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ABSTRACT BOOK 2015 Eastern Analytical Symposium & Exposition ANALYTICAL INNOVATION FROM BENCHTOP TO BUSINESS

Garden State Exhibit Center | Somerset, NJ November 16–18, 2015 Analytical eas.org Chemistry Opens Doors

2016 EASTERN ANALYTICAL SYMPOSIUM & EXPOSITION

SAVE THE DATE November 14–16, 2016 Garden State Exhibit Center Somerset, NJ

 Three-day technical program  State-of-the-art exposition featuring analytical equipment and services  Extensive selection of short courses and professional development workshops  Employment bureau, and more

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2016 EASTERN ANALYTICAL SYMPOSIUM & EXPOSITION eas.org Garden State Exhibit Center Somerset, NJ November 14–16, 2016

Call for Papers March 5–May 16, 2016 Abstracts received from May 16–Sept 12, 2016, will be reviewed for quality to be included in the poster session. You will be notified via email when/if the abstract is placed.

EAS seeks contributed abstracts in these and other analytical fields:

 Bioanalysis  Microscopy  Capillary Electrophoresis  Nanoparticles  Chemometrics  Near-Infrared (NIR) Spectroscopy  Conservation Science  NMR Spectroscopy  Environmental Analysis  Pharmaceutical Analysis  ESR Spectroscopy  Process Analytical Science  Food Analysis  Protein Analysis  Forensic Analysis  Quality-by-design  Gas Chromatography  Quality/Regulatory/Compliance  HPLC  Raman Spectroscopy  ICP/MS  Sample Preparation  Immunochemistry  Science Education  Industrial Hygiene  Sensors  Ion Chromatography  Size Exclusion Chromatography  IR Spectroscopy  Solid State Analysis  Laboratory Automation  Space Analytics  Laboratory Management  Supercritical Fluid Chromatography  Laboratory Miniaturization  Surface Science  Ligand Binding Assays  Very High-Pressure LC/  LC/MS, GC/MS Ultra High-Pressure LC  Microchemistry  Vibrational Spectroscopy

001646-EAS_8.5x11_CallForPapers_v5.indd 1 11/2/15 12:13 PM 2015 EAS Abstracts November 2015

2015 EAS Abstracts This volume contains the final abstracts for the oral and poster presentations which take place Monday, November 16, through Wednesday, November 18, 2015. Additional abstracts received after this volume was finalized are provided in the Addendum to the Final Program. If an abstract is not provided in this volume or the Addendum, then the presenting author did not supply an abstract. 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:

EAS Hotline 732-449-2280 EAS E-mail [email protected] EAS Web Site www.EAS.org

Eastern Analytical Symposium & Exposition, Inc. P.O. Box 185 Spring Lake, NJ 07762

Save the Date The 2016 EAS November 14 - 16, 2016 Somerset, NJ We want you to be a part of the 55rd Eastern Analytical Symposium! 2016 Call for Papers March 5 – May 16, 2016

1 000961-EAS_17x11_Spread_PREP.indd 1 10/20/14 8:43 AM 2015 EAS Abstracts November 2015

ABSTRACTS OF TECHNICAL PAPERS

1 Distance-of-Flight Mass Spectrometry: The Joy of Collaborating spray and of pH and surface activity is assessed. A model of droplet ionization has with Chris Enke been developed. The analytical properties and especially the enhanced sensitivity Gary Hieftje, Indiana University, 800 East Kirkwood Ave., Bloomington, to surface active species are discussed and compared with Chris Enke’s early treat- IN 47405, Christie G. Enke, Steven J. Ray, Alexander W. Gundlach- ment of surface active compounds in electrospray ionization. Graham, Elise A. Dennis, David W. Koppenaal, Charles J. Barinaga Chris Enke has been responsible for a host of innovations in analytical chemistry, in 4 Comprehensive Analysis and the Theory of Complex Mixtures areas ranging from electrochemistry to mass spectrometry, from electrospray ion- Christie Enke, University of New Mexico, 33 Vista de Oro, Placitas, NM ization to multicomponent analysis. In this presentation, recent work on one of his 87043, Alexander Gundlach-Graham, Alan G. Marshall innovations is outlined. This study, pursued collaboratively with Chris, involves the The responses obtained in the comprehensive analysis of many types of complex reduction to practice, characterization, and improvement of a new form of mass natural mixtures show an amazingly good fit to a log-normal distribution. Recent analysis: Distance-of-flight mass spectrometry (DOFMS), and several offshoots work in petroleomics, lipidomics, proteomics, and metabolomics have provided data from that study. Basically, DOFMS is very similar to time-of-flight mass spectrometry that substantiate this observation. This observed regularity provides the basis for (TOFMS), but tailored for folks who are impatient. As in TOFMS, ions to be mass-an- the development of a new area in chemistry which we will call “The theory of com- alyzed by DOFMS are accelerated to mass-dependent velocities. However, before plex mixtures”. The motivation to develop this new area comes, in part, from the the lightest ion to be measured emerges from the field-free flight tube, ions are sent possibility of predicting the number of potentially detectable compounds in a given sideways onto a position-sensitive detector. Thus, detection is truly simultaneous, analysis, how many compounds have been missed, and the responses they would there is no upper-mass limit, masses are spatially segregated and can be either have had, were they observable. From this, one could determine what aspect(s) of detected or collected at will, and no magnetic fields are needed. As importantly, de- the technique employed (sensitivity, dynamic range, resolution, S/N) that need to be tectors and electronics need not be capable of high time resolution, so direct charge improved and to what degree they would improve the fraction of compounds detect- integration (either analog or digital) is possible. Further, DOFMS can be coupled ed. Efforts to analyze comprehensive analysis data in this way have raised a num- with TOFMS in several beneficial ways, and principles embodied in DOFMS can be ber of questions. How is the distribution of responses related to the distribution of transferred to TOFMS to yield better spectral resolution. Recent activities in these concentrations? How do the compounds below the detection limit contribute to noise several areas are detailed, and plans for future studies are sketched. in the measurement? How do techniques of sample fractionation affect the range of detectable compounds? What is the basis for this distribution model in nature? 2 21 Tesla Fourier Transform Ion Cyclotron Resonance Mass This talk reviews the progress made to date in our efforts to answer these questions. Spectrometer: A National Resource for Ultrahigh Resolution Mass Analysis 5 Separation of Carbon Nanoparticles Alan G. Marshall, Florida State University, Ion Cyclotron Resonance Luis A. Colon, SUNY – Buffalo, Department of Chemistry, Buffalo, NY Program, NHMFL, Tallahassee, FL 32310, John P. Quinn, Nathan 14260, Zuqin Xue, Karina M. Tirado-González, Amaris C. Borges-Muñoz K. Kaiser, Donald F. Smith, Greg T. Blakney, Tong Chen, Chad R. Carbon nanoparticles have been the subject of much attention in recent years, with Weisbrod, Christopher L. Hendrickson, Steven C. Beu many suggested applications varying from metal ion sending and imaging to po- Tesla Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry offers tential adsorbents for chromatographic use. For example, the photoluminescence the highest achievable broadband mass resolving power and mass accuracy of any properties, biocompatibility, water solubility, and chemical reactivity of carbon dots mass analyzer. Resolving power and scan rate improve linearly, and mass accuracy (C-dots) nanoparticles are attractive features that have encouraged the study of and dynamic range improve quadratically with magnetic field strength, such that C-dots as an environmentally benign alternative to replace metal-based nanopar- resolving power greater than 1 million and mass accuracy better than 1 ppm be- ticles in various applications. The procedure to obtain carbon nanomaterials may come routine at sufficiently high magnetic field strength. We report here the design, vary, but in the majority of the cases the as synthesized nanoparticles are con- construction, and characterization of the first 21 tesla FT-ICR mass spectrometer. A tained in a heterogeneous mixture with nanoparticles varying in size and/or surface Thermo Fisher Velos Pro (modified for front electron transfer dissociation (FETD)) chemistry. In various cases, separation of the different components of the mixture is combined with a National High Magnetic Field Laboratory (NHMFL)-designed ex- becomes necessary. We have developed methodology based on chromatographic ternal linear quadrupole ion trap, radio frequency quadrupole ion transfer optics, and/or electrophoretic techniques that allow the separation of carbon nanomateri- and a novel dynamically harmonized ICR ion trap. The isotopic distribution for bo- als. The separated nanoparticles can then be studied in isolation, providing insight vine serum albumin (66 kDa) is resolved with a 0.38 s detection interval, facilitating into their contribution to the overall bulk sample properties. This presentation fo- top-down proteomic analysis of proteins up to 100 kDa by online liquid chroma- cuses on studies of carbon nanomaterials prepared by top-down and bottom-up tography-mass spectrometry (LC-MS). Longer detection interval generates higher synthetic approaches using chromatographic and electrophoretic techniques. resolving power, with RP > 1,000,000 routinely achievable for an isolated charge state of transferrin (78 kDa). The root mean square mass measurement error for 6 Magnetic Ionic Liquids in Bioanalytical Chemistry a mixture of ten peptides is <150 ppb. Electron transfer dissociation and collisional Jared Anderson, Iowa State University, 1605 Gilman Hall, Department dissociation combine to produce hundreds of fragment ions of carbonic anhydrase. of Chemistry, Ames, IA 50011 Overlapping isotopic multiplets are easily resolved, even at high (>1 Hz) scan rate. No abstract submitted by the author. A BaF2 window on the ICR cell flange enables future coupling of photodissociation induced by ultraviolet (193 or 266 nm), visible (532 nm), or infrared (10.6 micron) 7 Membrane Alterations and Additives to Improve Microdialysis lasers. Work supported by the NSF through DMR-1157490, CHE-1016942, CHE- Sampling Recovery 1019193, and the State of Florida. Julie Stenken, University of Arkansas, 246 CHEM Building, 345 N. Campus Drive, Fayetteville, AR 72701, Sarah J. Phillips, Thaddeus W. 3 Mass Spectrometry as a Method of Accelerated Small-Scale Vasicek Synthesis Microdialysis (MD) sampling is a diffusion-based separation method widely used R. Graham Cooks, Purdue University, 560 Oval Dr., West Lafayette, IN for in-vivo sampling over four decades. Despite the popularity of the technique and 47949, Michael Wleklinski, Yafeng Li, Ryan Bain, Christopher Pulliam, its straightforward sampling mechanics, the technique has significant limitations for Anyin Li, Xin Yan sampling either large molecular weight or lipophilic solutes. These limitations are im- This presentation deals with molecular and materials synthesis in charged droplets posed by the nature of the sampling process and by physicochemical properties of generated in the course of ambient ionization from paper or spray ionization from an the targeted analyte. Two common problems are the mass transport restrictions im- emitter. The time scale for synthesis is milliseconds and this allows on-line analysis posed by the dialysis membrane and loss of analyte due to non-specific adsorption. by mass spectrometry or off-line analysis by thin-layer chromatography or nuclear To address these problems, our research group has a long history of developing dif- magnetic resonance. The scale of the reactions is typically low milligrams. The de- ferent methods using additives to the microdialysis perfusion fluid including addition gree of reaction can be controlled by the extent of desolvation, allowing a distance- of cyclodextrins, heparin and microsphere-immobilized antibodies. In this present of-flight experiment to be done in which intermediates can be generated at short work, we describe our efforts toward developing affinity-based microdialysis mem- distances and products at long distances. The reasons for the rate enhancement will branes and inclusion of spherical bound polymer brushes (SPB) to serve as pro- be discussed together with examples ranging from simple derivatization, through tein capture agents within a hollow-fiber membrane separation. Laccase chemistry C-C bond formation to catalyzed reactions. The role of the voltage applied in paper has been used to specifically modify the polyethersulfone (PES) membranes used 2 2015 EAS Abstracts November 2015

