoroanthrylethanol via fluorescence. Studies are in also examining changes in surface chemistry as the nanocrystals are processed. It progress to test the ability of the CILs to discriminate between the pure enantiomers has also been shown that post synthesis surface washing of the CdSe nanocrystals of propranolol and ibuprofen. These results indicate that the CILs can act as chiral will cause a decrease in observed quantum yield. This has been studied previously solvents and show great potential to act as stationary phases in GC. The funding on PbS nanocrystals where the post synthesis washing had significant effect on the that supported this work was provided by St. John Fisher College. measured nonlinear optical properties. X-ray photoelectron spectroscopy experi- 260 The Application of a HS Trap with GC Olfactometry and Mass ments, collected at the Synchrotron Radiation Center of the University of Wiscon- Spectrometry for the Analysis and Aroma Characterization of sin-Madison, for these treated CdSe nanomaterials will be used to give measure Perfumes changes in chemical state and electronic structure at the nanocrystal surface. Initial David J. Scott, PerkinElmer, 710 Bridgeport Ave., Shelton, CT 06484, results show significant oxidation in the selenium core level features, the increase in Andrew Tipler, Sheila Eletto oxidation correlating to the loss of observed fluorescence. Perfume has a long history in human culture from ancient through to modern times. Incorporating Application Based Service Learning into an The understanding, manufacturing and technology of fragrance have evolved over 264 Instrumental Methods of Analysis Course time but the final choice of a fragrance remains a very personal thing. Headspace L. Jay Deiner, New York City College of Technology, 300 Jay St., (HS) sampling enables the sampling of many raw materials such as flowers, when Brooklyn, NY 11201, Gregory Galford, Gail Rowe, Nancy Trun a successful extraction may be difficult, or oils that may be challenging to inject di- Application based service learning (ABSL) is a pedagogy that brings original re- rectly onto a gas chromatography (GC) column. With the classical headspace tech- search and community service into undergraduate laboratory courses. The research nique, only a very small fraction of the total vapor is transferred to the GC column. 42 2013 EAS Abstracts November 2013

and service both focus on a local community problem. The ABSL methodology has diation efforts. However, the most commonly used methods for extracting PCBs and previously been incorporated into biology laboratory courses at Duquesne Universi- PBBs from sediment slurry in microbial dehalogenation studies are often expensive, ty and La Roche College. Preliminary assessments from these first implementations slow, and require drying the sediment. Thus, in this study a simple high-throughput show that the ABSL methodology increases students’ retention of content while giv- method for extracting and quantifying PCBs and PBBs from sediment slurry using ing them insight into the application of science research to solve real world prob- ultrasonic extraction and gas chromatography mass spectrometry was developed. lems. In spring 2012, the New York City College of Technology began the process This method is being used to compare the dehalogenation rates of hexaPCBs in the of remaking its Instrumental Methods of Analysis course using the ABSL pedagogy. presence and absence of hexaPBBs. The process of transitioning a traditional Instrumental Methods of Analysis course into an ABSL course entailed changing the syllabus to allow time for research and 268 Exploring the Heterogeneity of Oil in Sand Patties from the Gulf of service, selecting a community issue, designing in-class research, and engaging a Mexico community partner. In this poster, we describe strategies for accomplishing these Patrick Williams, Haverford College, Department of Chemistry, 370 changes through the use of student input and through work produced by the stu- Lancaster Ave., Haverford, PA 19041, Helen White dents in the course of scaffolded assignments. In addition, we describe how similar Oil-soaked sand patties originating from the Deepwater Horizon oil spill were col- strategies can be applied to incorporate other high impact practices typical of ABSL, lected from Gulf of Mexico beaches in June and July, 2012. Sand patties of various including the development of interdisciplinary partnerships that connect the Instru- color, size, thickness, and distance from the surf were compared to explore the het- mental Methods of Analysis course to other science and non-science courses. erogeneity of oil present. Gas chromatography-flame ionization detection (GC-FID) was used to determine the composition and quantity of oil present and the amount 265 Sample Preparation Optimization for GC-MS Characterization of of saturated, aromatic, and oxygenated fractions in extracts was quantified using Cell Culture Media thin layer chromatography−flame ionization detection (TLC−FID). The presence of Christina Joseph, Muhlenberg College, Chemistry Department, oxygen functional groups was examined via Fourier transform infrared spectroscopy 2400 Chew St., Allentown, PA 18104, Christine M. Ingersoll, John (FTIR) to provide greater insight into the degree to which the oil was weathered Cunningham, Adam M. Fermier by biodegradation and photooxidation. Significant variations in oil composition and Cell culture media are complex aqueous mixtures containing many nonvolatile con- degrees of oxidation were observed both between sand patties and within sand pat- stituents of interest. This presents challenges when characterizing media by gas ties. Current and future work focuses on using two-dimensional correlation analysis chromatography mass spectrometry (GC-MS); chemical derivatization is required to further explore how oil changes under various environmental conditions. Correla- prior to analysis, which leads to multifaceted sample preparation methods. This tion analysis of time-trial experiments will explain more clearly the mechanisms and work describes the process of developing an automated method used to carry out time dependencies of oxygenated functional groups in weathering oil. the entire sample preparation process, including a two-step derivatization followed by extraction, for the characterization of cell culture media constituents by GC-MS. 269 Mass Spectrometry for Assessing the Occurrence and Biological Specifically, a liquid handler is used to execute all reagent additions, agitation, Consequences of Oxidatively Induced Tandem DNA Lesions derivatization, and extract transfer, to yield the final samples suitable for analysis Yinsheng Wang, University of California-Riverside, Department of from the original aqueous media. Significantly better reproducibility is achieved in Chemistry, Riverside, CA 92508 less time compared to manual sample preparation. Design of experiments (DOE) is Endogenous aerobic metabolism and exposure to various environmental agents also used as a tool to optimize the automated sample preparation method. Factors lead to cellular production of reactive oxygen species (ROS), which can cause (and combinations of factors) involved in mixing, reagent additions, and reaction damage to DNA. Although substantial repair studies have been conducted for the conditions, are identified and optimized using a factorial design model. Results are ROS-induced single-nucleobase lesions, not much is known about how oxidatively reported for representative optimized conditions and successes and challenges are induced tandem DNA lesions are repaired in mammalian cells. Our liquid chroma- also presented. tography triple mass spectrometry (LC-MS/MS/MS) quantification results revealed that 8,5’-cyclo-2’-deoxyadenosine (cyclo-dA), 8,5’-cyclo-2’-deoxyguanosine (cyclo- 266 Identifying Crossover Points between Particle Sizes in the dG) and the GT intrastrand crosslink are present at significantly lower levels in tis- Optimization of Liquid Chromatography sues of repair-competent animals than in Ercc1-deficient animals. Likewise, we Adam Matula, Chemistry Department, University of Minnesota, 207 found higher levels of these lesions in brain tissues of xeroderma pigmentosum Pleasant St. S.E., Minneapolis, MN 55455, Peter Carr group A gene (XPA)-deficient patients than in repair-proficient individuals. Viewing Presently, the guiding principles in the optimization of high-performance liquid chro- that XPA and Ercc1 are required for nucleotide excision repair (NER), the above matography (HPLC) state that the absolute best separation possible at a given anal- results support that these DNA lesions are substrates for NER in-vivo. Additionally, ysis time and maximum system pressure is achieved by simultaneously optimizing we observed an age-dependent accumulation of these DNA lesions in mammalian column length, eluent velocity, and particle size. The results of this three-parameter tissues, suggesting that the accumulation of these DNA lesions may play an impor- optimization, known as the “Knox-Saleem limit,” are rarely practicable; only a lim- tant role in aging-related pathological conditions. We also found, from the LC-MS- ited number of commercially-available particle sizes exist, limiting whether ‘ideal’ MS and plasmid-based in-vivo transcription assay, that both cyclo-dA and cyclo-dG separation conditions can exist. Two-parameter optimization, often depicted with substantially blocked transcription in mammalian cells, and transcription bypass ef- isocratic “Poppe curves,” holds particle size constant, optimizing only column length ficiency is much lower in NER-deficient mammalian cells than repair-proficient cells, and eluent velocity simultaneously. This almost always results in separations that suggesting again the compromised repair of these DNA lesions in XPA-deficient are less efficient than those predicted by three-parameter optimization, though in cells. Moreover, the LC-MS-MS method allowed for the identification and quantifi- every case there is a particle size that minimizes this loss in efficiency. Although it is cation of mutant transcripts produced from the transcriptional bypass of the cyclo- not immediately obvious from two-parameter optimization which particle size should dA and cyclo-dG lesions. Together, our combined chemistry and biology approach be used, there exists a distinct and identifiable point between each two particle sizes led to important novel insights into the repair and biological consequences of the after which the most efficient separation is maintained by switching from one size radiation-induced tandem DNA lesions. to the other. Here we present simple, linear equations identifying these “crossover points” in terms of analysis time and plate count, allowing a practitioner to rapidly 270 Mass Spectrometry as a Tool for Structural Biology identify the correct particle size for use in tackling a particular separation problem. Lisa M. Jones, Indiana University-Purdue University, 402 N. Blackford Additionally, a formulation for comparing the results of two-parameter and three- St., Indianapolis, IN 46202 parameter optimization is given. Results generated using this formulation reveal Structural analysis of macromolecular complexes (e.g., antibody-antigen complexes that, provided an operator switches particle sizes at the prescribed crossover points, and viruses) is hampered owing to their size and complexity. High resolution bio- relatively little separation efficiency is lost as a result of the limited availability of dif- physical methods for structural analysis are not applicable to proteins that have a ferent particle sizes. high molecular mass. In order to analyze these large structures, methods based on mass spectrometry (MS) can be employed. MS-based methods are advantageous 267 Development of a Method for a Comparative Study of the Microbial because they are not limited by size and require only microgram quantities of pro- Dehalogenation of PCBs and PBBs tein. Footprinting methods coupled with MS are a powerful tool for studying higher Kathleen Naccarato, Swarthmore College, 500 College Ave., order structure of proteins. These methods can be used to monitor protein confor- Swarthmore PA, 19081, Alison Holliday mational changes and identify protein-protein and protein-ligand interaction sites. Many US and international waterways have been historically contaminated with ha- The protein footprinting method fast photochemical oxidation of proteins (FPOP), a logenated persistent organic pollutants, such as polychlorinated biphenyls (PCBs) radical footprinting approach, utilizes hydroxyl radicals to oxidize solvent-exposed in the Hudson River and polybrominated biphenyls (PBBs) in the Pine River Reser- residues on a short time scale. The irreversible label of FPOP has several advan- voir. This contamination has inspired research into the in-situ microbial degradation tages including the ability to purify proteins after labeling. This is important in anti- of these, as well as more contemporary halogenated pollutants, in hopes of devel- body systems because time is needed to reduce extensive disulfide bonds and in oping inexpensive and less environmentally costly alternatives to traditional reme- viruses where viral membranes need to be removed prior to MS analysis. We have 43 2013 EAS Abstracts November 2013

successfully used FPOP for epitope mapping as well as distinguishing structural signment of OS NMR spectra using mismatched Hartmann-Hahn conditions will be differences in isomeric antibodies. We have also used FPOP to elucidate protein- presented. This allows one to transfer magnetization between the dilute spins that protein interactions in the bacteriophage P22. This research marks the first time are over 6 Angstroms apart. oxidative labeling coupled with MS has been used to study antibodies and viruses. 274 Transmembrane Allosteric Coupling Between the Inner and Outer 271 Metal-Induced Structural Changes of Human Metallothionein-2A: A Gates of a K+ Channel in Lipid Bilayers Revealed by Solid-State Combined Theoretical and Experimental Structural Analysis of NMR Metal-Free and Partially Metallated Intermediates Benjamin J. Wylie, Columbia University, 3000 Broadway Department of David Russell, Texas A&M University, Department of Chemistry, College Chemistry, Havemeyer Hall Box 3132, New York, NY 10027, Manasi P. Station, TX 77842, Shu-Hua Chen Bhate, Ann E. McDermott Metallothioneins (MTs) are cysteine-rich (~ 30%), low molecular mass (~ 6 kDa) It has been hypothesized that transmembrane allostery is the basis for inactivation proteins with unusual metal-reactivity; cysteine thiolate groups coordinate a wide of the potassium channel KcsA: opening the intracellular gate is spontaneously fol- variety of metal ions. Atomic resolution (NMR) measurements of the fully-metal- lowed by ion expulsion at the extracellular selectivity filter. This suggests a corollary: lated form reveal a dumbbell-shaped molecule with two metal-centered domains following ion expulsion at neutral pH, spontaneous global conformation change of (Cd3β- and Cd4α-domain), but very little is known about the mechanism of metal- the transmembrane helices (potentially similar to the motion involved in opening) is lation or the structure(s) of the partially metallated speices. Chemical labeling with expected. Consequently, both the low potassium state and the low pH state of the N-ethylmaleimide (NEM) and tandem mass spectrometry experiments clearly show system could provide useful models for the inactivated state. New nuclear magnetic that the four metal ions of Cd4MT are located in the α-domain, and demetalation resonance (NMR) studies of full length KcsA (160 aa) in hydrated bilayers provide (by EDTA) of Cd7MT is step-wise process that involves metal removal from the strong evidence for such a mutual coupling across the bilayer: namely, upon remov- β-domain followed by metal ion removal from the α-domain. Ion mobility studies ing ambient potassium ions, changes are seen in the NMR shifts of the carboxyl- clearly illustrate the coexistence of compact and extended structures for the apoMT ates in the pH gate (118 and 120), in the hinges of the inner transmembrane helix and distinct metallation pathways involving metal ion-induced dimerization and (98-103), and in the selectivity filter, all of which resemble changes seen upon acid metalion-induced protein folding. The extended apoMT folds into a well-defined, induced opening and inhibition, and suggest that ion release can trigger channel compact conformation via multi-trajectory pathways as the number of bound metal helix opening. ions increases. Despite the structural variability of apoMT and partially-metallated intermediates, each species converges toward similar structure(s) as the number 275 Structure Studies of Amyloid Fibrils in Alzheimer’s Diseases, from of metal ion bounds increases. Independent all-atom MD simulations for hydrated Test Tube to Human Brain and dehydrated structures were carried out for all CdiMT (i = 0 − 7) at 300 K and Wei Qiang, National Institutes of Health, Rm. 406, Bldg. 5, 9000 at elevated temperatures (350 K). The simulations at 300 K show a consistent pro- Rockville Pk., Bethesda, MD 20892, Robert Tycko tein size where metal-sulfur interaction is replaced by the intramolecular interaction Solid-state nuclear magnetic resonance (NMR) studies on biological samples such upon removal of metal ions, whereas the simulations at 350 K reveal significant as proteins often involve the utilization of uniform isotope labeling. The quantitative structural changes where demetallation promotes unfolding to extended structures. evaluation of the cross peak volumes from multi-dimensional NMR spectroscopy is usually hindered by the dipolar truncation effect, where the polarization transfer 272 Mass Spectrometry-Based Protein Footprinting: A Tool for between weakly-coupled spin pairs are eliminated by the transfer between strongly- Biophysics coupled ones. We show that the stochastic dipolar recoupling techniques, which in- Michael L. Gross, Washington University - St. Louis, Department of troduce random amplitude and phase modulations to the homonuclear dipolar cou- Chemistry, Box 1134, St. Louis, MO 63130 pling Hamiltonian through chemical shift interactions, effectively recover the dipolar There is a compelling need to determine properties of proteins that do not crystallize coupling between weakly-coupled spin pairs. For microcrystalline GB1 with N spins, and are not suitable for high-resolution nuclear magnetic resonance (NMR) because the cross peak volumes in two-dimensional spectra can be described quantitatively they are too large or oligomerize at low concentration. Furthermore, the relevant by an NxN rate matrix, which are purely determined by inter-nuclear distances. Ad- properties of a protein are not in water but in unusual media (e.g., high lipid or ditional, the buildup of cross peak volumes also provide information of protein side protein concentrations) or in protein assemblies. Many low-resolution approaches chain torsion angles. exist (e.g., fluorescence, circular dichroism) but do not usually yield information at the peptide or amino-acid levels. Mass spectrometry (MS), on the other hand, as a 276 Insight into the Structure and Formation of Polyglutamine Amyloid middle-resolution method, can map protein interactions, follow folding and unfold- Aggregates from Solid-State NMR Spectroscopy ing, and determine binding affinities from chemical footprinting that uses the same Patrick van der Wel, University of Pittsburgh, Department of Structural analysis as is well developed for proteomics. To this end, we are developing fast Biology, 3501 Fifth Ave, BST3 Room 2044, Pittsburgh, PA 15260 photochemical oxidation of proteins (FPOP) and implementing HD exchange(HDX), Various human diseases are characterized by the misfolding and aggregation of and other covalent modification approaches to interrogate protein interactions, in- proteins. This is found perhaps most prominently in amyloid diseases, where these terfaces, and dynamics of folding/unfolding. For irreversible labeling, we developed processes lead to protein aggregates that feature intermolecular beta-sheets along FPOP, which has a significant advantage of speed, owing to the use of free radicals the length of long fibrillar assemblies. In the family of polyglutamine (polyQ) ex- to footprint proteins at sub-microsecond time and in a single conformation. Besides pansion disorders, disease and protein aggregation both correlate to the (genetic) •OH, other radicals can provide different selectivity for amino-acid residues. FPOP expansion of a polyQ segment in a disease-specific protein. Until recently, a lack of can also be the “probe” in a classic, two-laser “pump-probe” experiment whereby a structural data on the resulting aggregates led to divergent models of the internal temperature-jump perturbation is produced as a “pump” by one laser and a second amyloid configuration, which in turn has limited our understanding of the aggrega- laser initiates FPOP as the “probe” to afford residue-specific information. These tion processes accompanying these devastating diseases. To address this, we have experiments and related biophysics experiments can be done with bottom-up or employed solid-state NMR spectroscopy to provide unique atomic-level insights into top-down analyses. Examples to illustrate these general ideas are described in the the structure of various polyQ-containing aggregates. Despite additional challenges lecture. compared to its application to other amyloid-like fibrils, magic-angle-spinning NMR has provided site-specific constraints on the local molecular structure within the 273 Structural and Dynamic Studies of Membrane Proteins by Oriented- aggregates and identified localized solvent exposure and molecular motion. These Sample NMR new structural and motional data help clarify the aggregation mechanism, highlight Alexander Nevzorov, North Carolina State University, 2620 Yarbrough unusual features specific to polyQ amyloid, and have proved highly complementary Dr., Raleigh, NC 27695 to ongoing biochemical, mechanistic and biological studies into Huntington’s Dis- Oriented-sample nuclear magnetic resonance (OS NMR) has emerged as a pow- ease and other expanded polyQ disorders. erful technique for structure determination of membrane proteins in their native- like lipid environment. Several issues for improving the performance of OS NMR 277 Using the Fourth Derivative to Understand Changes in NIR Spectra are addressed. The first one is line narrowing due to the uniaxial ordering (mosaic Karl H. Norris, NIR Consultant, 11204 Montgomery Rd., Beltsville, MD spread) and rotational diffusion. We have performed lineshape simulations using 20705 the Stochastic Liouville Equation in the slow- and fast-motional regimes. The theory A number of mathematical tools are available for deconvolution of complex near- is exemplified by solid-state NMR spectra of Pf1 coat protein in the phage form infrared (NIR) spectra. Most of these involve derivatives, and this presentation con- and reconstituted in magnetically aligned bicelles. Second, we have developed a centrates on the use of the fourth derivative. The fourth derivative from different sensitivity-enhancement pulse sequence, which increases the signal-to-noise ratio computer programs does not provide identical results, but they all provide useful re- by as much as 80%. Moreover, the use of membrane-embedded radicals allows sults. The segment/gap derivative from vision and the gap derivative as well as the one to further speed up data acquisition. Finally, a method for the spectroscopic as- S. Golay derivative from Unscrambler 9.8 are used in this presentation. The focus is

44 2013 EAS Abstracts November 2013

on the spectral changes from sample temperature changes and the identification of of a polar primary column and a non-polar secondary for the analysis of Navy mobil- noise sources in reflection, transmission, and interactance measurements on differ- ity fuels. For fuels, this configuration is proving to be a powerful tool to resolve the ent samples with different instruments. numerous compound classes and better uses the two-dimensional chromatographic space. With the US Department of Defense’s efforts to incorporate more alternative 278 Are Non-Linear Regression Techniques Useful for NIR fuels into its supply, GCxGC–TOFMS has proven to be unmatched in the forensic Pharmaceutical Applications? A Pharmaceutical Tablet Assay analysis of fuels and fuel blends as well as detection and analysis of contaminants. Example Additionally, GCxGC–TOFMS will provide detailed compositional information to Benoit Igne, Duquesne University, 600 Forbes Ave., Pittsburgh, PA characterize and certify legacy petroleum and emerging alternative fuels through 15282, James K. Drennen III, Carl A. Anderson compositional modeling and compound class profiling. The second dimension of While non-linear regression techniques such as artificial neural networks and sup- separation of the fuel will enable construction of more accurate and robust models. port vector machines are being used for the determination of agriculture product The Navy will have the capability to understand how a new fuel or fuel blend will and feedstuff properties by near infrared spectroscopy, they have not been adopted perform before spending significant resources on fuel certification. in regulated environments such as the pharmaceutical industry. Until a year or two ago, little to none of the main stream software packages provided the algorithms 282 A Comparison of Heartcutting, Selective Comprehensive, and Fully and a very limited amount of literature exists on the topic. In addition, the reputa- Comprehensive Two-Dimensional Liquid Chromatography, with an tion of “black box” carried by these algorithms has pushed chemometricians to limit Emphasis on Practical Factors Important for Success their use when regulators have to review the method development process. While Dwight Stoll, Gustavus Adolphus College, 800 West College Ave., St. method validation are not discussed, this presentation shows how artificial neural Peter, MN 56082, Eric Talus, David Harmes, Kelly Zhang networks and support vector machines can be used for the prediction of pharma- Interest in two-dimensional liquid chromatography (2-DLC) continues to increase ceutical tablet active content. The complexity of their implementation is contrasted across a variety of application areas as users look to analyze increasingly complex with the performance they can provide, compared with the traditional partial least samples with a higher degree of confidence, and with shorter analysis times. Most squares regression. recently, the introduction of commercially available instruments for comprehensive 2-DLC (LCxLC) characterized by excellent performance and robustness has made 279 Addressing THE Problem with NIR the 2-DLC more accessible to a wider array of potential users than in the past. Howard Mark, Mark Electronics, 69 Jamie Ct., Suffern, NY 10901 The availability of sophisticated software with high potential for automation for both Modern near-infrared (NIR) analysis is subject to a near-universal, but widely ig- instrument control and data analysis has removed much of the tedium that has his- nored, problem. Chemometrics has covered up the problem and enabled calibration torically been associated with these important aspects of successfully implement- models that “worked.” But while valuable, this is game-playing, not Science. Expe- ing 2-DLC. Nevertheless, method development for 2-DLC remains complex and rienced practitioners of NIR sense that something is “wrong,” and the methodology full of pitfalls that can jeopardize the performance potential of these separations. In sensed as not being part of the universe of Science. Many characteristics if NIR fly this presentation we will use the 2-DLC separation of a degraded pharmaceutical in the face of conventional Science: 1) more variables (wavelengths or factors) are as a case study to compare the effectiveness of different 2-D separation modes, needed than any rational estimate indicates. 2) Algebra dictates that you should and illustrate the impacts of some critical method development decisions on the not need more variables or equations than variational sources. 3) Inability to repro- effectiveness of these methods. We find that a properly optimized online LCxLC duce calibrations from similar samples. 4) Inability to reproduce wavelength sets method has sufficient detection sensitivity in the second dimension separation to (for multiple linear regression (MLR) models) 5) Difficulty and/or inability to relate reveal low-level degradation products that would otherwise be obscured by higher wavelengths chosen (MLR) or prominent bands (polymerase chain reaction (PCR) concentration components when only a single dimension of high-performance liquid / partial least square (PLS)) to spectral features. 6) Calibration size exclusion chro- chromatography separation is used, even when very long analysis times are used. matography should drop precipitously to the noise level. 7) Models should cover the entire range of concentrations. 8) Calibrations should be extrapolatable. 9) Calibra- 283 Can Multidimensional-Like Separations Be Achieved in Liquid tion transfer should be easily and readily performed. A recent discovery seemed to Chromatography via a Sequential Use of Orthogonal Mobile have the right properties. This was a different kind of data transform, which differed Phases? in two key respects: 1) It was a transform of the concentration values; 2) it was Joe Foley, Drexel University, Department of Chemistry, 305 Disque Hall, based on known physical chemistry. A minimal algorithm applied to a very small set 3141 Chestnut St., Philadelphia, PA 19104, Adam Socia of specialized data supported the hypothesis. What was still needed was a large If performed correctly, comprehensive two-dimensional (2-D) chromatography can enough dataset to support conventional algorithms. A suitable dataset was found, provide a higher peak capacity and a greater probability of resolving the compo- and I will be presenting the results of applying MLR, PCR and PLS to that data. nents in a given sample than conventional one-dimensional separations. However, the instrumental setup is more complex, requiring two orthogonal stationary phases, 280 NIR Methods for Health Assessment: New Eyes on an Old Problem and analysis times can be much longer. In contrast to this stationary phase-centered David Burns, University New Brunswick, 3 Bailey Dr., Fredericton, NB approach to 2-D chromatography, sequential-elution liquid chromatography (SELC) E3B 5A3, Canada is a mobile-phase-centered approach to increasing the peak capacity and probabili- Medicine is the 21st century is changing dramatically. With the advent of many ty of a successful separation that only requires conventional high-performance liquid new non-invasive and minimally invasive monitoring methods, rapid point-of-care chromatography instrumentation. In SELC, a sample comprised of different classes diagnosis is becoming possible. Near-infrared spectroscopy has been shown to of compounds is introduced into a chromatographic system that is highly retentive have significant potential for in-vivo and in-vitro assessment of biological status. for all compounds because of a weak mobile phase that is initially employed. The This presentation gives an overview of recent advances in chemometric inspired highly-retained compounds are then subjected to a sequence of r selective mobile spectroscopic measurement for in-vitro assessments and focus on the principles phases, each designed to elute and separate one class of compounds. The result behind clinical use of several new methods to understand metabolism. An emphasis of SELC is the sequential elution and separation of all sample components by class on instrument simplification for field portable instrumentation is made. As an ex- and within each class. The benefits of SELC with r sequential elution modes are 1) ample, a new method to assess infection status in lactating mothers is presented. an r-fold increase in peak capacity; 2) a ln r-fold decrease in the disorder (entropy) One overarching principle, wavelength rationing, which has proven to be useful for of the separation, and 3) a significant increase in the probability that all sample comparisons of these measurements is discussed. Results demonstrate how these components will be resolved. The theory is summarized and a few applications are simple techniques provide a valuable tool for monitoring of patient status. reported, with an emphasis on an approach utilizing one or two pH gradients prior to a solvent gradient that can be employed to separate weak acids, weak bases, and 281 Comprehensive GCxGC Separation of Petroleum and Alternative neutral compounds from each other. Navy Mobility Fuels Thomas N. Loegel, NOVA Research, 1900 Elkin St., Ste. 230, 284 Two-Dimensional Liquid Chromatography in Pharmaceutical Alexandria, VA 22308, Kristina M. Myers, Robert E. Morris, Jeffrey A. Analysis: An Industry Perspective Cramer, Mark H. Hammond Kelly Zhang, Genentech Inc., One DNA Way, South San Francisco, CA The chromatographic analysis of jet and diesel fuels requires high separation effi- 94080, Yi Li ciency to resolve the hundreds of compounds typically found in petroleum products. Two-dimensional high-performance liquid chromatography (2-DHPLC) greatly in- Traditional one-dimensional gas chromatography – mass spectrometry (GC-MS) creases the separation power that cannot be achieved by one-dimensional HPLC. lacks the capability to fully explore a fuel sample in one analysis. Comprehensive Because of the high safety bar of pharmaceutical products and strict regulatory two-dimensional gas chromatography – time of flight mass spectrometry (GCxGC– requirements, complete impurity profiling, detection and quantification of low-level TOFMS) with thermal modulation allows the use of two GC columns with orthogonal impurities are critical in pharmaceutical analysis to ensure patient safety. The appli- stationary phases and cryo-focused peaks to analyze complex organic mixtures cation of 2-DLC in pharmaceutical analysis is getting more attention in recent years such as fuels. We present method development work that has centered on the use 45 2013 EAS Abstracts November 2013