during microdialysis sampling with verification by X-ray photoelectron spectroscopy target compounds. The methods evaluated were effective for the detection of the for the modified membranes. Modified membranes are capable of collecting large targeted compounds in commercial products and biological fluids such as whole molecular weight solutes such as fluorescein isothiocyanate (FITC)-labeled dex- blood and urine at toxicologically meaningful concentrations. trans (FITC-10 and FITC-20). SPBs are created using poly(N-isopropylacrlamide) (PNIPAM), a thermo-responsive polymer, displaying protein binding above 32 °C 11 The Detection of Organic Components Found in Gunshot Residue and reduced protein interaction below 32 °C. This talk covers these more recent by Use of LC-QQQ-MS to Assess Home Reloaded Ammunition attempts to create affinity-based membranes and segmented filamentous bacteria Kyle Brown, Duquesne University, Dept. of Chemistry & Biochemistry, (SFB) for improving sampling of proteins and other large molecular weight solutes. Pittsburgh, PA 15282, Leah Ali, Holly Castellano, Stephanie J. Wetzel In addition to the rise of gun violence in the past decade, gun sales broke records 8 Eolaíocht na Deighilte: Putting the Irish in Separation Science and ammunition shortages have plagued the United States. Because these recent Apryll M. Stalcup, Dublin City University, National Centre for Sensor ammunition shortages have driven sportsman and shooting enthusiasts alike to Research, Glasnevin, Dublin 9, Ireland reload their own ammunition, inorganic and organic gunshot residue (GSR) result- Understanding the relationship between intermolecular interactions and chemical ing from home-reloaded ammunition may become prominent physical evidence in separations has been a long-term scientific research goal of our group, indepen- firearm related crimes. However, because the uniqueness of inorganic GSRhas dent of the separation platform. The talk elucidates the evolution in our group from been brought into question and heavy-metal-free primers have been introduced, the traditional separation methods, relying primarily on a single molecular interaction analysis of organic components is mandated. Specifically, the stabilizer additives mode, to chiral separations and illustrate how the intermolecular interactions shap- are considered the most unique to organic GSR. Thus, an optimized method was ing the phase behavior of pure substances (e.g., dispersion, hydrogen bonding, developed for liquid chromatography with triple quadrupole mass spectrometry (LC- dipole-dipole, dipole-induced dipole, electrostatic, etc.) can be creatively exploited QQQ-MS) to examine the organic components of GSR. Through the optimization to accomplish the desired chiral or achiral separations. The global interactions be- of LC-QQQ-MS parameters, the seven most common organic components of GSR tween the solute and the separation system are reflected in the separation which were successfully separated and detected. Additionally, the developed method pro- acts as a molecular amplifier of these interactions. Further, separation techniques vided the limits of detection and quantitation for the seven organic components of are incredibly sensitive tools for investigating and amplifying subtle differences in interest. After GSR from both factory-manufactured and home-reloaded ammunition intermolecular interactions. Examples from current and past research are used to was extracted, differentiation between the two types of ammunition was achieved. illustrate these points. Therefore, because the distinction between these ammunitions was plausible, it was hypothesized that differentiation between factory-manufactured 9mm ammunition 9 The Analytical Investigation of Synthetic Street Drugs Known as and the seven most commonly used reload-powders for 9mm can be attained as “Bath Salts” well. Thus, by utilizing two extraction methods for unburnt and burnt powder, it was Amanda Kaspick, Agilent Technologies, 2850 Centerville Rd., possible to differentiate between factory-manufactured 9mm rounds and the pow- Wilmington, DE 19808, Frank Dorman, Philip B. Smith ders most commonly used for reloading 9mm ammunition based on the presence of Since the early 2000’s, synthetic “designer drugs” have flooded the drug market due certain stabilizer compounds in each powder’s resulting GSR. to their legality and high-like effects. Among these drugs were synthetic cathinones (marketed as “bath salts”) which entered the US in 2007 at an overwhelming pace. 12 Application of BioSPME for the Extraction of Illicit Substances By the time enough information was known about a drug to place it under tempo- from Urine rary or permanent scheduling, replacement compounds were already created and Kaitlyn Hess, Cedar Crest College, 100 College Dr., Allentown, PA readied for distribution. The potentially dangerous behavior and the risky side ef- 18104, Thomas A. Brettell fects caused by synthetic cathinones demonstrated that the analysis of “bath salts” Drug use has a well-known history dating back thousands of years in all parts of the and the determination of their composition was necessary to aid law enforcement world, beginning with the use of naturally occurring substances, and gaining popu- and understand what potential users may be subjected to. Ten synthetic cathinones larity with synthetic substances. New drugs, though they may be similar in structure were identified in fourteen separate street samples analyzed utilizing a variety of to commonly recognized drugs, often evade detection by specific techniques like im- techniques, including gas chromatography with mass spectrometric detection (GC- munoassays, making them difficult or nearly impossible to identify. A new BioSPME MS) and flame ionization detection (GC-FID). Additionally, preparatory high-per- fiber has been engineered and is being explored for its use in extracting drug ana- formance liquid chromatography (HPLC) for the fractionation of multi-component lytes from different biological matrices. The BioSPME fiber is stationed within a pipet samples and the use of direct infusion tandem mass spectrometry (MS-MS) was tippet and is functional in a 96-well format. Each fiber contains either mixed mode necessary to identify compounds which were not available as reference materials. hydrophobic and cation exchange particles or C-18 (reversed-phase) particles to These cathinones include 3,4-methylenedioxy pyrovalerone (MDPV), 3, 4-methy- enrich drug analytes of interest, leaving behind biological matrices. The fiber is di- lenedioxy-α-pyrrolidinobutiophenone (MDPBP), 4-fluoromethcathinone (4-FMC), rectly placed into a biological fluid for extraction, desorbed into solution, dried, and butylone, mephedrone, naphyrone, 4-methylethcathinone (4-MEC), ethcathinone, reconstituted for analysis by liquid chromatography tandem mass spectrometry (LC- α-pyrrolidinopentiophenone (α-PVP), and 3-methyl-α-pyrrolidinopropiophenone MS-MS). This experiment utilizes an ABI Sciex 3200 QTRAP triple quadrupole mass (3-MPPP). Concentrations of the active compounds varied between samples. For spectrometer interfaced with a Shimadzu LC system. Extracted samples are run in example, MDPV was determined to be the most common cathinone. It was found positive ionization mode using electrospray ionization (ESI), and chromatography is in five of the fourteen samples and ranged from 11 to 73 percent between samples. achieved using an Ultra C18 column (50 x 2.1mm, 3µm) Restek®. The strong mo- bile phase was 0.1% (v/v) formic acid in water and the weak mobile phase was 0.1% 10 Development of an Analytical Method for “Nootropic” Smart Drugs (v/v) formic acid in acetonitrile. Twelve drugs from different classes including mor- in Powders, Capsules and Biological Fluids phine, methamphetamine, cocaine, nordiazepam, and methylone are being stud- Mollie Mares, Arcadia University, 450 S. Easton Rd., Glenside, PA ied for their extraction efficiency from urine with this novel BioSPME method. Drug 19038, Barry Logan, Karen Scott, Donna Papsun analytes are quantified following LC-MS-MS analysis to determine their recovery. Smart drugs, also known as nootropics, are an emerging area drugs with implica- tions for forensic toxicology. The drugs have stimulant properties and are alleged 13 Withdrawn by the author. to boost brain function and cognition. The media attention on these drugs has in- creased within the last few years. The drugs have developed an underground fol- 14 Approaches to Quantitative 1-D and 2-D NMR Spectroscopy lowing and are commonly sold online and in illicit supply chains. Most have not been John L. Markley, University of Wisconsin-Madison, Dept. of Biochemistry, approved or scheduled in the US, and are therefore of concern to regulators such 433 Babcock Dr., Madison, WI 53705, Hamid R. Eghbalnia, Marco as Food and Drug Administration and Drug Enforcement Administration. There are Tonelli, William M. Westler, Roger A. Chylla, Kaifeng Hu, Ian A. Lewis ongoing investigations in the applications of smart drugs in the treatment of Alzhei- Quantification of molecules in solution is important in studies of metabolomics and mer’s disease, Huntington’s disease, and Attention Deficit Hyperactivity Disorder. natural products. One of the challenges is to quantify peaks in overlapped spec- The stimulant properties of the drugs have led to their use in academic doping and tra. We developed fast maximum-likelihood reconstruction (FMLR) to addresses as drugs of abuse. The goal of this project was to develop a single analytical method this problem in one-dimensional (1-D) and two-dimensional (2-D) spectra. More for screening, confirmation, and quantification of a series of the more widely known recently, we are finding that a Bayesian approach yields superior results. To quan- smart drugs in blood and urine. Gas chromatography/mass spectrometry (GC-MS) tify the relative amounts of ¹²C and ¹³C at specific positions in molecules we have and liquid chromatography/mass spectrometry (LC-MS) were investigated to de- developed a method called ITOCSY that can be applied to metabolic flux analysis, termine the optimum approach for sensitivity and ability to detect a broad range of isotope dilution studies, and metabolite quantification. In studies of complex spectra nootropic compounds, specifically piracetam, pramiracetam, aniracetam, modafinil, of metabolite mixtures or natural products, it is desirable to use 2-D ¹H-¹³C hetero- adrafinil, ciproxifan, and noopept. The thermal instability of these drugs precluded nuclear single quantum coherence (HSQC) spectroscopy to minimize peak overlaps their analysis by GC-MS. LC-MS provided a superior means of identification for the in the ¹H and ¹³C dimensions. In fully-relaxed 1-D ¹H and ¹³C nuclear magnetic

3 2015 EAS Abstracts November 2015

resonance (NMR) spectra all peak intensities (areas) are directly proportional to for noise reduction, color grading, object and edge detection. The use of kernels, concentration. Thus, they can be related to the intensity of a standard of known Fourier transforms and image morphology techniques for image enhancement and concentration. However, in 2-D ¹H-¹³C HSQC spectra this relationship does not hold. removal of artifacts are covered. Additionally, we demonstrate how automated anal- Theoretical approaches for the derivation of correction factors have been proposed, ysis of microscopy images can be of help in the design of new commercial polymer but we developed an experimental approach to the problem, which we have named materials, and how specialized image analysis techniques can be applied to assess HSQCₒ. Because HSQCₒ analysis yields the correction factors linking HSQC peak the visibility of scratches on surfaces of commercial materials and to measure the intensity and relative concentration, it needs to be applied only once for longitudi- coating thickness of thin films. If time permits, we will also briefly cover motion pic- nal studies. For quantification, HSQCₒ can be combined with FMLR or Bayesian tures with regards to how the immense amount of high resolution video data are analysis. We have applied this approach to a variety of problems, including natural compressed into digital files of a manageable file size and how special effects and products and plant cell wall profiling. video transitions can be generated with minimal skill to produce effective visual animations and video sequences. 15 Using NMR to Understand Pharmaceutical Drug Dissolution Andy Phillips, AstraZeneca, F40/14 Etherow, Silk Road Business Park, 18 Application of Field Microscopy to Emergency Response and Macclesfield SK10 2NA, United Kingdom, Steven R. Coombes, Leslie P. Military Analysts Hughes, Francesco Tres, Jonathan C. Burley Pauline Leary, Smiths Detection, 1934 Bulls Head Rd., Stanfordville, NY Dissolution testing is a key aspect of the analysis of pharmaceutical products such as 12581, John A. Reffner tablets and other oral immediate release dosage forms. Dissolution testing demon- The analysis of microscopic samples in the field continues to evolve to include both strates whether the active pharmaceutical ingredient (API) is released from the tab- light and vibrational spectroscopic methods. Instrumentation to perform these types let into solution. It provides confidence that the API will be available for absorption of analyses are available and streamlined to meet the needs of the field analyst. from the gastro-intestinal tract and so ensures the quality and consistency of the Consideration for field deployment, especially with regard to size and portability, are product. Conventional dissolution methods, however, provide little of the mechanis- especially important. Many different disciplines are successfully employing these tic information which could assist in product design and optimization. We present systems to provide on-scene diagnostics, investigative information, and presump- the use of 1H nuclear magnetic resonance (NMR) coupled with magnetic resonance tive identifications. Primary users include emergency- and military-response units. imaging (MRI) as a novel measurement approach for improved understanding of the Analysts working within these disciplines may be performing scene assessments, dissolution of conventional pharmaceutical tablets [1] and amorphous solid disper- identifying and mitigating threats, or providing other actionable information to inci- sions (ASDs) [2,3], which are used to deliver APIs with poor aqueous solubility. 1H dent command. These users frequently work under extremely stressful conditions NMR has benefits over the conventional UV measurement approach due to much because the data and results they generate may be used to make decisions having greater analyte selectivity and detection of non-UV absorbing tablet excipients such life-and-death consequences. They must be proficient in the use of their equipment, as sugars and polymers. With ASDs for example it is possible to monitor the dissolu- and they must be able to accurately interpret their visual observations and spec- tion, super-saturation and precipitation of API and excipients. MRI gives insight into troscopic data. Their methods must provide reliable information that addresses the complementary processes such as hydration and erosion. NMR and MRI data were questions they need answered. In many cases, microscopical evaluations are crit- obtained by a combination in-line flow cells and off-line sampling. ical and generate results that can’t be gleaned using other methods. This presen- References: tation describes success stories in the field where the application of microscopical [1] Coombes, S. R., Hughes, L. P., Phillips, A. R., Wren, S. A. C., Anal. Chem. 2014, methods was used in emergency- and military-response settings to perform scene 86, 2474-2480. assessments and mitigate threats. Analysis performed using light microscopy, as [2] Langham, Z., Booth, J., Hughes, L. P., Reynolds, G. K., Wren, S. A. C., J. Pharm. well as infrared and Raman microspectroscopy are included. Sci., 2012, 101, (8), 2798-2810. [3] Tres, F., Coombes, S. R., Phillips, A. R., Hughes, L. P., Wren, S. A. C., Aylott, J. 19 Pharmaceutical Glass Vial Interior Surface Defect Characterization W., Burley, J.C. submitted to Molecules. Using Microscopy Robert A. Carlton, GlaxoSmithKlein, 1250 South Collegeville Rd., 16 Circumventing the Pitfalls of Quantitative NMR: An Innovative Collegeville, PA 19426 External Standard-Internal Reference Approach Glass corrosion (commonly called delamination) can result in the generation of Yande Huang, Bristol-Myers Squibb, 1 Squibb Dr., New Brunswick, NJ glass related particulates in parenteral drug product. [1] There is an expectation by 08903 regulatory authorities that pharmaceutical manufacturers will understand the pro- Nuclear magnetic resonance (NMR) is well known as a powerful tool in structure pensity of their product to cause corrosion and to monitor corrosion on accelerated elucidation. It is also gaining popularity as a quantitative methodology (qNMR). Al- stability studies.[2] Evaluation of incoming and treated vials before filling with drug though qNMR is less popular as compared to other quantitative chromatographic product is a key element of glass corrosion evaluations both initially and on stability. methods, it is a unique quantitative method because NMR is a universal detector. Various optical and electron microscopy techniques have been applied to this prob- This allows qNMR to use non-specific internal and/or external standards to conduct lem along with several other analytical methodologies [1]. Two of the more common the quantitative experiment. Recently, qNMR has been used to certify organic ref- microscopy techniques applied to glass vial interior surface studies are differen- erence materials under accreditation guidelines. It has also been recommended to tial interference contrast (DIC) and scanning electron microscopy (SEM). DIC has the authors of the Journal of Medicinal Chemistry as an accepted method for the the advantages of examination of the whole vial with minimal sample preparation establishment of compound purity. However, there are pitfalls when using qNMR. whereas SEM has the advantage of high resolution even though the glass vial must For traditional qNMR, the internal standard coexists with the analyte. Therefore, be sectioned for analysis. In practice, DIC is used to locate possible regions of cor- compatibility, such as stability and resonance specificity, between the standard and rosion or of glass imperfections in the vial followed by breaking or cutting the glass the analyte is essential, which will limit the choice of internal standards. In contrast, in these regions and mounting on SEM stubs for examination. Surprisingly, there is the compatibility can be eliminated by using an external standard method, such as a wide variety of surface morphology among different vial manufacturers which can PULCON or electronic referencing. However, the later method has to overcome affect stability studies. instrument factors to achieve desired accuracy. In order to retain the advantages References: of the traditional internal standard (cancellation of instrument factors) and external [1] Iacocca R.G., Allgeier A., J Mater Sci 42, 2007 pp. 801-811. standard (avoidance of compatibility between the internal standard and the analyte), [2] Food and Drug Administration (FDA). 3-25-2011 a new qNMR method using a combination of external standard-internal reference (http://www.fda.gov/drugs/drugsafety/ucm248490.htm). has been developed. The exact purity and concentration of the internal reference may not be required if an appropriate sample preparation procedure is followed. 20 Problem Solving Using Light and Electron Microscopy The external standard-internal reference qNMR method is described and examples Rich Brown, MVA Scientific Consultants, 3300 Breckinridge Blvd., Ste. are presented. 400, Duluth, GA 30096 Forensic particle investigations involve quality control issues that affect product 17 What you can do with Images: Advanced Image Analysis and quality, consumer safety and manufacturing costs. The investigation must progress Processing for Microscopy and Beyond quickly as the laboratory results drive the investigation. Small foreign particles, one Gunter Moeller, Arkema Inc., 900 1st Ave., King of Prussia, PA 19406 millimeter or less, that caused the investigation can be difficult to characterize us- For most of us, the acquisition, processing, or distribution of digital images have ing bulk analysis or non-microscopical methods. Preliminary examination using low become an integral part of our lives. Digital images also play an important role in power stereomicroscopes, fluorescence microscopy, reflected brightfield andre- industrial applications for quality control (QC) and inspection. In this presentation we flected darkfield microscopy determines how many types of particles are present, if explain the technical steps involved in generating a digital image based on light in- the particles can be sub-sampled for multiple analyses to occur simultaneously and duced charges accumulated on an image sensor. We then cover various techniques to determine what type of testing will give the most information in the least amount

4 2015 EAS Abstracts November 2015

of time. A combination of scanning electron microscopy-energy dispersive X-ray 24 Characterization and Imaging of Archival Texts: Let’s Have a Word spectrometry (SEM-EDS), Fourier transform infrared microspectroscopy (FTIR), with Terahertz! Confocal Raman microspectroscopy (CRM) and polarized light microscopy (PLM) Tiphaine Bardon, University College London, 14 Upper Woburn Pl., is used to characterize the particle(s). The type of particle, (plastic, elastomeric, London WC1H 0NN, United Kingdom, Matija Strlic, Robert K. May, metal, oxide, mineral, organic) determines the analysis sequence in the laboratory Philip F. Taday, Gerrit De Bruin allowing multiple testing on a single particle. The information provided leads the in- Recent research has revealed the potential of in-depth imaging techniques, such vestigation to possible sources of the contamination. Case examples are presented as terahertz time-domain (THz-TD) imaging or X-ray phase contrast imaging, to demonstrating the application of this analysis procedure in investigations involving produce legible and well contrasted images of texts enclosed in complex documents hazardous working conditions, contaminated medical devices and coatings. (e.g., burnt scrolls, stained parchments, stack of overlaid papyri). Yet, such studies often present solutions specific to the heritage object under investigation. The pres- 21 Terahertz in Art and Cultural Heritage Inspection: Present and ent study introduces a systematic investigation of various historical documents and Future historically informed document models, which have been analyzed with different Albert Redo-Sanchez, Massachusetts Institute of Technology Media THz-TD set-ups and accessories, together with complementary analytical and imag- Lab, 75 Amherst. St., Cambridge, MA 02139, Barmak Heshmat, Salman ing tools, in order to explore the parameters that influence the contrast in terahertz Naqvi, Mingjie Zhang, Ramesh Raskar images of those documents. These parameters include dispersion and absorption In this paper we provide a review of current challenges of terahertz technology in spectral signatures of various archival materials, the physical structure of docu- art and cultural heritage inspection. Many publications show the potential of tera- ments, as well as the technical specifications of the particular imaging system used. hertz in this field but specific challenges need to be addressed with new methods Knowledge of the refractive index and absorption coefficients of the different mate- involving both hardware and software development. For example, the extraction rials constituting a document proves valuable in interpreting and enhancing contrast and reconstruction of deep layers in terahertz waveform is often challenging due to in terahertz images and it is argued that it should be made available in the online rapid signal-to-noise (SNR) degradation as the number of layer and depth increas- database thzdb.org. To provide a reliable interpretation of an image based on these es. Conventional deconvolution methods such as CLEAN and frequency-based optical parameters, their accuracy and precision should be reliably determined. To do not work well when SNR < 10 dB. We propose a probabilistic pulse extraction do so, recent advances in terahertz metrology should be taken into account and (PPEX) method that is capable to extract the position of layers from densely lay- compounds of different provenance, or prepared using different recipes, should be ered samples imaged with a terahertz time-domain system in low SNR conditions. analyzed to account for the diversity and complexity of archival materials. PPEX performance is notably better in both simulated and experimental data than conventional CLEAN and frequency-based deconvolution techniques in separating 25 Getting Closer: Making Quantitative Molecular Spectroscopy into a and localizing overlapping pulses in low SNR (10 dB) waveforms. The combination Metrical Science of PPEX with a clustering method allows identifying each layer in a stacked sam- Jerry J. Workman Jr., Unity Scientific, 117 Old State Rd., Brookfield, ple so that the reflected pulse amplitude can be mapped and the content of each CT 06804 layer recovered. The application of PPEX in terahertz time-domain data allowed Metrical sciences provide a basis for calibrating spectrophotometers such that us to extract the position and content of nine layers of stacked paper mimicking a calibration transfer is made automated and precise when compared to existing ad closed book. PPEX method can be used to reveal finer features in deep layers from hoc techniques. The use of metrical techniques allows the user and manufacturer paintings, old documents and wooden-based objects. The method can also be used of instrumentation to pay particular attention to the aspects of stable systems and to measure the effectiveness of surface and coating treatments for cultural object measurements over time based on first-principles, primary physical standards, with preservation and restoration. uncertainties clearly defined and characterized. The application of metrical science to spectroscopic measurements provides for accurate analysis and improved, re- 22 Development of Novel Systems Architecture in THz Imaging for the liable commerce on a global basis [1-3]. For calibrations used in quantitative or Analysis of Cultural Heritage Materials qualitative analysis to be stable and traceable, either the calibration must become Roxanne Radpour, University of California – Los Angeles, Materials dynamic to compensate for temporal instrument changes, or the instrument must Science and Engineering, 410 Westwood Plaza, 3111 Engineering be continuously corrected to a constant, precise steady state. The application of V, Los Angeles, CA 90095, Shijun Sung, Warren Grundfest, Ioanna a calibration model to a dynamic measurement system requires some routine sys- Kakoulli, Zachary Taylor tem adjustments in order to maintain measurement stability over time. To create Terahertz (THz) imaging is increasingly becoming a desired method of investigation long-term stability in a measurement system, primary physical, chemical, optical, or for scientists and conservators in the field of cultural heritage. The lower energy THz emissive standards are used; either resident within the spectrophotometer (or man- source along with its deep penetrating ability due to the longer wavelength near the ually measured in an external sample presentation mode). These standards, which far-infrared (IR) region, makes THz imaging a powerful analytical tool at revealing by definition are well-characterized and stable, provide primary correction of both hidden features and non-discernible iconography in multilayered structures. In the secondary standards and instrumentation. The process of using primary standards past several years, significant advancements and promising research were obtained applies for dynamic correction of multiple instrument parameters in real-time, for using THz time domain spectroscopy (THz-TDS). However, most THz research per- example: wavelength axis, photometric axis, line shape, and detector linearity [4-5]. formed in the field of cultural heritage has been done using commercial THz-TDS This concept is presented within this paper. systems whose development is tailored towards imaging targets that do not always References: represent the complex topographical, structural and compositional nature of objects [1] Jerome Workman, Jr., Howard Mark, Calibration Transfer, Part 2: The Instrumen- such as wall and panel paintings. This research, focusing on revealing hidden ico- tation Aspects,” Spectroscopy, 28(5), 12-25 (2013). nography (covered by layers of paint and/or plaster) involves the development of a [2] Jerome Workman, Jr., Howard Mark, Calibration Transfer, Part 3: The Mathe- direct detection, reflective pulsed THz system to address the challenges specific to matics of Wavelength Standards used for Spectroscopy”, Spectroscopy 29(6), cultural heritage materials such as uneven surfaces, multi-layered hierarchy, strong 18-27 (2014). scattering coatings and in situ environment. Based on the success using an in- [3] Jerome Workman, Jr., Howard Mark, Calibration Transfer, Part 6: The Mathe- house custom-made medical THz imaging system of similar technical design, the matics of Photometric Standards used for Spectroscopy, Spectroscopy 29(11), new system development takes advantage of the former’s fundamental features 14-21 (2014). while improving on optical design, portability, and system size reduction. The overall [4] Jerome. J. Workman, Spectrometer Reference Calibration, U.S. Patent goal is twofold: to push the boundaries of THz imaging instrumentation development 20,150,142,364, 2014. for cultural heritage purposes, and to apply this THz system architecture in-situ for imaging of wall paintings to overcome complex stratigraphy and reveal hidden ico- 26 Infrared Microscopy for Practical Cancer Imaging nography for improved comprehensive studies. Rohit Bhargava, University of Illinois at Urbana-Champaign, 405 N. Mathews Ave., Urbana, IL 61801 23 Terahertz Systems for Cultural Heritage We describe a combination of theory, model experiments and statistical analyses Philip Taday, TeraView Ltd., Platinum Building, St. John’s Innovation to devise fast infrared (IR) imaging protocols for cancer pathology. Conventional Park, Cambridge, CB4 0DS, United Kingdom Fourier transform infrared (FT-IR) spectroscopic imaging systems typically employ Over the last few years very rapid development has occurred in the application of an incoherent globar source and achieve spectral specificity through interferometry. terahertz sensor technology in for the investigation of cultural heritage studies. In Recent advancements in broadly tunable external cavity quantum cascade lasers this paper I will explore the design considerations required for a sensor optimized (QCL) offer new approaches and new possibilities for IR imaging. We present re- for acquiring terahertz images on objects. Equally as important is the data handling sults from a discrete frequency infrared (DFIR) microscope based on a QCL source a terahertz image consists of quasi-three dimensional information and quite easily and demonstrate wide-field imaging with high NA lenses and a sub-micron effective an experimenter can get into the realms of “big data”. I explore the recent advance- pixel size. The images demonstrate high resolution, diffraction limited capabilities ments in the data processing. as well as several unique features due to coherence effects from the laser source. 5 2015 EAS Abstracts November 2015

Precise outcome prediction is crucial to providing optimal cancer care across the 30 Method to Improve the Recovery of pH Labile Anti-Drug Antibodies spectrum of solid cancers. We report an approach to predict the risk of prostate during Acid Dissociation and Extraction cancer recurrence, at the time of initial diagnosis, using a combination of IR imaging Weifeng Xu, Bristol-Myers Squibb, Route 206 and Province Line Rd., and a diagnostic protocol that focuses on both the tumor and its microenvironment, Princeton, NJ 08543 and data analysis of frequent patterns in molecular expression. Dyes such as he- When large amount of a biotherapeutics drug is present in the clinical samples, matoxylin and eosin (H&E) and immunohistochemical stains have been widely used these drugs have to be dissociated and removed from anti-drug antibodies (ADA) to visualize tissue composition in research and clinical practice. We developed an so that ADAs can be detected by either ligand-binding assay or cell-based bioassay. alternative approach to obtain the same information using stain-free IR imaging and By screening a panel of more than 20 ADA positive control mAbs, we found that its combination with a computational prediction algorithm. Together, the develop- current widely used acid dissociation followed by biotinylated-drug extraction led to ment of IR imaging technology, theory, practical assays and deployable protocols is low recovery of more than 40% of these ADA positive controls, due to pH sensitivity. moving us closer to making the technology practical for users. Here we discuss the alternative methods for ADA extraction so that both pH labile and pH resistant species can be maximally recovered. This will increase the sensi- 27 Use of Near-IR and Hyperspectral Imaging in Analysis of tivity of immunogenicity testing. Agricultural Commodities Stephen R. Delwiche, United States Dept. of Agriculture, Agricultural 31 Sol-Gel Capillary Microextraction Coupled to HPLC for the Research Service, Building 303, BARC-East, 10300 Baltimore Ave., Preconcentration and Analysis of Biologically Important Molecules Beltsville, MD 20705 Abdul Malik, University of South Florida, 4202 E. Fowler Ave., Tampa, FL In the past ten years, hyperspectral imaging (visible and near-infrared) applications 33620, Abdulllah Alhendal, Emre Seyyal, MinhPhuong Tran, Sheshanka have expanded beyond remote sensing to now include the inspection of raw and Kesani processed food products. Initially reserved for scientific exploration, improvements Capillary microextraction (CME) [1] can be an effective analytical tool for the pre- in data capture rates and processing have moved this technology closer to analysis concentration of trace and ultra-trace analytes from complex matrices. In CME, a in real time. Recent research applications in the cereal grains include classification, surface-coated capillary is used to achieve analyte enrichment by simply passing purity or cleanliness assessment, milling quality assessment, and detection of vari- the sample through the capillary. In this process, the target analyte(s) get selective- ous forms of kernel damage including Fusarium, insect, sprout, black tip, and other ly extracted by the surface coating, leading to the desired enrichment. However, molds. Applications in fruits and vegetables include detection of cracks, bruises, stability of the surface coating may become an issue, especially for traditionally defects, and contamination from possible pathogenic bacteria. Research endeavors coated capillaries that lack chemical bonds between the coating and the capillary of the United States Food and Drug Administration personnel involved in the devel- surface. This makes such coatings susceptible to leaching out and degradation by opment of hyperspectral and multispectral image analysis, including aspects of data high-performance liquid chromatography (HPLC) mobile phases during CME opera- collection, processing, lighting, defining regions of interest and chemometrics for tion in hyphenation with HPLC. Sol-gel technology offers an effective solution to this data reduction, are presented. problem by providing in-situ created surface coatings that get chemically bonded to the capillary inner walls during their in-situ creation from a sol solution. Analysis of 28 NIR with Problem Data Sets biomolecules may also require acidic or basic media to achieve the enrichment and Franklin E. Barton, Light Light Solutions, 1101 Laurel Springs Ct., /or chromatographic analysis. Traditional silica-based materials may not provide Watkinsville, GA 30677, James A. de Haseth the needed pH stability. Luckily, sol-gel technology can be used to create non-sili- There are many approaches to developing a data set for near-infrared (NIR) calibra- ca surface coatings exhibiting exceptional pH stability [2]. In this presentation, we tions, some statistically valid, some not; but all with some interesting insights. There demonstrate how both silica- and non-silica based sol-gel hybrid organic-inorganic is the ideal situation with a large set of samples, ample laboratory resources for coatings can be created for online coupled CME-HPLC operation to enrich biologi- reference chemistry. Do you make a random selection or take every nth sample and cally important molecules including neuro transmitters and phosphopeptides. scan and analyze? Do you start scanning and analyzing and build your calibration References: data set gradually as you go, with the big question? When do I stop? Yes, you can [1] S. Bigham, J. Medlar, A. Kabir, C. Shende, A. Alli, A. Malik, Anal. Chem. 2002, use the reference data to select a diverse range of samples insuring that the ends as 74, 752-7561. well as the range are adequately sampled. Some chemometric software packages [2] S. Segro, J. Triplett, A. Malik, Anal. Chem. 2010, 82, 4107-4113. will choose the most diverse set of samples based on the spectra. This provides a stopping point for initial calibrations and limits the amount of reference chemistry 32 Proteomics Study to Identify the Protein Differences in Human you may have to do. However, it is often not the situations that arise in the NIR lab Breast Milk from Breast Cancer Patients and Controls Using Mass that provide the most strenuous challenges. Usually it is the real world samples from Spectrometry commercial operations that have their limitations based on the process. The bottom Devika Channaveerappa, Clarkson University, 8 Clarkson Ave., line is you get to work with what you are handed. We examine some of these data Potsdam, NY 13699, Roshanak Aslebagh, Costel C. Darie, Kathleen sets and the unique opportunities for model development. F. Arcaro Breast cancer is the second leading cause of cancer death in women. About 12% 29 Transferring Assays to CROs, Strategies and Processes for both women in the United States develop breast cancer. Breast milk can be assessed Small and Large Molecules to evaluate the risk of one getting breast cancer by comparing the proteomes of Xiaohui Xu, Bristol-Myers Squibb, 14 Walnut Ct., Cranbury, NJ 08512, breast milk from healthy and breast cancer suffering individual. This study makes Bruce Stouffer, Zoe Tzogas, Johanna Mora, Jim Shen, Pathanjali use of mass spectrometry based proteomics to identify the differences between Kadiyala, Dennis Stocker, Dennis Garner, Mark Arnold the control and cancerous samples which would further help in identifying potential Outsourcing is a vital component of the drug development process. Bioanalytical biomarkers for breast cancer. Firstly, SDS-PAGE was used to separate the proteins assay outsourcing and assay transfer to a contract research organization (CRO) from the whole milk sample. The gel bands for each sample was then excised and is an integrated step in the life cycle management of a bioanalytical method. This cut into small pieces. The gel pieces were trypsin digested to extract the peptides. presentation seeks to address strategies and processes needed for outsourcing Peptide mixtures were analyzed by liquid chromatography-tandem mass spectrom- both small and large molecule bioanalysis for PK and immunogenicity. Successful etry (LC- MS/MS). Raw data obtained were processed using ProteinLynx Global assay transfer to a CRO is critical for downstream quality data delivery for an out- Served (PLGS version 2.4) and then submitted to Mascot database search for pro- sourced program. CRO selection often starts with the review of available resource tein identification. For further analysis Scaffold version 4.1 software was used. In the and expertise at the lab to ensure appropriate pre-requisites have been met. For sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel, after a new CRO, a pilot project is often placed to evaluate scientific expertise and pro- Coomassie staining, the protein patterns did show minor differences. After LC-MS/ cess before more projects are placed. Sharing of appropriate information during the MS analysis, the proteins identified were compared for the relative ratio between transfer is equally important. Both individuals and organizations develop technical the proteins from the milk samples from control donors and the donors with breast habits that differ slightly from other laboratories. Differences between the laborato- cancer. There were significant differences identified in the proteomes of the two sets ries may exaggerate inherent method variability and cause a validation failure due of samples. Some of the proteins were upregulated in the breast cancer samples to unacceptable accuracy and precision. It is not uncommon for cross-validation to and some were down regulated when compared with the controls. Additional inves- fail because of slight changes in common buffers, kit solutions, columns, experiment tigation of more breast milk samples is ongoing. This study focuses on identifying execution. Therefore, a rigid process for the CRO to follow and hands-on monitoring biomarkers directly in the milk of donors with breast cancer. from the sponsor is critically important for a successful transfer. Qualified project managers at both sponsor and CRO labs with suitable scientific and project man- agement backgrounds who can critically evaluate and resolve issues in a timely manner are of particular importance. Overall, the process and strategies for CRO selection, method transfer, project monitoring as well as case studies are presented. 6 2015 EAS Abstracts November 2015