due to the advancement of HPLC hardware and software, and the commercializa- 288 High Spatial Resolution Infrared Spectroscopy and Imaging Using tion of the 2-DLC system. This presentation overviews the applications of 2-DLC AFM Detection in pharmaceutical analysis with case studies, such as peak co-elution, peak purity Curtis Marcott, Light Light Solutions, 2558 High Hammock Rd., assessment, impurities and degradation products profiling, and stability indicat- Seabrook Island, SC 29455, Michael Lo, Qichi Hu, Kevin Kjoller ing method development for both drug substance and drug product. The accurate Atomic force microscopy (AFM) and infrared (IR) spectroscopy have been com- quantification of a low-level impurity by 2-DLC is demonstrated. The advantages bined in a single instrument capable of producing 100-nm spatial resolution IR spec- of 2DLC-mass spectrometry in pharmaceutical analysis are also discussed. The tra and images. This new capability enables the spectroscopic characterization of authors also discuss current limitations and future perspectives of implementing domains in polymeric samples at levels not previously possible. A tunable IR laser 2-DLC method in the regulated environment for quality control. source generating pulses on the order of 10 ns was used for excitation of cast sample films or thin cross sections deposited on IR transparent ZnSe prisms. Short 285 Standardization of Raman Spectrometer Systems to Facilitate Data duration thermal waves, due to infrared absorption, were studied by monitoring the Comparison and Exchange resulting excitation of the contact resonance modes of the AFM cantilever. Differ- Aaron Urbas, National Institute of Standards and Technology, 100 ences in the IR spectra as a function of spatial position provide insight into polymer Bureau Dr., Gaithersburg, MD 20899, Steven Choquette blend miscibility, microdomain formation, and can lead to increased understanding Raman spectroscopy is an analytical technique enjoying rapid growth because it of how fibers and other nanomaterial additives affect the molecular structure and offers the potential for rapid analysis with a minimal of sample preparation. Some properties of polymer materials. Several example applications in the polymer and applications include confirmation of chemical identity, discrimination between poten- life sciences are presented. tially harmful and benign substances and the ability to distinguish counterfeits from real products. However, comparison of Raman spectra obtained on different spec- 289 The Emergence of Computational Photography: Interpretation, trometer systems is challenging because spectra can be significantly influenced Measurement, and the Quantification of Surface and Material by a variety of factors including, but not limited to, instrument response function, Property Change spectral resolution, laser excitation wavelength and sample orientation effects. This Mark Mudge, Cultural Heritage Imaging, 2325 3rd St., Suite 323, San talk focuses on instrument calibration and characterization approaches intended Francisco, CA 94107, Carla Schroer to facilitate spectral comparison across spectrometer systems. Specifically, relative Computational Photography extracts and synthesizes information from image se- intensity response correction, Raman shift standardization and compensation for quences to create a new type of image containing information not found in any spectral resolution differences will be addressed. Other instrumental obstacles to single image in the sequence. Examples of the existing and cutting-edge uses of inter-system spectral compatibility, such as polarization biases, and approaches at photography-based imaging are explored. The presentation discusses Computa- minimizing their impact are also discussed. Finally, comparisons of standardized tional Photography tools Reflectance Transformation Imaging, Algorithmic Render- spectra across several Raman spectrometer systems are presented to highlight ing, Structure from Motion Photogrammetry and Multi-spectral imaging examples. both the efficacy and limitations of these standardization practices. New research from Princeton University, Simon Frasier University, and the Georgia O’Keeffe museum demonstrate how these techniques can be used to track changes 286 Quality-by-Design and Spectroscopy: The Perfect Pair in object surfaces. We also explore the necessity for transparent evalaution of sci- John Wasylyk, Bristol-Myers Squibb, One Squibb Dr., New Brunswick, entific digital representations. The goal is to establish the conditions under which NJ 08901, Daniel Hallow, Ming Huang, Douglas McLeod, Robert a “real world“ subject can be digitally represented as a “digital surrogate”, which Wethman can reliably serve as a digital stand-in that can be used for subsequent scientific In the pharmaceutical industry, quality-by-design (QbD) can serve as a critical tool or scholarly examininations. Computational photography tools lend themselves to in the development of drugs and enhance the quality of the drug product during meeting these requirements. the manufacturing process. Spectroscopy plays a key role in enhancing process knowledge by gaining key information that allows the research scientist to estab- 290 Advanced Spectral Imaging for Noninvasive Preservation of lish critical design spaces. We have utilized in-line and off-line spectroscopy tools Cultural Heritage Materials that enable rapid analyses with minimal sampling. We will present examples which Fenella G. France, Library of Congress, 101 Independence Ave. SE, include in-line Raman Spectroscopy for following the progression of reactions, in- Washington DC 20540 cluding form control, and during the process development phase that not only define An ongoing challenge associated with the preservation of cultural heritage is the design space limitations but also provided valuable kinetic information. In addition, need for advances in non-invasive, non-destructive analytical techniques that can we have utilized off-line spectroscopy techniques to facilitate rapid evaluation of be used to identify and analyze substrates (paper, parchment) and media (inks, pig- process steps during the screening and evaluation stages. Both in-line and off-line ments, colorants). Spectral imaging systems developed for astronomical imaging spectroscopy techniques have played a key role in defining not only the knowledge and remote sensing have been adapted and customized for the cultural heritage transferred to manufacturing but have also provided insight as to whether or not field. The Library of Congress Preservation Research and Testing Division has de- specific in-line spectroscopy methods would be critical in manufacturing. veloped the application of hyperspectral imaging to the preservation and analysis of cultural heritage materials as a powerful, non-invasive technique with a range 287 Handheld Spectrometers: Providing Answers in the Field of capabilities. Advanced spectral imaging allows non-invasive characterization of Richard A. Crocombe, Thermo Fisher Scientific, 2 Radcliff Rd., materials, providing enhanced non-visible and visible information in high-resolution Tewksbury, MA 01876 registered digital images. This imaging non-invasively characterizes colorants, inks “Small” spectrometers fall into three broad classes: small versions of laboratory in- and substrates through their spectral response, monitors deterioration or changes struments, providing data, subsequently processed on a PC; dedicated analyzers, due to exhibit / environmental conditions, and assesses and tracks potential con- providing actionable information to an individual operator; and process analyzers, servation treatments. Additional capabilities include the detection and capture of providing quantitative or semi-quantitative information to a process controller. The obscured and hidden text, layering of information on the object, and capture of wa- emphasis of this paper is on handheld dedicated analyzers. Many spectrometers termarks on paper. The resulting image cube creates a new “digital cultural object have historically been large, possible fragile, expensive and complicated to use. (DCO)” – related to, but distinct from the original. The range of data this object The challenge over the last dozen years, as instruments have moved into the field, contains allows multidisciplinary collaboration for integration of preservation and has been to make spectrometers smaller, affordable, rugged, easy-to-use, but most cultural information. A range of case studies are utilized to discuss the challenges of all capable of delivering actionable results. Actionable results can dramatically associated with characterizing cultural heritage materials and the creation of a stan- improve the efficiency of a testing process and transform the way business is done. dardized data set for preservation scientists and scholars. The imaging program There are several keys to this handheld spectrometer revolution. Consumer elec- includes the integration of data from other non-invasive analytical techniques, to tronics has given us powerful mobile platforms, compact batteries, clearly visible create a full mapping of cultural heritage objects. displays, new user interfaces, etc., while telecomm has revolutionized miniature optics, sources and detectors. While these technologies enable miniature spectrom- 291 & Watching Paint Age I & II eters themselves, actionable information has demanded the development of rugged Melinda H. Keefe, Michael Linsen, The Dow Chemical Company, 727 algorithms for material confirmation, unknown identification, mixture analysis and 292 Norristown Rd., Spring House, PA 19477 detection of suspicious materials in unknown matrices. These algorithms are far The Dow Chemical Company has been conducting exposure tests for over 50 more sophisticated than the ‘correlation’ or ‘dot-product’ methods commonly used years, since its development of waterborne acrylic polymer dispersions. Dow sci- in benchtop instruments. Finally, continuing consumer electronics advances now entists employ its exposure stations as outdoor laboratories all over the world to enable many more technologies to be incorporated into handheld spectrometers, evaluate performance of exterior paints, coatings and caulks as well as the compo- including Bluetooth, wireless, WiFi, GPS, cameras and bar code readers, and the nents employed in such formulations. The data gained from exposure testing have continued size shrinkage of spectrometer ‘engines’ leads to the prospect of dual played and continue to play a vital role in the company’s effort to provide the latex technology or ‘hyphenated’ handheld instruments. architectural coating industry with technologically advanced products for exterior 46 2013 EAS Abstracts November 2013

paints. Through this research there has been in-depth analysis of varying failures of discovery-phase animal PK/TK studies. The potential for extending the universal observed in acrylic paints exposed outdoors. These failures include gloss and color assay approach into clinical studies is also discussed. retention loss, efflorescence, adhesion failures to varying substrates, cracking, flak- ing, soiling, and blistering, to name but a few. This presentation, divided into two 296 Quantitative LC-MS/MS Analysis of NanoRNAs in Escherichia Coli parts, provides an overview of the composition of water-borne acrylic paints, and Landon Greene, Rutgers University, 43 Putnam St., Somerville, NJ the exposure testing approach, image collection, and analytical analysis of paint 08876, Jeehiun Lee, Mu Chen performance utilized at Dow. Following this review, case studies utilizing analytical Although it is widely accepted that RNA transcription is initiated de novo (i.e.; RNA imaging techniques to probe acrylic paint performance are discussed. polymerase, RNAP, initiates RNA synthesis using free NTPs alone), significant in-vitro evidence supports that both eukaryotic and prokaryotic RNAPs can utilize 293 Quantitation of an Adenine Adduct of the Cytotoxin of an Antibody small RNA oligomers ranging in length from ~2-5 nucleotides, “nanoRNAs”, to prime Drug Conjugate BMS-936561 in Urine as Potential transcription initiation. Recently, studies have been completed which show that Pharmacodynamic Biomarker of DNA Binding nanoRNAs do prime transcription initiation also in-vivo, in prokaryotic cells. These John Lute, Bristol-Myers Squibb, PO Box 4000, Princeton, NJ 08543, high-throughput DNA sequencing experiments provided information into the rela- Jian Wang, Anne-Françoise Aubry tive abundance of various nanoRNAs species present during transcription initiation. Bristol-Myers Squibb (BMS)-936561 is an antibody drug conjugate (ADC) consisting However, the exact RNA species and absolute quantities of these nanoRNAs need- of a human monoclonal antibody targeting cluster of differentiation 70 (anti-CD70 ed to be determined by a specific and sensitive analytical method in order to fully mAb) conjugated to DNA minor groove binding agent MED-2460. BMS-936561 is support this theory. In this research, a specific and sensitive high-performance liquid designed to deliver its cytotoxic agent selectively to CD70-expressing tumor cells, chromatography tandem mass spectrometry (HPLC-MS-MS) method has been de- resulting in DNA alkylation, inhibition of cell proliferation, and subsequent cell death. veloped in order to fully study the nanoRNA species present throughout cell growth. MED-2526 is the active DNA binding moiety of this drug candidate. An adenine This method uses a hydrophilic interaction liquid chromatography (HILIC)-ion pair- adduct of MED-2526, designated as MED-2620, produced as a result of DNA ca- ing dual mode of chromatographic separation and tandem MS to generate specific tabolism, has been identified as a potential pharmacodynamic biomarker of BMS- and sensitive quantitative values. With a linear ion trap mass spectrometer, the de- 936561 binding to its target. A sensitive liquid chromatography tandem mass spec- tection limit of this method was determined to be ~ 5 pmole for several nanoRNA trometry (LC-MS-MS) method to analyze MED-2620 in urine has been developed. species in cell lysates. The method precision was shown to be <20% at the limit of The chromatography method and sample extraction procedure were screened and quantitation. Quantitation in cell lysates was performed by standard addition, due to optimized. Non-specific binding of MED-2620 in urine samples was evaluated. A limited resources. The results generated demonstrate the ability of the developed sample collection procedure was established to eliminate the adsorption issue by method to quantitatively identify various nanoRNA species during E. coli cell growth; the addition of a surfactant in urine samples. The presentation summarizes the which has been used to further study the role of nanoRNAs in transcription initiation. method development, qualification, and sample analysis for this unusual analyte in a proof of concept mouse study. 297 Characterization of Curcumin-Nicotine Interaction in Cetyltrimethyammonium Bromide Micelle 294 Detection of Fentanyl Analogs and Metabolites in Four Biological Maurice O. Iwunze, Morgan State University, 1700 E. Cold Spring Lane, Matrices; Urine, Plasma, Dried Urine Spots, and Dried Blood Spots Baltimore, MD 21254, Yongchao Zhang Rebecca L. Shaner, Centers for Disease Control and Prevention, 4770 UV-Vis spectrophotometric technique was used to characterize the interaction of Buford Hwy., Atlanta, GA 30341, Pearl Kaplan, Courtney T. Callahan, curcumin with nicotine in a cetytrimethylammonium bromide (CTAB) micelle. It is Elizabeth I. Hamelin, Rudolph C. Johnson observed in this medium that curcumin and nicotine interact in a 1:1 ratio using the Human exposure to chemicals, such as fentanyls, can be confirmed through the molar ratio technique. However, using the Hildebrand-Benessi equation the molar analysis of blood and/or urine. Methods of collecting, shipping and storing these absorptivity, ε, of the curcumin-nicotine complex was found to be 29145.15 and specimens can vary dependent upon available resources. Blood and urine speci- the association constant, Ka, was 4070.11/M. The free energy of association, ΔGa, mens are typically collected at a hospital visit, following an exposure, but may be was calculated to be -20.59 kJ, an implication that the complex is spontaneous and challenging to ship and store. For this reason biological matrices were evaluated in stable. both liquid and dried spot formats for the identification of nine fentanyls and three metabolites. Fentanyls, powerful narcotic analgesics with symptoms similar to other 298 Determination of Phthalate Metabolites, Bisphenol A Glucuronide, opioids, are both medically prescribed and illicitly used. A quantitative method using 1-Hydroxypyrene Glucuronide, Perfluorinated Chemicals and a pentafluorophenyl liquid chromatography column with tandem mass spectrometry Leukotriene E4 in Urine was developed to detect these compounds. Precision and accuracy were improved Shin-Hung Liu, National Taiwan University, 17 Hsu-Chou Rd., Taipei using select deuterated internal standards. For the urine and plasma samples, solid City, 10055 Taiwan, Chia-Yang Chen phase extraction was optimized and automated in a 96-well format, while the dried Biomonitoring on multiple biomarkers or compounds is an important approach for blood and urine spots were extracted directly from the paper. By creating one liquid human exposure. This study developed an analytical method that extracted sev- chromatography tandem mass spectrometry method for multiple sample types, we eral metabolites of compounds relating to anaphylaxis, including five phthalate me- are prepared to handle various samples that maybe available to confirm exposure tabolites, bisphenol A glucuronide, nine perfluorinated chemicals, 1-hydroxylpyrene to these compounds. glucuronide and leukotriene E4, in the total of 17 analytes from human urine using 96-well Oasis WAX μElution plates, and were analyzed by ultra-performance liquid 295 Universal LC-MS-MS Assays for Bioanalysis of Human Monoclonal chromatography/tandem mass spectrometry with negative electrospray ionization. Antibody Drug Candidates in Pharmacokinetic / Toxicokinetic Two ion transitions were recorded on each analyte using selected-reaction monitor- Studies ing and isotope-dilution techniques were used for the quantification. A fused-core Michael T. Furlong, Bristol-Myers Squibb, Route 206 & Province Line Ascentis Express C18 column (50 × 2.1 mm, 2.7 μm) was for chromatographic Rd., Princeton, NJ 08543 separation with acetonitrile and 0.04% acetic acid as mobile phases. One hundred Monoclonal antibody (mAb)-based therapeutic agents play a central role in the and fifty micro-liters of urine were mixed with the same volume of 10 mM ammo- treatment of many human diseases. The rapidly increasing number of mAbs enter- nium acetate aqueous buffer, and were spiked with stable isotope-labeled internal ing pre-clinical drug development represents a daunting challenge to bioanalytical standards in methanol; after vortex, the sample was passed through the conditioned laboratories, particularly during early pre-clinical animal studies wherein multiple plate. After two steps of wash, analytes were eluted with 0.1% ammonium hydroxide structural variants of each drug candidate are evaluated in various animal species to in methanol. This high-throughput assay took about 3 min averagely in sample prep- enable prioritization for further development. Liquid chromatography tandem mass aration and 7 min in the instrument analysis with no need of overnight enzymatic spectrometry (LC-MS-MS)-based assays for bioanalysis of human monoclonal anti- hydrolysis to cleave the glucuronide group. The limits of detection on the analytes body (mAb) drug candidates typically employ trypsin digestion of the matrix sample ranged from 0.1 to 37 ng/mL. followed by quantification of a surrogate “signature” tryptic peptide whose amino acid sequence is unique to the mAb analyte. A drawback of assays developed with 299 Imputation of Incomplete Data in Classification Analysis this approach is they can only be applied to one mAb, and thus each new mAb Yushan Liu, University of Delaware, Department of Chemistry and entering the bioanalytical laboratory requires the development of a new assay to Biochemistry, Newark, DE 19713, Steven D. Brown facilitate its quantification. To overcome this drawback, we have devised a surrogate Most chemometric methods work with complete datasets only. If a dataset is incom- “universal” peptide strategy as an alternative to the traditional signature peptide plete, an imputation method can be used to fill in the missing values and the imputed approach. This presentation describes: 1) the identification of universal peptides dataset is then ready for use. Various imputation algorithms have been shown to be found in the constant regions of the majority of human mAb drug candidates; 2) effective when imputing data missing under a Gaussian distribution model. How- incorporation of these universal peptides into discovery-grade universal LC-MS-MS ever, problems arise when imputation algorithms encounter an incomplete dataset assays; and 3) successful deployment of the universal LC-MS-MS assays in support consisting of two or more different groups having different distributions. We report 47 2013 EAS Abstracts November 2013

an imputation method for incomplete multivariate datasets drawn from a heteroge- 303 A “Built-in-Quality” Analytical Separation Method Obtained neous group of populations. Considering the different groups as components of a through QbD concepts: Method Development, NDA Validation and finite mixture, the new method fits the data to a finite-mixture model, estimating the Technique Transfer model parameters with the expectation-maximization algorithm (EM algorithm), and Li Li, Bristol-Myers Squibb, One Squibb Dr., New Brunswick, NJ 08903, then draws the missing values from the conditional, predicted distributions of the Peter Tattersall missing values. To assess the performance of this method, a comparison is made Quality-by-design (QbD) has been widely adapted to drug development in recent of the behavior of the new imputation approach to that of other, common imputation years. The application of QbD concepts to develop analytical separation methods methods on both simulated datasets and on real datasets. Special emphasis will be has increased as they are an essential part of the pharmaceutical product develop- given to a comparison of classification performance after imputation. The preserva- ment process. In this presentation we describe a systematic approach to develop tion of data structures upon imputation will also be investigated based on comparing reverse-phase high-performance liquid chromatography (RP-HPLC) methods using the principal component analysis results from the original, complete data with those QbD principles, which includes defining an analytical target profile (ATP), risk as- from the imputed data. sessment to determine areas of concern and unknown influences, the application of DryLab and Fusion AE chromatographic modeling and design of experiments (DOE) 300 Geospatial Pattern Recognition: Geographical Pattern Knowledge software to develop and optimize the method. In addition, Fusion AE was used to Discovered from Surface Water Data predict the operating range of key parameters such as mobile phase composition, Liyuan Chen, University of Delaware, 163 The Green, Newark, DE gradient slope, column temperature and amount of additives. These variables are 19716, Steven D. Brown discussed in terms of their impact to the method; mobile phase selection and dilu- Geographical pattern recognition is frequently used to identify origins of samples ent optimization with regard to selectivity will be highlighted. New drug application based on chemical measurements. However, the estimation of the geographical validation of the method was performed as predicted for both ultra-HPLC and HPLC location in terms of longitude and latitude of a sample is relatively unexplored. A versions of the method under the defined control strategy. Both UHPLC and HPLC systematic way combining geographical classification and regression is developed methods were transferred to three global sites successfully. As a result of these to identify samples’ location and the associated uncertainty of location based on studies, the operating range of the method is well understood, which has resulted in chemical signatures by using a hierarchical tree structure model. The methodology a consistent and controlled execution of the method in multiple labs. is applied to surface water data from US Geological Survey to estimate the locations of surface water samples and may be extended to other spatial data. By taking ad- 304 Determining Particle Size Distribution Effects on Acoustic vantaging of seasonal variations, the study is extended to permit more detailed and Emission reliable exploration of chemical signatures for more flexible geo-location estimation. Shikhar Mohan, Duquesne University, 600 Forbes Ave., Pittsburgh, PA This talk considers advances in the use of the tree-structured modeling of geospatial 15282, Benoit Igne, Carla A. Anderson, James K. Drennen III data, with the aim to geo-locate the samples based on chemical signature more High-shear wet granulation is one of the most commonly used granulation process- quickly and accurately with less uncertainty. es in the pharmaceutical industry. Advantages of high-shear wet granulation include low-friability, spherical, rapidly forming granules. Fast granulation growth can lead to 301 Examination and Dating of Silver Gelatin Fiber Based Photographic over-granulation if not carefully controlled. Few measurement systems are capable Papers Using Infrared Spectroscopy and PLS-GLSW Modeling of in-process determination of the quality parameters of high-shear granulation. In Donald B. Dahlberg, Lebanon Valley College, 101 N College Ave., this work, we examine effects of particle size on acoustic emission as a prelude to Annville, PA 17003, Ana Martins monitoring particle size during high shear wet granulation process using a piezo- Gelatin silver developing-out papers were introduced in the 1880s and became electric microphone sensitive in the audible range (20-2000 Hz). Passive acoustics the most prevalent photographic papers until their popularity faded with the advent measurement systems are used to analyze the acoustic emissions from the process. of color processes followed by the digital era. Attenuated total reflectance Fourier A high-shear granulator includes an impeller, for mixing and shearing purposes; and transform infrared spectra (ATR/FTIRS) were taken of a set of over 300 dated film a chopper, for breakage of larger granules. During this process, acoustic emissions stills of American movies selected from the MoMA Film Stills Archive. These photo- are being produced from particle-particle collisions, particle-wall collisions, particle- graphs spanned some seven decades and their compositions reflect changes and impeller collisions, particle-chopper collisions, and machine noise. To reduce the developments in the paper and photographic paper industry during this period. After effects of machine noise, dry mixing experiments were performed in a small scale dividing the samples into a calibration and test set, partial least squares (PLS) mod- simulator with microcrystalline cellulose spheres (Cellets). The design of experiment els were constructed to predict the date of the photographs. PLS models using only included three impeller speeds and six particles sizes of Cellets. Results from mul- multiplicative signal corrected (MSC) data resulted in a root-mean-square error of tivariate analysis (principle component analysis and partial least squares) showed prediction (RMSEP) of 8.7 years using 6 latent variables. The scores and loadings specific frequencies that were experimentally well correlated with particle size. With plots gave no interpretive results. Generalized least squares weighting (GLSW) is this approach, only specific frequencies were then monitored in a high-shear granu- a pretreatment method that deweights variables unrelated to the property being lation process where results demonstrated that acoustic emissions monitoring have predicted. The application of GLSW only slightly improved the RMSEP to 7.9 years, potential for use as part of a process analytical system for this unit operation. but required only 3 latent variables. Examination of the scores plots clearly revealed the dates of compositional changes in the production of the paper as well as a better 305 Characterizing and Optimizing the use of Flow Additives in understanding of the aging process of the paper. The application of GLSW pretreat- Powders and Powder Formulations ment can result in much more parsimonious models that reveal more interpretable Tim C. Freeman, Freeman Technology, One Miller Court, Severn Dr., information and more robust predictions. Tewkesbury GL20 8DN, United Kingdom, John Yin, Michael Delancy, Brian Armstrong, Katrina Brockbank 302 Raw Material Characterization for Mammalian Cell-Cultures Using Many pharmaceutical powders have poor basic flow properties; they stick in hop- Spectral Technologies pers, exhibit inconsistent or pulsatile discharge rates; adhere to the surfaces of pro- Nicolas A. Trunfio, University of Massachusetts-Lowell, One University cessing equipment and are difficult to mix with other materials. In some instances Ave., Lowell, MA 01854, Haewoo Lee, Seongkyu Yoon it is possible to intentionally modify the powders’ flow properties by means of flow One of the primary issues with characterizing the variability of raw materials used in additives, lubricants or glidants in order to improve their process-ability. The flow mammalian cell culture, such as wheat hydrolysates, is that the analyses of these additive reduces particle-particle friction and therefore changes the powder’s bulk materials can be time consuming and the results of the analyses are not straight- resistance to movement and ability to flow. However, adding too little flow additive forward to interpret. To solve these issues, spectroscopy can be combined with will result in insufficient improvement of the processing performance; adding too chemometrics to provide a quick, robust and easy to interpret raw material char- much will impact manufacturing costs and may even result in a powder that is unpre- acterization paradigm. Cell culture performance will increase as a result of being dictable and unmanageable (uncontrollable powder flushing is a distinct possibility). able to correlate the variability structure with cell culture performance indices. In this Understanding the precise impact of using a flow additive in any given formulation study, four spectroscopic technologies: infrared spectroscopy, Fourier transform in- is a key part of any Quality by Design analysis, especially when studies have indi- frared spectroscopy, Raman spectroscopy and fluorescence spectroscopy are used cated that optimum levels of additive may exist for a given flow regime and there is in conjunction with principal component analysis to characterize the variability of unlikely to be a single level that optimizes flow-ability in consolidated, conditioned wheat hydrolysates and demonstrate that classification of good and bad lots of raw and aerated or fluidized flow regimes. This study investigates how different powders material is possible. Then, the same spectroscopic platforms are combined with are affected by flow additives by using a powder rheometer to measure dynamic as partial least squares regressions to estimate cell culture critical quality attributes well as bulk and shear properties. The analysis of the data demonstrates how the (CQAs): integrated viable cell density (IVCD) and titer. The results show that all four optimum levels of flow additive for each powder type in each of these measurement technologies can be used to estimate the CQA’s with Ramen Spectroscopy being regimes can be identified and reviews the benefits and possible consequences of the best technology for estimating IVCD and fluorescence spectroscopy being the using flow additives in several applications or processes. best technology for estimating titer. 48 2013 EAS Abstracts November 2013

306 Survey of Practices and Recommendations for Toxicological 309 Case Reports in the Analysis of Designer Drugs Investigation of Drug-Impaired Driving and Motor Vehicle Fatalities Jeffery Hackett, United Chemical Technologies, 2731Bartram Rd., Kayla J. Lowrie, Center for Forensic Science Research and Education, Bristol, PA 19007, Albert A. Elian 2300 Stratford Ave., Willow Grove, PA 19090, Jennifer Turri, Jillian In this presentation attendees learn about two case reports involving newer de- Yeakel, Barry K. Logan, Jennifer Limoges, Amy Miles, Colleen Scarneo, signer drugs i.e., methoxetamine and AM-2201. These cases both involve driving Sarah Kerrigan, Laurel Farrell of motor vehicles under in the influence of drugs in US. It gives attendees an in- After attending this presentation, attendees are able to compare their laboratory’s sight into the behavior and analysis of these drugs from biological fluids. The first practices with peer laboratories and evaluate typical cutoffs used for drug screen- case report describes the pre-history and standard field sobriety test (SFST) reports ing and confirmation. This presentation describes data from a survey carried out to pertaining to the detention of the offender. The presentation discusses the extrac- evaluate the practices of forensic toxicology laboratories performing analysis in the tion and analysis of whole blood samples using solid-phase extraction (SPE) and investigation of driving under the influence of drugs (DUID) cases. The survey was liquid chromatography-tandem mass spectrometry (LC-MS-MS). The offender was sponsored by the National Safety Council’s Alcohol, Drug and Impairment Division found to have not only methoxetamine (10 ng/ mL) but also clonazepam (300 ng/ (formally known as the Committee on Alcohol and Other Drugs). This presentation mL), 7-aminoclonazepam (34 ng/ mL), carboxy-THC ( 6 ng/ mL), diphenhydramine also describes the review and update of a set of minimum recommendations for (47 ng/ mL), 3,4-Methylenedioxymethamphetamine (MDMA) (83 ng/ mL) and MDA. the toxicological investigation of suspected alcohol and drug impaired driving cases These results are discussed in light of other findings. In the second case report, a and motor vehicle fatalities involving drugs or alcohol. The recommendations are pre-history and results of SFST’s are presented. In this case, the SPE extraction and based on a survey of practices in US laboratories performing this kind of analysis, LC-MS-MS analysis of not only the urine but also associated plant material will be consideration of existing epidemiological crash and arrest data, and practical con- presented. The offender’s urine was found not to contain any common drugs apart siderations of widely available technology platforms in laboratories performing this from carboxy-THC, subsequent analysis determined the presence and quantity of work. The final recommendations were derived from a consensus meeting of ex- AM-2201 metabolite. Details of the confirmation and quantification of the particular perts recruited from survey respondents and the membership of the National Safety metabolite gives attendees a better understanding of the toxicology of this drug. Council’s Alcohol, Drug and Impairment Division. The recommendations have the goal of ensuring that a consistent set of data regarding the most frequently en- 310 The Analysis of Challenging Samples Using Ion Analysis countered drugs linked to driving impairment is collected for practical application in Techniques the investigation of these cases, and to allow epidemiological monitoring and the Peter Bodsky, Thermo Fisher Scientific, 1228 Titan Way, Sunnyvale, CA development of evidence-based public policy on this important public safety issue. 94085, Art Fitchett, Rong Lin, Sheetal Bhardwaj, Kannan Srinivasan, Chris Pohl 307 SPE in Forensic Toxicology The steady supply of good quality drinking water is very important to the general Michael J. Telepchak, United Chemical Technologies, 2731 Bartram public health. Drinking water comes from many different sources. They typically Rd., Bristol, PA 19007 go through purification and disinfection before released for public consumption. To In the field of forensic toxicological analysis, providing the correct result(s) for drugs ensure the quality of drinking water, the drinking water has to meet the various and metabolites is the aim of all analysts. In order to isolate them from challeng- federal and state standards. Ion chromatography is a commonly used technique for ing matrices such as whole blood, tissues and (increasingly) alternative matrices monitoring the ionic species. However, many of the contaminants present in drink- such hair, nails, and insects, they must use methods that will extract them not only ing waters are in low ppb range, while the matrix ions are in the ppm range. This efficiently but cleanly and reproducibly too. In this presentation given by one of the poses a separation challenge as the resolution between the analytes of interest innovators of solid-phase extraction (SPE), attendees learn about the principles and the matrix ions deteriorates and the quantitation of the analytes becomes diffi- underpinning the chemistry of SPE and its phases, applications relating SPE and cult. Recently several new techniques have been introduced to meet this challenge, forensic toxicology. The mechanisms of SPE discussed here go to the heart of the including a new ion analysis methodology termed two-dimensional matrix elimina- extraction process, leading to the attendees having a better understanding of the tion ion chromatography. This methodology uses a large format column in the first relationship between chemical structures, their Pka values, and the SPE method dimension to separate the analytes from the matrix ions. The suppressed effluent optimized for that extraction. This presentation discusses the application of acid, portion containing the analytes is then concentrated onto a concentrator column neutral, and basic drug extractions and their relationships to general forensic toxi- and subsequently analyzed in the second dimension using a small format column cological analysis, and give attendees a better insight into why SPE is the most with a different selectivity, thus resulting in enhanced sensitivity and selectivity. In efficient analytical extraction technique in forensic toxicological analysis. this presentation, we show the application of this new methodology to the analysis of various trace contaminants in drinking waters. 308 Simultaneous Quantification of Opiates and Polar Metabolites in Urine and Blood by SPE and HILIC/MS/MS 311 Improved Determination of Trace Concentrations of Perchlorate in James N. Anasti, Cedar Crest College, Department of Chemistry, 100 Drinking Water with Analytical/Capillary Two-Dimensional Ion College Dr., Allentown, PA 18104, Thomas A. Brettell Chromatography Hydrophilic interaction liquid chromatography (HILIC) has become a chromatogra- Peter Bodsky, Thermo Fisher Scientific, 1228 Titan Way, Sunnyvale, CA phy tool to better retain polar analytes as compared to reversed-phase chromatog- 94085, Art W. Fitchett, Lillian Chen raphy. HILIC uses a mixed-mode system involving a polar stationary phase and a Perchlorate is identified as an environmental contaminant found in drinking, ground, mostly organic mobile phase with some water. The polar water favors the hydro- and surface waters. Perchlorate contamination is attributed to the manufacture philic moieties of the column packing and creates a stagnant layer in which a parti- and use of ammonium perchlorate in solid propellant for rockets, missiles, and fire- tion occurs with the mostly organic mobile phase allowing for the polar analytes to works. Research on perchlorate has received a lot of attention because perchlorate interact more with the column and elute last. In this presentation, zwitterion-HILIC is found to impair normal thyroid function by interfering with iodine uptake by the demonstrates the ability to analyze low concentrations of polar metabolites with- thyroid gland. Ion chromatography (IC) has been recognized as an effective tool for out sacrificing selectively between the parent opiates. Analytes chosen are mor- the determination of perchlorate in drinking water. The United States Environmental phine and codeine and their glucuronides including morphine-6-glucuronide and Protection Agency (EPA) Methods 314.0, 314.1, and 314.2 describe the determina- morphine-3-glucuronide, structural isomers. These two metabolites when analyzed tion of trace perchlorate in drinking water using IC with suppressed conductivity separately can provide intoxication levels and predisposition of abuse. Also hydro- detection. This work presents an improvement to EPA Method 314.2 by using an and oxy- derivatives of MOR and COD could be selectively separated from their analytical/capillary two-dimensional (2-D) ion chromatography system. Some drink- associated parents. 6-MAM the primary metabolite of heroin was also included. The ing water sources contain high concentrations of anions, such as chloride, sulfate, high organic mobile phase proved highly compatible with ionization in the electron and carbonate. This can pose a challenge in determining perchlorate at µg/L con- spray ionization interface of the triple quadruple detector. SPE was used to increase centrations with accuracy and precision. Resolving the analyte from the sample signal to noise without the use of a C18 sorbent. This required a two-step elution in- matrix in the first dimension immediately followed by preconcentrating the analyte volving a more aprotic solvent to elute the glucuronides. This presentation describes before the separation in the second dimension using a reagent-free ion chroma- a validated method can determine heroin abuse from regular pain management tography (RFIC) system enhances the level of automation and improves precision. programs involving commonly prescribed morphine, hydrocodone, codeine, oxyco- Significant enhancement in sensitivity is achieved due to a large increase in concen- done, hydromorphone, and oxymorphone. tration factor using the capillary format in the second dimension. The 2-mm/0.4-mm 2-D configuration is operated with a lower flow rate compared to the 4-mm/2-mm 2-D configuration in Method 314.2, thus greatly reducing reagent consumption and system maintenance. This method demonstrates a MDL of 0.005 µg/L and good recovery results of perchlorate in different sample matrices were obtained.