33 Mass Spectrometry-Based Protein Biomarker Discovery in scribes the analytical challenges that were overcome to develop robust methods for Neurodevelopmental Disorders monitoring these genotoxic impurities at low parts per million (ppm) levels in both Kelly L. Wormwood, Clarkson University, 8 Clarkson Ave., Potsdam, the input triazole and the key intermediate. NY 13699, Armand G. Ngounou Wetie, Emmalyn J. Dupree, Alisa G. Woods, Costel C. Darie, Laci Charette, Jeanne P. Ryan 37 The Determination of the Physico-Chemiscal Properties of Neurodevelopmental disorders are a group of disorders in which the development of Nanoemulsion the central nervous system is disturbed. These are very common with approximately Maurice O. Iwunze, Morgan University, 1700 E. Cold Spring Ln., 15% of children in the United States ages 3 to 17 being affected by at least one Baltimore, MD 21251 disorder. Examples include Autism Spectrum Disorder (ASD) and Smith-Lemli-Opitz Nanoemulsion (formulated by water:oil:surfactant and co-surfactant, in appropriate Syndrome (SLOS). ASD affects approximately 1/63 children in the United States ratios) technology has come of age and its physico-chemical properties need to be and is characterized by repetitive behaviors, communication deficits and impair- determined and documented. Such properties include dielectric constant, polari- ments in social interactions. There is currently no biological diagnosis or known ty, refractive index, viscosity, density and electrical conductance. These properties cause of ASD. SLOS is characterized by a cholesterol deficiency due to a mutation have been determined in this work. The determination of such parameters and will on the 7DHCR gene. Approximately 1/20,000 babies are born with SLOS. Diagno- play a significant role in efficiently designing and tailoring its/their uses in academic sis is achieved by measuring cholesterol and 7-dehydrocholesterol (7DHC) levels research, industry and in medicine. Although its/their uses in these disciplines have in the blood, however, there is currently no proven treatment for SLOS. Because been recorded, determinations of its/their physico-chemical parameters are needed of this, research is increasing to determine biomarkers for these disorders. Here, to tailor its/their applications effectively to specific targets. In addition to the physi- samples from people with ASD (sera and saliva) and SLOS (saliva), and matched co-chemical properties mentioned above, the acidity and the basicity of a specific oil controls were analyzed using a combination of gel electrophoresis (Tricine-PAGE, in water (o/w) nanoemulsion are also determined and discussed. SDS-PAGE and Blue Native PAGE), in gel digestion or insolution digestion and nanoliquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) to in- 38 Mechanistic Understanding of Local pH for Acidified Formulation vestigate differences between the proteomes of people with these neurodevelop- of Weakly Basic Drug Using Surface Dissolution Imaging mental disorders and matched controls. Several alterations in protein expression Sanjay Patel, Merck, 126 E Lincoln Ave., Rahway, NJ 07065, Lei Zhu, were identified. These differences may lead to potential biomarkers for diagnosis, Leah Xiong, Navneet Sharma possible therapeutic targets and an altogether better understanding of the disorders. The dissolution of the weakly acidic or basic drug increases when it is combined with alkaline excipients or organic acids respectively. It has been claimed that these 34 A Multi-Class Drug and Metabolite Screen by LC-MS/MS excipients alter the local pH around the drug which increases the dissolution. The Sharon Lupo, Restek, 110 Benner Circle, Bellefonte, PA 16823 aim of the present proof of concept study was to investigate pH effects in relation to The use of pain management drugs has been steadily increasing. As a result, hos- the dissolution of Compound A. Compound A is a weakly base drug with steep pH pital and reference labs are seeing an increase in patient samples that must be dependent solubility. Formulations were made with acidifying approach to enhance screened for a wide variety of drugs to prevent drug abuse and to ensure patient solubility in combination with hydroxypropyl methylcellulose (HPMC) or Polyvin- safety and adherence to their medication regimen. Therapeutic drug monitoring can ylpyrrolidone (PVP) hydrophilic polymer. Two different formulations were created be challenging due to the low cut-off levels, potential matrix interferences and iso- using polymer and acid. To measure local pH in the vicinity of the solid drug com- baric drug compounds. To address these challenges, many drug testing facilities are pact as well as monitoring drug dissolution was developed using UV/vis imaging. turning to liquid chromatography coupled with mass spectrometry (LC-MS/MS) for Intrinsic dissolution rates were monitor using UV imaging. Real-time measurement its increased speed, sensitivity, and specificity. The Raptor™ Biphenyl column was of pH was achieved by using a pH indicating dye with visible imaging probe. The developed to complement high-throughput LC-MS/MS analyses by combining the acidifying formulation containing HPMC demonstrated superior intrinsic dissolution increased efficiency of superficially porous particles with the resolution of Ultra Se- performance compared to a PVP formulation. The slower drug diffusion across the lective Liquid Chromatography™ column technology. In this presentation, I present HPMC gel barrier is the predominating mechanism to maintain acidic local pH and the methodology for a 230 compound multi-class drug and metabolite screen and to enhance release. The UV-imaging approach is considered useful to understand discuss the challenges one must consider when developing a large screening as- pH dependent dissolution phenomena in dissolution testing. say. Topics of the discussion include mobile phase considerations, isobar resolution, drug interferences, and instrumentation. 39 An In-Vitro Evaluation of Micelle-Partitioning Effects Upon Flux Using the Pion® µFlux Platform 35 Analysis of Electrochemical Oxidation Products of Albendazole Gregory A. Johnson, Merck, 770 Sumneytown Pike, West Point, PA Amos Mugweru, Rowan University, 201 Mullica Hill, Glassboro, NJ 19486, Walter Wasylaschuk, Justin Pennington, Paul Harmon 08028, Samantha Gibson, Geoffrey Kamau In-vitro flux models for potential correlation with pharmacokinetic (PK) food effects Methyl [5-propylthio-1-H-benzimidazol-2-yl]carbamate (albendazole) is widely used have shown limited or mixed success. A model compound (“Cpd A”) for example, as anthelmintic for the control of gastrointestinal and lung nematodes. The efficacy has shown a negative food effect in PK studies, while no such indication was ob- of this drug is thought to be partly due to the formation of Oxfendazole OFZ (methyl served during outside testing with an in-vitro membrane partitioning assay. As a N-[6-(benzenesulfinyl)-1-H-1,3-benzodiazol-2-yl] carbamate. After oral administra- crude model for the effects of micelle partitioning upon flux, the Pion® µFlux was tion, albendazole (ABZ) is transformed by liver microsomal enzymes into albenda- used in house to evaluate the flux of Cpd A in a fasted state simulated intestinal zole sulphoxide (ASOX), a pharmacologically major active metabolite. Subsequent- fluid (FaSSIF) and fed state simulated intestinal fluid (FeSSIF) biorelevant media ly, this metabolite is further transformed by cytochrome P450 enzyme (CYP2C) to through Pion GIT-0 lipid membrane, and into acceptor sink buffer (ASB), with UV dip an inactive metabolite, albendazole sulphone (ASON). In this work, electrochemical probes used to monitor the active pharmaceutical ingredient (API) in each chamber. transformation of albendazole was investigated using amperometric technique. Compared to FaSSIF, a 4.5-fold reduction in flux is observed with FeSSIF, agree- The products of oxidation/metabolism of this drug were analyzed using high-per- ing with negative food effect observations in PK studies. Differences in salt and formance liquid chromatography with 59:27:14 acetonitrile, methanol, and distilled pH were shown to have only a minor effect, suggesting that micelle partitioning is water as the mobile phase and using a C-18 column. The products of electrochem- the major factor in the reduced flux. Evaluation remains ongoing for evaluating the ical oxidation and method development for the metabolic products are discussed. potential correlation of observed PK food effects (APIs showing positive, negative, and neutral effects) with in-vitro biorelevant media-dependent flux. The establish- 36 Development of Key Analytical Methods to Define the Control ment of such a correlation would be a valuable tool for developing early formulation Strategy for Genotoxic Hydrazine Impurities in a Novel Drug strategies. Candidate James Chadwick, Bristol-Myers Squibb, Reeds Lane, Moreton CH46 40 Withdrawn by the author. 1QW, United Kingdom, Ian Hale, Michelle Haslam, Emma Quirk A new drug candidate currently in Phase 3 development at Bristol-Myers Squibb 41 Quantification of Polysorbate 80 in Biotherapeutic Formulations is synthesized via a key intermediate which is generated from an Ullman coupling Using LC-MS with a triazole starting material. The triazole starting material is manufactured from Dilusha S. Dalpathado, Bristol-Myers Squibb, 104 Georges Rd., New hydrazine and as such the impurity profile of this starting material can contain a Brunswick, NJ 08901, Joseph Valente, Kathleen A. Kelly, Mark S. Bolgar number of hydrazine derivatives, many of which are confirmed genotoxins. Ana- Polysorbate 80 (PS-80) is a non-ionic surfactant which is commonly included in bio- lytical methodology was required to monitor these genotoxic impurities in both the therapeutic formulations to prevent aggregation and increase solubility and stability. triazole and the key intermediate to determine the fate of the hydrazines and subse- There has been an increased attention to PS-80 analysis within the pharmaceutical quently define an appropriate control strategy. Development of these methods was industry in response to health authority concerns regarding the long term stability challenging due to the number of different genotoxic substrates and the physical of PS-80 as well as the potential for loss of PS-80 during drug product processing. properties of both the hydrazines and the key intermediate. This presentation de- Published approaches to PS-80 assays are limited by interference from high protein 7 2015 EAS Abstracts November 2015

concentrations and often incorporate extensive sample preparation procedures to 45 Modern Particle Designs for U/HPLC, Past, Present and Future remove high concentrations of protein. This presentation describes the develop- David S. Bell, Supelco, Division of Sigma-Aldrich, 595 North Harrison ment of a rapid and specific liquid chromatography mass spectrometry (LC-MS) Rd., Bellefonte, PA 16823, Richard A. Henry method for the analysis of PS-80 which requires no sample preparation beyond a Although liquid chromatography is often considered a mature technique, there have simple dilution of samples. Using a mixed mode column the protein is eluted in the been numerous advances over the past decade. The quest for speed of analysis void volume at the initial conditions. A step gradient is then employed to elute the has generated great interest in improving the efficiency of high-performance liq- PS-80 components as a single peak. Electrospray ionization with in-source colli- uid chromatography (HPLC) separations. To achieve this much attention is due to sion induced dissociation is used to generate a fragment ion common to PS-80 the design of HPLC particles. Initially, the development of commercial sub-2-µm components resulting in high specificity and sensitivity. The selected fragment ion particles in 2004 (along with the instrumentation required to effectively use them) incorporates the critical oleic acid - polyethylene glycol ester bond and as such provided improved efficiencies over common 3-µm and 5-µm materials. Novel 2.7- allows for the differentiation of intact PS-80 from its hydrolyzed forms. Using this µm superficially porous particles (SPP) were then introduced in 2006 that provided method, PS-80 has been quantified in different formulations having a wide range of greater efficiencies than similarly-sized fully porous particles (FPP) and were com- protein concentrations. parable in performance to sub-2-µm particles. Columns packed with SPP particles could thus be utilized in place of smaller particles to obtain similar efficiencies but 42 The Need for Speed: Efficiency Gains of UHPLC vs. High without the burden of high backpressures. Although the SPP architecture was ini- Performance Column Technology tially designed to improve mass transfer kinetics, further research has revealed that Jonathan E. Clark, Procter & Gamble, 8700 Mason Montgomery Rd., the increased efficiencies observed for small molecules has been largely dueto Mason, OH 45040 improvements in both eddy diffusion and longitudinal diffusion. This improvement is The advent of ultra-high-pressure liquid chromatography (UHPLC or UPLC) brings at least partly due to much narrower particle size distribution resulting from the SPP the promise of faster separations, shorter equilibration times, improved sensitivity construction process. These discoveries have led to a revival of research and devel- and higher resolution. The advantages of UHPLC are clear but implementation of opment concerning the importance of monodisperse particle technologies for liquid this relatively young technology requires capital investment and extensive training chromatography. The most important developments in particle technology, device for inexperienced users. Liquid chromatography (LC) column-based innovation has designs and instrumentation are highlighted in this presentation. Particular attention been occurring in parallel with the development of UHPLC technology. Smaller (<2- is paid to how sub-2-µm and superficially porous particles have improved the HPLC µm), more homogeneous column packing materials, as well as, superficially porous and hyphenated HPLC landscape. Newer developments in monodisperse particle (or “Core-Shell”) columns can provide increased performance similar to UHPLC and technology and how it is expected to fit into the future of HPLC are also covered. only require lower pressure (<400 bar) HPLC systems. Data comparing the through- put and performance of instrument vs. column based innovation is presented. Dis- 46 Column Pore-Size as a Critical Variable in HPLC Method cussion of the challenges of implementation of each new technology into research Development and development and quality control environments is also included. Richard A. Henry, Consultant, 983 Greenbriar Dr., State College, PA 16801 43 The Impact of Different UPLC Sample Managers on Dissolution Screening and optimizing stationary and mobile phase now receives primary at- Results: A Case Study tention in developing high-performance liquid chromatography (HPLC) methods for Vincenc Camaj, Celgene Corporation, 86 Morris Ave., Summit, NJ small molecules. Popular HPLC column particles have an average pore-diameter 07901, Meijia Chen, Yali Sun of about 100 angstroms, allowing small solutes to easily enter the pores and equil- An ultra-performance liquid chromatography (UPLC) method using a Waters Acqui- ibrate with stationary phase. The surging interest in separating peptides, proteins ty UPLC system was developed for the assay of drug product dissolution samples. and other large molecules, however, has created a strong need for HPLC columns Two models of a Waters UPLC Sample Manager were evaluated during method de- with particles that have larger pores. If pores are not adequately large, molecules velopment, the Classic Sample Manager and Sample Manager – FTN (flow through may not gain full access to stationary phase, causing retention loss and peak broad- needle), both utilized by Waters Acquity UPLCs. It was found that a difference in ening due to restricted mass transfer. The optimum column pore-diameter for low sample assay was observed between the results generated with Classic Sample molecular weight (MW) solutes (up to several kDa) has proven to be 80-120 ang- Manager and those generated with the Sample Manager – FTN. Based on this stroms, while the optimum pore-diameter for medium MW solutes (between 5- 50 study, it is recommended that the application of the UPLC system and correspond- kDa) increases to 150-300 angstroms. If solute MW exceeds 50 kDa, HPLC col- ing sample manager for the assay of dissolution samples should be carefully eval- umns with 400-500 angstrom pores or even larger are needed to optimize sepa- uated prior to its selection. ration performance. Guidelines for column selection and method development will be expanded to include the pore-size variable, which may become dominant when 44 The Impact of Instrument Design Characteristics on Reversed- samples include large solutes. Solute size and MW are compared and discussed in Phase HPLC and UHPLC Methods Transfer relation to pore-diameter and surface area of HPLC particles. Practical MW limits Paula Hong, Waters Corporation, 34 Maple St., Milford, MA 01757, are established for optimizing separation performance with columns of different pore Patricia R. McConville diameters. Transfer of established reversed-phase methods across both high-performance liquid chromatography (HPLC) and ultra-high-performance liquid chromatography 47 Monolithic Silicas in High-Performance Liquid Chromatography: (UHPLC) chromatographic instrumentation requires careful consideration of the op- The Alternative to Conventional Packed-Particle Columns erating parameters and design of each instrument. For example, gradient formation Egidijus Machtejevas, Merck KGaA, Frankfurter Str. 250, Darmstadt can be influenced by binary or quaternary mixing, dwell volume, viscosity changes 64293, Germany in the mobile phase, the gradient shape, residual volumes, and many other factors In contrast to conventional packed-particle columns, monolithic silica columns are that can vary across different pumps. The mechanism for sample injection, as well made of a single continuous-bed rod of high purity porous silica that is then bond- as the detector, can influence linearity and quantitation. Lastly, thermostatting can ed with C18, C8 and the other modifications that chromatographers have known alter retention- whether due to frictional heating affects, mobile phase pre-heating and trusted to solve their separation needs through the history of high-performance or thermal gradients. To understand the effect these factors may have on methods liquid chromatography (HPLC). For the first time, the physics of the HPLC column transfer, both instrument characteristics and specific method conditions must be have been completely reengineered, providing dramatically reduced backpres- factored and evaluated when transferring HPLC and UHPLC methods. In this pre- sures due to the open flow-through design, while maintaining the high resolution sentation, studies evaluate transfer of a single United States Pharmacopeia method and performance that chromatographers expect today. Monolithic columns remove across multiple instruments and instrument vendors and the impact of instrument backpressure as the primary consideration in method development and give back attributes- including solvent delivery and temperature control - on the separation the flexibility of choices in flow rates for much higher throughput, column lengths fidelity. The transferability of the method will be assessed through system suitability for superior resolution, and solvent choices for optimum selectivities that smaller criteria (relative retention, % area, etc.). The analysis will employ the same column and smaller sized packed-particle columns have slowly taken away over the de- across multiple instruments. An additional set of experiments will evaluate the im- cades. Because they have no individual particles to shift or break, column perfor- pact of system attributes on method scaling, in which the l/dp ratio will remain con- mance is very consistent over much longer lifetimes, making them ideal for method stant, and the stationary phase will range from 5-μm to sub 2-μm particles. In both transfer into production quality control or multiple sites. Their high permeability also sets of examples, consideration will be made to conduct method transfer in accor- makes them very forgiving of shortcuts and timesaving in sample preparation as dance with regulatory guidelines for allowable adjustments to compendial methods. well as easier to aggressively flush out to re-equilibrate. This presentation guides you through the world of monolithic silica materials. Benefits are demonstrated with many application examples including pharmaceutical and bioanalysis separations (proteomics, peptidomics, etc.), calibration curves, recovery calculations, and meth- od robustness overviews. 8 2015 EAS Abstracts November 2015

48 High-Throughput LC-MS-MS Measurement of 17-Hydroxyproges- based on column switching between a loading trap (2x10 mm C8 guard cartridge) terone in Human Blood Serum for Research Purposes and 2x50 mm fused core reversed phase column (Bioshell A160 Peptide C18 from Joseph M. Di Bussolo, Thermo Fisher Scientific, PO Box 2081, West Supelco) via a low dwell volume ultra-high-performance liquid chromatography Rhe- Chester, PA 19380, Ian White, Raidiri Castillo, Amit Shah, Hashim odyne valve. Each column elutes by an independent binary gradient pump. The Othman system was successfully tested for up to 100 µl injections of 5x diluted urine sam- 17α-Hydroxyprogesterone (17-OHP) is a biosynthetic precursor to other ples without any pre-treatment, filtration or precipitation. The real advantage of this steroids such as corticosteroids, androgens and estrogens. Researchers need to method is its ability to accommodate any harsh buffer or high concentrations of measure 17-OHP within an analytical range of 10 to 1000 ng/dL of blood serum. salts (4-8 M Urea or Guanidine chloride) for sample preparation. Using this platform This was achieved using ultra-high-performance liquid chromatography (UHPLC) we were able to assess the degree of pure C-peptide binding to glass surfaces. utilizing a solid-core column packing coupled to a triple-quadrupole mass spectrom- Lyophilized C-peptide standards were reconstituted in water or in 8M Guanidine eter (MS) with atmospheric-pressure chemical ionization (APCI). Sample prepara- chloride to avoid analyte binding to glass and analyte area in a both type of samples tion involved extracting specimens with methyl-t-butyl ether after spiking them with was successfully compared. Of note, high salt samples are not compatible with an internal standard (17-OHP-D8). After evaporating and reconstituting the extracts single column LC-MS analysis. to concentrate them two-fold, injections were made into a 4-channel UHPLC sys- tem. A 4-minute water-to-methanol gradient eluted the analyte and internal standard 52 Comparison of Chemical Additive Preservation Methods for Total into the APCI source. Selective-reaction monitoring within a 1-minute data window Mercury Analysis in Human Urine by ICP-MS produced quantitation and conformation peaks with intra-assay and inter-assay pre- Shing Nam Lau, University of New Hampshire, Depart. of Chemistry, cisions < 6% and carryover < 0.5%. Correlation of 47 specimen results with refer- Parson Hall, #29, 23 Academic Way, Durham, NH 03824, Mamta Dua, ence-lab results showed Deming regression R2 = 0.9961, slope = 0.979, intercept = Julianne Nassif, Sterling Tomellini 2.8 and standard error of estimate = 10.6%. Sample throughput was 14, 28, 42 or 56 Quantitation of total mercury has been of great interest not only in environmental injections per hour when multi-channeled across 1, 2, 3 or 4 channels, respectively. fields, but also in medical research. This is because mercury (Hg) one of the most 17-OHP batches were also multi-channeled with batches for analysis of methylma- toxic elements, and is also a well–known global pollutant. Researchers strive to lonic acid (MMA), which utilized the same MS source conditions. understand how toxic elements bioaccumulate in both aquatic ecosystems and ter- restrial systems, along with the effect on human health. Numerous studies and ana- 49 High-Throughput LC-MS-MS Measurement of Pregnenolone in lytical methods have been published for the assessment of mercury in many differ- Human Blood Serum for Research Purposes ent sample matrices. Accurate analysis of these complex matrices is dependent on Joseph M. Di Bussolo, Thermo Fisher Scientific, PO Box 2081, West accurate and reliable analytical standards. Yet, little research has been performed Chester, PA 19380, Ian White, Emily Herman, Raidiri Castillo, Amit to assess variables that affect standard stability.[1] The objective of this study was Shah, Hashim Othman to evaluate the stability of the standard mercury solutions under various prepara- Pregnenolone is a biosynthetic precursor to other steroids such as corticosteroids, tion and storage conditions using inductively coupled plasma mass spectrometry androgens and estrogens. Researchers need to measure pregnenolone within an (ICP-MS). Mercury standards were prepared in the low parts per billion concentra- analytical range of 10 to 500 ng/dL of blood serum. This was achieved using a tion range. Three preservative solution combinations were investigated: 1% (v/v) of multi-channel ultra-high-performance liquid chromatography (UHPLC) utilizing concentrated nitric acid with 1% (v/v) sulfamic acid and 1% (v/v) of concentrated solid-core packed columns with an alkyl-aromatic bonded phase coupled to a tri- hydrochloric acid; 2% (v/v) of concentrated nitric acid with 1% (v/v) sulfamic acid; ple-quadrupole mass spectrometer (MS) utilizing heated electro-spray ionization and 5% (v/v) of concentrated nitric acid with 1% (v/v) of sulfamic acid. Standards (HESI). Sample preparation involved extracting specimens with methyl-t-butyl ether were stored in sealed polypropylene containers at three temperatures (-28˚C, 1˚C after spiking them with an internal standard (Pregnenolone-D4). After transferring and room temperature). The ultimate goal of this study is to extend the storage life- and evaporating each extract to respectively labeled tubes, the residue of each time of the standards, provide high recoveries, and minimize the mercury memory was reacted with hydroxylamine to form oxime derivatives of pregnenolone. Each effect which will improve the applicability of biomonitoring studies for human health tube was then dried and the residue was reconstituted with water and methanol risk assessment related to mercury exposure. and transferred into autosampler vials. Injections were then made into a 4-channel Reference: UHPLC system. A 4.5-minute water-to-methanol gradient eluted the analyte and [1] Bornhorst JA, Hunt JW, Urry FM, McMillin GA. Am. J. Clin. Pathol. 2005 Apr; internal standard into the HESI source of the MS. Selective-reaction monitoring 123(4):578-83 within a 1-minute data window produced quantitation and conformation peaks with intra-assay and inter-assay precisions < 6% RSD and carryover < 0.5%. Correlation 53 Effect of Cytosine Methylation on the Chemical Selectivity and of specimen results with reference-lab results are in progress. Sample throughput Rate of Nucleobase Adduction on Exon 7 Fragment of p53 Tumor was 13, 26, 39 or 52 injections per hour when multi-channeled across 1, 2, 3 or 4 Suppressor Gene channels, respectively. Pregnenolone batches were also multi-channeled with es- Spundana Malla, University of Connecticut, 55 North Eagleville Rd., trogen batches which utilized the same MS source conditions. Storrs, CT 06269, Karteek Kadimisetty, You J Fu, Dharamainder Choudhary, James F. Rusling 50 The Analysis of Common Antiepileptic Drugs in Human Urine by Cytosine – phosphate-guanine (CpG) sites are regions of DNA where cytosine is LC-MS-MS next to a guanine. In mammals 70-80 % of cytosines in the CpG sites are meth- Randall L. Romesberg, Restek Corp., 110 Benner Circle, Bellefonte, PA ylated. 30% of p53 mutations are found at CpG dinucleotides. Five major hot spot 16823, Sharon Lupo, Cathy Hetrick codons 175, 245, 248, 273 and 282 observed in most cancers are all CpG sites with The use of liquid chromatography coupled with mass spectrometry (LC-MS-MS) in cytosine methylated. In this study a 32 base pair exon 7 fragment with all cytosines therapeutic drug monitoring and toxicology labs has increased significantly over the methylated except for the one cytosine at the restriction enzyme recognition site of years. LC-MS provides sensitivity, speed, and the ability to simplify sample prepara- NlaII will be used. Benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), reactive tion. The Raptor™ Biphenyl column was developed to complement high-throughput metabolite of bezo[a]pyrene, a well known carcinogen will be used for nucleobase LC-MS-MS analyses by combining the increased efficiency of superficially porous adduction. CpG sites are known to enhance the rate of formation of BPDE-DNA particles (SPP) with the resolution of Ultra Selective Liquid Chromatography™ adduct formation at these sites. This study would emphasize on the change in se- (USLC™) technology. In this example, a simple dilute and shoot method was devel- lectivity and rate of DNA damage on methylated DNA fragment vs. unmethylated oped for 14 common antiepileptic drugs in urine using a Raptor™ Biphenyl column. DNA fragment using liquid chromatography tandem mass spectrometry. This is the first study that focuses on comparison of rate of reactivity between codons within the 51 Direct Analysis of Human C-Peptide from Urine by Two-Dimensional ds-oligonucleotide. In addition to this rate of reactivity of each codon is compared Liquid Chromatography and Mass Spectrometry (LC-MS) with its methylated version of each codon. Alex Borovoy, Clarkstown North High School, 151 Congers Rd., New City, NY 10956, Eduard Rogatsky, Daniel T. Stein 54 MALDI-TOF MS Analysis of Sequence Effects on Abiotic RNA Direct LC-MS analysis of biological fluids is a challenging goal in modern clinical Polymerization research. Previously reported attempts to use LC-MS measurements of human Kristin M. Coari, Rensselaer Polytechnic Institute, 304 Cogswell, 110 8th C-peptide, a biomarker of insulin secretion in diabetic patients, from urine have been St., Troy, NY 12180, Rebecca C. Martin, Kopal Jain, Linda B. McGown slow, requiring over 15 minutes per assay due to strong matrix effects. In addition, The origins of life on Earth may potentially be found in the RNA World Hypothesis, laborious and expensive sample preparation was required to achieve success. We which posits that ribonucleic acid (RNA) was the first major biological molecule of report for the first time a 6 minute LC-MS method of measuring human C-peptide life due to its dual abilities to code genetic information and catalyze chemical reac- using direct urine analysis. We developed a novel, fast method for direct analysis tions including auto-replication. However, the formation of RNA without cellular ma- of human C-peptide from urine through two dimensional liquid chromatography/ chinery is not well understood, including the selection of the nucleobases. Modern mass spectrometry. This method was established on an Agilent 1100 system and is RNA comprises of only the four canonical bases – guanine (G), cytidine (C), adenine 9 2015 EAS Abstracts November 2015