49 2013 EAS Abstracts November 2013

312 Determination of 100 Pesticides in Water Using Ultra-Performance 315 Withdrawn by the author. Liquid Chromatography-Tandem Mass Spectrometry with Electrospray Ionization Ming-Tsung Lu, National Taiwan University, 17 Hsu-Chou Rd., Taipei 316 Preconcentration and Quantitative Determination of Venlafaxine City, 10055 Taiwan, Chia-Yang Chen HCl Present in Water Pesticide residues in water are a serious concern to the aquatic ecosystem. This Prakash B. Samnani, The Maharaja Sayajirao University of Baroda, study developed and validated a method to analyze five classes of pesticides us- Department of Chemistry, Faculty of Science, Vadodara 390002, India, ing high-flow solid-phase extraction and reversed-phase ultra-performance liquid Santosh K. Koppula chromatography-tandem mass spectrometry with electrospray ionization. Analytes A simple method for pre-concentration of venlafaxine HCl from its aqueous solu- included 62 insecticides, 8 fungicides, 28 herbicides, 1 acaricide and 1 nematocide. tion has been developed. The method is based on solid-phase extraction on com- Five hundred milliliters of water samples were extracted with 50-mm PolarPlus C18 mercially available polystyrene divinyl benzene polymer, used in the form of 8% Speedisk at a flow rate of 70-80 mL/min using an automated solid-phase extraction cross-linked macroporous beads. The drug was recovered from the polymer using system and analytes were eluted with methanol and dichloromethane. The eluent methanol followed by quantitation using high-performance liquid chromatography. was concentrated to 5 mL for the instrumental analysis. Quantification was done Various experimental parameters for extraction were studied and optimized so as to using a matrix-matched calibration curve. Analytes forming positive ions and nega- achieve a maximum pre-concentration factor of 10. The method enables determina- tive ions were separated on a Kinetex PFP column (50 mm x 2.1 mm, 2.6 ¼ m) and tion of the drug in water sample over the linear concentration range of 0.03 – 0.95 an Ascentis Express C18 column (50 mm x 2.1 mm, 2.7¼m), respectively, and the ppm (mg / L) and limit of detection and limits of quantitation of 0.03 and 0.24 ppm chromatographic time required only 8 min for each run including re-equilibrium. The respectively. The method was then applied to treated sewage water which did not recoveries ranged from 62.4% to 99.8%, and the precision in % relative standard show presence of the target drug at these concentration levels. deviation was less than 30%. The limits of quantitation (LOQs) were from 0.50 to 2.5 ¼ g/L. A total of 35 water samples were collected from three rivers in southern 317 Online Implementation of NIR – Lessons Learned (or not) Taiwan, a reservoir as a drinking water source, raw water and finish water from a Susan Foulk, Guided Wave, 3033 Gold Canal Dr., Rancho Cordova, drinking water treatment plant, and influent/effluent from a sewage treatment plant; CA 95670 most analytes were either not detected or with levels lower than the LOQs. Among Near-infrared (NIR) spectroscopy applications span a variety of fields including as- positive samples, the most frequently detected were acephate, fonofos, phosmet, tronomy, agriculture, medicine, remote sensing, pharmaceutical, and chemical to cartap, carbendazim, thiophanate-methyl and pendimethalin in the river water, and name a few. The use of NIR for online chemical process measurements should the highest concentration was carbendazim (8.7¼ g/L) in the river water. now be considered a mature analyzer technology. Several decades of experience in design, implementation and installation of NIR analyzers in process environments 313 Fracking, Boldly Going where no Ion Analysis has Gone Before reveals varying levels of success in NIR analyzer projects. Success for these proj- Stuart J. Procter, Metrohm USA, 6555 Pelican Creek Circle, Riverview, ects relies on multidisciplinary coordination of a number of areas including project FL 33578, Jay Gandhi, Johnson Matthew management, process chemistry, process engineering, sampling, process control, Oil and natural gas as fossil fuels are valuable diminishing resources that are uti- business goals, and safety along with overall instrument performance and method lized for energy and materials production. These naturally occurring products cannot development (chemometrics) tasks. Looking at project success from an analyzer be easily replenished, so fracking is becoming of increasing importance to extract vendor point of view over time brings out a list of best practices for successful natural gas and oil from the shale formation around the globe. For the USA this local analyzer implementation. This presentation reviews several NIR process analyzer resource can have a significant impact on long term economic stability. Recently de- projects that highlight common issues and pitfalls that are encountered. In theory, veloped new fracking technology represents a doorway to vast untapped reserves, learning from the experience gained in these projects would seem straightforward. but with it comes environmental concerns and analytical challenges that need to be A related quote (no attribution) would seem to apply, that ‘theory and reality are addressed. Therefore, analytical techniques for the long term monitoring of ground only theoretically related’, because we tend to repeat the same learning exercises. and surface water are necessary for environmental conservation to help aid in the detection of accidental leakage from bored wells. Furthermore, for waste water 318 Monitoring, Online and in Real Time, the Coating of an Active treatment, the analysis of flow-back waters and discharge water from the fracking Solution onto Tablets by Near-Infrared Spectroscopy process is required. The waters from the fracking process can have up to 30% Benoit Igne, Duquesne University, 600 Forbes Ave., Pittsburgh, PA sodium chloride as an ionic matrix. This type of water matrices can be challenging 15282, Hiroaki Arai, Hanzhou Feng, James K. Drennen III, Carl A. for analytical chemists and instrumentation. This talk presents new advances in the Anderson field of ion chromatography (IC) analysis that utilizes modern instrument technolo- Tablet coating is primarily employed in the pharmaceutical industry to give solid gies to minimize these challenges. These new methods enable the simultaneous dosage forms an aesthetic appearance, mask undesirable taste, and control the analysis of anions, cations and organic acids that are of environmental interest in a rate of drug release. It can also be used to coat an active ingredient onto a tablet 30% sodium chloride matrix. Furthermore, we discuss improvements in automation core when a specific release pattern is required or to enhance stability. Typically, technology that enables the combining of standard wet chemistry techniques (titra- a polymer solution/suspension is sprayed onto tablets in a fluidized-bed or a pan- tion) with that of the more sophisticated IC instrumentation. coating system. The coating unit operation is traditionally monitored by performing at-line measurements of quality attributes such as tablet weight gain. More recently, 314 Environmental Impact and Testing Requirements Required in the near-infrared spectroscopy has been used to perform at-line and on-line tablet qual- Process of “Fracking” with a Focus on the Analysis of Methane, ity measurements, consistent with a process analytical technology paradigm. In this Ethylene, and Ethane in Water by Headspace/Gas Chromatography study, the coating of acetaminophen onto tablet cores in a pan coater was monitored (HS/GC) with Flame Ionization Detection (FID) in real-time by near-infrared spectrometry. Processing parameters were set accord- Lee D. Marotta, PerkinElmer Instruments, 710 Bridgeport Ave., Shelton, ing to a design of experiment where spray speed, active concentration, and the CT 06484, Bill Hahn, Cindy Liang coating solution polymer concentration were varied. Near-infrared measurements The rapid development of natural gas from unconventional sources in North Amer- were performed on-line as well as off-line and UV measurements were used to ica has created an energy “gold rush” not seen in contemporary times. The advent develop models and confirm results. Online near-infrared spectroscopy was dem- of horizontal drilling technologies and hydraulic fracturing has made this production onstrated to effectively monitor the coating of the active ingredient and allowed the economical and presents an energy source of sufficient magnitude that could last control of the process, in real time. The accuracy of online measurements was con- 100 years. The technology presents a number of analytical challenges. The wells firmed by off-line spectroscopic evaluations and UV analysis. are drilled vertically through aquifers on their way to the deep shale deposits thou- sands of feet under the surface. In the process of drilling the wells and preparing 319 Multivariate Optical Computing in Oilfield Exploration them for production (including “fracking” to optimize production), opportunities arise Michael Myrick, University of South Carolina, 631 Sumter St., Columbia, for contamination of the clean drinking water aquifers with methane and other low SC 29208 molecular weight organics (e.g., propane and ethane). This presentation discusses Chemical properties of reservoir fluids, such as the gas/oil ratio (GOR), partly deter- how headspace-gas chromatography is an optimum solution to this challenge. Such mine whether a petroleum reservoir can be produced economically. The traditional optimization includes a simplified technique of sampling; ease of instrument op- method of analyzing GOR and other chemical properties is to capture a representa- eration; and enhanced precision and accuracy. In addition, this solution provides tive sample of fluid at the reservoir level down-hole and bring it back to the surface, aggressive detection limits required by this important application. Instrumentation, and then send it to a laboratory. There are a number of problems with this approach, method parameters, data and results are discussed and achieving Environmental including changes in the properties of the fluid as it cools to surface temperature, Protection Agency criteria is demonstrated. and the time required getting an analysis back. While waiting for the laboratory anal- ysis, equipment is tied up in place, leading to costs associated with frozen capital. A

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solution to this inefficiency is down-hole sampling with a tool capable of measuring 323 Recent Innovations in Core Shell HPLC Columns for the Separation chemical properties. Spectroscopic methods with chemometric analyses are among of Biomolecules the best methods for remote chemical measurement, but the high temperatures and Jason Anspach, Phenomenex, 411 Madrid Ave., Torrance, CA 90501, confined space associated with down-hole measurements are a particular challenge Michael McGinley, Lawrence Loo, Jeff Layne for most instruments. Multivariate optical computing (MOC) provides an approach Trends in pharmaceutical development in recent years have seen an ever increas- to making instruments that rely entirely on specially-designed interference filters. ing investment and efforts into bio based therapeutics. Some reports are forecast- In this presentation, we report results from the design, fabrication and testing of an ing that as much as 50% of all new drugs will be protein based. Despite these MOC instrument at temperatures ranging between 150 oF and 280 oF. Analyzing research efforts, there have only been a few innovations in separation tools for three very different oils charged with a range of volatile components to GOR values large and small biomolecules. Up until recently these innovations have centered on between 0-1500 units, we demonstrate an accuracy below 100 GOR units and the the use of sub-2-µm fully porous packing materials. One of the drawbacks to these ability to create optical elements that are independent of temperature over wide miniaturized materials is the significant increase in backpressure that is needed to temperature ranges. operate these columns, necessitating the installation of new instrumentation. The higher backpressure also limits flexibility in terms of optimizing columns dimensions. 320 Near Infrared Spectroscopy for Assessment of Fetal, Maternal and Six years ago saw the reemergence of core-shell based high-performance liquid Infant Health chromatography (HPLC) particles. This latest generation of core-shell materials has David Burns, University of New Brunswick, 3 Bailey Dr., Fredericton, NB been shown in the literature to provide 30 – 50% higher efficiencies than fully po- E3B 5A3, Canada rous materials of an equal size, allowing for the use of significantly larger core-shell Medicine is the 21st century is changing dramatically. With the advent of many new particles without compromising on performance. In this presentation we explore how non-invasive and minimally invasive monitoring methods, rapid point-of-care diag- the higher efficiency per unit back pressure can help in peptide separations. We also nosis is becoming possible. Near-infrared spectroscopy has been shown to have investigate how the core-shell technology can be adapted to create columns specifi- significant potential for in-vivo and in-vitro assessment of biological status. This cally targeted for the separation of large proteins. presentation gives an overview of recent advances in chemometric inspired spec- troscopic measurement for in-vitro assessments and focus on the principles behind 324 Challenges and Rewards for Fast and High-Performance clinical use of several new methods to understand metabolism. As examples, new Techniques for Protein Separations: An Application Point of View methods to assess health such as growth and infection status during pregnancy Phu T. Duong, Agilent Technologies, 2850 Centerville Rd., Wilmington, are presented using a range of biofluids from amniotic fluid to blood plasma. The DE 19808, James Martosella, Andrew Coffey, Keeley Mapp general hypothesis for these measurements is that hydroxyl radical modifications of Size-exclusion chromatography, ion-exchange chromatography, and reversed- underlying constituents such as proteins, lipids and carbohydrates can be detected phase chromatography are popular choices for the analysis of proteins and pep- by biospectroscopy and are selective for biological condition. Results demonstrate tides. Many analytical columns have been developed for fast separation in recent how these simple techniques provide a valuable tool for monitoring of patient status. years, due to increasing demands to save time and resources spent on analysis. These many column options can cause confusion for users when selecting the most 321 High Resolution LC/MS Peptide Separations with Formic Acid appropriate column for method development, because each method requires a thor- Mobile Phases Using Charge Surface Modified C18 Columns ough understanding of the column’s capacity, limitations, and the compatibility of Matthew A. Lauber, Waters, 34 Maple St., Milford, MA 01757, Stephan the column and instrumentation. However, these different parameters also provide M Koza, Kenneth J. Fountain analysts with opportunities to optimize method development for a specific chroma- Chemical modification of a reversed-phase stationary phase can have significant tography. We discuss here the challenges involved in selecting the right column for impact on the peak capacity of a peptide separation. We have investigated the util- different types of chromatography. Such challenges include compatibility between ity of a C18 sorbent with a surface modified to bear a positive charge under acidic columns and instrument systems, buffers, and mass spectrometry compatibility, conditions. It has been found that, unlike conventional C18 columns, a C18 column which can maintain peak resolution, efficiency, and reproducibility. In addition, we modified with a low level positive surface charge provides excellent peak shape with compare modern high-performance and traditional techniques, to highlight the re- either trifluoroacetic acid (TFA) or formic acid (FA)-containing mobile phases. Com- wards of using fast and high-performance methods and columns for protein and pared to unmodified C18 columns, a column packed with charged surface modified peptide analysis. C18 can provide 20% greater peak capacity with 0.1% TFA and 90% greater peak capacity with 0.1% FA. Since a positive surface charge can reduce the amounts 325 Impurities in Drug Products: Recent Updates of mass spectrometry (MS) signal suppressing, ion-pairing additives needed, such Antonio Hernandez-Cardoso, United States Pharmacopeial Convention, as TFA, to achieve high peak capacity separations, a charge surface modified C18 12601 Twinbrook Parkway, Rockville, MD 20852 column was used to develop a high resolution peptide map of the therapeutic an- In an United States Pharmacopeia (USP)/Food and Drug Administration (FDA)- tibody, trastuzumab. High peak capacity separations were obtained at the mass sponsored workshop performed in 2011, attendees explored some key challenges loads needed to detect low abundance modifications, while using a mobile phase posed by over-the-counter (OTC) drugs with regard to quality—one critical factor be- (0.02% TFA/0.08% FA) that provides high MS sensitivity. With these conditions, ing the large number of dosage forms associated with a single drug substance. For electrospray ionization-MS signal obtained with a quadrupole time-of-flight mass example, currently in USP–NF, there are 37 different monographs for acetamino- spectrometer matched or exceeded the signal from an in-series UV detector. Thus, phen dosage forms alone. USP is looking to its chapter approach to help streamline an advantage of applying this column technology is that the same chromatographic some of the quality-related issues inherent to OTCs. General Chapter <1086> Im- conditions are equally suited to UV-based quality control analyses and MS-based purities in Drug Substances and Drug Products includes definitions associated with characterization studies. impurities which are aligned with those established by other pharmacopeias and the International Conference on Harmonization (ICH). Proposed revisions to chapter 322 Advances in Sample Preparation and Chromatographic Separations <1086> are currently being addressed by an Expert Panel with FDA’s participation, in Bioanalytical Analyses which will provide recommendations to the USP’s Chemical Analysis Expert Com- David Bell, Supelco Division of Sigma-Aldrich, 595 North Harrison Rd., mittee, and will include the implementation of a new chapter named <476> Organic Bellefonte, PA 16823, Craig R. Aurand, Gaurang Parmar Impurities in Drug Substances and Drug Products and suggestions to update the For modern bioanalytical studies, it is important for analysts to have access to fast USP General Notices, under the number 5.60 Impurities and Foreign Substances. and robust analytical methods for accurate measurements. Industrial trends toward Both chapters: revised <1086> and new <476>, will take into account ICH Q3A and highly specific liquid chromatography mass spectrometry applications have resulted Q3B guidelines. A stimuli article is being prepared to provide the rationale, strategy in the need for high-speed chromatographic assays along with effective and efficient and understanding of the complexity of the revisions in addition to the information sample preparation procedures. Often the limitation of a bioanalytical technique is already provided in the USP website, under Monograph Modernization: http://www. based upon the effectiveness of the sample preparation technique. Plasma and usp.org/usp-nf/key-issues/monograph-modernization. Also, a survey was launched serum samples are susceptible to irregularities due to matrix-induced interferences. in June 2013 to obtain feedback and comments from stakeholders regarding this Additionally, traditional front-end separation approaches may result in long run times initiative. A summary of the results are presented. and/or insufficient retention and selectivity. Effective and efficient sample prepara- tion and solid chromatographic performance are paramount toward this objective. 326 Dissolution/Drug Release Testing - Compendial Updates This report discusses several modern sample preparation approaches that minimize Erika Stippler, United States Pharmacopeial Convention, 12601 matrix interference while maintaining efficient throughput. In addition, alternative Twinbrook Parkway, Rockville, MD 20852 approaches toward retention and separation are presented. Several applications, This presentation gives a summary on the ongoing United States Pharmacopeia’s especially in the realm of therapeutic drug monitoring, are used to illustrate the ef- (USP) initiatives to revise existing general chapters related to dissolution. Addition- fectiveness of combining these modern tools. ally new general chapters are currently under development at USP to evaluate the

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performance of different dosage forms. Some of the General Chapters undergoing 330 Spatially Resolved Quantitative Magnetic Resonance Imaging revisions are: the section in the General Chapter <711> which refers to the use of Studies of API Behavior during In-Vitro Dissolution of Solid Dosage enzymes in the dissolution medium for dissolution test of gelatin capsules and gela- Forms tin coated tablets; the Performance Verification Test (PVT) of dissolution assemblies Michael D. Mantle, University of Cambridge, Department of Chemical (<711>) is used periodically to show that the dissolution assembly is performing as Engineering & Biotechnology, Pembroke St., Cambridge CB2 3RA, expected. Recent changes to the PVT require the demonstration of consistency of United Kingdom the results by evaluating the geometric mean and %CV. The USP General chapter The use of nuclear magnetic resonance imaging (MRI) as an analytical tool to facili- <1092> will be expanded to include more information on filter selection and compat- tate our understanding of the behavior of solid pharmaceutical dosage forms during ibility, determination of the effect of media, pH on drug solubility, choosing suitable dissolution testing is an area of increasing activity in the pharmaceutical research dissolution medium and apparatus, sample handling and the quantitation of the dis- community.[1] MRI is a quantitative, motion sensitive, nuclear specific technique[2] solved drug. A section on the automation will be added and the validation section that can provide spatially resolved information on length scales of 10s-100s of mi- will be expanded. The General Chapter <2040> which applies for dietary supple- crons. This talk shows how it is possible to map water concentration, water self- ments includes a new specification for the beakers used in the disintegration test, diffusivity and water hydrodynamics during pharmaceutical tablet dissolution testing disintegration of delayed-release soft shell capsules, dissolution testing of vitamin A in a SotaxTM USP-IV dissolution cell in less than three minutes, thereby providing tablets and of lipid filled soft shell gelatin capsules. The development of several new complementary information for the traditional drug dissolution profile obtained from General Chapters are also presented, such as <1724>, <1090>, <1094>. UV-visible spectroscopy data. Until recently, MRI studies of the simultaneous be- havior of dissolution media (water) ingress and active pharmaceutical ingredient 327 USP Packaging Standards - Recent Changes and those to Come (API) egress during dissolution testing has been problematic due to the API having Desmond G. Hunt, United States Pharmacopeia, 12601 Twinbrook a much lower 1H concentration than that of the water. In order to circumvent this Parkway, Rockville, MD 20852 problem the talk will show how we have recently developed and combined fluorine Packaging systems constructed from plastic, glass, and metal materials and compo- (19F) API imaging with 1H imaging to allow simultaneous tracking of both disso- nents are used to package therapeutic products (pharmaceuticals, biologics, dietary lution media ingress and drug egress in real-time during the dissolution process. supplements and devices). Therapeutic products come into direct contact with pack- Results from both model and commercially available pharmaceutical tablets are aging systems and their materials of construction as the product is manufactured, discussed to highlight the potential of MRI as a quantitative tool to evaluate drug stored and administered. Such contact may result in an interaction between the release mechanisms from solid pharmaceutical dosage forms, in-vitro, under phar- therapeutic products and the packaging systems and its materials or components macopeial conditions. of construction. These interactions must be such that the suitability for use (includ- References: ing its safety and efficacy) of the therapeutic product and the packaging systems is [1] M.D. Mantle, ‘NMR and MRI studies of drug delivery systems’. Current Opinion not adversely impacted by the interaction. Obtaining such a necessary and desir- in Colloid and Interface Science’18(3), 214-227, 2013. able outcome is facilitated by the use of well-characterized materials of construction [2] M.D. Mantle, ‘Quantitative magnetic resonance micro-imaging methods for phar- in components, containers and packaging systems and by the appropriate testing maceutical research’. International Journal of Pharmaceutics 417, 173–195, of packaging systems. The presentation discusses newly developed and revised 2011. United States Pharmacopeia (USP) packaging standards related to glass, plastic, and metal packaging materials. 331 A Miniaturized Instrument for Rapid In-Vitro Release Testing David M. Goodall, Paraytec, York House, Outgang Ln., Osbaldwick, Revision to Spectroscopy Chapter - Recent Proposals 328 York YO19 5UP, United Kingdom Horacio N. Pappa, United States Pharmacopeia, 12601 Twinbrook Current methodology for in-vitro release testing (IVRT) of semi-solid pharmaceuti- Parkway, Rockville, MD 20852 cal formulations uses the Franz diffusion cell, with experiments typically taking 6 United States Pharmacopeia is in the process of revising all the spectroscopy chap- hours to complete. Experiments normally involve sampling from the receptor com- ters. This includes the creation of separate chapters for those techniques covered partment, followed by high-performance liquid chromatography (HPLC) analyses. in Chapter <851> Spectroscopy and Light Scattering. The new chapters include The aim of the work reported here was to develop a miniature version of the Franz sections on instrument qualification and procedure validation cell to be used with direct imaging of transfer of the active ingredient from donor to receptor compartments to allow rapid measurements with small amounts of sample. UV Surface Imaging Studies of Dissolution Behaviors of an 329 The presentation covers design of miniature diffusion cells and their integration with Extended Release Drug Formulation an ActiPix UV imaging detector (Paraytec Ltd) and a Sirius SDI (surface dissolution Christopher A. Zordan, Bristol-Myers Squibb, One Squibb Dr., New imager) (Sirius Analytical Instruments Ltd). Use of the miniaturized instrument will Brunswick, NJ 08903 be exemplified with applications including: 1) release of tetracaine from PEG for- Dissolution of an oral solid dosage form is the first step towards achieving bioavail- mulations. In this study, the active pharmaceutical ingredient (API), which is a local ability of the active pharmaceutical ingredient (API). Dissolution testing is an impor- anaesthetic, was present in the donor phase containing polyethylene glycol (PEG), tant analytical procedure required by United States Pharmacopeia and regulatory and permeation was through a silicone membrane into water. 2) Release of keto- agencies to ensure the appropriate release of the drug in-vitro. Compendial dissolu- profen from agarose gels. Here the API, which is a non-steroidal anti-inflammatory tion testing measures bulk behavior but do not generate insight into the surface- drug, was released from a gel donor phase with a direct interface to phosphate level dissolution behavior of the API or formulated blends. Recent developments in buffered saline (PBS) as the receptor fluid. Benefits are that the tests require only 10 spectroscopic imaging have enabled examination of dissolution phenomena at the µg API or 10 µL sample volume, partitioning into ~1 mL of receptor media, and that surface of the API or solid dosage form, which could provide mechanistic under- results can be obtained in minutes rather than hours. We are currently seeking to standing of the dissolution process and help in formulation design. In this presenta- put together a consortium with end user partners to develop a stand-alone system. tion, applications of UV optical imaging for the study of dissolution behaviors of a pharmaceutical formulation are discussed. The surface dissolution imaging instru- Understanding Dissolution Inconsistency Through In-Situ Study of ment used in the studies is Actipix SDI 300. The drug intrinsic dissolution rate (IDR), 332 Disintegration Mechanisms Using FBRM® drug diffusion, and excipient swelling, which provides key information for selection Jack Shu, Des O’Grady, Mettler Toledo Autochem, 7075 Samuel Morse of an appropriate drug formulation. The extended release formulations studied in Dr., Columbia, MD 21046 this work are metformin blends. In order to control the release of the drug, methylcel- Particles play a critical role in final tablet quality. Upstream granule distributions from luloses with various viscosities and molecular weights were blended with the API to granulation to the tablet press directly affect dissolution and disintegration rates. find a desirable formulation. The effect of these different excipient viscosities on the The particle distribution is critical to ensure consistent content uniformity from table drug release and dissolution behaviors were examined and compared. Comparator to tablet. Dissolution testing with in-situ particle characterization is a powerful tool APIs with different solubility characteristics from metformin were further analyzed for quality-by- design (QbD). Formulators use FBRM® in-line particle characteriza- to examine impact of API solubility on the swelling and dissolution behavior of the tion to link tablet disintegration and dissolution mechanisms to upstream formulation excipient blends. process steps and to the initial active pharmaceutical ingredient (API) and excipi- ent particle size. FBRM® can speed process development by providing immediate understanding as to whether the root cause of dissolution profile inconsistency is due to the formulation or raw materials. Tracking tablet and granule disintegration and dissolution mechanisms in real-time helps uncover the root cause of disso- lution test inconsistencies. Mapping dissolution and disintegration throughout the design space with FBRM® PAT tools can easily identify critical process parameters.

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By investigating the mechanism for particle disintegration and dissolution, FBRM® 336 HILIC (Hydrophilic Interaction Chromatography) Chromatography- is used to correlate release inconsistencies to the upstream source of variability ELSD (Evaporative Light Scattering Detection) Detection for the including changes in raw materials, granulation inconsistency, or tableting irregulari- Quantitative Analysis of Amino Acids ties. Process analytical technology (PAT) tools promote faster and more thorough Nicolas J. Hauser, Sigma-Aldrich, 2931 Soldier Springs Rd., Laramie, mapping of experimental design space, enable consistent scale-up and tech trans- WY 82070, Andy Ommen, Carmen T. Santasania fer, and allow control of the process to a desired endpoint that assures downstream The traditional, non-specific and, at best, semi-quantitative methods used to ana- critical product quality and process efficiency. lyze amino acids are problematic for the assessment of the purity content of an individual amino acid. Instead of using a direct comparison analysis technique with 333 Platform Technologies for Pharmaceutical Counterion Analysis the use of a certified reference standard to assign an assay value, traditional amino Michael W. Dong, Genentech, 1 DNA Way, S. San Francisco, CA 94080, acid assays are carried out by titration. Due to the standard titration system that is Ross Woods used, the assay value can easily be overestimated as the most common impurities Counterion analysis is a mandatory assay for release testing of pharmaceutical of amino acids (similar amino acids) are titrated within the same system. For impu- salts. While ion chromatography (IC) using ion-exchange chromatography (IEC) rity analysis, amino acids have classically been evaluated by a thin layer chroma- with suppressed conductivity detection is the traditional methodology, this highly tography (TLC) procedure which makes quantitation of the impurities difficult due to sensitive technique appears to suffer long system equilibration, frequent component the lack of sensitivity of the procedure. In this talk, we describe a high-performance failures and non-optimum method accuracy when IC system is used sporadically liquid chromatography-evaporative light scattering detector (HPLC-ELSD) method for quality control release testing. In this paper, we present evaluation results on a that was developed to overcome the shortfalls of the traditional methods, in order to comparative study of six analytical platform technologies. These are: 1) ion chroma- perform a direct comparison assay as well as a quantitative impurity analysis. Amino tography with suppressed conductivity detection; 2) mixed mode chromatography acids, a majority of which lack a chromophore, are typically difficult to analyze by with charged aerosol detection; 3) mixed mode chromatography with SQ-MS detec- HPLC-UV methods without a preparatory derivatization step. However, the ELSD tion; 4) ion exchange chromatography with inversed photometric UV detection; 5) provides an alternative detector for analyzing amino acids without the requirement ion exchange chromatography with conductivity detection; 6) microtitration. We dis- of any derivatization. The use of hydrophilic interaction liquid chromatography (HIL- cuss the comparative advantages and potential shortcomings of these techniques in IC) mode LC allows for better retention and separation of each amino acid individu- terms of method performance (accuracy, precision, sensitivity, linearity, specificity) ally. In particular, the smaller, more polar amino acids, which generally constitute the and other important attributes such as, minimum sample size, system reliability and impurities, are easily resolved. HILIC LC-ELSD provides an accurate, specific, and availability, ease of use and system qualification, potential applicability to all New reliable method for performing a quantitative analysis of amino acids. Chemical Entities and analysis speed. Chloride, the most common pharmaceutical anion, was used as a model anion for this study. The results of this research will offer 337 Resolution of Antibody Charge Variants by Ion Exchange options to pharmaceutical labs to improve productivity for this problematic release Chromatography assay and provide useful comparative data for the industry. Hillel Brandes, Supelco Division of Sigma-Aldrich, 595 North Harrison Rd., Bellefonte, PA 16823, Dave S. Bell, Roy Eksteen 334 Determination of Anions in Dried Distillers Grains with Solubles Monoclonal antibodies (mAbs) have become a major class of biotherapeutics in (DDGS) development of effective and better targeted treatment of cancer. While the effec- Art W. Fitchett, Thermo Fisher Scientific, 1228 Titan Way, Sunnyvale, tiveness of approved mAbs is generally not in question, there is a need for optimum CA 94085, Peter Bodsky, Kassandra Oates characterization of these high molecular weight entities as individual production DDGS are a co-product of the biofuel process and have been added to animal feed batches may vary slightly due to truncation, oxidation, deamidation, phosphoryla- for nearly a century. Because the nutritional composition of DDGS can vary, the tion, glycosylation or other post-translational modifications. The distribution of com- distillery industry is interested in determining its composition to properly formulate positional variety of a mAb is referred to as charge variants or charge heterogeneity. a nutritionally balanced diet for the animal feed industry. We determined chloride, Thus, bioanalytical chemists are challenged to improve existing or develop new sulfate, and phosphate in DDGS using a hydroxide-selective anion exchange col- techniques to better characterize mAbs. Such improvements ultimately will lead to umn with suppressed conductivity detection in < 10 min. The method demonstrates changes in process design that will result in higher drug purity as it is traditionally un- good accuracy with spiked recoveries between 96-107% and good sensitivity with derstood by chemists. Heterogeneity such various species is typically assessed by limits of detection from 1.7-5.7 µg/L. Polyvalent anions, such as inositol phosphate ion exchange chromatography. Thus, resolution is controlled by a variety of factors: (InsP6), are also of interest in DDGS. During fermentation and processing, phos- the buffer system, pH, the mode of elution, the choice of the exchangeable ion in the phate groups are released from InsP6 to produce InsP5-InsP1 and free phosphate, case of salt elution, and of course the particle design and chemistry employed in the increasing phosphorus bioavailability to non-ruminant animals. Using a high resolu- chromatographic separation. We report on the impact of these various parameters tion strong anion exchange column under acidic conditions, 25 InsP isomers are in achieving the best results in a reasonable time frame. separated and detected by UV after postcolumn reaction. 338 Assaying Gentamicin Sulfate by HPLC-Charged Aerosol Detection 335 Mixed-Mode Chromatography for Pharmaceutical Analysis with an Ion-Pairing Reagent Gradient Peter Bodsky, Thermo Fisher Scientific, 1228 Titan Way, Sunnyvale, CA Michael Hvizd, Thermo Fisher Scientific, 1228 Titan Way, Sunnyvale, 94085, Xiaodong Liu, Chris Pohl, Michael G. Hvizd, Art W. Fitchett CA 94085, Peter Bodsky, Lipika Basumallick, Jeff Rohrer Selectivity, mainly governed by column chemistry, is the key in high-performance Aminoglycoside antibiotics are proven human and veterinary medications with liquid chromatography (HPLC) separation. Although reversed-phase columns (e.g., broad-spectrum activity, particularly against gram negative bacteria. Many of these C18) are most commonly used in pharmaceutical applications, they often fail to antibiotics are manufactured by microbial culture (fermentation) processes. These retain highly polar molecules (e.g., counter ions), and offer limited selectivities. processes can produce a mixture of antibiotic compounds. One such example is Mixed-mode chromatography provides a viable solution to these challenges by gentamicin, which is produced by Micromonospora echinospora (Micromonospora using both reversed-phase and ion-exchange retention mechanisms. One major purpurea). Gentamicin sulfate is a mixture of four major compounds: gentamicins advantage of this approach is that the column selectivity can easily be modified by C1, C1a, C2, and C2a. In addition, gentamicin C2b is commonly present as a minor adjusting mobile phase ionic strength, pH and/or organic solvent concentration. As component. These gentamicins, as well as fermentation impurities and degrada- the result, not only can a mixed-mode column provide selectivity complementary tion products, are structurally similar and do not possess chromophores, making to reversed-phase columns, but it also allows for multiple complementary selec- chromatographic separation and subsequent detection challenging. Alternate de- tivities on a single column under different chromatographic conditions. Mixed-mode tection techniques are available for non-chromophoric compounds. In this work a chromatography is also well-suited to retaining ionic analytes, hydrophobic (e.g., method has been developed to separate the five gentamicin sulfate congeners and Naproxen) or hydrophilic (e.g., Na+ and Cl- ions), and requires no ion-pairing agents sisomicin, a common impurity, by reversed-phase high-performance liquid chroma- in the method, significantly improving MS compatibility. This presentation provides tography with charged aerosol detection. This detection technique does not rely on an overview of state-of-the-art mixed-mode chromatography technology and exam- the presence of a chromophore, does not require sample derivatization for detec- ples of pharmaceutical applications by mixed-mode chromatography, such as active tion, is compatible with volatile ion-pairing reagents, and readily detects non-volatile pharmaceutical ingredient and counter ion, surfactants and glycans. analytes such as gentamicin. The method was used to quantify gentamicin sulfate in ointments, solutions, and creams with a total analysis time of 15 min using a gradient of ion-pairing reagents. Method precision, as relative standard deviation, for individual replicates ranged between 0.77-4.86 for triplicate injections. The de- termined amounts in the samples were consistent with the label claim for gentami- cin present in the products. Gentamicin sulfate recoveries ranged from 80-113%, indicating consistent response and the absence of interfering matrix components.