(A), and uracil (U) – yet other nucleobases such as hypoxanthine (which is in ino- monomers and non-ester radical-coupled bonds with phenolics. Ferulic acid can sine nucleoside, I), were also likely to have been present on early Earth. While it is also link by ester bonds to glycans and act in cross-linking of polysaccharides and unclear when sequence selection began, the nucleobases are likely to have played lignins. Fatty alcohol/ω-hydroxyacid hydroxycinnamoyl transferase (FHT) is a BADH a role in early polymerization. By activating nucleotides with an imidazole group acyltransferase responsible for the formation of alkyl-ferulates in suberin biosynthe- attached to the phosphate (ImpX, where X = G, C, A, U, and I) and reacting these sis. In previous studies, periderm from FHT knock-down tubers showed a significant nucleotides in the presence of catalytic montmorillonite clay, short RNA oligomers decrease in suberin ferulate esters and an increase in soluble phenolics. To further can be formed abiotically. By using matrix-assisted laser desorption/ionization time- investigate the consequences of FHT silencing in potato periderm, metabolite pro- of-flight mass spectrometry (MALDI-TOF MS), we can study the length and nucleo- filing of polar extracts of native and wound tuber periderm were conducted by liquid base composition of polymers formed from mixtures of nucleotides to see if there chromatography mass spectrometry (LC-MS) and solution nuclear magnetic reso- are preferences toward inclusion of particular nucleobases and how the presence of nance (NMR) spectroscopy. A principal component analysis (PCA) of wild type and one nucleobase can affect incorporation of another. Tandem MALDI-TOF-TOF MS FHT-RNAi potato showed clear distinctions among metabolites present in the per- may also be capable of determining the sequence of the polymers. iderm extracts. Orthogonal partial least squares discriminant analysis (OPLS-DA) revealed potential biomarkers with significantly enhanced amounts of six phenolic 55 Pyrethroid Identification and Quantification by GC- Ion Trap Mass amines in FTH-RNAi native periderm, including feruloyl tyramine, feruloyl putrescine Spectrometric (ITMS) Analysis and caffeoyl putrescine, which are derived from feruloyl-CoA. WT and FHT-RNAi Nicole Renkel, Environmental and Health Occupational Sciences wound-healing periderm showed a convergent metabolomics process. Institute, Rutgers University, 170 Frelinghuysen Rd., Rm 328, Piscataway, NJ 08854, Hilly Yang, Min Liu, Brian Buckley 58 LC-MS Analysis of Electrochemical Oxidation of Albendazole Pyrethroid pesticides are regularly used agriculturally, residentially, and commer- Zahilis Mazzochette, Rowan University, 201 Mullica Hill Rd., Glassboro, cially. Current studies of this class of compounds vary in their selection of specific NJ 08028, Geoffrey Kamau, Amos Mugweru analytes, while typically covering both type I and type II pyrethroids. The ten pyre- Albendazole (ABZ) is a widely used anthelmintic drug for the treatment throids in this study, allethrin, resmethrin, tetramethrin, phenothrin, lambda-cyhalo- of parasitic worms and diseases caused by such infestations. Albendazole is poorly thrin, permethrin, cypermethrin, cyfluthrin, fenvalerate, and deltamethrin, have not absorbed in-vivo and hence high concentrations of this drug are administered. The all been quantified simultaneously in one assay to date. The method described in poor absorption is due to its low solubility in aqueous media. After oral administra- this presentation used solid-phase extraction (SPE) to extract the analytes from hu- tion, albendazole is transformed by liver microsomal enzymes into albendazole- man serum, testing two SPE cartridges and two solvent elution schemes for method sulphoxide (ASOX), a pharmacologically major active metabolite. Subsequently, optimization. A Varian Star 3400CX gas chromatograph coupled with a Varian Sat- this metabolite is further transformed by cytochrome P450 enzyme (CYP2C) to an urn 2000 ion trap mass spectrometer was used with a DB-XLB column for the iden- inactive metabolite, albendazole sulphone (ASON). We are trying to understand the tification and quantification of these pyrethroids. Chromatographic resolution was nature and properties of this drug, using different analytical techniques. In this work one of the key optimization parameters as there was co-elution of two analytes. The we use liquid chromatography mass spectrometry (LC-MS) and LC-ultraviolet to limit of detection (LOD) was between 200 – 500 ppb for all analytes, while having a monitor the electrochemical oxidation and reduction of ABZ. linear range between the LOD and 5 ppm. The presentation describes the details of the method development and show results for individual analytes as well as discuss 59 Method Development for 2C and NBOMe Drugs in Urine via LC-MS- the intended application quantitation of pyrethroids in blood. MS Bria L. Lind, Arcadia University, 450 S. Easton Rd., Glenside, PA 19038, 56 GC-MS Metabolomics Analysis of Natural and Feruloyl Transferase- Mandi Mohr, Barry Logan Silenced Potato Suberin Non-Polar Extracts The N-2-benzyl-methoxy (NBOMe) derivatives of the hallucinogenic 2C series Qing Cai, City University of New York - City College, 85 St. Nicholas drugs have seen increased use over the last four years among young, recreational Terrace, CDI Bldg., Rm. 1320F, New York, NY 10031, Wenlin Huang, drug users. This chemical modification of the 2Cs increased hallucinogenic potency Olga Serra, Mercè Figueras, Marisa Molinas, Ruth E. Stark by increasing binding affinity for the 5-HT2A serotonin receptors. The NBOMes have Specialized terrestrial plant tissues formed in the periderm phellem cells contain demonstrated serious side effects such as tachycardia, hyperthermia, dissociation, suberin, a biopolymer that acts as a barrier to control water diffusion, mechanical violence, and possibly death. To date, there is little information on the metabolic breakdown, and pathogenic invasion. Ferulic acid esters are considered as the most fate of NBOMe drugs in humans, and no established method for detecting such important linkage between aliphatic and aromatic suberin domains. Silencing of a drugs and their metabolites in biological samples. However, the 2C compounds potato gene encoding a fatty ω-hydroxyacid/fatty alcohol hydroxycinnamoyl trans- have been proposed as metabolites since they are precursors to the NBOMe’s. The ferase (FHT), can prevent ferulic acid from becoming esterified to ω-hydroxyfatty method development for this study included infusion and a percent recovery study. acids or alcohols. In order to complete the suberin biosynthesis pathway map and The infusions determined the detected mass, cone voltage, and collision energy for define the suberin macromolecular structure, metabolite profiling was conducted on each compound. The percent recovery was necessary to evaluate the extraction soluble non-polar extracts from native periderms of wild-type and FHT-RNA interfer- method. The method was seen to be inefficient at extracting the 2C compounds ence silenced potato samples using gas chromatography mass spectrometry (GC- using NaOH to create the basic environment. By using NH4OH instead, plus HCl to MS). In this study, principal component analysis (PCA) shows consistency among stabilize the compounds, the percent recovery greatly improved. Results from this biological replicates and discrimination between wild-type (WT) and FHT-RNAi study show that the NBOMe’s and the 2C compounds are easily separated by this extracts. Orthogonal-projection to latent structure-discriminant analysis (OPLS- method through liquid chromatography on a BEH C18 column with no interference DA) was applied to extract the potential biomarkers; 25 of the 31 compounds were or co-elution. The cone voltage was 20V for each 2C compound and the optimum identified in the non-polar extracts. The classes of highly abundant WT metabolites collision energy was either 15 or 20eV. The percent recovery improved from less include alkanes, caffeic acid, and fatty acids (C16:0, C18:0, C18:2). By contrast, than 30% to greater than 68%. the high-abundance FHT-RNAi metabolites include long-chain saturated fatty ac- ids, long-chain primary alcohols, methyl esters and 1-monohexadecanoyl glycerol. 60 Electrochemical Reduction of Artemisinin: Chromatographic These results indicate that blockage of this key step in suberin formation can result Analysis of the Metabolites in up-regulation of precursor compounds. It is also possible that the ‘orphan’ ω-hy- Minxue Shi, Rowan University, 201 Mullica Hill Rd., Glassboro, NJ droxyfatty acids in FHT-RNAi silenced periderms are reduced, or that the aliphatic 08028, Geoffrey Kamau, Amos M. Mugweru glycerol esters are hydrolyzed. Artemisinin (ART) is mainly used as an anti-malaria drug in some countries. It is also suggested to have biological relevance with the anticancer activities. ART is a 57 Metabolomic Profiling of FHT-RNAi Silenced Potato Periderm naturally occurring sesquiterpene lactone, containing endo-peroxide bridge, which Tissues with LC-MS and Solution NMR together with its synthetic derivatives constitute an important new class of antima- Liqing Jin, City College of New York, CUNY Graduate Center and larial drugs, which is effective against chloroquine- and multidrug-resistant strains of Institute for Macromolecular Assemblies, Department of Chemistry, CDI Plasmodium falciparum. Direct electrochemical reduction of ART involves electron Bldg., Rm. 1320D, 85 St. Nicholas Terrace, New York, NY 10031, Wenlin being transferred to moiety to reduce the endo-peroxide group. We have investigat- Huang, Olga Serra, Mercè Figueras, Marisa Molinas, Ruth E. Stark ed electrochemical reduction of ART and potential adduct formation with DNA bas- Potato native periderm forms an effective barrier to protect the tuber from dehydra- es. We have used liquid-chromatography mass spectrometry (LC-MS) to investigate tion and pathogen infection. Tubers injured during growth, harvest or seed cutting the ART-guanine adduct formation a first step in most anticancer agents. Adenine will form a wound periderm that shares many characteristics with native periderm. thymine and cytosine did not show any quantifiable adduct formation withART. The protective function of the periderm is attributed to suberin, a complex cross- linked biopolymer that contains polyaliphatic and lignin-like aromatic domains. Cur- rent models describing the macromolecular structure of suberin assume that ferulic acid cross-links both domains, as it may form carboxyl ester bonds with aliphatic 10 2015 EAS Abstracts November 2015