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Between-day analysis precisions for the assay are within 10%, showing good preci- 342 Method Development, Method Validation, Specifications and Data sion. This assay provides a direct-detection method for gentamicin product analysis Reporting for RTRT that is fast and reproducible. Henrik T. Rasmussen, Vertex Pharmaceuticals, 130 Waverly St., Cambridge, MA 02139, Kelly A. Swinney, David E. Nadig 339 HPLC Column “Standardization” Process for Pharmaceutical The evolution of process analytical technology (PAT) techniques such as Raman Analysis and near-infrared spectroscopy and particle size measurements, have allowed for Michael W. Dong, Genentech, 1 DNA Way, S. San Francisco, CA 94080, the potential of measuring critical quality attributes in-situ during pharmaceutical Midco Tsang, Dawen Lou, Roberto Garcia manufacturing. To move from measurements to real-time release testing (RTRT), Our laboratory, like many other pharmaceutical development laboratories engaging the collective measurements must be demonstrated to provide suitable manufac- in Chemistry, Manufacturing and Control (CMC) activities to support new small mol- turing control and collectively demonstrate that the pharmaceutical product meets ecule drug development, uses hundreds of high-performance liquid chromatogra- requirements for potency, purity and bioavailability. This paper provides an overview phy (HPLC) columns every year. While the number of columns in stock continues to of how PAT instruments can be integrated into continuous manufacturing, in the escalate because of the varieties of brands and types preferred by individual scien- context of an overall quality-by-design (QBD) approach to process development. tist (e.g., particle sizes, lengths, inner diameters, types of bonded phases, micropar- Specifically, an overview is provided of the instrumentation utilized for successive ticulates or core-shells), it appears that one can never readily find the exact column steps of the manufacturing process, how methods evolve from off-line to on-line ap- in the lab when a new drug candidate entering the development cycle. We believe plications, how methods are validated to demonstrate the suitability of use, and how while each new chemical entity may have some unique physicochemical properties, the collective measurements translate to product specifications and certificates of there are sufficient commonalities to warrant the use of a common column set for analyses suitable for product disposition. column screening. Because this problem is not unique in our department, we decid- ed to form a focus group to “standardize” our column use in method development. 343 Application of an Advanced Process Controller to a Continuous The goal of this group is not to stifle creativity or individual preferences, rather to Mixing / Direct Compression Process find a set of “best” column choices via a consensus-building process. The potential Daniel Blackwood, Pfizer, Eastern Point Rd., Groton, CT 06340, benefits are convenience (stocking of a limited number of the “best” columns), cost Salvador Garcia-Munoz, Yang A. Liu, Koji Muteki, John N. Mack saving (negotiating larger discounts via bulk orders) and productivity enhancements A continuous mixing, direct compression process is introduced. This system con- (encourage best practice by information sharing and reduce “column proliferation”). sists of three gravimetric feeders, an in-line powder mixing device, an at-line near The focus is on finding a set of achiral reversed-phase HPLC columns for stability- infrared probe, an advanced process controller and a rotary tablet press. Engineer- indicating (purity) methods for active pharmaceutical ingredient and raw materials. ing models, focusing on powder mixing, residence time distribution and powder lu- This paper is a summary of our findings and our consensus conclusions on particles brication phenomena, are described for this system. A series of experimental trials size, length, inner diameter, C18 and other “orthogonal” phases. varying process parameters such a mixer impeller speed (RPM), target mixer hold Abstracts 340 - 469 up mass (g), and gravimetric feeder set point (g/min), were completed. The impact of process parameter set point changes to quality attributes, such as blend concen- 340 PAT Options for Measuring Induction Seals and Blister Packs tration by near-infrared (NIR), mixer hold up mass and tablet press hopper level, Robert A. Lodder, University of Kentucky, 789 South Limestone St., was assessed. Using these experimental results, a preliminary control model has Lexington, KY 40536, Jarred Williams been developed to enable closed loop control of the gravimetric feeders to achieve The pharmaceutical industry has made significant advancements in container clo- a target blend potency concentration by NIR. The control model was subsequently sure integrity (CCI) testing. Some of the accepted CCI testing methods, such as dye challenged and experimentally verified by set point changes to the total mass flow testing, are destructive to the package being tested, and can be slow and increase rate from the gravimetric feeders as well as changes to the target blend concen- the cost associated with packaging and sealing. Instrumentation-based technolo- tration. The result of this work highlights the integration of continuous processing gies, such as high voltage leak detection, vacuum/ pressure decay, mass extraction, equipment, engineering models, at-line process analytical technology devices, and and tracer gas detection (helium, oxygen etc.), have emerged and demonstrated advanced process controllers as part of an advanced manufacturing process for improved detection capabilities in some products compared to conventional dye pharmaceutical, solid-oral dosage forms. and microbial ingress methods. The ability to detect leaks quickly, as small as 10 micrometers in diameter, is desired by the industry. A nondestructive test method 344 Why Small is Good: Selected Applications of Small Volume NMR would be useful in order to maintain the original package being tested and reduce Probes in Pharmaceutical R&D waste. Thermal emissivity, thermal conductivity, and acoustic eigenvalues are three Michael D. Riley, Bristol-Myers Squibb, PO Box 4000, Princeton, NJ options that could provide a nondestructive test method for CCI testing. These pro- 08540 cess analytical technologies (PAT) have been used for on-line 100% inspection and/ Nuclear magnetic resonance (NMR) has enjoyed a long history of adding significant or drug product stability CCI testing. In this presentation, we review data collected value as an analytical tool in pharmaceutical research and development. It is the for three different types of packages: high-density polyethylene pill bottles that are only practical tool for providing solution state atomic resolution structural informa- induction sealed, foil laminate stick packs, and blisters with foil backing. The results tion, making it indispensable for reaction monitoring in medicinal chemistry, struc- are used to suggest a systematic approach for CCI testing method selection, devel- tural elucidation of pharmaceutically important molecules such as proteins, metabo- opment, and validation. lites, degradation products and impurities, studying dynamic interactions and for discovering biomarkers. The Achilles heel of NMR; however is poor sensitivity. This 341 A Look at RTRT Nearly One Decade Following Approval requires exorbitant amounts of time for signal averaging, or large amounts of mate- Steven M. Short, Merck, 556 Morris Ave., Summit NJ 07901, Nathan C. rial. Time is an increasing scarce commodity in this industry and frequently charac- Pixley, Monoharan Ramasamy, Gert Thurau, Busolo Wabuyele terization must be carried out on precious and scare materials. This has driven the A robust pharmaceutical quality system ensures that drug products are reliably of development of NMR equipment that allows faster data acquisition on smaller sam- the highest possible quality. Further, the quality system serves as the foundation ples and the industry has adopted this new technology with open arms. Whereas the for integrating strategic initiatives such as quality-by-design (QbD), which itself is most common NMR platform utilizes samples of 500-600 microliters, this presenta- becoming somewhat of an unwritten prerequisite for new drug applications (NDAs). tion provides selected examples of the benefit of reducing this to between 7 and Highlighted in International Conference on Harmonization (ICH) Q8, a benefit of 30 microliters in specially designed room temperature and cryogenic NMR probes. the QbD paradigm is that for certain drug products, it affords the opportunity to use alternative testing approaches within the control strategy framework to ensure 345 NMR Analysis of [3H]Radiopharmaceuticals (beyond what traditional testing can offer) the consistent manufacture and release of Peter G. Dormer, Merck, PO Box 2000, Rahway, NJ 07065, David J. quality products. One such alternative testing approach is real-time release testing Schenk (RTRT), which offers many scientific and business drivers. For example, the larger Tritium-labeled compounds are used across drug discovery and development. Prior sampling of critical process and quality attributes leads to enhanced process char- to use in metabolism studies, these materials must be analyzed using a suite of acterization and ultimately greater assurance of product quality. Merck & Co., Inc. instrumental methods including radio-high-performance liquid chromatography received approval for its first RTRT methods in 2005 as part of the Food and Drug (HPLC), LC-mass spectrometry, solution count, and nuclear magnetic resonance Administration’s QbD pilot program. Nearly a decade later, it seems fitting to review (NMR) to measure/determine radiochemical purity, specific activity, tritium label the RTRT methods to discuss the perceived benefits of and hurdles to RTRT from location(s), and radioactivity per volume. We present an NMR-based method to the vantage point of the patient, the regulator, and the industry. These discussions measure specific activity, radioactivity per volume, tritium location, and determine will hopefully open up greater lines of communication between all vested parties in radiochemical purity. an effort to help establish clearer expectations for RTRT methods. Candid dialogue is instrumental in ensuring that RTRT methods are successfully implemented in future NDAs. 54 2013 EAS Abstracts November 2013

346 Small Molecule Applications of Optimized Acquisition-Based addition, we have incorporated sample submission, tracking and reporting. This pre- Sensitivity Enhancement sentation covers the selection, implementation of the notebook, sample processing David A. Rovnyak, Bucknell University, Chemistry Department, One and our experiences in using the system. Dent Dr., Lewisburg, PA 17837 Unraveling the true structures and conformations of complex small molecules usu- 351 Managing for Increased Productivity in a Rapidly Evolving ally requires the application of many multi-dimensional (nD)-nuclear magnetic reso- Analytical Landscape nance (NMR) experiments to delineate bonding and spatial relationships among all Richard Durand, Sun Chemical, 631 Central Ave., Carlstadt, NJ 07072 nuclei. When facing sensitivity-limited conditions due to the availability of very small Bringing a new capability into the laboratory which has evolving business benefit masses of samples, for example, then sufficient resolution and sensitivity may not can increase expectations and the overall demand for services. As a result, merely be obtainable in many nD-NMR experiments. The common practice of uniformly being capable to carry out the work is not enough. There are ever increasing stress- incrementing the indirect evolution periods of nD-NMR experiments is increasingly es on maintaining the timelines for this potential higher volume of work even when viewed as a serious limitation to acquiring nD-NMR spectra on sensitivity-limited all the technical nuances still need to be covered as well. Thus, work processes samples. In its place, non-uniform incrementation of indirect evolution periods in need to be re-examined to provide an acceptable path forward which insures quality nD-NMR has been widely characterized as a time-efficient means to obtain high of output and staff availability to meet these challenges. A specific case study is pre- resolution in the indirect dimensions of nD-NMR when paired with modern meth- sented which is related to assessing the migration of compounds from ink/coatings ods of spectral reconstruction. Although many investigators for over two decades on printed food packaging. This type of analysis has become increasingly important have considered that sensitivity gains in nD-NMR by non-uniform sampling may to assessing the safety of food packaging and provides key data that supports that be possible, only recently has the exact theory describing this effect been provided ink/coating products will be suitable for a variety of application areas. These studies along with rigorous experimental validation. We have extended prior work to give often are critical to testing new print processes and chemistries which drive pack- new insights into the optimization of the absolute sensitivity of non-uniform sam- aging design. This case study illustrates how creative uses of various resources pling. Application of optimal non-uniform sampling to a range of complicated small related to personnel, equipment , processes and software are necessary for the molecule targets is described, where the ability of non-uniform sampling to change lab manager to manage in order to maintain the quality and timeliness of services the detection limit of nD-NMR is demonstrated. It is evident that small molecule needed for capturing new business opportunities. two-dimensional-NMR spectroscopy is well suited to benefit from acquisition-based sensitivity enhancement. 352 Tools for Knowledge Retention and Transfer Scott Hanton, Intertek Chemicals and Materials, 7201 Hamilton Blvd., 347 Online NMR Reaction Monitoring in Pharmaceutical Process RD1, Dock #5, Allentown, PA 18195 Development Knowledge retention and transfer (KRT) is an important part of management re- Brian Marquez, Pfizer, 445 Eastern Point Rd., Groton, CT 06340, David sponsibility. While most managers understand the importance of developing indi- Foley viduals, it is more complex to sustain that development and organizational learning The use of online nuclear magnetic resonance (NMR) technology in the develop- as the staff changes. Effective KRT tools will enable the protection of key, business ment of organic reaction processes provides information rich data, which can be specific knowledge. The KRT process has two primary steps: 1) identifying the most used to provide a deeper understanding of the process under investigation. When important knowledge in the organization, who has it, and what risk they are to leave; coupled with other process analytical technology techniques, it allows chemists and 2) identifying effective tools to transfer that knowledge to others to retain it within the engineers to make informed decisions based on a suite of analytical information, organization. This presentation shows tools for both steps and discusses some of ultimately leading to more robust processes. The development of an NMR reaction our experience using these tools. monitoring platform is discussed, including a novel flow cell designed for increased flexibility of use, compared to fixed flow cells. The implementation of this technology 353 Determination of Choline in Infant Formula and Other Food is also demonstrated, citing specific examples of reactions developed and optimized Samples by IC employing on-line NMR. Ken Kirkbride, Thermo Fisher Scientific, 1228 Titan Way, Sunnyvale, CA 94085, Peter Bodsky, Michael G. Hvizd, Kassandra Oates 348 CRAFT: Redefining “High-Throughput” NMR Choline is a water-soluble micronutrient vital to cell membrane integrity, support of Krish Krishnamurthy, Agilent Technologies, 5301 Stevens Creek Blvd., methyl group metabolism, and nervous system activity. Therefore, it is important to Santa Clara, CA 95051 determine the choline content in common foods. The Association of Analytical Com- The intrinsic quantitative nature of nuclear magnetic resonance (NMR) is being in- munities method 999.14 describes the determination of choline in infant formula creasingly exploited in areas ranging from complex mixture analysis (as in metabo- and milk using an enzymatic colorimetric method. Here, we present an improved lomics and reaction monitoring) to quality assurance/control (QA/QC). Extraction of method where two modified sample preparation procedures were evaluated prior to quantitative information generally involves significant prior knowledge and/or opera- ion chromatography to separate choline from other cations in the sample. For the tor interaction to deconvolute peaks of interest. We discuss a workflow that achieves first sample preparation modification, syringe filtration was shown to have a signifi- a complete reduction to amplitude frequency table (CRAFT) in an automated and cant time-saving advantage in filtering the hydrolysate compared to using the filter highly time-efficient fashion – thus converting the fid to a frequency-amplitude table. papers described in the official method. The second modification used microwave The CRAFT tables can be used further for data-mining of quantitative information digestion to hydrolyze the samples, which significantly reduced the sample prepa- using fingerprint chemical shifts of compounds of interest. Example application from ration time from 3 hours to <30 min. The method is further simplified by using an metabolomics, reaction monitoring and food sciences are presented. electrolytically generated methanesulfonic acid eluent to avoid manual preparation of the eluent. The cation-exchange column with an optimized selectivity for small 349 Coaching your Team to Improved Performance hydrophilic amines provided high efficiency for choline with an analysis time of < 20 Stephanie Olexa, Lead to the Future, 2425 Butter Creek Rd., min. The choline linear range is 0.5-50 mg/L with a correlation coefficient of 0.9999 Quakertown, PA 18951 and a detection limit of 2.3 µg/L. The intra- and between-day peak area precisions Laboratory managers wear many hats: supervisor, mentor, team leader, and scien- are <0.8% and <1.6%, respectively for all the tested food samples. In addition, the tist, to name a few. But one of the most impactful roles a laboratory manager can National Institute of Standards and Technology 1849 a selected reaction monitoring fill is coach. In this session we discuss the difference between the roles, especially for infant formula was evaluated and the results from this study were determined the difference between being a mentor and a coach. Through illustration and in- to meet the certified value for choline, which demonstrates the method’s accuracy. teraction we demonstrate the key skills of coaching, active listening, appreciative Furthermore, the recoveries for infant formulas, adult nutritionals, egg powder, and inquiry, mindful reflection, engagement, goal setting and accountability. As a coach- soy flour samples were between 90-100%. ing leader, a laboratory manager empowers the team to reach their best potential. 354 Rapid Measurement of Pomegranate Juice Adulteration with 350 Experiences with an Electronic Notebook: A Manager’s Perspective. Minimal Sample Preparation Using DSA/TOF Dennis F. H. Swijter, International Flavors & Fragrances, 1515 State Avinash Dalmia, Perkin Elmer, 710 Bridgeport Ave., Shelton, CT 06484 Highway 36, Union Beach, NJ 07735 Food adulteration or food fraud occurs when an ingredient is replaced partially or The storage and accessibility of data are common problems. Automation and prog- fully with something different – without the knowledge of consumer. Most of the ress in analytical instrumentation continuously produces increasing amounts of data time, food adulteration occurs to improve profits but in some cases, there can be which then have to be processed and stored. In the past that meant paper and mi- serious health consequences. Fruit juices are among the top 10 adulterated foods. crofilm; nowadays it is the use of electronic media. Although a fact of life, these me- In the past, methods employing gas chromatography-mass spectrometry (GC-MS) dia are also constantly changing. Nevertheless, the advantages of electronic data and high-performance liquid chromatography (HPLC) hyphenated to MS (HPLC- management are obvious. Paper notebooks were, and often still are, the first critical MS) have been implemented for measuring food adulteration. These methods are step. At IFF, we have replaced the paper notebook with the electronic version. In time consuming, expensive and require extensive sample preparation. Recently, 55 2013 EAS Abstracts November 2013

we have developed direct sampling analysis (DSA) ionization source which utilizes prior to the determination of phthalates in solid samples by GC-MS. We discuss field free atmospheric pressure chemical ionization source. The advantages of this how these approaches can save up to 90% on solvent usage, time and labor costs method, compared to conventional techniques, are direct sampling with minimal or associated with traditional sample preparation techniques. no sample preparation with high throughput. In this work, DSA coupled with high resolution and accurate mass capabilities of time-of-flight (TOF)-MS was used for 358 Preparation and Analysis of Imported Children’s Toys for Toxic measurement of adulteration of pomegranate juice with grape juice and apple juice. Metals In literature, it has been reported that tartaric acid is present in grape juice but ab- Patricia L. Atkins, SPEX CertiPrep, 203 Norcross Rd., Metuchen, NJ sent in pomegranate juice and malic acid levels are about 10 times higher in apple 08840 juice in comparison to pomegranate juice. Therefore, using DSA-TOF, we demon- Over the past decade the regulations regarding potentially hazardous elements and strated that the presence of tartaric acid and elevated levels of malic acid in pome- compounds have increased in scope and decreased the restricted compound limits. granate juice can be used for measuring its adulteration with grape juice and apple Children’s consumer products have been an active focus for study and regulation juice with minimal sample preparation and measurement time less than 30 seconds. due to their ability to expose infants and children to potentially damaging and dan- gerous chemicals. In regards to organic chemicals, certain phthalates and Bisphe- 355 Determining Flavors and “Defects” in Beer by Headspace Trap/Gas nol-A (BPA) have been either restricted or banned in children’s plastic products. As Chromatography/Mass Spectrometry (HS Trap/GC/MS) for inorganic contaminants, there is recognized list of toxic metals (As, Pb, Hg, Cr, Lee D. Marotta, Perkin Elmer, 710 Bridgeport Ave., Shelton, CT 06484, Cd, Sb), as well as, potentially hazardous elements that can be found in children’s Andrew Tipler, Bill Hahn, Cindy Liang products. This study looks at the concentration of hazardous inorganic elements de- Beer is a popular beverage produced by the fermentation of hopped malt extracted tected in imported children’s toys by inductively coupled plasma mass spectrometry from barley and other grains. Some compounds (flavors) have a positive effect on (ICP-MS). These toys were previously studied for restricted phthalates and BPA by aroma (attributes) and some have a negative effect (defects). This presentation gas chromatography-MS and were found to have high levels of hazardous organic focuses on a new method that enables the investigation and characterization of chemicals that were at concentrations at or far exceeding the current United States flavors and defects of beer in one analysis using headspace trap-gas chromatog- guidelines. This second study is a continuation of the examination of these imported raphy mass spectrometry (GC-MS). Classically, this analysis is performed on four toys to determine the inorganic elemental composition and determine if a correlation separate detectors. This new method employs one detector (MS) to provide these exists between toys which were identified as high in restricted organic compounds solutions required for the production and the testing of beer. The outcome is a more as having high restricted elemental contaminants. cost effective, accurate means to ensure the validity and the quality control of their product. Other benefits include enhanced productivity, attaining more information 359 Analysis of Terpene Hydroperoxides Related to Allergic Contact from a single analysis, and requiring less bench space. The following experiments Dermatitis Caused by Fragrance Use and results are discussed: 1) quantitation of dimethyl sulfide (DMS), 2,3-butanedi- Michael J. Calandra, Firmenich, P.O. Box 5880, Princeton, NJ 08543, one (diacetyl), 2,3-pentandione and t,2-nonenal; 2) characterization of several types John A. Impellizzeri of beers; 3) fermentation profiling; 4) analysis of raw materials; 5) aging studies. Limonene and linalool are unsaturated terpenes found as major components in es- sential oils and fragrances; both compounds easily react with atmospheric oxygen 356 Rapid Detection of Food Contaminants Using Surface Enhanced to form mixtures of isomeric hydro peroxide derivatives. The resulting terpene hydro Raman Spectroscopy (SERS) peroxide mixtures are sensitizers capable of causing allergic contact dermatitis. The Lili He, University of Massachusetts, Chenoweth Lab, Department of sensitizing potency of each individual component within these mixtures varies, de- Food Science, Amherst, MA 01003 pending on the parent terpene and the hydro peroxide group position. Due to the Food contamination problems have become globalized and are the causes of many varying potencies, analytical methods that are incapable of species differentiation health problems and economic losses. There is an increasing need for rapid detec- would not be expected to give results that correlate well with a sample’s dermato- tion of food contaminants in foods and on food processing surfaces, as traditional logical sensitizing potency. Iodometric titration methods, currently used to measure detection methods, such as plating for microbes and high-performance liquid chro- peroxide value (POV) levels in essential oils, give only a total level of all oxidants, in- matography mass spectrometry for chemicals, are usually time consuming. Here cluding but not specific for the targeted hydro peroxides. This creates a need for an I demonstrated the superior capacity of surface enhanced Raman spectroscopy analytical method capable of differentiating individual isomers of limonene- and lin- (SERS) for rapid detection of food contaminants, such as pesticides, allergens, and alool-derived hydro peroxides. A high-performance liquid chromatography method bioterror agents in foods within 30 min, research funded by the US Department of using a post-column reaction to produce chemiluminescence via luminol oxidation Agriculture and the US Department of Homeland Security. SERS is a combination was developed to fill this need. The method separates two linalool hydro peroxide technique of Raman spectroscopy and nano-techniques. The use of nanostructures isomers and six limonene hydro peroxide isomers commonly found in autoxidized like silver dendrites increases the Raman signals by more than 10^6. Contaminants citrus oils. The analytes are expressed as equivalents of tert-butyl hydro peroxide can be either extracted from the foods or captured by specific antibody or aptamer because pure analyte standards are currently unavailable. The repeatability is 4.4% agents, then rapidly measured using a Raman instrument. Our results show SERS RSD for total linalool hydro peroxides and 2.6% relative standard deviation for total can detect multiple pesticides in apple juice and on an apple surface at parts per limonene hydro peroxides. Spiking experiments gave 104.1% recovery for total lin- million (ppm) levels, 1 ppm egg allergenic protein in milk and 5 µg egg protein on a alool hydro peroxides and 106.0% recovery for total limonene hydro peroxides. The stainless steel surface, 0.25 ppm ricin in milk and 0.1 ppm ricin in orange juice, and limit of detection is below 2 picomoles of organic hydro peroxide. 10^4 Bacillus anthracis spores in orange juice. All detections can be done within 30 min. including the sample preparation. In summary, the use of SERS shows great 360 Firearms and Fabric success in rapid detection of various food contaminants. Peter Diaczuk, John Jay College, 524 W 59th St., New York, NY 10019, Jack Hietpas 357 Rapid Phthalate Screening Techniques for Environmental and Fabrics are arguably the most common substrates that a bullet will encounter in Consumer Product Samples a shooting incident. Only when a victim is shot in the relatively small areas of ex- Ken Kirkbride, Thermo Fisher Scientific, 1228 Titan Way, Sunnyvale, CA posed skin will some type of fabric not be involved. Thus it is useful to examine 94085, Peter Bodsky, Jennifer Peterson the clothing at and near a bullet impact site for the purposes of reconstructing the Phthalates are used as plasticizers in polymers and are found in numerous consum- event. Muzzle-to-target distances can sometimes be estimated by the presence and er products like medical devices, toys, shampoos, and cosmetics. Several of these density of the discharged propellant pattern. In cases where more than one shot compounds have been banned in the United States and European Union, and the was fired, the sequence of shots may be of importance. Alternatively, a projectile US Environmental Protection Agencey has listed some phthalates on the Chemicals can enter, exit and re-enter, occasionally creating more holes than shots that were of Concern list. Determining phthalate levels in these mentioned products is of inter- fired. Sometimes it is even necessary to determine whether a hole in a garment is est because some of these compounds have been linked to hormone disruption in from a ballistic event or from a different mechanism such as mechanical damage or some studies. Sample preparation prior to chromatographic determination can be wear. This presentation covers several examples of bullet and shotgun pellet impact time-consuming and labor- intensive, particularly for ppb levels of contaminants. damage to garments of differing manufacture (knits and weaves). It also compares The various steps of preparing samples for analysis often end up being the rate- and contrasts garment damage produced from differing bullet types (full metal jacket limiting step in the overall analytical workflow. In this presentation, we discuss two and hollow point). A cursory inspection of the garment damage produced from these sample preparation approaches that improve the overall productivity for the deter- bullet types may not appear significantly different from one another. However, a de- mination of phthalates. The first is automated solid-phase extraction (SPE), using tailed microscopical examination reveals differences that, when properly recognized the AutoTrace 280, prior to the determination of phthalates in aqueous samples by and interpreted, can contribute to a more accurate shooting scene reconstruction. gas chromatography mass spectrometry (GC-MS). The AutoTrace 280 can process Properly directed scientific assessments during the early stages of an investigation up to 6 large volume samples (20 mL to 20 L) in parallel using either SPE cartridges avoid costly and time-consuming efforts during the adjudicative stage. or disks. The second approach is the use of accelerated solvent extraction (ASE) 56 2013 EAS Abstracts November 2013

361 Microanalysis of Environmental Particulates column initial screening configuration using a C18 column in water/acetonitrile and Randy Boltin, MVA Scientific Consultants, 3300 Breckinridge Blvd., a phenyl column in water/methanol. If the initial separation shows promise with the Suite 400, Duluth, GA 30096 C18 column, the mobile phase is adjusted or the column selection in narrow down Problems associated with the wide variety of microscopic particulate that resides further with a C4, C8, or cholester (cholesteryl functional group) column. If the phe- in indoor and outdoor environments include health and aesthetic issues. Examples nyl column shows better separation, further mobile phase refinement can be done include discoloring agents, friable asbestos-containing bulk or debris materials that or other columns with stronger π-π interactions such as π naphthalene functional may yield airborne respirable fibers, lead-bearing paint particles, mold, soot and group or pyrenylethyl functional group can be tested. The increased retention on the voluminous amount of entrained and settled material generated by natural or unsaturated or conjugated small molecules could save in the analysis time and ease human-induced events (e.g., volcanic eruptions and dust generated by the collapse in method transfer effort. Examples of method development using this two-column of the World Trade Center towers). The identification of specific dust or debris con- screening system are shown. stituents for comparison with potential source materials constitutes the basis for many environmental forensic studies. Identification of particles of interest can usu- 365 Optimization of Reversed-Phase HPLC Separations of ally be accomplished using one or relatively few microanalytical methods. Basic Biomolecules examination by light microscopy is critical for determining the physical attributes of Stephanie A. Schuster, Advanced Materials Technology, 3521 Silverside fine particulate (color, size, shape, homogeneity, deformation characteristics) and Rd., Quillen Bldg., Ste. 1-K, Wilmington, DE 19810, Barry E. Boyes, provides clues as to which additional procedures, if necessary, may be appropri- Brian M. Wagner, Joseph J. Kirkland ate for more specific identification. Spectroscopic analysis using methods such as Superficially porous particles were originally created with pore sizes and particle micro- Fourier transform infrared or Raman spectroscopy are useful in the identifica- sizes that were suited for small molecule analytical separations. To improve re- tion of microscopic polymer particles. Energy dispersive X-ray spectrometry coupled versed phase high-performance liquid chromatography (HPLC) separations of with electron microscopy is useful in classifying a variety of particles, including soot, biomolecules, which were known to have slower mass transfer kinetics, thus to ex- metals and minerals. hibit broad peak shapes, modification in the particles are required. To resolve these limitations, we introduce superficially porous silica particles with properties that are 362 Does Your UV-Visible Microspectrometer Have Intrinsic specifically designed for larger biomolecules. New particle designs with specially Polarization? selected bonded phases for peptide and protein separations are discussed. Fast, Dale K. Purcell, Chaminade University of Honolulu, Wesselkamper high resolution separations of a variety of peptides and proteins demonstrate the Science Center, 3140 Waialae Ave., Honolulu, HI 96816, John A. advantage of the superficially porous particle design. The high temperature stability Reffner, Thomas A. Kubic, Fran Adar of the columns of these new particles and bonded phase characteristics are ideally Grating and prism-based dispersive spectrometers produce a degree of polariza- suited for the conditions that are often used for analytical and small scale prepara- tion. This intrinsic polarization can be detected by recording the change in intensity tive biomolecule separations. Examples of high molecular weight protein separa- when a linear polarizer is rotated through 360°. If the spectrometer has no intrinsic tions highlight the advantages of using columns of superficially porous particles with polarization, then the intensity will be constant as the linear polarizer is rotated. 400 Å pores. Using a spectrometer without knowing its intrinsic polarization can lead to serious problems. Placing a single linear polarizer either before or after the sample provides 366 Larger Superficially Porous Particles: The “Long and Short of It” the necessary control to orient the radiation’s electric field. Since most microscope for HPLC Separations spectrometers operate in a single-beam mode it is necessary to record a back- Stephanie A. Schuster, Advanced Materials Technology, 3521 Silverside ground for each orientation of the polarizer. The intrinsic polarization of several com- Rd., Quillen Bldg., Ste. 1-K, Wilmington, DE 19810, Thomas J. Waeghe, mercial microscope spectrometers and various dispersive elements is presented to William L. Johnson, Joseph J. DeStefano, Joseph J. Kirkland illustrate the magnitude of this problem. There is a mistaken belief that if you are Since 2006, modern sub-3-μm superficially porous particles have been shown to not using a polarizer then there is no dichroic affect. The intrinsic polarization of the deliver high speed separations comparable to sub-2-μm particles, in addition to spectrometer is introducing an unseen variable when measuring the absorbance high resolution separations for challenging samples. In 2012 larger particle size spectrum of a dyed fiber, for example. superficially porous particle (SPP) for small molecule analyses were introduced. These 5-μm particles can deliver performance comparable to, or better than, 3-μm 363 The Statistical Significance of the Aggregate Trace Evidence found particles - at the backpressure of 5-μm particles. Such features and benefits now in Household Dust Specimens bring the advantages of superficially porous particles to all applications of the high- Nick Petraco, John Jay College, 524 W 59th St., New York, NY 10019, performance liquid chromatography (HPLC) instrument user. Notably, these new Nicholas D. K. Petraco, Anna Duggar, Mary Eng 5-μm SPP columns have initially fit a very useful niche for LC-mass spectrometry This paper continues the discussion of the statistical significance of household dust (MS) users, in that they can be used for high-throughput analyses with narrow-bore specimens. Results of the original and new household dust specimens obtained columns operated at high flow rates (> 1 mL/min) at normal HPLC pressures (< 400 from locals around the New York City Tri-State region are presented. The contents bar/6000 psi). Such high-speed analyses are not achievable with sub-2-μm columns of each new dust specimen were sorted into groups of similar looking: hairs, fibers, unless UHPLC/UPLC® instrumentation is used. Moreover, some users have found and particulate materials. Aggregate groupings of fibrous and particulate materials that peaks produced by the sub-2-μm columns were, in fact, too narrow for full fidel- were teased out of each dust specimen and examined. Each fibrous and/or particu- ity with their fastest LC-MS scan rates. The slightly broader peaks obtained with late subset was mounted on a 7.5 cm x 5cm microscope slide in Cargille® 1.540 5-μm SPP columns did not have such a problem. In addition to high throughput ap- high dispersion (HD) refractive index oil. Finally, each fibrous and/or particulate plications, 5-μm SPP columns also bring high resolution separations to the realm of subset aliquot was characterized and identified utilizing stereo and polarized light HPLC users. The much lower pressure of the larger particle size, coupled with high microscopy. Three aliquots were examined from each household dust specimen. efficiency, permits longer columns or tandem columns to be applied for challeng- The resulting information for each new specimen was collected on a revised dust ing HPLC separations. In this paper, we demonstrate some of the advantages of tabulation data sheet. The newly acquired data and the data collected in the prior these new particles and columns using several high-throughput and high resolution study were combined and re-subjected to principal component analysis, support examples versus comparable sub-2-μm separations. vector machines and partial-least squares discriminant analysis in order to build statistical models of the composition profiles. These statistical models were then 367 Application of Fusion AE™ and DryLab® Software in HPLC Method subjected to a test set of randomly selected unlabeled dust samples in order to com- Development on a New Drug Substance (DS) pute estimates of misidentification rates (error rates). The methods used to compute Lily Du, Boehringer Ingelheim, 900 Ridgebury Rd., PO Box 368, these error rates were hold-one-out cross validation and bootstrapping. A number of Ridgefield, CT 06877, Fenghe Qiu interesting trends such as the ubiquitous occurrence of certain natural and synthetic This work developed a method to determine potential and actual impurities from a fiber types as well as the frequent occurrence of several classes of biological and new DS. Due to the complexity of synthetic process, about 10 intermediates, poten- particulate materials are extensively discussed. tial degradation products and unknown process impurities have to be considered in the analysis. Further, these compounds have a broad range of polarity and gener- 364 Fast Two-Column Initial Screening in HPLC Method Development ally lack of chromophore; therefore, development of a method with UV detection Ken Tseng, Nacalai USA, 10225 Barnes Canyon Rd., Ste. A103, San analyzing impurities ≤ 0.5% w/w was challenging. Design of experiment (DOE) was Diego, CA 92121, Toshi Ono, Tsunehisa Hirose, Kazuhiro Kimata carried out using Fusion AE in screening columns, modifiers and mobile phases. Selectivity is arguably the most influential factor on peak separation and conse- Further optimization was achieved by using DryLab software modeling. The final quently the resolution. When developing better separation methods, the use of high-performance liquid chromatography (HPLC) method used a HALO C8 column orthogonal selectivity in high-performance liquid chromatography (HPLC) column (4.6 x 150 mm, 2.7 µm), a UV detector and a gradient mobile phase in four linear screening is an effective way for selecting the right column. We propose a fast two- steps. Mobile phase A was 0.1% phosphoric acid in water and B, a mixture of metha-

57 2013 EAS Abstracts November 2013

nol/ acetonitrile at 25/75 (v/v). The method was sensitive and robust. Specificity was scientists. Furthermore, certain PGIs are unstable in typical reverse-phase high- validated through stress testing. The quantitation limit was 0.05 % w/w. The DS performance liquid chromatography (HPLC) conditions, creating more difficulty to and all impurities were baseline separated in 18 minutes. Retention times from the quantify the PGIs in pharmaceutical compounds accurately. A trace-level LC-mass modeling and from data acquisition agreed. A method was developed quickly and spectroscopy (MS) method was developed to directly analyze residual 1-ethyl-3-(3- efficiently by using Fusion AE and DryLab software. This work demonstrated the util- dimethylaminopropyl)carbodiimide (EDC). EDC converts to the inert urea product, ity of combining the strengths of two pieces of software in a challenging real system. (N-ethyl-N-(3-dimethylaminopropyl)urea, EDU in low pH polar protic conditions. Hy- drophilic interaction liquid chromatography (HILIC) conditions with high pH mobile 368 Very High-Pressure Liquid Chromatography (vHPLC) Method phase conditions were employed to prevent on-column hydrolysis of EDC to EDU. Development for the Determination of Purity and Impurities of Drug Using a single quadrupole with single ion monitoring, this method was validated Substance A by Utilizing Computer Simulation with suitable sensitivity, specificity, precision, linearity, and accuracy for the direct Rushin Jadawala, Bristol-Myers Squibb, One Squibb Dr., New quantitation of EDC in multiple drug substances. Brunswick, NJ 08901, Mohan Kanhasamy, Yan Ma, Harshad Patel vHPLC is widely used by pharmaceutical industry for the purity and impurities analy- 372 Direct Observation of Dissociation Behavior of Organic Salts in sis of active pharmaceutical ingredients (API). It is beneficial compared to tradi- Preparative SFC tional HPLC because of its high-throughput, lower dwell volume and lower solvent Mickey Rego, Averica Discovery Services, One Innovation Dr., Biotech consumption. This presentation describes the method development and optimiza- 3, Worcester, MA 01605, Paul Lefebvre, John H. Tipping, Jeffrey P. tion process for the determination of purity and impurities of drug substance A. The Kiplinger key challenge during method development included the separation of nine process Many synthetic pharmaceutical compounds requiring chromatographic purification impurities and degradants from the main peak and from each other. A systemat- are organic salts. At the drug discovery stage, salts may be prepared quickly, and ic approach for method development was followed by screening various column over salted or partially salted lots of compound are common. Preparative super- chemistries, mobile phase composition, column temperature and gradient. A Wa- critical fluid chromatography (SFC) is a valuable technology to obtain pure material ters UPLC™ system with multiple column-compartments was used for automatic for development testing. While it is often assumed that salts dissociate when dis- column screening. Mobile phase compositions were also evaluated and optimized solved in chromatographic eluents, the extent of salt dissociation under SFC condi- for the desired separation. The Drylab™ modeling tool was used to optimize the tions can vary. The level of dissociation impacts both preparative chromatography column temperature and mobile phase gradient to obtain the final method condi- performance and the resulting isolates. Many simple salts use counterions with no tions. Method validation was performed to demonstrate sufficient specificity, linear- UV signature, so that their elution profile is not detected by UV monitoring of the ity, sensitivity, precision, and robustness of the method. The method is planned to preparative chromatogram. Problems include multiple chromatographic bands of support chemical process development, stability studies, and release testing of clini- dissociated and undissociated material and differing amounts of counterion in frac- cal material. tions. Salt counterions may also form different salts with buffers added to the chro- matographic eluent, resulting in contamination of one or more fractions. We have 369 Development of a 2-Butanol Background Electrolyte for Non- examined the dissociation behavior of a variety of salts under preparative SFC con- Aqueous Chiral Capillary Electrophoresis and the Separation of ditions, varying solvents and eluent buffers and comparing salt forms with free acid/ Charged Pharmaceutical Enantiomers base forms of analytes. To detect non-UV active dissociated counterions we have Erin J. Ennis, Drexel University, 3141 Chestnut St., Philadelphia, PA coupled preparative SFC with evaporative light-scattering detection (ELSD). Ion 19104, Joe P. Foley chromatography was also used to evaluate material collected in fractions collected Non-aqueous capillary electrophoresis (NACE), an alternative to aqueous electro- using stacked-injection chromatography. This work demonstrates that consideration kinetic chromatography, is a technique that utilizes organic modifiers to enhance of salt form and salting equivalence in synthetic samples can offer significant ben- the separation of charged hydrophobic compounds. NACE can be applied to the efits to the chromatographer. separation of charged enantiomers by utilizing chiral additives or an enantiopure solvent, although the latter has received less attention. The separation of charged 373 Investigation of an Unknown Degradation Product Found in a Drug enantiomers is examined at various concentrations of chiral non-aqueous solvent Product Batch Release Test in order to optimize experimental methods. While traditionally performed with high Frances F. Liu, Novartis, One Health Plaza, East Hanover, NJ 07936, concentrations of organic solvent, this work examine 2-butanol as a potential NACE Sung Yoon-Kim background electrolyte at both low (mainly aqueous) and high (mainly non-aque- An unknown peak was found in a drug product during releasing test. The structure ous) concentrations. Experimental emphasis is on the optimization of resolution, of the peak was proposed based on the analysis results of high-performance liq- migration time, and enantioselectivity for pharmaceutical compounds of varying hy- uid chromatography-electron spray mass spectrometers (quadrupole ion trap mass drophobicity. Chromatographic figures of merit are examined and applications are spectrometry and time-of-flight mass spectrometry). This degradation product is a explored for future work. reaction product of drug substance with formaldehyde. It is known that formalde- hyde is an impurity of methanol (sample solvent) and some of excipients. Quantita- 370 Identification of Ephedrine Analogs in Lipodrene Products Using tive gas chromatography mass spectrometry (GC-MS) methods were developed to Capillary Electrophoresis determine the formaldehyde contents in methanol and excipients. Formaldehyde Michael Fletcher, Drexel University, 3141 Chestnut St., Philadelphia, PA reacts with pentafluorobenzylhydroxyl amine (PFBHA). The PFBHA derivative was 19104, Joe P. Foley analyzed by direct GC-MS for methanol and static headspace GC-MS for excipi- Lipodrene is a commercially available diet pill containing a variety of ingredients, ents. Eight logs from two brand names’ methanol were tested. The formaldehyde including extracts from the ephedra plant. The label on the lipodrene bottle reports contents in different lots with the same brand name were very similar. But they were ephedra extract as the primary ingredient, but does not report the quantities of the approximately 5 times higher in one brand methanol than the other one. Formalde- individual components of the extract. These extracts are primarily composed of hyde was not detectable in the six excipients used in current formulation. Therefore, ephedrine and derivatives of ephedrine. Due to its high efficiency, low reagent con- the degradation peak was generated during sample preparation. The sample solu- sumption, and speed of analysis, capillary electrophoresis (CE) is used to provide tion stability was re-evaluated. the separation and identification of the ephedrine derivatives in lipodrene. Standard solutions of the individual ephedrine stereoisomers are prepared in order to analyze 374 HPLC Method Development for Analysis and Release of a Multi- these samples for their purity and batch-to-batch variability. In addition, since all Component Drug Product ephedrine derivatives are chiral, the pharmacological and psychoactive effects of Kalyan Potluri, Almac, 20 Seagoe Industrial Estate, Co Armagh BT63 the enantiomers may differ, methods to identify and quantify each are necessary for 5QD, United Kingdom, John Wood, Abul Kalam Azad proper evaluation. The use of chiral agents such as crown ethers and cyclodextrins To develop a single HPLC method for assay / related substances and dissolution for aids in the enantiomeric separation of these compounds. multi component drug product containing active ingredients at very different concen- trations (tablets containing Aspirin 500 mg, Codeine 5 mg and Magnesium Oxide 70 371 Development of a Trace Level LC-MS Method for the Direct mg). Reverse phase high performance liquid chromatography (HPLC) method with Quantitation of EDC (1-ethyl-3-(3-dimethylaminopropyl) gradient flow was used. The results show that a single HPLC method with a total carbodiimide) in Pharmaceutical Compounds run time of 60 minutes was developed and validated to reliably quantify 6 known im- Timothy Nowak, Merck, 126 E. Lincoln Ave., Rahway, NJ 07065, Ryan purities for Aspirin and 15 known impurities for Codeine making a total of 21 known Cohen, Yong Liu, Renee Dermenjian impurities. In addition a rapid HPLC method with a total run time of 20 minutes Potential genotoxic impurities (PGIs) are chemical compounds that can potentially was developed and validated to determine the dissolution rates, assay and con- cause DNA damage. Trace level control and quantitation of these compounds in ac- tent uniformity of Aspirin, Codeine and Magnesium Oxide in the tablets. Magnesium tive pharmaceutical ingredients (APIs) have proposed challenges to pharmaceutical Oxide was determined by atomic absorption spectroscopy. The poster describes

58 2013 EAS Abstracts November 2013

some of the challenges encountered and solutions to overcome these challenges. phases: IC, ID, IE and IF. The increase in the size of the immobilized column library Developing a method for separating, identifying and quantifying 21 impurities by has made it possible to use an expanded range of solvents. The presentation sum- chromatography using a single run requires very high technical expertise. However marizes the results of the systematic investigation of 11 non-conventional solvents by achieving this, the need for having multiple methods for the analysis and release and mixtures thereof with methanol using six immobilized chiral stationary phase of two active pharmaceutical ingredients in the drug product is removed leading to columns from Chiral Technologies and nine commercially available racemates. We substantial reduction in time and resource spent. demonstrate the utility of using non-conventional solvents adjust retention and to address solubility and/or stability issues for both analytical and preparative super- 375 The Importance of Thresholding in Imaging Analysis of Protein critical fluid chromatography. Aggregates Lew Brown, Fluid Imaging Technologies, 200 Enterprise Dr., 378 Extraction of ß-blockers from Small Volume Biological Fluid Scarborough, ME 04074 Samples using a new Versatile SPE 96-well Plate Format Dynamic imaging particle analysis (DIPA) shows much greater sensitivity to trans- Matthew Cleeve, Kinesis Ltd., 9 Orion Ct., Ambuscade Rd., St. Neots parent particles, such as protein aggregates, than light obscuration can. While not PE19 8YX, United Kingdom yet fully accepted in industry compendia, DIPA is being used increasingly in the Developments in liquid chromatography tandem mass spectrometry (LC-MS-MS) formulation process for characterization of sub-visible particulates in biologics. DIPA detection levels have seen biological fluid sample volumes reduced to such an ex- measures particle size and shape by first creating a binary image based upon a de- tent that conventional solid-phase extraction (SPE) formats are not always suitable, fined threshold from the background value for each pixel in the image. The resulting or provide considerable analyte dilution and extended evaporation times. When ex- binary image is used for all particle measurements, so the choice of thresholding tracting from sample volumes of less than 100 µl, it is important elution volumes and technique is critical to the validity of the measurements. This poster demonstrates the sample prep format are fully compatible with the original sample volumes being how different thresholding techniques can produce dramatically different character- processed. The new TELOS MicroPlate format from Kinesis is a modular small vol- ization of protein aggregates. To do this, a base set of camera images are acquired ume bioanalytical sample prep format that provides both flexibility in terms of the for a therapeutic protein containing aggregates. These “raw images” can then be number of samples to be processed and allows the use of elution volumes that run through image processing software using different thresholding techniques to minimize analyte dilution. The TELOS MicroPlate can be used either partially or fully quantify and measure the aggregates contained. By using the same set of images populated, depending on assay numbers. The poster summarizes the extraction for each thresholding process, we insure that any variation in results can only be of three drugs from human plasma, Metoprolol, Oxprenolol and Propranolol using caused by the thresholding process. The results show striking differences caused TELOS neo PRP, a water-wettable polymer-based SPE sorbent. Analyte concentra- by the thresholding variation, thereby supporting the importance of proper thresh- tions range from 1 to 100 ng/ml. The data highlights the minimum elution volumes olding when using DIPA. that can be achieved from both 5 and 10 mg sorbent masses. Also reported are quantitative recoveries from a range of sample volumes using both sorbent masses. 376 Evaluation and Application of Hybrid Quadrupole-Time-of-Flight In addition to minimum sample and elution volumes, data is also presented on the Mass Spectrometry in Discovery Quantitative and Qualitative maximum sample volumes that can be applied to the 5 and 10 mg sorbent masses Analysis in the TELOS MicroPlate format. For the sample volumes uses, elution volumes and Hongwei Zhang, Bristol-Myers Squibb, 311 Pennington-Rocky Hill Rd., recoveries are reported, ensuring throughput is maximized. Pennington, NJ 08543, Baomin Xin, Timothy Olah A high resolution quadrupole time-of-flight (Q-TOF) mass spectrometer (MS), Triple- 379 Simplifying Safety Inspections in Chemistry Laboratories through TOF™ 5600 has been evaluated for its performance and suitability in conducting an iPAD Application both quantitative and qualitative analysis in discovery bioanalytical workflow. TOF Tilak Chandrasekaran, Merck, 126 E. Lincoln Ave., Rahway, NJ 07065, MS and three MS-MS data acquisition modes including multiple reaction monitoring David Waterhouse, Roy Helmy, Gabriel Graffius, Shaun Vandoren, high resolution (MRM-HR), SWATHTM and intel dynamic acceleration (IDA) have Girish Rao, Sivadasan Plakote, Ashish Das been applied in our discovery work and their advantages have been demonstrated Data collection during monthly safety inspections in chemistry laboratories can be a for different applications. TOF MS, MRM-HR and SWATH have been evaluated for tedious process. The findings are often collected manually with the data then sum- quantitative determination of small molecule compounds, peptides and proteins in- marized in a central system for the purpose of report creation and dissemination. volved in discovery programs. The assay performance was assessed by lower limit In order to simplify this process, collaboration between Merck Analytical Chemistry of quantitation, linear dynamic range, signal stability, accuracy and precision and and Real Soft, Inc. (RSI) led to the development of an iPAD application to use dur- compared with a triple quadrupole (API-4000) in MRM mode. The typical sample ing safety inspections. This application, known as ‘SAIL’ (safety audit inspection for preparation and ultra high performance liquid chromatography (UHPLC) methods labs), has been piloted in our chemistry labs. ‘SAIL’ is an innovative, touch-screen used in discovery settings were applied. A set of cyno thymidine kinase (TK) study application that simplifies data collection during the laboratory safety inspection pro- samples were analyzed on both Triple TOF 5600 and API 4000 and the exposure cess. In addition to data collection, it also enables safety reps/inspectors to seam- data were compared. In addition, Triple TOF 5600 along with in-line UV detection lessly click-n-attach pictures to signify non-compliances. Additional useful capabili- has been successfully applied to identify and semi-quantitatively determine metabo- ties of this application include dynamic lab creation and configuration, standard and lites involved in a TK study. The metabolite was identified and confirmed by accurate customized reports, 5S-certification, laboratory compliance trend analysis (historical mass and IDA MS-MS experiment. Our work has shown that Triple TOF 5600 is a data analysis), email integration, security, and customized questionnaires. In this practical instrument for conducting both quantitative and qualitative analysis in sup- poster we highlight the time savings, increased accountability, simplified reporting, port of discovery pharmacokinetic-pharmacodynamic studies. data trending, easy operability and portability for safety inspections using this state- of-the-art iPad application. 377 Evaluation of Non-Conventional Polar Modifiers on Immobilized Chiral Stationary Phases for Improved Resolution of Enantiomers 380 New Wide Pore C18 Phase for Fast and Efficient Purification of by Supercritical Fluid Chromatography (SFC) Peptides by Flash Chromatography Jimmy O. DaSilva, Merck, 126 E. Lincoln Ave., Rahway, NJ 07065, Chitra Sundararajan, Grace Discovery Sciences, 2051 Waukegan Brandyce Coes, Lisa Frey, Ingrid Mergelsberg, Ray McClain, Lisa Road, Deerfield, IL 60015, Nogle, Christopher J. Welsh Peptides and proteins are becoming increasingly popular for their potential use as As the diversity of drug intermediates and active pharmaceutical ingredients (APIs) therapeutic drugs. To earn and maintain a share in the fast-growing peptide market, increase, solubility has become a big concern. These compounds are less and peptide researchers and manufacturers are constantly trying to improve and opti- less soluble in the conventional solvents used in chiral SFC like methanol, ethanol, mize the various steps in peptide synthesis. One of the main bottlenecks in peptide 2-propanol, and acetonitrile and in carbon dioxide itself. Chlorinated solvents have synthesis is the purification step. Techniques such as fast protein liquid chroma- typically been used to aid in the dissolution of samples with poor solubility character- tography and preparative high-performance liquid chromatography are limited by istics. Only a small percentage, usually 10-20% of chlorinated solvent in the diluent, small loading amounts, long separation times, poor recoveries and high costs. In can be used with standard coated chiral columns without washing the coating away. this work, we demonstrate that flash chromatography can be a powerful tool in the In some cases the chlorinated solvent has been added, at a very small percentage fast and efficient purification of a diverse range of peptides. A new wide pore C18 (< 5%), to the mobile phase to increase solubility. Use of non-conventional solvents phase expands flash purification capabilities to larger sized peptides, while provid- can cause the coating to be washed away. In the past there were very few chiral ing better resolution. We present data to show the benefits of higher loading and stationary phases which were immobilized onto silica: Whelk-O, ULMO, and the faster purifications in peptide purification. This rapid purification technique ensures Chirobiotic line. In the last few years Chiral Technologies has released two new col- less degradation of peptides and provides better recovery, yields and purity. umns which are immobilized versions of two of their more popular coated phases, IA (immobilized AD) and IB (immobilized OD), as well as four novel immobilized

59 2013 EAS Abstracts November 2013

381 Improved Isolation and Purification of Natural Products by and high-throughput instrumentation as the system can process up to 24 samples Automated Flash Chromatography simultaneously. The MARSXpress Plus vessels with disposable glass liners were Chitra Sundararajan, Grace Discovery Sciences, 2051 Waukegan utilized in the extraction of semivolatile organic compounds from spiked soils follow- Road, Deerfield, IL 60015, Melissa Wilcox, Ben Hayes ing US EPA Method 3546. The solution containing the extracted compounds was Natural products play a dominant role in the development of new drugs for medici- analyzed with gas chromatography mass spectrometry. The spike recovery results nal purposes. A crude extract typically contains lead compounds requiring multiple are discussed. steps to isolate and purify. Conventional flash purification techniques either fail to detect compounds that are non-chromophoric or lack the necessary sensitivity often 385 Determination of Aflatoxins in Dried Fig, Dried Apricot and Red required for detecting small concentrations of vital components. Modern automated Pepper in Turkey flash chromatography systems with integrated evaporative light scattering detection Sevgi Kocaoba, Omer Ozcan, Yildiz Technical University, Faculty of can detect and fractionate both chromophoric and non-chromophoric sample com- Art and Science, Department of Chemistry, Davutpasa Cad. No:127, ponents. In this work we demonstrate how automated flash chromatography with 34210-Esenler, Istanbul 34210 Turkey integrated evaporative light scattering detection can save time and produce higher Aflatoxins B1, B2, G1 and G2 (AFB1, AFB2, AFG1 and AFG2, respectively) are purity and sample recoveries than traditional UV-based flash systems. toxic metabolites generated by Aspergillus flavus and Aspergillus parasiticus. These toxins have been reported to be associated with acute liver damage, liver cirrhosis, 382 Measurement of Density/Porosity in Pharmaceutical Tablets Using induction of tumors and teratogenic effects. Contamination of food such as rice, Terahertz Pulsed Spectroscopy nuts, peanut, dried fruits and spices especially chili by aflatoxins is widespread. A Wiphusanee Dendamrongvit, Advantest America, 508 Carnegie Center, rapid and simple method using ultra high-pressure liquid chromatography (UHPLC) Ste. 102, Princeton, NJ 08540, Mark Sullivan with fluorescence detection for the determination of aflatoxins in dried fig, dried apri- The purpose of this work is to determine tablet porosity using terahertz (THz) spec- cot and red pepper has been developed. Samples were collected from different troscopy without the requirement of measuring tablet thickness. Porosity is one of supermarkets and spice markets. Aflatoxins were quantified by using HPLC with the most important factors contributing to tablet mechanical strength that can af- fluorescence detector after sample preparations. The individual limit of detection fect product performance during handling and storage. It may also influence the (LOD) values, within each red pepper samples, ranged from 0.47 to 1.09, limit of diffusion rate of the active pharmaceutical ingredient which ultimately affects drug quantitation (LOQ) values from 0,62 to 1.36 (for B1 and total aflatoxins), for dried dissolution and bioavailability. In this study, tablets were made from microcrystalline fig samples, LOD values ranged from 0.45 to 1.07, LOQ values from 0.59 to 1.35 cellulose (Avicel® PH 102) using 5 different compaction forces of 5, 8, 10, 15, and 20 (for B1 and total aflatoxins) and for dried apricot samples, LOD values ranged from kN. Our preliminary results from terahertz time domain spectroscopy have shown a 0.45 to 1.08, LOQ values from 0.60 to 1.37 (for B1 and total aflatoxins), respectively. strong correlation between peak height and porosity which is independent of tablet According to the Turkish legal limits, reported for red pepper for B1 is 5 µg/kg for thickness. Thus, terahertz spectroscopy can potentially be applied as a fast, non- total aflatoxins are 10 μg/kg, for dried fig and apricot for B1 is 8 μg/kg, total aflatoxins destructive, process analytical technology tool for pharmaceutical development and are 10 μg/kg. Non-packed samples were higher than legal limits (12-15 μg/kg) but manufacturing. packed samples were below than these values (1-2 μg/kg). The results obtained from this study indicate that aflatoxins are common contaminants of food, and can 383 Detecting Trace Amounts of Impurity in Methylene Chloride - be considered to be a main concern for public health. Sample Preparation Matters Subhra Bhattacharya, Thermo Fisher Scientific, One Reagent Ln., Fair 386 A New NIST Standard Reference Material to Support Unbiased Lawn, NJ 07410, Eric Oliver, Chad Goldman, Apollo Quezada, Mark Perchlorate Measurements in Drinking Water and Other Matrices Jasko, Deva H. Puranam, Stephen C. Roemer Therese Butler, National Institute of Standards and Technology, 100 Methylene chloride is widely used as an extraction solvent in pharmaceutical, en- Bureau Dr., Gaithersburg, MD 20899, Michaele Winchester, Lee Yu, vironmental, food and chemical industries. To produce the finest quality methylene Rabia Oflaz chloride, trace amounts of impurity must be detected and removed from this solvent In 2011 the United States Environmental Protection Agency decided to regulate the which requires careful sample preparation technique. Detecting trace constituents amount of perchlorate as well as other toxic contaminants in drinking water. The in a solvent typically involves concentrating a substantial amount of neat solvent to reason for the regulation is that perchlorate has an impact on iodine uptake that may achieve the desired sensitivity before instrumental analysis. Care should be taken affect children later on in life. Perchlorate occurs naturally, but is also anthropogenic, during the concentration procedure so that the solvent retains all of the constituents. being used in fireworks, road flares, rocket fuels, bleaches, and fertilizers. Research We report a sample preparation technique for methylene chloride wherein the neat indicates that perchlorate may impact the thyroid, causing improper development sample is spiked with parts per trillion concentration of lindane and recovery of lin- during gestation and early childhood. In 2013 National Institutes of Health (NIST) is dane assessed using different concentration methods such as rotary evaporator, adding SRM 3188 Perchlorate Anion Standard Solution to the series. The SRM an- Kuderna-Danish (K-D) apparatus, and a combination of both. The K-D concentra- ion solutions are directly traceable to the International System of Units (SI), enabling tor showed better recovery compared to the rotary evaporator, however, the most their use as a vital and effective traceability link between commercial reference ma- consistent data was observed using a hybrid method of solvent concentration in terials and the SI. The availability of the new perchlorate SRM will underpin perchlo- which a combination of rotary evaporation followed by K-D concentration was used. rate measurements needed to ensure public and environmental health and safety. Gas chromatography- electron capture detector data showed consistently > 92% NIST has developed a high-performance technique that has now been successfully recovery of lindane with the hybrid method whereas the K-D technique showed only translated to ion chromatography (IC). Certification of the perchlorate anion mass 65% recovery. To our knowledge, this is the first report of combining K-D and rotary fraction in SRM 3188 was performed partly on the basis of this high-performance evaporation in to a high quality, time-effective concentration technique for analyzing IC methodology. In this poster presentation, the new SRM 3188 Perchlorate An- trace constituents in methylene chloride. ion Standard Solution is described, and data is presented to illustrate the use of high-performance IC in the certification. Also, the impact of the SRM on sustainable 384 High-Throughput Microwave Assisted Solvent Extraction of Semi- green chemistry and engineering are discussed. Volatile Organic Compounds Using US EPA Method 3546 Jason Keith, CEM Corporation, 3100 Smith Farm Rd., Matthews, NC 387 Withdrawn by the author. 28106, Daniel Iversen, Michael Karney Conventional extraction techniques with shakers, Soxhlet, or sonication for the preparation of environmental organic samples are time consuming, generate large 388 Nutrients and Dissolved Oxygen in Monmouth County Coastal volumes of solvent waste, and expose workers to toxic organic solvents for ex- Waters: Sustainability Concerns tended periods of time. Pressurized solvent extraction uses less solvent, but can Ellen Rubinstein, Monmouth University, 400 Cedar Ave., West Long only process six samples per hour and is limited by sample size. Microwave as- Branch, NJ 07764, Sarah-Pearl Siganporia sisted solvent extraction (MASE) is a proven technique that is fast, uses significantly Previous studies found nitrate concentrations in several Monmouth County coastal less solvent than conventional techniques, and is cost-effective. For these reasons, lakes to be higher than the minimum eutrophic level. Negative effects on the aquatic MASE is an approved procedure for the extraction of semi-volatile organic com- ecosystem include decreased levels of dissolved oxygen and damage to fish popu- pounds from soil, sediment, clay, sludge, and solid waste as listed in United States lations. Due to the impact of Superstorm Sandy on the coast of Monmouth County, Environmental Protection Agency (EPA) Method 3546. Most environmental labora- NJ on October 29, 2012, breaches occurred in the narrow land masses separating tories that follow US EPA Method 3546 will prepare and analyze several hundred the ocean from some coastal lakes. This project extends our earlier work to examine samples per week. These laboratories require instrumentation that is more sophis- the effect the storm may have had on nutrients and pH in these lakes. In addi- ticated and user-friendly for higher throughput. The recent introduction of MARSX- tion, it incorporates measurements of salinity and dissolved oxygen. Water samples press Plus vessels with disposable glass liners meets their needs for user-friendly were collected at the eastern ends of several coastal lakes. Dissolved oxygen and