61 Absolute Quantitation of Semen Specific Biomarkers from Post- removal of the NBOMe moiety. This project assists in allowing identification of spe- Coital Samples cific metabolic markers of use for NBOMe’s in human biological samples. Catherine Brown, Arcadia University, 405 S. Easton Rd., Glenside, PA 19038, Masha Signaevsky, Heather McKiernan, Kevin Legg, Phillip 64 Identification of Synthetic Cannabinoids Using GC-MS at the NYPD Danielson Laboratory Sexual assault is a prevalent crime in today’s society; however, the forensic testing Donald G. Brown, New York Police Department Police Lab, 150-14 utilized for sexual assault evidence is costly and time consuming, resulting in a Jamaica Ave., Queens, NY 11423, Maria K. Petela backlog of untested evidence. The enzyme-activated and antibody immunochro- Synthetic cannabinoids are a large family of chemically unrelated structures func- matographic tests used by forensic examiners for the identification of seminal fluid tionally similar to Delta-9-THC. Users often display signs and symptoms of excited provide presumptive results, due to false positives with non-biological material and delirium and experience withdrawal on par with that of a heroin addict. The ease positive results with non-target fluids. A negative serological test may prevent the with which one can purchase or produce synthetic cannabinoids is astounding. With sample from receiving additional DNA testing, regardless of if the protein was pres- simple ingredients and a quick search on-line, one can produce his/her own brand of ent in a degraded form. The presumptive nature has deterred scientists from reliably synthetic cannabinoids. At the New York City Police Department Police Laboratory, determining a post-coital interval. The development of a confirmatory technique for there has been an increase in the number of cases with alleged synthetic canna- the identification of seminal fluid would allow for the post-coital detection of semen binoids. Law enforcement has been struggling with confiscating brightly colored, to be determined and would aid in the analysis of sexual assault evidence. The “kid-friendly” packets that are always labeled “NOT FOR HUMAN CONSUMPTION” goal of the current research was to validate a quantitative variation of a previous- and dealing with unruly, disruptive users. The chemical compounds are currently ly developed qualitative method that established the sensitivity of semen-specific not controlled in New York State and therefore, prosecuting individuals often re- protein. The current method employs high-performance liquid chromatography with sults in a violation along with a fine. It was first hypothesized that the packaging scheduled multiple reaction monitoring on an Agilent© 6430 triple quadrupole mass would predict the exact type of synthetic cannabinoid it contained. However, after spectrometer. Using synthetic peptide standards, a linear calibration model was de- creating a database tracking the specific packaging, it was soon shown that there veloped and mock casework samples were used to analyze the multiplex assay’s was no correlation between the packaging and the contents it contained. To identify sensitivity and limits of detection, in addition to a comparison of the specificity and what synthetic cannabinoids are present on the vegetative matter, gas chromatog- selectivity to the immunochromatographic assays. This study provides evidence raphy-mass spectrometry (GC-MS) is used. A 5:1 (mg/mL) ratio is used to ensure that mass spectrometry produces greater sensitivity and a more reliable method for that a representative sample is obtained. One major issue that the laboratory has the detection of semen in sexual assault samples. encountered is the constant changing of chemicals sprayed on the vegetative mat- ter and obtaining the standards to identify them. 62 Method Development and Optimization of Detection of Decomposition Products in Soil Using HS-GC-MS 65 GC-MS Carrier Gas Conversion from Helium to Hydrogen Amanda L. Haggerty, Arcadia University, 450 S. Easton Rd., Glenside, Parag K. Shah, New York Police Department Police Lab, 150-14 PA 19038, Kimberlee Moran, Heather Harris, Kenneth Clarke Jamaica Ave., Jamaica, NY 11432, Sue Chen, Erika Chen, Michelle For years, detection of clandestine burial sites has relied on the use of volatile or- Rockwell ganic compounds (VOCs). However, without specific instrumentation, detection of The majority of the forensic laboratories use helium as a carrier gas in their gas VOCs can be difficult. This research investigates the possibility of using headspace chromatography mass spectrometry (GC-MS) analysis. Limited supply and the gas chromatography mass spectrometry (HS-GC-MS) to detect VOCs in a soil ma- rising cost of helium made us consider hydrogen as the carrier gas. The current trix. With little to no sample preparation, HS-GC-MS is a faster and easier method helium methods at the New York Police Department Police Laboratory for analyzing when trying to detect decomposition products. The five VOCs of interest in the proj- controlled substances, fire debris, explosives, and general unknown analyses are ect were dimethyl disulfide, heptanal, 1-hexanol, nonanal, and 1-octen-3-ol. Based being replaced by hydrogen as a carrier gas. Purity, safety, analysis time, quality of on previous literature research, these five compounds were found to be in both pig chromatography peaks and mass spectral data were evaluated and possible mass and human decomposition in higher frequencies. Since piglets were being used as a spectral conversions were monitored, during the development phase. Different col- human cadaver substitute, having this crossover can relate back to the compounds umn sizes were assessed and certain standards were analyzed more than 30 times, that a decomposing human body will release. Once the piglets reached the required over several days, for qualitative analysis and certain quantitative techniques. Re- accumulated degree days (ADDs) associated with the corresponding decomposi- sults showed shorter run times with equivalent separation. tion state, a 33 inch AMS® unplated soil probe was used to collect samples. The collected soil was retained in precut foil squares and labeled appropriately. Samples 66 Linking Suspects to the Scene of a Crime: Identification of Tear were then placed on ice for transport until they could be placed into a freezer for Gas Lachrymators on Clothing storage. Collected samples were analyzed on an Agilent® 6890 HS-GC using a Laura McGregor, Markes International, Gwaun Elai Medi-Science CTC Analytics® Combi Pal autosampler coupled with an Agilent® 5973 Network Campus, Llantrisant, RCT, CF72 8XL United Kingdom, Steve Smith, Mass Selective Detector. All compounds were successfully separated with good Chris Hall, Charles Haws resolution and peak shape using the HS-GC/MS. Lachrymators are compounds present in tear gas and self-defense sprays (such as pepper spray and ‘Mace’), which invoke a burning sensation and cause the eyes to 63 Metabolic Profile Determination of NBOMe’s Using Human Liver water. Dissolved in solvent and contained within an aerosol dispenser, lachryma- Microsomes (HLM) tors are generally used by the police to control large crowds, but are available to Keith-Dane H. Temporal, Arcadia University, 450 S. Easton Rd., purchase for self-defense in some countries. Illegal use of lachrymators has driven Glenside, PA 19038, Barry K. Logan, Amanda Mohr a need for forensic analysts to assess crime-scene materials for their presence, in N-benzylmethoxy phenethylamines (NBOMe’s) are a class of emerging halluci- order to link a suspect to a crime. A range of complex matrices are encountered in nogenic designer drugs recently associated with cases of acute and fatal intoxi- these analyses, but this work will focus on the example of clothing extracts. The cations. The first synthesized NBOMe was 25I-NBOMe (2-(4-iodo-2,5-dimethoxy- highly complex nature of such samples demands a highly resolving chromatograph- phenyl)-N-[(2-methoxyphenyl)methyl]ethanamine) reported by Ralf Heim at the Free ic technique, and comprehensive two-dimensional gas chromatography coupled University of Berlin in 2003. This was derived from the substituted phenethylamine with time-of-flight mass spectrometry (GC×GC–TOF MS) is an ideal choice. The 2C-I (2,5-dimethoxy-4-iodophenethylamine). The “NBOMe” designation describes benefits of this approach are illustrated for trace-level quantitation of lachrymators substitution of a 2-methoxybenzyl group at the 2C amine. NBOMe’s are potent se- in these challenging matrices. rotonergic agonists whose effects mimic those of the classical hallucinogen LSD (lysergic acid diethylamide). NBOMe’s adverse effects include episodes of intense 67 Simultaneous Determination of Prescription Benzodiazepines and psycho-stimulation, violent outbursts, and fatal intoxications. These acute and fatal Designer Benzodiazepines in Urine by SPE and LC-MS-MS intoxications frequently occur in cases where NBOMe’s are mistakenly ingested as Tina Fanning, United Chemical Technologies, 2731 Bartram Rd., Bristol, LSD by recreational drug users, resulting in adverse effects as a result of their much PA 19007, Michael Telepchak greater potency. Many current drug screening methods do not include the NBOMe Benzodiazepines, frequently referred to as “Benzos”, are prescribed for the treat- drug class or their metabolites in their scope. We developed a method for the iden- ment for a wide variety of symptoms from anxiety to seizure-prevention on account tification of NBOMe’s and their metabolites using in-vitro metabolism preparation of their ability to depress the central nervous system. Generally, these drugs are and analysis by liquid chromatography quadrupole time-of-flight (LC-QTof). Four deemed safe and highly effective when used properly and for short durations of NBOMe drugs (25I-, 25H-, 25C-, and 25B-NBOMe) were incubated with pooled time. However, Benzodiazepines are also recurrently utilized as illegal recreational human liver microsomes (HLM) and a nicotinamide adenine dinucleotide phosphate drugs. Similar to other commonly abused compounds, such as cannabinoids or am- (NADPH) generating system to activate cytochrome (CYP) P450 enzymes gener- phetamines, “legal” alternatives have been developed for Benzos as well in an at- ating biomarkers specific to each drug. The project successfully identified several tempt to bypass the controlled substances act. These new designer drugs are struc- Phase I metabolites for each the NBOMe’s studied, including O-demethylation and tural or functional analogs of the controlled substance designed to not only mimic 11 2015 EAS Abstracts November 2015

the pharmacological effects of the original drug, but also avoid illegal classification was chemically modified via solution phase reaction of CnH2n+1Si(CH3)2N(CH3)2 and/or detection in a standard drug test. By utilizing United Chemical Technologies’ where n = 1, 4, 8, 18. The energies of adsorption, surface area, and pore volumes (UTC) XCEL I extraction columns in conjunction with UCT’s Selectra DA high-perfor- of modified substrates were calculated using nitrogen adsorption isotherm. Graft- mance liquid chromatography (HPLC) column, both prescription Benzodiazepines ing density of bonded ligands was determined from the weight percent of carbon. and synthetic Benzodiazepines levels can be monitored simultaneously reducing Comparison of the excess adsorption isotherms measured on these columns, and both analyst time and instrument time. Good absolute recoveries were observed expressed in surface specific form demonstrates significant similarity of the adsorp- (77-90%) with a minimal matrix effect for the 16 analytes representing both prescrip- tion properties for all columns. This allows us to introduce “standard adsorption tion and designer Benzos. The increasing need to keep up with the ever changing isotherm” for reversed-phase alkane type columns and suggests that adsorption de- designer drug market has proven to be a real challenge for laboratories across the pends on the type of ligands and bonding density of the ligands and it is independent country. The universal nature of this extraction method should make it amenable to on the adsorbent geometric morphology, pore shape, and distribution. other synthetic Benzodiazepines and metabolites, which is important since there is no sign of slowing down on the development of new variations. 72 Sequential Elution Liquid Chromatography-Mass Spectrometry Using a Wide-Range pH Gradient 68 Validation of the HPLC Method for Determination of Stability of Catherine Kita, Drexel University, Dept. of Chemistry, 3141 Chestnut St., Sodium Pentobarbital in Diluted Marketed Euthanasia Solutions Philadelphia, PA 19104, Joe P. Foley Oksana Leidy, Merck, 770 Sumneytown Pike, WP78A-31, West Point, Sequential elution liquid chromatography (SE-LC) is a novel approach for the liquid PA 19486, Hal Grosso, Lara D. Penn chromatographic separation of samples comprised of two of more classes of ana- Sodium pentobarbital euthanasia solutions such as Nembutal Sodium, Sleepaway, lytes via the use of multiple selective elution modes. By employing a wide-range pH and Fatal-Plus are frequently used in in-vivo animal studies conducted in universi- gradient in the mobile phase, a sample composed of weak acids and weak bases ties, medical schools, and pharmaceutical companies.[1] Sometimes there is need can be separated by class and within class (i.e., each analyte from another). Pre- to prepare lower concentrations of sodium pentobarbital which puts the resulted vious work showed a higher probability of a successful separation utilizing SE-LC solutions in a category of “mixtures of articles with carriers” per GLP Regulations 21 compared to conventional high-performance liquid chromatography (HPLC), due to CFR Part 58.113, requiring determination of the stability of the article in the mixture the increased peak capacity and decreased separation disorder of the former.[1] before the study initiation or concomitantly with the study. As the result of these In this work, a mass-spectrometry-compatible pH gradient based on a wide-range requirements, validated analytical procedures for determination of pentobarbital so- buffer system is employed for the sequential-elution mediated separation of ion- dium along with impurities and degradation products in diluted euthanasia solutions izable compounds by liquid chromatography-mass spectrometry. The compounds would be used in the pharmaceutical industry. A validation of a high-performance examined thus far include small organic molecules and amino acids. Experimental liquid chromatography (HPLC) stability-indicating method and stability determina- emphasis is on the optimization of chromatographic figures of merit and on the tion of Sleepaway and Fatal-Plus euthanasia solutions is discussed. The chromato- viability of the proposed gradient for the analyte detection by mass spectrometry. graphic separations were achieved using Zorbax 300SB – C18, 5 microns, 4.6 mm Reference: x 150 mm column along with gradient elution and UV detection at 210 nm. The [1] A. Socia and J. P. Foley, J Chromatography A, 2014, 1324, 36-48. method is sensitive, selective and reproducible. Long-term chemical stabilities of 65 mg/mL Sleepaway and 65 mg/mL Fatal-Plus solutions were established at ambient 73 Optimization of Assay/Degradation Method for Triple Combo Drug conditions protected from light for at least 101 days and for at least 86 days, respec- Product Using DryLab tively. Freeze-thaw and short-term ambient light chemical stabilities were examined Jagruti Patel, Merck, 126 E. Lincoln Ave., Rahway, NJ 07065, Ye Tian as well. DryLab was used to optimize a gradient reversed-phase high-performance liquid Reference: chromatography (HPLC) method for the determination of assay/degradation of a [1] AVMA Guidelines for the Euthanasia of Animals: 2013 Edition. American Veteri- triple combo fixed dose combination drug product. The primary method utilizes Wa- nary Medical Association. Version 2013.0.1 ters Atlantis T3 column with 100% aqueous mobile phase A at pH 7.0 and aceto- nitrile mobile phase B. Among the three active pharmaceutical ingredients, two of 69 Investigation of Two-Dimensional High-Performance Liquid them are highly polar compounds and the retention is susceptible to mobile phase Chromatography Approaches for Reversed-Phase Resolution of pH and column temperature. Mobile phase pH also exerts great influence on peak Warfarin and Hydroxywarfarin Isomers shape, i.e., causing peak tailing and peak splitting at lower pH. Therefore, three key Erik L. Regalado, Merck, 126E. Lincoln Ave., PO Box 2000, Rahway, NJ factors, gradient time, mobile phase pH, and column temperature, were selected to 07065, Christopher J. Welch, Joseph A. Schariter determine the effects on separation. DryLab automatically generated an experiment In this presentation we describe several off-line and on-line two-dimensional high-per- design with 12 experiments, includes three pH values (6.8, 7.2 and 7.6), two column formance liquid chromatography (2-D HPLC) methods for the reversed-phase reso- temperatures (20 °C and 40 °C), and two gradients (1.25% and 2.5% mobile phase lution of a complex mixture of closely related warfarin and hydroxywarfarin isomers. B change/min). After execution of experiments, peaks were tracked to optimize the By combining reversed phase achiral/chiral HPLC separation with UV-triggered conditions by DryLab. It demonstrated that a slower gradient with higher mobile fraction collection and subsequent chiral/achiral reversed phase HPLC analysis of phase pH at lower column temperature would provide acceptable chromatography. collected fractions, complete resolution of all 12 components of the mixture was The application of DryLab has significantly reduced the development time and pro- possible. In addition, a faster method was developed from on-line 2-D HPLC anal- vided solid scientific guidance in optimizing chromatographic conditions. ysis where multicomponent fractions from the first dimension are simultaneously chromatographed in the second dimension. 74 Columns with 2.0-µm Fused-Core Particles Generate Highest UHPLC Performance 70 Analysis of Tri-Lysine by Hydrophilic Interaction Liquid Richard A. Henry, Supelco/Sigma-Aldrich, 595 North Harrison Rd., Chromatography with ELS Detection Bellefonte, PA 16823, David S. Bell, Hugh Cramer, Gaurang Parmar Timothy Brockman, Integra LifeSciences, 313 Enterprise Dr., Plainsboro, New Ascentis Express 2.0-µm columns are perfectly matched to ultra-high-perfor- NJ 08536, Steve Bennett, Thomas Twardowski mance liquid chromatography (UHPLC) instruments and make it practical to over- A purity assay for tri-Lysine peptide in borate buffer, pH 10.2 was developed for come certain back-pressure disadvantages of smaller, sub-2-µm particles. Ascentis ultra-high pressure liquid chromatography with evaporative light scattering (ELS) Express columns with 2.0-µm superficially porous particles (SPP) match or exceed detection. The assay also established that tri-Lysine is stable at the high pH of the efficiency of sub-2-µm totally porous particles (TPP) and SPP columns while op- borate buffer, and that these conditions do not result in degradation to mono-Lysine erating at much lower pressures under identical flow conditions. Plots of reduced or di-Lysine subunits. This method doubles as an assay for tri-Lysine concentration plate height versus flow velocity show that new 2.0-µm Fused-Core particle columns and exhibits excellent linearity from 0.75 to 8.0 mg/mL, (r2 = 1.0), precision, (< match the van Deemter performance of pioneering 2.7-µm Fused-Core particle col- 3.0%), and accuracy (100-106%). umns that started a particle design revolution in 2006 by delivering h values that dropped below 2 and remained remarkably low even at extremely high flow veloc- Comparison of Excess Adsorption of Binary Aqueous Organic 71 ities. Operation at high performance with lower pressure permits faster methods Mixtures on Classical Packing Material and Core-Shell SBA-15 and assays on any instrument, extends useful column life, and minimizes chance Modified with Alkylated Ligands of selectivity change or performance loss caused by frictional heating within the Margaret Figus, Seton Hall University, 400 South Orange Ave., South packed bed. The Ascentis Express 90Å Fused-Core column line for small molecules Orange, NJ 07079, Yuri V. Kazakevich, Alexander Y. Fadeev is now completely scalable in performance with 5-µm particles that bring better per- The excess adsorption isotherms of acetonitrile from water were measured on formance to routine quality control, original 2.7-µm particles that bring sub-2-µm four in-house packed columns with different adsorbent geometry. A classical 10- performance to any HPLC method, and new 2.0-µm particles for highest speed, µm pours silica particle and SBA -15 10-µm nonporous spherical particle that was resolution and ruggedness in UHPLC methods. A complete line of classic phases is prepared using polymer-templated sol-gel synthesis and were well characterized available for all particle sizes. via low temperature nitrogen adsorption, TGA, and STEM. The adsorbent’s surface 12 2015 EAS Abstracts November 2015