60 2013 EAS Abstracts November 2013

temperature were measured on-site with Vernier sensors. Nitrate ion concentration, timeline perspective into direct mercury analysis technology, principle and applica- pH, and salinity were measured with Vernier electrodes on site or in the laboratory tions, a comparison of direct mercury analysis and conventional techniques and with the LoggerPro computer program. In the laboratory, UV-visible spectroscopy the development of extended range analysis with data covering several industrial was used for phosphate ion analysis. Our data contributed to understanding the matrices. health of our local lakes after Superstorm Sandy, and indicated locations for pos- sible remediation. It is recommended that future studies include periodic sampling 392 Novel Extraction and Analysis of 18 EPA PAHs from Mussel Tissue: and data analysis throughout the year, to observe any trends in salinity, nutrient, and Baseline Resolution of All PAHs in 6 Minutes with No Post dissolved oxygen concentrations with changing time and temperature. Extraction Clean-Up Ken Kirkbride, Thermo Fisher Scientific, 1228 Titan Way, Sunnyvale, CA 389 UPLC-UV Analysis of PAHs in Avian Egg Samples Collected after 94085, Peter Bodsky, Art W. Fitchett, Michael G. Hvizd, Michael Martin the Crude Oil Spill in the Gulf of Mexico with Rapid QuEChERS Sample preparation for the determination of trace level organic pollutants in solid Extraction and Novel 96-Well Phospholipid Cleanup samples can be time consuming, labor intensive and require large volumes of sol- Anthony A. Provatas, University of Connecticut, 3107 Horsebarn Hill Rd., vents. Accelerated solvent extraction (ASE) was developed to address these issues. Building 4 Annex, U-4210 Storrs, CT 06269, Aliaksnadr V. Yeudakimau, Since ASE uses elevated temperatures and pressures, sample extractions can be Gina M. Guerrera, Christopher R. Perkins, James D. Stuart performed with less solvent, time and labor as compared to traditional techniques. Our research involves the development of a rapid and novel method for extrac- However, for samples containing lipids or other co-extractable materials, clean-up tion and quantification of polycyclic aromatic hydrocarbons (PAHs) in avian egg procedures are often required after the extraction and prior to analysis. These clean- samples. PAHs are toxic organic compounds consisting of two or more fused aro- up procedures can also take time and copious amounts of solvents to complete, matic rings and are found in crude oil. They are a serious environmental concern adding to the overall cost and time of the total analytical workflow. If we modify due to their carcinogenic and mutagenic effects on higher organisms. As a result, the ASE process slightly by adding adsorbent material to the extraction cell along the United States Environmental Protection Agency has classified 16 un-substituted with the sample, properly optimized conditions can produce extracts essentially free PAHs as priority pollutants. The development of a rapid method for the quantification from interferences. This ASE in-cell clean-up procedure can be used to eliminate of PAHs is increasingly relevant since the widespread contamination of the Gulf of secondary chromatographic clean-up techniques and improve productivity. In this Mexico due to the Deepwater Horizon offshore rig explosion. QuEChERS (quick, paper we present results from our studies of the use of ASE with in-cell clean-up easy, cheap, effective, rugged, and safe), in conjunction with a novel Waters Ostro® for the determination of trace level PAH in marine animal tissue samples. Data will cleanup approach was utilized as an alternative to traditional extraction and clean- be shown demonstrating the impact of ASE operational parameters on analyte re- up procedures. This method greatly reduced sample preparation time from the 2-3 covery. With the properly optimized conditions, extracts were free from interfering days process to just hours compared to more traditional techniques that include co-extractables compounds. liquid-liquid extraction and alkaline digestion followed by gel permeation chroma- tography (GPC) and solid-phase extraction (SPE) cleanup. Analysis was conducted 393 Chromium (III) and Chromium (VI) Content in Agricultural Products on a Waters® UPLC-UV chromatographic system. Additionally, there was no loss in in Vicinity of Chromium Smelters sensitivity or selectivity observed during the sample analysis. The method detection Isiaka Alade Owolabi, Tshwane University of Technology, Department limit for PAHs ranged from 1.73 to 8.71 ng/mL based on UV detection (λ = 200-450 of Chemistry, PO Box 56208, Arcadia 0007, Pretoria GA 0001, Georgia nm) and a spiking concentration of 25 ng/mL. The analytes concentration recoveries Chromium (Cr) is one of the most and important trace metals which can be pres- were in the range of 87-105% and showed good linear correlation with % relative ent in two oxidation states: toxic Cr(VI) and non- toxic Cr(III). Cr(III) is an important standard deviation being between 1.1 and 3.5%. microelement for plant and animal nutrition and essential for the maintenance of glucose as well as for the lipid and protein metabolism. With regard to human health, 390 Methods for Determining Sugars and Hydroxymethyl Furfural in Cr(III) is a required nutrient, with 50–200 µg per day recommended for adults. On Biomass the contrary, Cr(VI) is toxic and carcinogenic for the human body, leading to lung Michael Hvizd, Thermo Fisher Scientific, 1228 Titan Way, Sunnyvale, cancer, skin allergy and probably also to asthma and renal diseases. A toxic effect CA 94085, Peter Bodsky, Lipika Basumallick, Jeffrey Rohrer for the biological systems is attributed to the ability of Cr(VI) to migrate across the Common biomass sugars and hydroxymethyl furfural (HMF) are routinely analyzed cell membrane, thus enhancing the intracellular chromium concentration. Hexava- during biofuel processing. It is important to monitor sugars to optimize processes lent chromium is rarely found in nature and is generally man-made, especially in for maximizing ethanol yield. There is also a growing interest in monitoring HMF as: fumes generated during the ferrochrome production. The permissible exposure 1) it is a ‘platform compound’ that can be used to synthesize several compounds limit (PEL) of chromium in air is 5 μg m-3 measured as Cr(VI). For this study, the (solvents, fuels etc.) that are currently derived from crude oil, and 2) it inhibits en- samples of industrial dust, as well as samples of such agricultural products as corn, zymes used in the fermentation process. This work describes high-performance tomato and potato were collected. Samples of food were washed with deionized anion-exchange chromatography with pulsed amperometric detection methods for water for the Cr(VI) determination. All measurements were carried out using a the determination of (i) sugars (ii) HMF in acid-hydrolyzed corn stover using electro- Perkin Elmer atomic absorption spectrometer model AAnalyst 600 with Zeemann lytically generated hydroxide eluent, separation on strong anion-exchange columns, background correction. It was found that Cr(VI) concentration in industrial dust was and electrochemical detection with disposable gold working electrodes. Biomass rather high, in the range 120-320 µg g-1, which contaminates the surface of to- samples often have sugar concentration over 100 g/L, and the common sugars mato. The internal concentration of Cr(VI) in tomato, corn and potato was about are fucose, sucrose, arabinose, galactose, glucose, xylose, mannose, and fructose. 0.6 ng g-1. The reason for that could be the presence of antioxidant components The method for sugar analysis has a short run time (8 min), while resolving all in agricultural plants. the sugars, linear range of 0.5-2 g/L, retention time precisions of 0.01-0.12%, peak area precisions of 1.7-2.7%, and acceptable recoveries (70-112%). The described 394 A Preliminary Examination: Historical Records of Polycyclic method is capable of handling the high concentration biomass samples with minimal Aromatic Hydrocarbons in Corals from the South China Sea sample preparation (i.e.; the strong acid hydrolysate just needs to be diluted and the Carolyn M. Poutasse, Haverford College, 370 Lancaster Ave., Haverford, column is not affected by the amount of sulfate in the diluted sample). The proposed PA 19041, Helen K. White, Nathalie F. Goodkin method for HMF has a broad linear range (0.1-1000 µg/mL), a low detection limit A preliminary examination of the presence of polycyclic aromatic hydrocarbons (0.04 µg/mL), high precision, and good recovery (112%). These methods require no (PAHs) was performed on Porites corals collected off of the coast of Nha Trang, eluent preparation and due to short analysis time, good sensitivity, and consistent Vietnam. PAHs, derived from petroleum, have carcinogenic and mutagenic proper- response, can be used for routine sugar and HMF analyses. ties and we hypothesize that they become incorporated into the corals by a com- bination of atmospheric transport and urban runoff. Corals from the pre-industrial 391 Expanding the Capabilities of Direct Mercury Analysis: A Look into period (ca. 1650), post-industrial period (ca. 1965), and modern period estimated Evolution, Detection Ranges and Sample Types (ca. 2004) were solvent extracted and the extracts analyzed by gas chromatography Sumedh P. Phatak, Milestone Inc., 25 Controls Dr., Shelton, CT 06484 mass spectrometry operated in selected ion monitoring mode for the quantification Over the last twenty years, mercury analysis has been studied by a wide variety of of PAHs. Preliminary findings indicate changes in the concentrations of PAHs ob- research groups. Recently however, direct mercury analysis has gained popularity served at different years. as an alternative to traditional techniques for mercury determination in solids, liquids and gases. Even with these technical advances, detection ranges for mercury vary greatly from one industry to another and at times, lie beyond the capabilities of standard direct mercury analyzers. Milestone’s direct mercury analyzer, DMA-80, has been modified to study an industry-mixed range of matrices to cover mercury concentrations from ppt to hundreds of ppm. This presentation provides an overall

61 2013 EAS Abstracts November 2013

395 A Regional Academic, Biodiesel Testing Laboratory Following 399 High Throughput Analysis of Harmful Algal Toxins in Freshwater ASTM Methods Samples Utilizing Simple Preparation Followed by UPLC Tandem James D. Stuart, University of Connecticut, 3107 Horsebarn Hill Rd., Mass Spectrometry Storrs, CT 06269, Joseph S. Impellizeri, Robert D. Morrin, Anthony A. Anthony A. Provatas, University of Connecticut, CESE, 3107 Horsebarn Provatas, Christopher R. Perkins Hill Rd., Storrs, CT 06269, Elizabeth R. Guerrera, Syed U. Ali, Aliaksandr This poster focuses on the effective operation of an academic Biodiesel Testing V. Yeudakimau, James D. Stuart, Christopher R. Perkins Laboratory at the University of Connecticut. Fifteen out of the sixteen American So- Water quantity and quality play a critical role in determining vital characteristics ciety for Testing and Materials (ASTM) tests, as required by the National Biodiesel of marine and freshwater ecosystems, and contribute significantly to human well- Board, have been implemented, except for Cetane Number which requires the use being. Consequently, physical, chemical, and biological characteristics of water of a diesel testing engine. The parameters for the following key tests that certify the represent key indicators of both ecological and human health. Human activities quality of biodiesel are presented: acid number, cloud point, cold soak filtration, free such as urbanization, industrial development, agricultural practices, and fuel use and total glycerine and oxidation stability. To validate the accuracy of the testing significantly alter many characteristics of water, resulting in changes in quality. Due methods and personnel, we participated in the triannual ASTM Biodiesel Laboratory to increasing urbanization and changes in climate, aquatic environments can re- Cross-Check Program over a five year period. A summary of the tests implemented ceive inputs of nitrogen and become eutrophic, supporting high concentrations of and our results for the Laboratory Cross-check Program are presented. Cyanobacteria. Microcystis and Anabaena spp. can produce known algal toxins, mi- crocystin and anatoxin, which have been shown to be lethal to wildlife and humans. 396 Nanopore Stochastic Detection of an Anthrax Biomarker As a result, several methods have been developed to detect the presence of these Yujing Han, Illinois Institute of Technology, 3101 S. Dearborn St., compounds. These methods required multiple sample preparation steps including: Chicago, IL 60616, Shuo Zhou solid phase extraction (SPE), filtration, sonication, and repeated freeze/thaw cycles, Pyridine-2,6-dicarboxylic acid (DPA), a substance uniquely present in bacte- which translate to lengthy processing times. A high-throughput method has been rial spores, is an important useful biomarker in the detection of anthrax spores. developed to detect and analyze microcystins in water. A Waters Acquity ultra-per- In this work, a sensitive method for the detection of DPA is developed by using formance liquid chromatograph equipped with a tandem mass spectrometer (UPLC- an engineered alpha-hemolysin protein ion channel in the presence of a molecular MS-MS) was used to identify and quantify the microcystins. The targeted analytes probe. This method employs single-channel recording with the planar lipid bilayer are microcytin -LR, -RR, -YR, -LA, and anatoxin-a. A Genevac EZ-2 evaporator has technique, where current modulation represents individual binding events. In the been utilized for sample evaporation to simplify this process. The internal standard absence of DPA, the molecular probe only produces a major type of events. In used for this analysis is ketoprofen-d3 and the surrogate compound is leucine en- contrast, in the presence of DPA, a new type of current blockage events with sig- kephalin. This methodology is presented in this poster presentation. Data from the nificantly different residence times and amplitudes is identified. Further, the concen- method detection limit and precision accuracy studies are also presented. tration of DPA could be related to the frequency of the new events. Our nanopore sensing method could detect DPA at nanomolar levels, and should find a potential 400 The Determination of Organophosphate Degradation Products in application as a low-cost screening sensor to monitor anthrax in powder and liquid Water, Soil and Wipe Samples by LC-MS-MS samples. Anthony G. Gugliotta, CSS-Dynamac, 2890 Woodbridge Ave., Edison, NJ 08837, Alexander E. Bleich, Julia Capri, Lawrence Kaelin 397 Nanopore Detection of Copper and Zinc Ions A fast, reliable method for the analysis of nerve agent degradation products of VX, Shuo Zhou, Illinois Institute of Technology, 3101 S. Dearborn St., GB, R-VX., GF and GD is presented. The liquid chromatography tandem mass Chicago, IL 60616, Xiyun Guan, Liang Wang spectrometry (LC-MS-MS) method is suitable for water, soil and wipes samples, The activities of modern society have led to ubiquitous contamination by metals, following the appropriate extraction steps, without the need for derivatization. Pre- some of which are toxic, and some of which are essential to life, but these same viously published methods in American Society for Testing and Materials Interna- elements can be toxic in larger quantities. Here, we report a sensitive and selec- tional specify the use of a reversed-phase high-performance liquid chromatography tive nanopore stochastic sensing method for copper(II) and zinc(II) ions by using (HPLC) column with a 20 minute run time. Here, the analysis time is reduced by peptides as chelating reagents. The sensing element is an engineered staphylococ- using ultra-performance liquid chromatography tandem mass spectrometry with a cal alpha-hemolysin protein ion channel and employs single-channel recording with hydrophilic interaction liquid chromatography (HILIC) technique. The mass spec- the planar lipid bilayer technique, where current modulations represent individual trometer is operated in electrospray positive and negative ion modes in the same binding events. In the absence of copper and zinc ions, only peptide events were analysis, and the total cycle time for nine analytes was less than 9 minutes. Average detected. However, upon addition of copper or zinc ions, a new type of events with recoveries for the degradation products ranged from 87% to 102%, and method significantly different durations and different amplitude blockages was additionally detection limits values were less than 20 µg/L for waters, less than 30 µg/kg for soils identified, which may be attributed to the metal-peptide complex. Further, copper and less than 10 ng/wipe for wipe samples. and zinc ions were able to be conveniently differentiated and eve! n simultaneously quantitated due to the quite different event residence times and amplitudes of their 401 HPLC Separation of 10 Fat-Soluble Vitamins in a Single Run metal-peptide complexes. The method could detect copper and zinc ions at nano- Ken Tseng, Nacalai USA, 10225 Barnes Canyon Rd., Ste. A103, San molar levels, and should find a potential application in identifying and quantifying Diego, CA 92121, Toshi Ono, Tsunehisa Hirose, Kazuhiro Kimata copper ions in an aqueous environment. Knowing the exact amount of fat-soluble vitamins in food consumption is important for our health, because these essential nutrients can be toxic at high levels. How- 398 Isolation and Quantification of DOSS from Deep-Sea Sediments ever, high-performance liquid chromatography (HPLC) separation of structurally Shelby L. Lyons, Haverford College, 370 Lancaster Ave., Haverford, PA similar fat-soluble vitamins is often a challenge to chromatographers using a C18 19041, Sarah J. Harrison, Helen K. White, Elizabeth B. Kujawinski HPLC or UHPLC column, especially if a complete separation in a single run is desir- In response to the 2010 Deepwater Horizon oil spill, dispersants were applied to able. In this study, we use a novel HPLC column with cholesteryl functional group surface waters and at depth to promote the mixing and degradation of oil in the to achieve superior selectivity of 10 fat-soluble vitamin analogs including D2, D3, water column. A major compound present in the dispersant used, is the anionic cis- and trans-vitamin K1. In addition, examples are shown for this column’s better surfactant dioctyl sodium sulfosuccinate (DOSS). DOSS was observed to persist in shape selectivity in resolving molecules with subtle structural differences. the water column for 64 days after the dispersant application, but its fate in deep- sea sediments has not yet been explored. In this study, we developed a method to 402 Migration of USEPA Method 8270 from Helium Carrier Gas to extract DOSS from sediment samples in order to analyze and quantify it via liquid Hydrogen Carrier Gas chromatography with tandem mass spectrometry (LC-MS-MS). To reduce the in- Paul V. Macek, Shimadzu Scientific Instruments, 7102 Riverwood Dr., terference from fine sedimentary material, a 1:1 MeOH:H2O solvent system was Columbia, MD 21046, Cindy Burkhardt used for extraction in conjunction with two centrifugation steps. With this method, Helium is by far the most commonly used carrier gas for gas chromatography mass 104% (+/- 15%) of the DOSS added to spiked-sediment samples was recovered. spectrometry (GC-MS). Warnings from the American Chemical Society (ACS), These methods will be implemented to examine the presence of DOSS in impacted commercial helium vendors, and others predicting a helium shortage have forced sediments collected in December 2010, 11km from the site of the Macondo well. laboratories to explore the use of hydrogen as an alternate carrier gas for GC-MS. Findings from this study provide a more complete understanding of the fate of dis- United States Environmental Protection Agency (EPA) Method 8270 is used for persants in Gulf of Mexico deep sea environments. the analysis of a wide variety of compounds ranging from phenols to anilines and polynuclear aromatic hydrocarbons (PAHs). Hydrogen is known to react with some Method 8270 compounds. For example, nitrobenzene reduces to aniline when hy- drogen is used as the carrier gas on a GC-MS. Hydrogen is often thought of as a superior carrier gas, and often is an excellent carrier, but some unexpected results

62 2013 EAS Abstracts November 2013

were obtained for some compounds when the method was switched over to hydro- preparation is simplified, without the need of derivatization, since the detector only gen carrier. For many compounds the conversion to hydrogen carrier is almost trivi- requires a non-volatile analyte to provide a response. Samples were simply diluted al, but for some compounds, results deviated from what is obtained with Helium car- and analyzed. Examples of surfactant determinations or characterizations within rier. For example, some acidic compounds such as phenols, under some conditions, different product samples, aqueous and in mineral oil are described. exhibit a reduction in response factor and high % relative standard deviations across the calibrated range compared to calibration curves analyzed with helium carrier. 406 Fast Ion Determinations and Efficient Separations of Complex Some progress has been made through the use of on-column injection (OCI), but Samples Using Smaller Particle-Size Ion Exchange Columns that technique introduces some new challenges. Modifications to the OCI method Greg Malek, Thermo Fisher Scientific, 1228 Titan Way, Sunnyvale, CA are explored. It has been suggested that injection port reactions involving water, free 94085, Peter Bodsky, Terri Christison chlorine, and hydrogen may be causing some of the effects in the standard injection The demand for increased sample throughput and speed of results often neces- port. Accordingly, pH effects will be studied as will the use of alternative solvents. sitates sacrificing chromatographic peak resolution which in turn can compromise the accuracy and reproducibility of quantitation results. Recent advances in ion ex- 403 Impurity Analysis Using a New High Sensitivity UHPLC Detector change chemistry permit the reduction of resin particles to 4-µm from the previously William Hedgepeth, Shimadzu Scientific Instruments, 7102 Riverwood available 7–9-µm particle sizes. These 4-µm particle-size ion-exchange columns Dr., Columbia, MD 21046, Ken Tanaka have high theoretical plate counts, typically on the order of 8,000-12,000, which The use of ultra high-performance liquid chromatography (UHPLC) continues to increase chromatographic resolution of critical pairs and can permit faster separa- grow throughout the analytical industry as researchers increase their need for faster tions while maintaining excellent resolution. High-efficiency ion-exchange columns LC separations with more sensitive detection requirements. Recently, a new high- can accommodate increased flow rates yielding faster chromatographic separations sensitivity detector was introduced that provides improved UHPLC system per- without sacrificing resolution. For example, an increase of up to 3 times standard formance with new features that improve workplace efficiency. The new UHPLC flow rates utilizing 150 mm column formats proportionally reduces sample run times detector features a total reflection capillary cell to reduce system dispersion and to less than 4 minutes while maintaining baseline separation of all analytes. The allow ultra-high speed analysis. The high sensitivity of the detector combined with its use of 150 and 250 mm columns containing smaller-particle size resins will result wide dynamic detection range allows simultaneous analysis of a main peak and an in higher operating back pressures, often up to 3,000-4,500 psi, requiring a high- impurity peak with a 0.005% concentration. A new feature of this detector allows for pressure capable ion chromatography system. This presentation discusses recent peak deconvolution analysis with a new integration method (i-PDeA). This function advances in 4-µm particle size ion-exchange columns, fast ion determinations and allows complete separation of co-eluted peaks and addresses an important problem high efficiency separations using capillary and microbore formats on environmental, for researchers concerning the purity determination of a peak. By analyzing spectral beverage, and petrochemical samples. differences between co-eluting peaks, one of the co-eluted peaks can be eliminated to allow visualization of the remaining peak. Use of this feature can speed method 407 Improved Universal Approach to Measure Natural Products in a development and is a complement to quantitative integration. Another new function Variety of Botanicals and Supplements of the detector provides extended dynamic range (i-DReC). This function enables David Thomas, Thermo Fisher Scientific, 22 Alpha Rd., Chelmsford, MA an extension in apparent dynamic range by shifting the spectrum wavelength when 01824, Ian N. Acworth, Bruce A. Bailey, Qi Zhang, Marc Plante the linearity of the calibration curve cannot be obtained due to intensity saturation of Botanicals contain a great diversity of compounds that exhibit wide variation in their the detector. Potential analyst error is also avoided in the dilution procedure. Results physicochemical properties. Although no single analytical method is available to presented demonstrate the increased sensitivity of the new detector. Examples of measure all potentially active components, high-performance liquid chromatogra- peak deconvolution capabilities and the detector’s extended dynamic range are also phy (HPLC) with charged aerosol detection (CAD) is a nearly universal approach presented. that nonselectively measures any nonvolatile and many semi volatile compounds; that is, CAD does not require that analytes be ionizable (as required for mass 404 Examining the Capabilities and Benefits of a UHPLC Method spectrometry) or contain a chromophore (as required for UV spectrophotometry). Development Scouting System Presented are several HPLC/ultra-HPLC methods with charged aerosol detection William Hedgepeth, Shimadzu Scientific Instruments, 7102 Riverwood that have been improved to increase speed and sensitivity. The improved methods Dr., Columbia, MD 21046, Ken Tanaka were evaluated for the measurement of phytochemicals extracted from a variety of The scouting and optimization of method conditions in high-performance liquid chro- botanicals including: triterpenoid glycosides from Bacopa monniera; steroidal and matography (HPLC) requires a considerable amount of time and effort. Often, most pregnane glycosides from Hoodia gordonii; oxypregnane glycosides from Caral- of this time and effort may be devoted to the initial column tested before concluding luma fimbriata; steroidal lactones from Ashwagandha (Withania somnifera); flavo- it will not be suitable for the needed separation. Also, samples that may contain nolignans from milk thistle (Silybum marianum); triterpene glycosides from black unknown impurities or natural substances comprised of many components tend to cohosh (Cimicifuga racemosa); and ginsenosides from ginseng (Panax ginseng). be constructed by a trial and error approach with a number of columns and mo- Analytes showed consistent response independent of chemical structure (typically bile phases. The use of ultra HPLC has been proven to be beneficial in reducing < 10% variability between compounds corrected for gradient elution). All methods overall method development times by shortening individual runtimes by a factor of had a wide dynamic range (~four orders of magnitude), good sensitivity (typically 10x. However, a large amount of the analyst’s time is spent evaluating each run, low ng levels of detection), and excellent reproducibility (relative standard deviations programming new conditions, and changing columns and mobile phases. Method typically < 2%) even at low detection levels. Comparative data from evaporative light development times are also extended by the amount of time the analyst is away scattering detector and UV detection are also discussed. from the lab. Recently, the use of method development systems has become more popular. The capabilities and benefits that can be obtained with use of a system 408 A Fast and Easy Way for Determining the Level of Oxidative Stress are presented. Using UHPLC with Boron Doped Diamond Electrochemical Detection 405 Quantitation of Surfactants in Products by High-Performance Bruce A. Bailey, Thermo Fisher Scientific, 22 Alpha Rd., Chelmsford, MA Liquid Chromatography and Corona Charged Aerosol Detection 01824, Marc Plante, David Thomas, Qi Zhang, Ian N. Acworth Marc A. Plante, Thermo Fisher Scientific, 22 Alpha Rd., Chelmsford, MA Aminothiols (e.g., cysteine, glutathione [GSH], homocysteine), disulfides (e.g., cys- 01824, David H. Thomas, Bruce A. Bailey, Ian N. Acworth, Qi Zhang tine, glutathione disulfide [GSSG], homocystine), and thioethers (e.g., methionine) High-performance liquid chromatography is the ideal tool for the determination play many important biochemical roles in-vivo. GSH is a major cellular antioxidant of surfactants in samples. However, since surfactants do not typically possess a and plays a vital role in protecting the mitochondria organelle, which provides cel- chromophore, sensitive detection by ultraviolet or fluorescence is difficult. Universal lular energy production. The cellular ratio of GSH to GSSG is often used as a mea- detectors, such as refractive index and evaporative light scattering exist, but each sure of cellular toxicity and decreases in this ratio can be used as an indicator of has significant limitations on sensitivity and capability. Mass spectrometry is a useful mitochondrial dysfunction, which can lead to cell death associated with degenera- tool for these analytes but is more expensive to own and operate in a quality control tive diseases. Cysteine is also a cellular antioxidant and serves as a precursor to environment than a simpler detector, such as the Corona charged aerosol detector. glutathione. It is often found as part of a disulfide link in proteins. Methionine is Charged aerosol detectors are sensitive, mass-based detectors, especially well- an essential amino acid and serves as a methyl donor when incorporated in s- suited for the determination of non-volatile and many semi-volatile analytes with adenosylmethionine. Although a variety of high-performance liquid chromatography sensitivity to low nanograms on column. Analyte response is largely independent (HPLC) techniques have been developed for the measurement of thiols, disulfides of chemical structure, providing clear relationships among different analytes in a and thioethers most of these exhibit technical issues resulting from derivatization, sample. This sensitivity, combined with the specificity of the Acclaim Surfactant Plus and artifactual changes in the GSH/GSSG ratio that can occur during sample prepa- column for reversed-phase analysis, provides a unique analytical solution for sensi- ration. These compounds can be detected directly using sensitive electrochemical tive, reproducible, and routine analysis of surfactant-containing samples. Sample techniques using boron doped diamond electrodes. The rapid ultra-HPLC-Box-

63 2013 EAS Abstracts November 2013

Behnken design method presented is capable of determining several aminothiols method to resolve the degradant from the compound of interest. Combining HPLC and related compounds in less than 4 minutes. Rapid sampling and analysis mini- with UV detection the compound solubility was determined without interference. mizes artifactual changes in the GSH/GSSG ratio. An example showing the analysis of GSH and GSSG from blood samples using a simple “dilute and shoot” protocol 413 Withdrawn by the author. is presented. 409 Evaluation of Herb and Fruit Juice Adulteration and Authenticity by 414 Comparing Electrochemical Detection and Derivatization- Coulometric Array Detection and Pattern Recognition Analysis Photometric Detection in Analyzing Sulfur-Containing Compounds Qi Zhang, Thermo Fisher Scientific, 22 Alpha Rd., Chelmsford, MA Using Cation Exchange Chromatography 01824, Bruce A. Bailey, Marc Plante, David Thomas, Ian N. Acworth Jinmo Huang, The College of New Jersey, PO Box 7718, Ewing, NJ Although the adulteration of herbs and fruit juice is a frequent phenomenon, there 08628, Samantha Mascetti are few simple methods available for the screening of large numbers of commercial Sulfur-containing amino compounds including cysteine, cystine, reduced glutathi- batches of product. The challenge arises from the complexity and variability of genu- one (GSH), oxidized glutathione (GSSG), homocysteine, homocystine, and methio- ine material combined with the unrelenting conduct of adulteration. Herb and fruit nine are often coexisting in biological samples. Simultaneous determination of these variety, growing region, season, ripeness, and processing methods all contribute to compounds is critical in biochemical, clinical, and medical applications. In this work, the variability of the authentic product, making unambiguous characterization dif- cation exchange chromatography is used to separate sulfur-containing compounds. ficult. Currently, the most reliable and applicable authentication methods are based Both postcolumn ninhydrin-derivatized photometric detection and integrated pulsed on analytical chemical fingerprinting (metabolomic) techniques. High-performance amperometric detection are used to quantify these compounds. The results includ- liquid chromatography (HPLC) with coulometric detection is particularly suitable for ing detection limit, sensitivity, and linear range of calibration for these compounds generating chemical fingerprint for herb and fruit juices that contains endogenous are compared and discussed. electrocitive components essential to their flavor, stability, color and aroma. An ex- tension of phytochemical fingerprinting utilize statistical program such as principal- 415 Development and Validation of a Stability-Indicating UHPLC component analysis and pattern recognition to evaluate the authenticity of a given Method for Determination of Anastrozole and Anastrozole Related sample by comparing its chromatogram with a compiled population of authenticated Compounds in Pharmaceutical Dosage Forms reference sample. We investigated the approach of using HPLC-Coulometric Array Tatiana Kim, ENDO Pharmaceuticals, 8 Clarke Dr., Cranbury, NJ 08512, detection with pattern recognition software to identify potential oregano herb and Alex Schwarz, Scot Thoroughman, Joemon Varughese fruit juice adulteration. Oregano adulteration by blending with adulterants such as A single, stability-indicating ultra high-performance liquid chromatography (UHPLC) thyme and marjoram, were readily detected. In a second study, fruit juice adultera- method was developed and validated for the simultaneous determination of anas- tion using dilutions with another juice, or the inclusion of orange peel / pulp-wash trozole and anastrozole related compounds. The developed method separates an- could readily be evaluated using this technique. The specificity of this approach astrozole from five specified related compounds within a run time of 16 minutes. even allows the classification of orange juice samples by varietal and geographical The chromatographic separations were achieved using a gradient mixture of wa- region. ter, methanol, acetonitrile, and trifluoroacetic acid pumped at 1.0 mL/min through a Phenomenex Kinetex XB column (C18, 75 mm, 3.0 mm, 2.6 µm) held at 30 °C, 410 Withdrawn by the author. and a wavelength of detection of 215 nm. The developed UHPLC method was vali- dated according to the International Conference on Harmonization guidelines. The limit of detection and limit of quantitation were estimated at 0.002% and 0.007% 411 HPLC Method Optimization for Large Molecule in an Inhalation respectively, with respect to a sample concentration of 2 mg/mL. The described Dosage Form method was linear over a range of 0.2-30 μg/mL for anastrozole, 0.1-15 μg/mL for Craig D. Oakum, MicroDose Therapeutx, 4262 US Route 1, Maple impurity ANZRC01, 0.2-19 μg/mL for impurity ANZRC02, 0.2-21 μg/mL for impurity Shade, NJ 08852, Eugene R. Reynolds ANZRC03, 0.2-19 μg/mL for related Compound A, and 0.2-16 μg/mL for impurity An high-performance liquid chromatography (HPLC) method for a large molecule ANZRC05 (related compounds nomenclature provided by Hetero Laboratories). was developed for inhalation dosage test samples. MicroDose Therapeutx develops The accuracy range of the method has been established from 50-150% at assay dry powder inhalers (DPIs) that are tested for aerodynamic particle size distribu- level and 0.03-1% at the related compounds level. Due to its simplicity and rapid- tion (APSD, USP<601>). APSD generates large numbers of samples per dose. For ness, the proposed UHPLC method provides a significant improvement over the two high-throughput, preparations must be simple and the HPLC run time must be short. separate HPLC methods provided by the United States Pharmacopeia, and may be An HPLC method was initially developed with a run time of 2.2 minutes for samples used for determining anastrozole and anastrozole related compounds in the bulk prepared using a single dilution. Peak interference was observed with the method drug substance and other pharmaceutical dosage forms. at ≥80 µg/mL concentration. Continued use of this method would require multiple dilutions of samples, which would limit throughput. The mobile phase was adjusted 416 Development of Low Extractables Syringe Filters for Liquid from 72% to 74% aqueous which separated interfering peaks in concentrations up Chromatography-Mass Spectrometry Applications to 125 µg/mL. A single sample dilution step can now be used for the range of sample Gilbert Tumambac, Pall Corporation, 25 Harbor Park Dr., Port concentrations obtained. The run time increased to 4 minutes which still allowed Washington, NY 11050 for high-throughput testing. Decreasing the injection volume from 40 µL to 15 µL In analytical sample preparation, extraction and leaching of chemical compounds reduced peak tailing from 3.0 to 1.8. The limit of quantitation (LOQ) increased from from syringe filters are major concerns particularly when using liquid chromatogra- 0.5 µg/mL to 1.5 µg/mL, which is still sufficiently sensitive to support APSD testing. phy mass spectrometry (LC-MS) for analysis. An underlying issue when filtration is The linear range shifted from 0.5-50 µg/mL to 1.5-125 µg/mL. The optimized method used for sample preparation is the need to reduce the amount of extractable and developed for an inhalation dose of a large molecule demonstrated that multiple leachable compounds in the syringe filter effluents that may ultimately jeopardize sample concentrations can be prepared in a single dilution, and then analyzed with analytical results and data interpretation. Two common issues related to filtration for an HPLC run time of 4 minutes, meeting the requirement for high preparation and LC-MS sample preparation are the presence of extraneous chromatographic peaks analysis throughput. and ion suppression/enhancement. To address these concerns, Pall Corporation has developed new and unique syringe filters called Acrodisc® MS syringe filters 412 Solubility of a Drug Unstable to Buffer which are certified for low extractables using a highly sensitive LC-MS method. John H. Tipping, Averica Discovery Services, One Innovation Dr., Three These filters exhibit excellent compatibility, are made of high quality water-wettable Biotech, Worcester, MA 01605, Jeffrey P. Kiplinger, Paul M. Lefebvre, polytetrafluoro-ethylene (WWPTFE) membrane, and high-density polyethylene Michael J. Rego (HDPE) housing materials. In this study, the development of Acrodisc MS syringe The most popular and effective method for measuring a drug’s solubility is by direct filters is presented. In addition, the cleanliness of the effluents using Pall Acrodisc UV absorbance, which utilizes the linearity of concentration against UV absorbance. MS syringe filters was compared with a commercially available alternative syringe Occasionally the direct response from an alternative detector is used, such as Che- filter from two vendors by evaluating the presence of extractable/leachable com- miluminescent Nitrogen Detection (CLND) or electrochemical detection, as these pounds in the filter effluents under the same filtration and LC- electrospray ionization detectors are fairly consistent in response within a similar chemical series. While (ESA)-MS analytical test conditions. Moreover, the matrix effects (ion suppression/ we commonly use direct UV absorbance detection for these experiments, we often enhancement) due to potentially interfering extractable/leachable compounds were encounter compounds where direct UV absorbance is not a sufficient means for studied for the analyses of three drug substances) by comparing the MS signal in- analysis. A case in point is a compound which is unstable in the buffer solution tensity of each drug substance spiked into the filter effluent against a control solution used in the solubility measurement. As discussed herein, such a case illustrates the using fast gradient LC-ESI-MS. importance of developing a rapid high-performance liquid chromatography (HPLC)

64 2013 EAS Abstracts November 2013

417 Understanding the Health Safety and Environmental Implication of filtration membrane which divides the two chambers of the dialysis cell. The sample Formulating Solutions in the Laboratory Environment solution is continuously pumped through the first chamber of the cell passing over Anthony R. Kemperman, Honeywell, Burdick and Jackson, 1953 S. the filtration membrane. The residual monomer in this solution diffuses across the Harvey St., Muskegon, MI 49442, Jeremy N. Diringer semi-permeable membrane of the dialysis cell into the stationary acceptor solution, Laboratories are tasked with making blended formulations for use in environmental, where concentration equilibrium is set up. The equilibrated acceptor solution, which pharmaceutical and biopharmaceutical applications for use with high-performance now contains the residual monomer, is subsequently injected directly into the IC liquid chromatography (HPLC), gas chromatography and LC-mass spectrometry. system. Size exclusion chromatography was employed to monitor the extent of the These formulations are often developed without a full Health Safety and Environ- removal of the high molecular weight material from the samples. In summary, the mental (HS&E) review. The hazards can come from the neat chemicals used to inline dialysis cell provides an automated cost effective “green” sample preparation prepare the formulation, blend or buffer. They can also come from the new hazards step that greatly reduces the overall analysis time and plastic waste. associated with the formulation. HS&E groups tend to focus on the neat chemical or ingredient used to make the formulation. HS&E issues from the process of making 422 NMR in Structural Biology: Synergy of Solid- and Solution-State buffers and chemical solutions can reduce productivity and create hidden cost within Approaches a company. The poster examines the hazards associated with preparing formula- Dennis A. Torchia, National Institutes of Health, Bldg. 5, Room 128, Five tions in the lab. One focus is the hazards associated with the neat components Center Dr., Bethesda, MD 20892 used to make formulations. The other focus is on the lesser understood hazards of The past forty years have witnessed great advances in the sophistication and utility the new formulation for use in an application. These unknown dangers can have a of solid- and solution-state nuclear magnetic resonance (NMR) spectroscopic ap- direct impact to worker safety, chemical inventory management and productivity. A plications in structural biology. Both the dynamics and structure of a myriad of bio- solution to this dilemma is to utilize commercially prepared formulations. Honeywell molecules have been elucidated at atomic resolution, thereby providing a molecular LabReady Blends by Burdick & Jackson is an example of an offering that is specifi- basis for understanding function. Due to their distinct instrumental requirements, cally suited to address these factors. When organizations choose to “buy” versus solid- and solution-state studies are often segregated; however, the two approaches “make” their formulations, they are able to have a clearly defined product that meets are closely related and often provide highly complementary physical and biochemi- the various requirements of governing bodies of lab chemical hygiene. The poster cal information. I illustrate this using example from my past work which exploited the demonstrates how adopting Honeywell LabReady Blends can decrease the hazards synergy of the two approaches. I anticipate that this material will serve as a useful of preparing formulations in-house. The poster also shows how risk can be mitigated introduction to the three subsequent talks in this symposium, which describe cur- through the purchasing of commercially prepared formulations. rent state-of-the-art solid- and solution-state NMR approaches in structural biology. 418 Advantages of Reduced Surface Activity (RSA) Glass Autosampler 423 High Frequency Dynamic Nuclear Polarization Vials for Basic Solutes Robert Griffin, Massachusetts Institute of Technology, Francis Bitter Josh E. Young, MicroSolv Technology, PO Box 4, South New Berlin, NY Magnet Laboratory and Department of Chemistry, Cambridge, MA 13843, William Ciccone, Joseph J. Pesek, Maria T. Matyska 02139 Three test solutes were assayed by high-performance liquid chromatography (thia- Over the last few years we have developed cyclotron resonance maser (a.k.a. gyro- mine, benzoic acid, and phenol) in two different types of glass autosampler vials tron) microwave sources that operate at frequencies of 140-527 GHz that dynamic at two different time intervals. Thiamine showed a 32.6% reduction in peak area permit nuclear polarization (DNP) enhanced nuclear magnetic resonance (DNP- after four hours in the regular glass vial but only 3.1% in the reduced surface activ- NMR) experiments in magnetic fields of 5-18.8 T (1H NMR frequencies of 211-800 ity (RSA) glass vial. Benzoic acid and phenol, which do not have basic functional MHz, respectively). We review the instrumentation used for these experiments, groups, were not significantly affected in terms of peak area using either vial. The which include new NMR probe designs and tunable gyrotron oscillators and ampli- data illustrates how the basic functional groups were responsible for the loss of fiers. The latter should be useful for electron decoupling that eliminates undesirable analyte and most importantly how RSA vials lead to significantly less adsorption for paramagnetic broadening. In addition, we discuss two mechanisms used for DNP basic solutes compared to ordinary vials. experiments in solids at high fields – the solid effect and cross effect -- and the polar- izing agents appropriate for each. These include metals, biradicals and mixtures of 419 Separation of 1,3-Dimethylamylamine and Other Polar Compounds narrow line radicals that enable increased enhancements at reduced concentrations in Dietary Supplement Formulation Using Aqueous Normal Phase of the paramagnetic center. The newest nitroxide biradicals are yielding ε=400 @ Chromatography with Mass Spectrometry 90 K in standard samples. In addition, we discuss applications of DNP/NMR that Josh E. Young, MicroSolv Technology, PO Box 4, South New Berlin, NY illustrate its utility in enhancing signal-to-noise in MAS NMR spectra of a variety of 13843, Rosie Le, Maria T. Matyska, Joseph J. Pesek biological systems including membrane and amyloid proteins whose structures are Five ingredients (caffeine, L-arginine alpha-ketoglutarate, creatine, beta-alanine, of considerable scientific interest. Finally, we report the initial observation of Over- and 1,3-dimethylamylamine (DMAA)) from a workout supplement were separated hauser effects simultaneously present with solid effects in insulating solids. by high-performance liquid chromatography (HPLC)-UV and LC-mass spectrom- etry (MS) using a silica hydride-based analytical column operating in aqueous 424 Seeing the Invisible by Solution NMR Spectroscopy normal phase (ANP) mode. While reversed-phase methods were observed to be Lewis Kay, University of Toronto, One King’s College Circle, Toronto, ON inadequate for the analysis due to low retention, ANP chromatography was able to M5S 1A8, Canada readily retain the analytes. After method development on the HPLC-UV system, the Many biochemical processes such as ligand binding, enzyme catalysis, molecu- conditions were successfully transferred to an LC-MS system for analysis. Based on lar recognition and protein folding proceed through the formation of functionally calibration curve data, estimates of 12,697.4, 3003.4, and 2247.8 mg/serving were important intermediates that escape detection using traditional structural biology obtained for creatine, L-arginine, and DMAA respectively. The results suggest that methods. A nuclear magnetic resonance (NMR) approach for seeing such ‘invisible’ the conditions presented in this work could be applied towards a validated analytical states is described. A related problem is one where ‘near invisible’ systems, such method in future studies. as supra-molecular machines, are studied by solution NMR. New NMR approaches and applications that bring such systems into focus are presented. 420 Withdrawn by the author. 425 Can a Static Ubiquitin Structure “Out Score” a Dynamic Ensemble? Ad Bax, National Institutes of Health, Bldg 5, Room 126, Five Center Dr., 421 Inline Dialysis of Acrylate Polymers Prior to Residual Monomer Bethesda, MD 20892 Analysis The antibiotic squalamine, originally isolated from dogfish shark, forms a lyotropic Andrew R. Falls, Ashland Specialty Ingredients, Listers Mill, Beamsley liquid crystal at very low concentrations in water (0.3-3.5% w/v), which remains Rd., Bradford, BD9 4SH, United Kingdom, Nigel Crabtree, Fan Wu stable over a wide range of temperature (1-40 °C) and pH (4-8). Squalamine is This technique was created due to the need for a simple and quick sample prepa- positively charged, and comparison of the alignment of ubiquitin in this medium ration step for the removal of large molecular weight polymers from our research relative to 36 previously reported alignment conditions shows that it differs sub- samples. The analysis in question is the measurement of residual monomer levels stantially from most of these, but is closest to liquid crystalline cetyl pyridinium bro- in polymer products using ion chromatography (IC). Previously the samples were mide. High precision residual dipolar couplings (RDCs) measured for the backbone diluted in water then centrifuged through a 3000 molecular weight cut off filter for 1H-15N, 15N-13C’, and 13C’-13Ca one-bond interactions in squalamine medium fit well 1.5Hr at 3000 rpm. The filtrate is then manually transferred to a Metrohm Autos- to the static structural model previously derived from nuclear magnetic resonance ampler. The centrifuge technique is time consuming, costly and produces a large data. Inclusion of the new RDCs into the structure refinement procedure resulted amount of plastic waste. The new process just requires dilution of the sample in in improved agreement between alignment-induced changes in 13C’ chemical shift water prior to dialysis followed by IC analysis, therefore enabling the process to and experimental values, thereby validating the high quality of the static structural be fully automated. The main component of the dialysis cell is the Millipore ultra- model. Our result indicates that fitting of a single model to experimental data can 65 2013 EAS Abstracts November 2013

provide a better description of the time- or ensemble-averaged conformation than 429 An Evaluation of Alternative Carrier Gases for GC-MS analysis in do ensemble representations, whereas the latter can capture dynamic aspects of a the Flavor & Fragrance Industry protein, thus making them valuable complements to one another. Stephen Toth, International Flavors & Fragrances, 1515 State Highway 36, Union Beach, NJ 07735, Sharon Brown 426 Developments in Particles, Columns and Phases for HPLC and Helium is becoming scarce and therefore, more expensive to use as a carrier gas UHPLC for gas chromatography (GC) and GC-mass spectrometry (MS) analysis. Recently, Richard Henry, Consultant, 983 Greenbriar Dr., State College, PA 16801 there have been many articles written on the use of hydrogen as carrier gas when Columns filled with spherical particles have become the standard for high-perfor- using mass spectrometry. While most praise its speed and lower expense, there are mance liquid chromatography (HPLC) analytical methods; however, very significant concerns with safety as well as issues with background noise associated with the changes have occurred in columns and instruments during the past decade. HPLC scrubbing effect Hydrogen can have on the source. This conversion can, and has column efficiency had been stalled at 100,000 plates/meter for about two decades, been done successfully in many labs. An alternative to hydrogen is nitrogen as a placing limits on speed and resolution. Particle and column research continued un- carrier gas. Although often overlooked, nitrogen can be used as a cheap alterna- til commercial columns with much higher efficiency appeared in about 2004. Col- tive to hydrogen. The use of nitrogen will adversely affect the speed of separation, umns with significantly higher performance required HPLC instruments with higher but to what degree? This talk covers the evaluation of hydrogen and nitrogen as operating pressure and lower dispersion, which introduced the era of ultra HPLC alternatives to helium as a carrier gas in GC-MS analysis with respect to the flavor or UHPLC. Advances in smaller porous and solid-core particles that led to higher & fragrance industry. performance HPLC columns are briefly reviewed. While efforts have been focused on improvements in column efficiency, research has been deflected from devel- 430 Application of Quality-by-Design Strategies to Analytical Method oping new stationary phases with improved selectivity, peak symmetry, and sta- Development bility. Higher performance UHPLC columns are being offered with essentially the Serena Wang, Merck, 2000 Galloping Hill Rd., Kenilworth, NJ 07033, same stationary phases to allow chemists to use existing knowledge and improve Yuetian Chen, Shara Dellatore, Beth Junker, Daisy Richardson separation speed and sample throughput by using shorter columns. When desired, Robust manufacturing and reliable analytical testing are both required ensuring greater resolution can also be accomplished by using the same or longer columns. product quality. Quality-by-design (QbD) concepts were originally proposed to im- Advantages and disadvantages of transferring methods and developing new meth- prove the robustness of manufacturing processes. The concept can also be ap- ods using UHPLC components are described. Some suggestions on how to take plied to improve the reliability of analytical methods. A continuum scheme of QbD advantage of recent improvements in HPLC column and instrument technology are for analytical methods framework (A-QbD) is adapted from the traditional analyti- offered. cal method development approach by including a strong emphasis on the realiza- tion of the product requirements and target product profile. This strategy is then 427 Carbon-Based Phases Revisited: New Chemistries, and a Fresh used as guidance for the application of QbD in analytical method development. Perspective on Old Chemistries The structured step-by-step procedures utilized in the unified scheme illustrate a Dwight Stoll, Gustavus Adolphus College, 800 West College Ave., systematic method development approach that includes understanding the method St. Peter, MN 56082, Tuan Tran, Ian Gibbs-Hall, Paul Young, John requirements, evaluating and scouting of potential methods, performing risk assess- Danforth, Jonathan Thompson ment of the candidate methods, exploring the design space, and defining a method Over the past few years we have prepared a number of stationary phases based procedure with appropriate controls. The scheme provides a methodical thought on high purity porous silica by deposition and/or bonding of a variety of carbon process to method developers during each step in method development to obtain nanomaterials. Our interest in these materials is driven both by the attractive posi- optimal method performance and ensure the fulfillment of quality and performance tive characteristics of similar, existing materials (e.g., porous graphitic carbon), and targets for the method. The practical application of QbD concepts together with the deficiencies of these same materials. Specifically, the unique selectivities of Merck A-QbD are examined using a case study of N-glycan profiling of monoclonal existing materials that enable isomer separations and enhanced retention of polar antibodies. The analysis of glycan profile involves lengthy sample handling pro- and polarizable compounds are particularly useful in two-dimensional liquid chro- cedures, and therefore high variability of the test results is usually observed. With matography (2-DLC) where stationary phase chemistries that are complementary to assistance of Merck A-QbD, the critical steps in the method procedure were deter- existing bonded phases are needed for successful 2-D separations. However, exist- mined and optimized. Increased understanding of assay variability and multivariate ing materials have several weaknesses for separations of some compound classes interactions was further obtained by the use of statistical tools together with design that seem to be insurmountable in spite of decades of research, including: prohibi- of experiments. tively high retention, poor peak shape, poor efficiency, and high substrate chemical activity. In this presentation we review the physical and chemical characteristics of 431 Quality-by-Design: Specifications Setting for Biological Drug the materials we have prepared, and demonstrate how they complement existing Development materials. We present selectivity and efficiency data using simple probe molecules, Nanda Subbarao, Biologics Consulting Group, 400 N. Washington St., and demonstrate the application of the recently prepared materials in 2-DLC sepa- Suite 100, Alexandria, VA 22314 rations of molecules that cannot be analyzed using existing carbon-based stationary The traditional procedure to set specifications for products involves obtaining test phases. In lieu of the ideal carbon-based stationary phase for high-performance LC, data from the batches manufactured prior to regulatory filing. In contrast, the quality- which remains elusive, a more complete understanding of the properties of old and by-design approach is to set the specifications based on prior knowledge of the new carbon-based phases continues to enable separations using these phases, attributes relationship to product’s safety and efficacy profile. Attributes that have a and continues to inform the development of new stationary phase materials. significant impact on safety and efficacy are more tightly controlled than attributes which are known to not have a significant impact. Approaches for control strategy for 428 The Development and Utilization of Sub-2 Micron Chromatography raw material, in-process lot release and stability testing are discussed. Columns Matthew Przybyciel, ES Industries, 701 S. Rt 73, West Berlin, NJ 08091 432 Analytical Methods Life Cycle Management in Quality-by-Design Reversed-phase high-performance liquid chromatography (HPLC) is widely used Paradigm for Biological Drug Development and Commercialization for separation of many pharmaceutical compounds. A majority of these separations Robert W. Donaldson, Genzyme, 45 New York Ave., Framingham, MA are based on octadecylsilyl (ODS) type columns. However, retention and separation 01701 of various compounds have proven to be a challenge. Many of these types of com- In biologics, process evolution proceeds along a continuum from early phase de- pounds are unretained, poorly retained or not separated on most conventional ODS velopment through clinical trials, scale up for registration, to commercialization. The reversed-phase columns. In many cases the separation of these type compounds/ evolution of the analytical methods is propelled by this process change pathway in mixtures are not achievable even with ODS column are packed with highly efficient order to reflect the product quality resulted from the changes. In the early clinical sub 2 particles. Fortunately, to deal with these types of compounds other stationary phases, there is a focus on safety and efficacy with characterization methods devel- phases are available such as polar, fluorinated, non-hydrocarbon and alternative oped and qualified. Once in-place, expanding the focus from release and stability hydrocarbon phases. However, many of these alternative phases have not been testing of drug substances, drug products to increased testing for upstream and commercially available in a sub 2 micron particle format which deprives the chroma- downstream allows implementation of a defined process control strategy. This is tographer of the advantages of sub 2 micron columns. These advantages include a central component of Analytical Life Cycle Management (ALCM) which expands fast analysis and high resolution separations. It is the goal of this study is to develop the space of each method to include specific steps in the process and allows for an array of sub 2 micron particle based stationary phases that are not conventional changes in components or platforms along the continuum. Linking critical quality at- ODS phases. These phases include C30, hydrophilic interaction liquid chromatog- tributes and critical process parameters to analytical methods as a quality by design raphy, and fluorinated phases. We provide data on the performance of these unique approach sets criteria for analytical life cycle management. Quality-by-design (QbD) stationary phases and chromatographic applications on the developed phases. is used to define alert limits for key process parameters and to simplify product release testing by validating out specification testing through process control. QbD 66 2013 EAS Abstracts November 2013

is also applied to evaluating component or platform changes. There are significant 437 A Rapid, Nondestructive Method to Measure Layer Thickness in demands on the method development and validation group in evaluating the vari- Pharmaceutical Beads Using Terahertz Waves ous changes for the various in-process materials which can be lessened through Richard R. McKay, Advantest America, 508 Carnegie Ctr., Princeton, NJ incorporation of QbD. In this presentation, several examples are presented on how 08540, Edward E. King, Eiji Kato, Mark Sullivan, David Heaps, Akiyoshi we at Genzyme, as part of ALCM, apply quality by design approach to modernizing, Irisawa, Motoki Imamura redevelopment and revalidation of the analytical platforms to accommodate evolu- Many pharmaceutical products are administered in multi-particulate drug delivery tionary changes in process globally. systems for their benefits in bioavailability and safety over monolithic tablets. These beads are typically less than a millimeter in diameter and can have multiple drug 433 Case Study on Application of DOE to Formulation Development for and coating layers. Variations in bead diameter and layer thicknesses can have a Biologics dramatic effect on the release profile of the active pharmaceutical ingredient. There- Vishal C. Nashine, Bristol-Myers Squibb, One Squibb Dr., New fore, the manufacturing process must be monitored and controlled to assure the Brunswick, NJ 08903, Aastha Puri, Jose Tabora, Monica Adams desired therapeutic effect. We present two methods for rapid, accurate and non- As drug development paradigms and regulatory expectations continue to evolve, destructive measurement of multiple functional layers for a collection of individual there is increasing emphasis on utilization of science-driven approaches to ensure beads in a multi-particulate drug delivery system. One method utilizes time-of-flight that quality is an integral component of drug product development and design for terahertz reflection measurements to identify the layer interfaces and accurately cal- biologics. In this context, we present two case studies on application of design-of- culate layer thicknesses. Another makes use of scattering information from terahertz experiment (DOE) approaches in the early phase formulation development. In both spectroscopy. The terahertz data are compared to more traditional, but destructive cases, the designs were constructed based on understanding of the physical and and time-consuming, methods of bead layer thickness analysis. chemical attributes of the active molecules as well as the desired product profiles. In the first case study, initial solution screening and a subsequent DOE study enabled 438 Withdrawn by the author. development of a flexible drug product configuration to support intravenous admin- istration over the broad dose range required for the early trials. In the second case study, utilization of a DOE approach allowed minimization of the levels of impurities 439 Pulsed Terahertz Time-Domain and FT Spectroscopy for the formed via two competing instabilities (i.e., aggregation and deamidation) and se- Molecular, Microscopic and Bulk Characterization of lection of appropriate quality attributes necessary for the development of a subcuta- Pharmaceutical Tablets neous biologic product. As an extension of this study, we also present limitations of Mark J. Sullivan, Advantest America, 508 Carnegie Ctr., Princeton, NJ Arrhenius extrapolations towards prediction of end-of shelf-life levels of deamidation 08540, Edward E. King, Richard R. McKay, David Heaps, Eiji Kato products and describe the utility of modeling tools to estimate these levels from the Terahertz pulsed spectroscopy (TPS) is a versatile, non-destructive technique for accelerated stability data. the characterization of multilayered solids. One can measure spatial depth profiles of coating and layer thicknesses, chemical and physical composition of individual 434 Best Practice in Cascade Impactor Measurements constituents and bulk properties of the entire matrix. TPS offers significant advan- Volker Glaab, Boehringer Ingelheim GmbH & Co.KG, Binger Str. 173, tages over many complementary spectroscopic techniques in terms of its depth of Ingelheim am Rhein 55216, Germany, Terrence Tougas, Jolyon P. penetration down to several millimeters and its inherent ability to measure layer Mitchell, Svetlana Lyapustina thickness without external calibration. In this paper we present several applications For orally inhaled products (OIPs) such as pressurized metered dose inhalers, dry of non-destructive analyses on pharmaceutical solid dosage forms. These examples powder inhalers and nebulizing systems, one of the key attributes needed to assure demonstrate how the measurement of critical quality attributes on individual tablets quality, safety and efficacy of the drug product to the patient is aerodynamic particle can significantly improve statistical correlations with their performance properties. size distribution (APSD), which ensures that the OIP delivers a consistent inhalable mass fraction of active pharmaceutical ingredient (API) throughout its entire lifetime. 440 Applications of Differential Scanning Calorimetry High-Pressure APSD measurements are usually conducted by using cascade impactors (CI) that Crucibles are described in the respective Pharmacopeias. The results of these measurements Richard S. Kwasny, Fauske & Associates, 16W070 West 83rd St., Burr could be reported as a fine particle dose (as required by the European Pharma- Ridge, IL 60517, Kenneth Kurko, Zachary Hackmeister copoeia) or as stage groupings (as required by the United States Food and Drug Differential scanning calorimetry (DSC) analysis can only be used to identify and Administration). One of the advantages of the CI method is that it provides chemical quantify hazardous energy releases if there is no sample loss during the test, which identification of the API in specified size ranges based on aerodynamic diameter, typically is performed at a linear heat rate of 0.25-6.00 °C/min between 25-400 °C. and therefore linked with likely deposition in the respiratory tract. On the other hand, The test crucible must be able to withstand pressures at the material’s critical tem- CI measurements have been long recognized to be prone to bias, high method vari- perature, which can be up to 217 bar (3,190 psi). Aluminum pans and low-pressure ability (relative to the expected product variability) and error associated with their cells are not capable of containing such pressures. A number of classic decom- set-up and operation. These methodological errors and measurement uncertainties position reactions such as di-tertiary butyl peroxide, azobisisobutyronitrile, ammo- could be minimized or avoided through the use of Good Cascade Impaction Prac- nium nitrate, and nitrocellulose will be presented. The integrity of the high-pressure tices augmented under certain circumstances by the careful use of more rapid and crucibles will be demonstrated using water, which develops a pressure of 217 bar simpler-to-execute non-CI particle sizing methods. This presentation describes both at 374 °C. Since the DSC data is reliable for highly energetic decompositions, a the background problems and suggested solutions. demonstration of Yoshida’s correlation is provided, which estimates if a material can be shock sensitive and/or explosion propagating. Using a series of DSC tests 435 Withdrawn by the author. for the decomposition of azodicarbonamide, thermokinetic software was used to extract kinetic parameters by the differential isoconversional approach. These ki- netic parameters were then applied for the prediction of the chemical’s behavior 436 Physical Stability of Nanoparticle Dispersion on the kilogram scale. The results of the determination of decomposition kinetics Jonathan B. Denis, Formulaction Inc., 12555 Orange Dr., Ste .101, of azodicarbonamide are presented and applied to evaluate the self-accelerating Davie, FL 33330 decomposition temperature for a 50 kg package. Results obtained by the presented Nanoparticle applications in the industry are getting more and more important and method are compared to those determined by other methods. concern many different fields (drug delivery, carbon nanotubes, display technolo- gies, etc.). In the last decades a vast amount of scientific research has been de- 441 The Use of Natural-Abundance Stable Isotopes in the veloped to improve the understanding of these complex dispersions. Using new Characterization of Bio/Pharmaceutical Compounds and Synthetic analytical techniques, it is now possible to control and tailor properties of suspen- Pathways sions and to get a better understanding of time behavior. Following this idea, stability John P. Jasper, Nature’s Fingerprint, 8 Old Oak Ln., Niantic, CT 06357, measurements, which were commonly done by simple visual observations, can now Martin B. Pavane, Dean Eyler, Ila Sharma, Albert C. Lee be performed automatically via an optical device. This instrument is based on mul- The ambient distribution of light stable isotopes in biopharmaceutical synthetic path- tiple light scattering and is associated to a vertical scanning of the sample. It enables ways permits the identification and differentiation of potentially-infringing pathways. to identify and quantify instability phenomena before they are visible to the operator After reviewing three cases of product identification, we examine three cases of (up to 200 times earlier). Physical parameters and kinetics can be computed in process authentication: one of false advertising and two of process patent infringe- order to facilitate and improve sample comparison. Examples of carbon nanotubes ment. The three cases of product authentication demonstrate the dynamic range dispersibility measurements are presented using this technique and enable to fine of the light stable isotopes in differentiating sources of pharmaceutical materials. tune the dispersion in terms of solvent, surfactant content etc. The false-advertising case was substantially a product case because the green- tea L-Theanine and the client- and competitor L-Theanine were markedly differ- ent in carbon- (~15‰) and nitrogen isotopic (~10‰) composition. The competitor 67 2013 EAS Abstracts November 2013

L-Theanine process was revealed from court documents and chemical-isotopic 446 The Role of Chemometrics in Chromatography insight. The competitor was accused of falsely advertising the source of their L- Brian G. Rohrback, Infometrix, 11807 North Creek Parkway South, Theanine. The second case of process patent infringement was a straightforward Bothell, WA 98011 case of infringement. An infringer used synthetic intermediates that were readily As analytical chemists, nearly every sample we examine is a mixture. As a result, available on the market to produce the infringing product. When confronted with the much of our effort is focused on understanding or controlling composition. There- isotopic evidence of process infringement, an out-of-court, business resolution was fore, we employ chromatography as the most direct method of characterizing these reached. The third case of process infringement was a case of wrongful accusation samples. As chemometricians, we realize that computers can be tasked with more of infringement. The carbon-isotopic records of both the product and process stud- than just speeding throughput, spewing reports and storing results. Application of ies show that: 1) the products are of different origins and 2) the defendant had in multivariate approaches to signal processing and data interpretation allows us to fact used a different synthetic pathway so that he was not infringing the patent of re-engineer chromatographs to make them significantly more responsive and cost- the plaintiff. effective. The input for chemometric analysis comes in two forms: results tables and raw chromatograms. Processing the table is straight-forward, but does not handle 442 Challenges in Developing Robust Analytical Methods for Consumer inconsistent numbers of peaks easily. Raw data has its challenge in that chroma- Products tography is subject to fluctuation in retention time, which forces the operator to re- Jacqueline Erickson, GlaxoSmithKline, 1500 Littleton Rd., Parsippany, view and occasional intervene to insure precise, reproducible peak identification NJ 07054 and quantitation. A multivariate correlation approach is very successful at correcting Consumer healthcare products can vary widely in product format and often have these shifts in retention time and allows quantitative and qualitative chemometric complex formulations due to the addition of ingredients such as flavors and dyes, in processing be run as part of an unattended sequence. Interestingly, the proper han- order to meet consumer needs. In addition, the regulatory classification of consumer dling of retention time shifts also simplifies how we do calibration for most applica- healthcare products may differ in various regions of the world. Developing a robust tions. The primary benefits of the chemometric approach are found in performing analytical method to meet regulatory requirements can be challenging with complex objective data interpretation in real time, and in making the distribution of methods matrices such as those found in consumer products. This presentation discusses and applications much more plug-and-play. In this way, analytical experience gained some of the challenges and provide examples where methods have been devel- in one location on one instrument is transferable worldwide. As the technology is a oped for complex formulations. software-only solution, it can be applied to new installations and as simple retrofits. 443 Modernization of the USP Monograph with a Focus on the 447 TBA Consumer Products Karl Brooksh, University of Delaware, 2 Lammot Dupont Laboratory, Leonel M. Santos, United States Pharmacopeia, 12601 Twinbrook Newark, DE 19716 Parkway, Rockville, MD 20852, Clydewyn M. Anthony No abstract submitted by the author. A key initiative at United States Pharmacopeia (USP) is the modernization of mono- graphs across all compendia. Monograph modernization includes replacing test 448 On the Relationship Between Whitened Principal Components methods that are non-specific, contain outdated technology, and non-value added Analysis and Maximum Autocorrelation Factors test. It also includes addition of an organic impurities test and an identification test Neal Gallagher, Eigenvector Research, 160 Gobblers Knob Lane, when not present in the monograph. Collaboration between Food and Drug Admis- Manson, WA 98831, Jeremy M. Shaver tration and USP to focus on modernizing over-the-counter (OTC) product mono- Maximum autocorrelation factors (MAF) and the used of de-cluttering algorithms graphs is ongoing. Expert panels are formed to focus on OTC and prescription have been gaining popularity recently in the chemometrics. Generalized least products. The list of monographs that will undergo modernization is available online squares weighting, developed by Aitken c. 1935, is a popular and useful de-clut- and is continually updated. tering approach that can be used to “whiten” data prior to principal components analysis (PCA). The relationship between MAF and whitened PCA is elucidated 444 Method and Application of Using a Flow Cell Apparatus for with the objective of ‘demystifying’ the MAF and MAF-like algorithms. The insight is Visualizing Additive Deposition on Consumer-Relevant Substrates intended to provide a fundamental understanding of the methods such that they can Ian D. Henry, Procter & Gamble, 11511 Reed Hartman Hwy., Cincinnati, be employed to best effect and that related methods can be easily developed using OH 45241, Dirk W. Domaschko, Renee D. Bolden a similar relationship. Examples are shown for hyperspectral images In the consumer products world no adage rings truer than, “to see is to believe.” When trying to understand the technology and benefit of new formulas, the im- 449 Implementation of Out-of-Scope Diagnostic Tools in a pact of an image or visual captured in a relevant context cannot be understated, Pharmaceutical Environment especially for the Beauty consumer. To this end, the Modified Flow Cell Apparatus Dongsheng Bu, Bristol-Myers Squibb, One Squibb Dr., New Brunswick, was developed for inspecting the interaction and deposition of product additives NJ 08903, Gary McGeorge, Douglas Both on substrates. Based on the Focht flow-cell design, this apparatus affords the abil- Out-of-Scope measurement detection is a key element in successful pharmaceuti- ity to perform microscopy under controlled flow conditions similar to consumer in- cal quality monitoring by multivariate models and multi-channel measurements. We use conditions. In particular, substrates such as hair are fixed such that fluid flows have been developing and implementing multivariate models which can be used around all sides of the shaft, as opposed to being sandwiched, affording a more to predict properties of interest to ensure product quality and process robustness. relevant snapshot of the interaction of formulas and their additives with hair and In the present work we evaluated Hotelling-T2, Q-residual statistics and prediction other substrates as product mixtures flow across. This patented apparatus is shared uncertainty approaches as applied to partial least squares models that were applied in detail, along with example work illustrating key concepts. to a pharmaceutical tablet assay method incorporating near-infrared spectroscopy. We discuss algorithms available in several software packages, confidence limit set- 445 Implementation Challenges of Near IR Analysis in the Consumer ting for outlier measurement detection, and model maintenance strategy during the Products Industry method development and implementation. Ramon Santana, Colgate-Palmolive Company, 909 River Rd., Piscataway, NJ 08855 450 The ACS Approval Process - A Preview of Coming Attractions Innovation is a key element of the consumer products industry. Formulation revi- Cynthia K. Larive, University of California – Riverside, Department of sions from such innovation create a problem for the establishment of lasting quality Chemistry, Riverside, CA 92521 controls. Unlike the pharmaceutical field, changes in consumer product formulations The American Chemical Society (ACS) operates an approval program for chemistry happen with amazing frequency and speed and it is necessary to find a solution to undergraduate programs as a mechanism for fostering excellence in the training of keep up with the changing environment. Near-infrared spectroscopy (NIR) rapid chemists in the United States. The ACS Committee on Professional Training (CPT) methods have proven to be a very useful tool for this purpose. This presentation administers the ACS approval program and also has as its charge collecting and demonstrates how the factors of time and resources are addressed in the solution making available information concerning trends and developments in modern chem- to maintain robust methods. The discussion of the calibration development process ical education. An overview of the current ACS Guidelines for Approval of Bachelor’s begins with the more complex design of experiment (DOE) calibration through the Degree Programs and the processes followed are presented along with proposed simplification of the mathematical model through composite and liquid calibration to changes to the Guidelines currently under consideration. Proposed revisions cover- the very effective dry calibration model. To demonstrate robustness and efficiency, ing aspects of program infrastructure, the frequency of course offerings, laboratory calibration methods were compared statistically using the same samples for the experiences and instrumentation, student skills, and safety are discussed. method validation and the final plant qualification.

68 2013 EAS Abstracts November 2013

451 The Role of Research in the American Chemical Society Certified 455 VOC Mixture Composition Analysis: Quantification of an EPA VOC Degree Standard Mixture Using the BrightSpec Chirped-Pulse Fourier Thomas Wenzel, Bates College, 5 Andrews Rd., Lewiston, ME 04240 Transform Spectrometer Undergraduate research provides an experience in which students can gain and ex- Robin L. Pulliam, BrightSpec, 675 Peter Jefferson Parkway, ercise critical thinking and problem solving skills. An effective research experience Charlottesville, VA 22911, Brent J. Harris, Matthew Muckle, Dave also provides students with the opportunity to integrate chemistry concepts learned McDaniel, Brooks H. Pate in classes into the broader context of a research question. Finally, it provides stu- BrightSpec introduces benchtop chirped-pulse millimeter wave spectroscopy for gas dents with the opportunity to investigate previously unanswered questions or to mixture analysis. The detection and quantification of volatile organic compounds synthesize and study new molecules. In recognition of the important role research (VOCs) is of particular importance for environmental monitoring and headspace plays in the education of professional chemists, the guidelines for bachelor’s degree residual solvent analysis. Fourier transform infrared (FTIR), gas chromatography programs allow research to count for up to 180 of the 400 laboratory hours or for as (GC)- flame ionization detection, and GC-mass spectroscopy have significant limi- much as four semester credit hours toward the four in-depth courses required for tations in terms of absolute specificity and trace level detection of small volatiles. certification. This talk explores the role of research in the undergraduate curriculum Even with separation and purification, fingerprinting structurally similar species re- and the expectations of the experience for certified chemistry majors. mains difficult for these techniques even with vast libraries of molecular fingerprints in existence for these analytical techniques. We present the composition analysis 452 Boots on the Ground: The Role of the Visiting Associate in the ACS of standard Environmental Protection Agency (EPA) VOC mixture using the Bright- Approval Program Spec spectrometer without column separation. The BrightSpec CP-FT instrument Richard Dallinger, Wabash College, Department of Chemistry, takes advantage of intrinsic qualities of rotational spectroscopy, such as its depen- Crawfordsville, IN 47933 dence on the three-dimensional structure of a molecule and the high-resolution of When a chemistry department applies to the American Chemical Society (ACS) millimeter-wave spectroscopic techniques. As such, distinctions between isomers, Committee on Professional Training (CPT) for approval of its program, the CPT conformers and isotopologues are easily addressed. Linearity and detection limits often recommends a site visit by a member of its panel of Visiting Associates. The (< 100 ppb) of VOCs are demonstrated and composition analysis of a standard EPA Visiting Associate spends approximately two days on campus gathering information VOC mixture is presented. about the program. The Visiting Associate then evaluates the program within the framework of the guidelines for approval established by the CPT and reports the 456 GPC-IR Deformulation of Complex Polymer Mixtures with IR findings to the CPT to inform their final decision. This presentation describes the site Spectral Database Search visit process and explain the work of the Visiting Associate in the program approval Ming Zhou, Spectra Analysis Instruments, 257 Simarano Dr., process. The focus is on how the Visiting Associate evaluates the applicant program Marlborough, MA 01752, Dana Garcia, Farrel N. Borden, Tracy Phillpott with respect to both the letter and the spirit of the CPT guidelines. Mid-infrared spectroscopy is a well-established analytical technique to characterize chemical compositions of individual polymers and additives. Coupling an on-line 453 ACS Guidelines as a Catalyst for Institutional and Curricular infrared detector with gel permeation chromatography (GPC) made it possible to Change take Fourier transform infrared (FTIR) snapshot spectra of each polymer component Kimberley A. Woznack, California University of PA, Department of and additive in a complex polymer mixture and then identify them one at a time by Chemistry, 250 University Ave., California, PA 15419 IR spectral database search. GPC separates the complex polymer mixture into high The “ACS Guidelines for Bachelor’s Degree Programs” outline the expectations of molecular weight (MW) polymer components and low MW additives according to the American Chemical Society (ACS) with regards to high quality undergraduate their hydrodynamic sizes. The GPC-IR system removes the solvent and then depos- programs. Obtaining the ACS approval of an undergraduate program is a desirable its the chromatographic eluents as a continuous track on an IR-transparent ZnSe outcome for students, faculty and administrators. The chemistry faculty members at disc. The built-in interferometer simultaneously captures a set of time-ordered trans- California University of Pennsylvania have successfully utilized the ACS Guidelines mission IR spectra every 0.5 seconds from the solid-phase deposit. The snapshot as a point of advocacy for improvements to the department’s personnel, infrastruc- IR spectra at each elution time can be used for the IR database search to identify the ture, instrumentation, and curriculum. While some ACS Guidelines are very specific, chemical compositions. A case study is presented to deformulate a complex adhe- others are currently written in a way to allow departments to find creative solutions to sive sample into 3 polymers and 3 additives by a single GPC-IR run and various IR manage institutional challenges while meeting the ACS guidelines. Cal U is a pub- database search approaches: 1) direct IR library matching for the baseline-resolved lic institution with five full-time chemistry faculty members who have been actively peaks. 2) For the partially separated polymer peaks, the snapshot IR spectra at working towards ACS approval for several years. The overall quality of our program their leading/tailing edges can be used for the component library search. Spectral is far superior to our starting point. Cal U is currently deep in the initial approval subtraction and principle component analysis can confirm the results. 3) In the low- process with our site visit schedule to occur before the 2013 EAS. The perspective MW region where most additives come out at similar elution times, the snapshot IR of our program, that is going through the ACS approval process, is shared. spectra in the highly overlapped area can be used for multiple component search to identify each additive component (up to 4 sub-components by BioRad’s KnowItAll 454 Chirped-Pulse Millimeter Wave Spectroscopy of EPA TCL Volatiles software). Mix 5 Standard: Advantages of Coherent, Pure Rotational Spectroscopy for Trace Level Volatile Mixture Analysis 457 USP Spectral Library Pilot Brent J. Harris, BrightSpec, 675 Peter Jefferson Parkway, Charlottesville, Michael Dotlich, Eli Lilly, Lilly Corporate Center, Indianapolis, IN 46285, VA 22911, Matthew Muckle, Amanda Steber, Brooks H. Pate Bei Ma Conventional methods for gas mixture analysis do not address the need for multi- The USP is pursuing the development and implementation of an authoritative and component, trace level analysis of complex mixtures. Chirped-pulse millimeter wave comprehensive food and drug informatics database, known as the United States spectroscopy has been recently introduced to the field molecular spectroscopy and Pharmacopiea Spectral Library. Once fully developed and operational, the library promises new analytical capability for gas mixture analysis by utilizing the spec- will be a valuable tool in the development of quality standards using spectral images tral signature of pure molecular rotation. Originally developed at the University of as well as for material identification and rapid, non-invasive screening of counterfeit Virginia, chirped-pulse spectroscopy has become widely accepted by the chemi- and substandard foods and drugs. A precursor to the final development and imple- cal physics community as a highly sensitive, broadband technique for determining mentation of the Spectral Library includes a pilot study that has been designed to structure of molecules and complexes at microwave wavelengths. Recent advances focus on materials and technologies with a high probability of success, e.g., Raman, in solid state terahertz devices and high-speed digital electronics have enabled the Fourier transform infrared (FTIR) and near-infrared. The pilot has been designed to application of chirped-pulse spectroscopy to the millimeter wave spectrum where assess both the spectroscopic technology and the database infrastructure. As part benchtop, room temperature measurements of small volatile organic compounds is of the first pilot, materials were tested by handheld Raman and portable FTIR. Data highly favorable. The transformative advantages of chirped-pulse millimeter wave evaluation is used to demonstrate reproducibility and repeatability across material stem from the high resolution (0.0006 cm-1), absolute specificity to molecular struc- suppliers, collaborative testing laboratories, analytical instruments and analysts. ture, and coherent, time domain measurements. As a result, high dynamic range composition analysis can be accomplished without chemical separation and with 458 Quantitative Analysis of Feed and Agricultural Samples Using a an essentially zero false positive rate. A suite of time domain measurements can Portable NIR Instrument be used to enhance the analysis with double resonance identity verification, mass Suzanne Schreyer, Thermo Fisher Scientific, 2 Radcliff Rd., Tewksbury, estimation, and dipole estimation leading ultimately to library-free identification of MA 01876, Lin Zhang unknown spectral carriers. Advantages and fundamentals of chirped-pulse spec- Near-infrared (NIR) spectroscopy has been widely utilized in the food and agricul- troscopy are introduced and demonstrated on an Environmental Protection Agency tural fields for all stages of production. Its utility lies in its relative ease of use, requir- standard mixture of Hydrofluorocarbons. ing little or no sample preparation, to generate spectra that are characteristic of a material. NIR has been successfully employed in authentication and identification 69 2013 EAS Abstracts November 2013

of materials, determination of quantitative quality parameters, and food/feed inspec- presentation discusses the evaluation of a novel sample extraction apparatus that tion for adulteration or food safety issues. The recent advent of handheld instrumen- employs homogenizing solid oral dosage forms in parallel that significantly improve tation has allowed these applications to be taken from the laboratory environment the sample extraction time and increase sample throughput, thus achieving faster directly to the field, warehouse or process facility for direct, rapid quality testing at sample turnaround time, resource savings and ensuring extraction ruggedness and the point of need. NIR spectroscopy is well recognized as a reliable instrument to robustness for the pharmaceutical solid dosage forms. predict value added parameters such as moisture, protein and fat in food or agricul- tural samples. We present results obtained from the quantitative study of quality pa- 463 Class-Selective Extraction of Streptomycin and Related Drugs rameters in feed and ingredients based on the AuNIR Ingot library, implemented on from Cell Culture and Peptide Solutions Using Moleculalrly- a portable, handheld instrument. Further work was performed in-house to develop Imprinted Polymer SPE and LC/MS/MS algorithms for robust predictions including bias correction, model optimization and Emily Barrey, Supelco Division of Sigma-Aldrich, 595 North Harrison calibration transfer between instruments. Rd., Bellefonte, PA 16823, Olga Shimelis, Michael Ye Streptomycin and related aminoglycosides belong to a well-known class of antibiot- 459 Measurements of Key Cotton Fiber Properties Using a Small ics, and are widely used to control bacterial growth in pharmaceutical and biotech Portable NIR Analyzer preparations. Compounds in the aminoglycoside class are very polar and not read- James Rodgers, United States Department of Agriculture, 1100 Robert ily soluble in organic solvents. Due to the unique properties of amino glycoside E. Lee Blvd., New Orleans, LA 70124, Chris Delhom, Xiaoliang Cui compounds, researchers have had difficulties with current extraction and analytical Most of the cotton produced in the United States is exported overseas, placing in- methods. Recently, a new sample preparation method was developed using mo- creased emphasis on improved laboratory and field cotton quality measurement lecularly imprinted polymers (SupelMIP) solid-phase extraction (SPE). Target ana- systems in order to compete in the international market. Interest has been ex- lytes bind to the synthetically created cavities within SupleMIPs by multiple interac- pressed in the use of small, cost effective portable instruments that can rapidly tions. The strong retention between the MIP phase and its analytes allows for use and accurately monitor key cotton fiber quality properties, both in the laboratory of stronger washes during the SPE procedure, subsequently resulting in superior and at-line/in the field. Near-infrared (NIR) instrumentation has shown the ability sample clean-up. Here the extraction of aminoglycosides from “spent” mammalian to meet these requirements for several cotton fiber properties. Although fast and cell culture and model peptide solutions was tested using SupelMIP SPE – Amino- accurate, concerns were expressed on the size and cost of the initial NIR units glycosides. Ten antibiotic compounds were spiked into cell culture and extracted used. A new, very small (micro-size) NIR analyzer has been introduced, using linear and analyzed via liquid chromatography tandem mass spectrometry (LC-MS-MS). variable filter (LVF) technology. A program was implemented to determine the capa- Evolutions of recoveries, detection limits, ion suppression effects and class selectiv- bilities of the new micro NIR analyzer to measure key cotton fiber properties, with ity are reported. emphasis on fiber micronaire and its components (maturity and fineness). Labora- tory feasibility measurements were performed first (prior to field measurements). A 464 Investigating Matrix Interference in Bioanalysis of Antiarrhythmic well-defined cotton sample set (over 100 samples) was measured, and the results Cardiac Drugs were compared to the reference methods and previous NIR measurements. Instru- Craig Aurand, Supelco Division of Sigma-Aldrich, 595 North Harrison mental, sampling, and operational procedures for cotton fiber measurements were Rd., Bellefonte, PA 16823, Dave S. Bell, Lori Fields determined. Distinct spectral differences between samples were observed. Very en- For efficient therapeutic drug monitoring, it is important for clinicians to have ac- couraging results were obtained, with few outliers, low residuals, and good overall cess to fast and robust analytical methods for accurate assessment of drug efficacy. agreement with the previous results from the larger NIR units. The potential for Industrial trends toward highly specific liquid chromatography mass spectrometry at-line and field fiber measurements with the micro NIR analyzer is very promising, (LC-MS) applications over traditional enzyme-linked immunosorbent assay type im- and field trials are underway. munoassay have resulted in the need for high-speed chromatographic assays along with simplified sample preparation methods. In this study, hydrophilic interaction liq- 460 A New Truly Easy-to-Use Dedicated Infrared Microscope uid chromatography (HILIC) chromatography along with novel sample preparation Thomas J. Tague, Jr., Bruker Optics, 19 Fortune Dr., Billerica, MA 01821 techniques are evaluated for analysis of antiarrhythmic agents lidocaine, flecainide, A new stand-alone infrared microscope (LumosTM) has been developed for the rapid amiodarone and metabolites. The goal is to demonstrate the utility of HILIC over tra- analysis of small samples. The new microscope was developed with the intent of ditional reversed-phase chromatography for both sample introduction and analyte providing state-of-the-art microanalysis capabilities with a truly easy-to-use user in- detection in positive electrospray ionization LC-MS. In addition to chromatographic terface. The visual image quality of the Lumos is excellent so the important first step separation, sample preparation techniques are compared for analyte recovery along in the analysis, visualization, is easily accomplished. The Lumos utilizes a unique with sample matrix reduction. The impact of matrix ion-suppression from plasma objective design, where the numerical is low for sample viewing and high for the samples is evaluated using both sample prep techniques. Standard protein precipi- infrared data collection. This makes it very easy to locate and view the sample tation methods along with novel phospholipids depletion technology were utilized to without sacrificing infrared performance. The novel Wizard user interface controls demonstrate the impact of sample matrix on the detection of these analytes. This all aspects of the microscope and guides the user through the analysis process. study continues to supports the need to eliminate matrix effects in LC-MS to ensure The sample stage, sample focus assembly, condenser, aperture, polarizers, and at- reliable and accurate bioanalytical results. tenuated total reflectance (ATR) mode are controlled in the software providing true “point and shoot operation”. ATR microanalysis is accomplished by simply clicking 465 Withdrawn by the author. on the area of interest in the software to center it and selecting ATR. Area reflection, transmission, and ATR images are collected by simply drawing the desired analysis and starting the desired acquisition. The image processing software interface pro- 466 Simplifying Complex Robotic Methods Using Visual Basic vides research quality analysis tools with an intuitive interface. The Lumos also has Sam Abdelhamid, Purdue Pharma, 6 Cedar Brook Dr., Cranbury, NJ a unique ability to readily analyze samples with traditional sampling accessories. A 08512 port is provided to attach accessory modules from Bruker’s Alpha Fourier transform Technology has come a long way since the early days of automated liquid han- infrared (FTIR) Series. Standard ATR, transmission, reflection, and even gas cell dlers. However, most of the smaller benchtop systems haven’t really changed in the analysis can be readily conducted with the Lumos. Lastly, the Lumos comes with a way they interact with the user. Graphical user interfaces still demand a lot of input comprehensive validation package to support any range of validation requirements. from the user to create efficient methods. Once a method is written and verified, it becomes stagnant. By using visual basic (VB) to create custom user forms, we 461 Withdrawn by the author. are now able to create dynamic protocols that change to each user’s demands. Ex- amples showing VB forms on a Perkin Elmer Zephyr to automate pharmacokinetic samples are discussed. 462 Reducing Sample Extraction Time While Increasing Sample Throughput 467 Robotic Rheometry: A Novel Approach to High-Throughput Khanh Ha, Bristol-Myers Squibb, One Squibb Dr., New Brunswick, NJ Material Research 08903, Martin Schwalm James P. Eickhoff, Jr., Anton Paar USA, 10215 Timber Ridge Dr., Sample extraction of pharmaceutical solid oral dosage forms is a critical step of any Ashland, VA 23005, Prajakta Kamerkar, Maxine Quitaro assay analytical method. Typically the extraction process is challenging and very Automated rheology has garnered increasing interest in recent years in order for time consuming. Traditionally, agitation such as shaking and, or sonication is used companies to remain competitive in both material research and product develop- to facilitate dispersion of the solid dosage form over a given period of time. In addi- ment. Conventional material characterization techniques provide answers to intrigu- tion, a well-developed extraction method needs to be rugged and robust with short ing research questions but over very long development cycles. Thus a robotically sample preparation times to ensure accurate and precise method execution by dif- operated rheometer was developed to meet such needs. This automated solution ferent analysts at different facilities over the life of the pharmaceutical product. This was developed around a conventional rheometer, thus offering measurement flex- 70 2013 EAS Abstracts November 2013

are now able to create dynamic protocols that change to each user’s demands. Ex- 469 A Novel Engineered Carbon Intended for Pigmented Sample amples showing VB forms on a Perkin Elmer Zephyr to automate pharmacokinetic Cleanup 2013samples EAS are discussed. Abstracts Dwight Stoll, Gustavus Adolphus College, 800November West College Ave., 2013 St. Peter, MN 56082, Conor Smith, Jonathan Thompson 467 Robotic Rheometry: A Novel Approach to High-Throughput Activated carbon and graphitized carbon adsorbents are widely used in solid-phase ibility (rotationalMaterial and Researchoscillatory tests), high reproducibility in measured results, as 469extraction andA Novel matrix Engineeredcleanup work. Carbon A major Intended use is infor pigment Pigmented removal Sample during well as meetingJames high P. throughputEickhoff, Jr., demands. Anton Paar Incorporating USA, 10215 such Timbera high-throughput Ridge Dr., QuEChERSCleanup extract cleanup. These all carbon adsorbents, while high in surface solution allowsAshland, for rheological VA 23005, data Prajakta to be Kamerkar, collected 365Maxine days Quitaro a year unsupervised. area, sufferDwight from issues Stoll, suchGustavus as low Adolphus mechanical College, stability, 800 poor West planar College pesticide Ave., St.re- TheAutomated presented rheology high-throughput has garnered solution increasing is beneficial interest to insupport recent futureyears materialin order exfor- covery, andPeter, the shedding MN 56082, of fines. Conor A Smith,new type Jonathan of carbon Thompson adsorbent (CarbonX Plus) plorationscompanies in to the remain coating competitive industry as in well both as materialminimize research current production and product cycle develop times.- Activatedwith a porous carbon metal and oxidegraphitized core carboncoated adsorbentswith graphitized are widely like carbon used in is solid-phase presented ment. Conventional material characterization techniques provide answers to intrigu- extractionwhich improves and matrixupon allcleanup of these work. issues A majorwhile retaininguse is in high pigment surface removal areas andduring re- 468ing researchGel questions Point Determination but over very Thankslong development to Microrheology cycles. Thus a robotically QuEChERScoveries of pesticides. extract cleanup. The metal These oxide all corecarbon greatly adsorbents, increases while mechanical high in strengthsurface operated rheometerJonathan wasB. Denis, developed Formulaction to meet Inc., such 12555 needs. Orange This automated Dr., Ste. 101, solution Avie, area,and density suffer fromallowing issues for cleanersuch as sample low mechanical and solid-phase stability, extraction poor planar tubes pesticide and auto re-- was developedFL 33330 around a conventional rheometer, thus offering measurement flex- covery,mation ofand tube the filling. shedding Due ofto fines.the manufactured A new type of nature carbon of adsorbentthe particles, (CarbonX planar pestiPlus)- Nanoparticleibility (rotational applications and oscillatory in the tests),industry high are reproducibility getting more andin measured more important results, and as withcide recoverya porous ismetal also greatlyoxide core increased coated without with graphitized the need for like toxic carbon chemicals is presented such as concernwell as meetingmany different high throughput fields (drug demands. delivery, Incorporatingcarbon nanotubes, such adisplay high-throughput technolo- whichtoluene, improves as is usual upon for all graphitized of these issues carbons. while Fines retaining are alsohigh eliminatedsurface areas decreasing and re- gies,solution etc.). allows In the for last rheological decades data a vast to beamount collected of scientific 365 days research a year unsupervised.has been de- coveriesthe clogging of pesticides. of frits and The increasing metal oxide throughput. core greatly QuEChERS increases (quick, mechanical easy, cheap, strength ef- velopedThe presented to improve high-throughput the understanding solution ofis thesebeneficial complex to support dispersions. future materialUsing new ex- andfective, density rugged, allowing and safe)for cleaner extract sample cleanup and with solid-phase CarbonX Plus extraction also increases tubes and column auto- analyticalplorations techniques,in the coating it industryis now possible as well asto minimizecontrol and current tailor production properties cycle of suspen times.- mationlifetime ofand tube decreases filling. Due liner to foulingthe manufactured for gas chromatography nature of the massparticles, spectrometry planar pesti run- sions and to get a better understanding of time behavior. Following this idea, stabil- cidesamples. recovery is also greatly increased without the need for toxic chemicals such as Gel Point Determination Thanks to Microrheology ity468 measurements, which were commonly done by simple visual observations, can toluene, as is usual for graphitized carbons. Fines are also eliminated decreasing Jonathan B. Denis, Formulaction Inc., 12555 Orange Dr., Ste. 101, Avie, now be performed automatically via an optical device. This instrument is based on the clogging of frits and increasing throughput. QuEChERS (quick, easy, cheap, ef- FL 33330 multiple light scattering and is associated to a vertical scanning of the sample. It fective, rugged, and safe) extract cleanup with CarbonX Plus also increases column Nanoparticle applications in the industry are getting more and more important and enables the operator to identify and quantify instability phenomena before they are lifetime and decreases liner fouling for gas chromatography mass spectrometry run concern many different fields (drug delivery, carbon nanotubes, display technolo- visible (up to 200 times earlier). Physical parameters and kinetics can be computed samples. ingies, order etc.). to facilitate In the last and decades improve asample vast amount comparison. of scientific Examples research of carbon has nanotubebeen de- dispersibilityveloped to improve measurements the understanding are presented of theseusing complexthis technique dispersions. and enable Using to newfine tuneanalytical the dispersion techniques, in termsit is now of solvent, possible surfactant to control content and tailor etc. properties of suspen- sions and to get a better understanding of time behavior. Following this idea, stabil- ity measurements, which were commonly done by simple visual observations, can now be performed automatically via an optical device. This instrument is based on multiple light scattering and is associated to a vertical scanning of the sample. It enables the operator to identify and quantify instability phenomena before they are visible (up to 200 times earlier). Physical parameters and kinetics can be computed in order to facilitate and improve sample comparison. Examples of carbon nanotube dispersibility measurements are presented using this technique and enable to fine tune the dispersion in terms of solvent, surfactant content etc.

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