75 Transferring and Scaling Methods Among a Variety of Superficially 79 Evaluating Mass Overload on Superficially Porous Particle Porous Particle Columns Randall L. Romesberg, Restek Corp., 110 Benner Circle, Bellefonte, PA Anne E. Mack, Agilent Technologies, 2850 Centerville Rd., Wilmington, 16823, Sharon Lupo, Cathy Hetrick DE 19808, William J. Long, Maureen Joseph, Xiaoli Wang Superficially porous particles (SPP) are a powerful analytical tool for achieving fast Superficially porous particles offer improved efficiency and performance over sim- liquid chromatography analyses. The solid, impermeable core present in these par- ilarly sized traditional totally porous particles. Higher efficiency leads to improved ticles increases the column efficiency by decreasing the diffusion path. However, resolution and possible time savings with superficially porous particles, hence their the solid core also significantly reduces the surface area that is typically available growing popularity for high-performance liquid chromatography (HPLC) analyses. in traditional fully porous materials (FPP). There is a potential concern that arises Columns using superficially porous particles are currently available in a wide variety from a reduction in surface area: column loading ability. Column overloading (mass of particle sizes, pore sizes and stationary phase chemistries to meet most analysts’ overload) occurs when the amount of material injected onto the column exceeds needs. This work shows applications that are transferred from totally porous particle the available active sites of the stationary phase. The purpose of this study was to HPLC columns to superficially porous particle columns. Methods are geometrically evaluate a series of analytes and determine the effects the solid core has on the scaled, as column volumes are adjusted, preserving chromatographic integrity. As loading ability of superficially porous particles. methods are adjusted to higher efficiency columns with smaller column volumes, instrument hardware and software is adjusted accordingly. Some examples include 80 Analysis of Vanillin in Maple Sap for the High School Laboratory United States Pharmacopeia (USP) methods, which are transferred to superficially Tiffany Hatstat, University of New Hampshire, Dept. of Chemistry, 23 porous particles from their prescribed totally porous particle columns. Results are Academic Way, Durham, NH 03824, Douglas Baker, Walter Shortle, compared against the permissible adjustments found in the recently revised USP Elizabeth Brady, Martha Carlson, Barrett N. Rock, Stephen Hale, Martin General Chapter 621. McCrone, Sterling Tomellini The goal of this research is to develop a reversed-phase high-performance liquid 76 Utilizing pH as a Method Development Tool to Control Selectivity of chromatography (HPLC) method and associated classroom materials for the anal- Ionizable Compounds with Superficially Porous Columns ysis of vanillin in maple sap for high school laboratories. The analysis of maple sap Anne E. Mack, Agilent Technologies, 2850 Centerville Rd., Wilmington, has broad applications and addresses different content areas including: chemistry, DE 19808, William J. Long, Maureen Joseph, Xiaoli Wang biology, environmental science and mathematics. The students will conduct an ex- Superficially porous alkyl phases are widely used for reversed-phase high-perfor- periment using HPLC instruments that are made available for loan to high schools mance liquid chromatography (HPLC). These columns show comparable efficiency throughout New Hampshire by the UNH Leitzel Center. Previous work in our group to sub 2 micron particles with about half the back pressure. However, analysts oc- has analyzed maple sap to determine seasonal trends in phenolic compounds, in- casionally encounter difficult separations for which selectivity, ruggedness or repro- cluding vanillin, employing HPLC gradient conditions that utilize chemicals that are ducibility is not easily obtained under initial method development conditions. Varying not compatible in a high school setting.[1] The modified method utilizes isocratic pH can affect selectivity, as pH controls the ionization state of ionizable compounds mobile phase conditions consisting of very dilute isopropyl alcohol and acetic acid and, thereby, their retention times. With more pH stable superficially porous particle to separate vanillin in maple sap within the constraints imposed by the high school columns, a wider variety of buffers is available for use without sacrificing lifetime at schedule. Sample preparation and processing are simple and limited to filtering the high pH compared to silica based columns. Use of high pH is especially advanta- sap samples prior to injection into the instrument. Lesson plans and operation man- geous for bases. When the pH is increased over the pKa of a compound, increased uals are being designed to incorporate the principles found in the Next Generation retention time allows higher loading and in many cases better peak shape. Having a Science Standards (NGSS). The development of this laboratory experience will high pH stable column such as Poroshell HPH C18 available to use in bicarbonate allow students to use technology to collect, analyze and model data using mathe- and phosphate buffers is important. pH by itself can increase the orthogonality of a matics and qualitative reasoning as presented in the NGSS standards (HS-PS4-5, separation, but when used together with organic modifier choice and column selec- HS-PS4-1).[2] tivity, orthogonality can be further enhanced. Generic gradients with a wide range of References: acidic, basic and neutral compounds are run to determine the degree of difference [1] Brady, Elizabeth. 2014. Evaluation of Phenolic Compounds in Sap from New in chromatographic selectivity between two methods. The effects of buffer (pH and Hampshire Sugar Maple Trees by Liquid Chromatography/Mass Spectrometry. type) and organic solvent are studied. Retention factors are measured for all com- Dissertation. University of New Hampshire, Durham, NH. pounds using various LC method parameters. Correlation coefficients and slopes [2] NGSS Lead States. 2013. Next Generation Science Standards: For States, By from linear regression data are used to measure orthogonality. States. Washington, DC: The National Academic Press. 77 A Multi-Class Drug and Metabolite Screen of 231 Analytes by LC- 81 Highly Efficient Purification of Enantiomers Using Polysaccharide MS-MS Type Chiral Stationary Phases and Recycle Purification Technology Randall L. Romesberg, Restek Corp., 110 Benner Circle, Bellefonte, PA Ernest Sobkow, YMC America, 941 Marcon Blvd., Allentown, PA 18109, 16823, Sharon Lupo, Cathy Hetrick Keiko Kihara, Hideo Gabari, Takashi Sato, Saoko Nozawa, Noriko Shoji, The use of pain management drugs has been steadily increasing. As a result, labs Noritaka Kuroda, Takatomo Takai are seeing an increase in patient samples that must be screened for a wide variety The need for chiral separations in small molecule pharmaceutical development is of drugs to prevent drug abuse and to ensure patient safety and adherence to their important. Selective, fast, and efficient analytical and preparative separations are medication regiment. Therapeutic drug monitoring can be challenging due to the an essential part of the drug development tool kit. Key hurdles to be overcome in low cut-off levels, potential matrix interferences and isobaric drug compounds. To preparative separations involve improving the productivity of a given separation and address these challenges, many drug testing facilities are turning to liquid chro- the difficulty in cost effectiveness of the purification. We have recently developed matography coupled with mass spectrometry (LC-MS-MS) for its increased speed, various coated and immobilized chiral stationary phases with polysaccharide deriv- sensitivity, and specificity. In this example, a method was developed for a multi-class atives. The coated phases give great resolution and the immobilized phases offer drug and metabolite screen containing 231 compounds. a wider range of solvent compatibility. A separation method developed at analytical scale can be easily and linearly scaled up to purification from milligrams to gram 78 Rapid and Accurate LC-MS-MS Method for the Analysis of Nicotine, scale (and beyond) by using a preparative scale column and the liquid chromatog- Nicotine Metabolites, and Minor Tobacco Alkaloid in Urine raphy (LC)-Forte/R preparative LC system. In this poster we show that the efficien- Randall L. Romesberg, Restek Corp., 110 Benner Circle, Bellefonte, PA cy of purification is improved by applying recycle chromatography. This recycling 16823, Sharon Lupo, Cathy Hetrick method is widely applicable to cases where ideal resolution is not achieved at the A variety of chromatographic methods have been developed for nicotine metabolite method screening stage. analysis. Most of the modern methods adapt the usage of high pH chromatography with a relatively high concentration of additive reagents, which may not be applica- 82 SFC Analytical Method Development for Vitamin D3 and Related ble to all liquid chromatography mass spectrometry (LC-MS) instrumentation. The Compounds intent of this study was to develop a method for the analysis of nicotine related Ernest Sobkow, YMC America, 941 Marcon Blvd., Allentown, PA 18109, compounds in urine using “friendly” LC-MS-MS solutions and the highly efficient Junko Kawabata, Roland Spaegele, Toshikazu Adachi, Noritaka Kuroda and selective Raptor™ Biphenyl column. The clinical applicability of the method By utilizing the advantages of supercritical fluid chromatography (SFC) such as was demonstrated by accurate and reproducible analysis of fortified analytes in the high permeability and diffusibility, we could generally achieve higher resolution by urine of a non-tobacco user. SFC analysis in a shorter run time than by high-performance liquid chromatography (HPLC). Thus a combination of achiral columns and SFC is one of the effective strategies for reducing analysis cycle time of natural products such as fat-soluble vitamins and terpenoids. It has currently been recognized that some normal-phase

13 2015 EAS Abstracts November 2015

HPLC methods to assay Vitamin D3 and three related compounds (Pre-Cholecal- The unique multi-modal surface chemistry of the zirconia-based, polyethyleneimine ciferol, 5,6-trans-Cholecalciferol, Tachysterol3) with various impurities in nutritional coated, ZirChrom®-SAX phase can be helpful in the analysis of complex ionic sam- products are insufficient because analysis times of them are slightly longer and ples. A discussion of the surface chemistry and multi-modal separation mechanisms integration of them into a single method is difficult. In this poster, we describe the of the phase will be presented. Finally the method development for a separation of development of a robust and efficient SFC method to solve these problems. six water soluble vitamins will be included as an example of how the multi-modal selectivity of ZirChrom®-SAX can resolve a complex ionic mixture. 83 Simultaneous Determination of Protein and Carbohydrate Used in Bioformulations by High-Performance Liquid Chromatography 86 Technical Evaluation of Next Generation HPLC Instrument Marc Plante, Thermo Scientific, 22 Alpha Rd., Chelmsford, MA 01824, Weidong Tong, Merck, 126 E. Lincoln Ave., Rahway, NJ 07065, Lin Wang Bruce Bailey, David H. Thomas, Qi Zhang, Rainer Bauder, Daniel The high-performance liquid chromatography (HPLC) instrument is one of the Kutscher largest capital investments across pharmaceutical industry. Facing the challenges Many of today’s pharmaceutical formulations use proteins and other biotechnolog- of currently outdated HPLC fleet, the HPLC Life Cycle Management Team was ical active pharmaceutical ingredients (APIs). Along with these newer formulations established to develop a global HPLC replacement strategy at Merck. As part come different excipients, namely surfactants, amino acids, salts, and carbohy- of this evaluation, the team evaluated Waters H-class and Agilent 1290 Infinity drates. Carbohydrates are used to assist in the stabilization of the proteins in solu- quaternary ultra-PLC systems. The instruments were evaluated on the basis of tion, and are used in different amounts, depending on the protein and the product performance (speed, sensitivity, pressure capability, efficiency, system flexibility requirements. A method was developed that separates the carbohydrate excipient and transferability, reproducibility and robustness), software, ease of use and from the protein, and quantifies both simultaneously. The method uses a combined non-technical parameters. Overall results demonstrate that both systems meet C18 - hydrophilic interaction liquid chromatography (HILIC) process, with linear expectations and show comparable performance. Details on the evaluation results calibrations and selection of detectors, using Corona charged aerosol detection are presented in this poster. (CAD) for the carbohydrates and either a second CAD or ultraviolet to determine the protein amount. Linear calibration curves for all three detectors were >0.999 87 The Challenge to Determine Particle Size and Specific Surface Area and sensitivity values (ng on column) were: Sucrose, limit of detection (LOD) = 12 of an Agglomerated Drug Substance in the Late-Stage Development and limit of detection (LOD) = 35, Protein (BSA), LOD(UV) = 15 and LOQ(UV)= 46, Shasad Sharif, Bristol-Myers Squibb, 1 Squibb Dr., New Brunswick, NJ LOD(CAD) = 3 and LOQ(CAD) = 9. Recoveries on a mock formulation, consisting of 08902, Mario Hubert sucrose and BSA, were performed finding recovery values for sucrose of 93.8% and Solid-state powder properties of a drug substance, alternatively called active phar- for the BSA of 84.5% (UV) and 83.6% (CAD). Other carbohydrates, typically used in maceutical ingredient (API), such as particle size and specific surface area, play bioformulations, have been analyzed using this method, including sorbitol, mannitol, a critical role in late-stage pharmaceutical development. Specifically, surface area lactose, maltose, and trehalose, allowing the use of this method for the quantitation and particle size of APIs can impact bioavailability and dissolution of the solid oral of protein and carbohydrate in a wide variety of products. dosage form. Further, these parameters are used to establish process robustness. Naturally, there is a need to determine particle size and specific surface area during 84 Monograph Modernization and Impurity Profiling HPLC Methods API manufacturing. This presentation focuses on the particle size and surface area for Pharmaceutical Drugs following New United States method development for an API that is agglomerated to enhance the flow of the ma- Pharmacopeia (USP) Guidelines terial. Furthermore, this material is a non-stoichiometric hydrate that is particularly David Lentz, EMD Millipore, 290 Concord Rd., Billerica, MA 01821, challenging in terms of method development. In particular, surface area measure- Patrik Appelblad, Gora Sharangi ments require an initial degassing of the material, which could alter the hydration USP high-performance liquid chromatography (HPLC) methods often utilize column state of this API. The difficulties and challenges to measure the primary particle size, technologies that are several decades old and often don’t take advantage of the agglomerate size, and specific surface area of this agglomerated drug substance many advances and breakthroughs in modern columns that can shorten analysis are presented. The effect of the agglomerated API and its hydration state on the time and allow system suitability tests to be more easily passed when column effi- particle size and surface area as well as their implications for dissolution and bio- ciencies are borderline. Instead of waiting for long-range monograph modernization availability is discussed. implementations (many older USP HPLC methods are not even on this list), the USP is addressing this situation now by modifying General Chapters wording to al- 88 Residual Sulfate Analysis of Collagen and Wash Water by low more flexibility and freedom in transferring official methods over to state-of-the- Spectroscopy, Chromatography, and Opalescence Methods art column technologies like smaller particles, core-shell, ultra-HPLC and monolithic Hao Fu, Integra LifeSciences, 311 Enterprise, Plainsboro, NJ 08536, silica. One example of this is, as of November 2014, the USP has extended the gen- Timothy Brockman, Thomas Twardowski eral descriptions to include “or a monolithic rod” in HPLC packings definitions of L8, Several assays for the determination of residual sulfate in processed insoluble col- L10, L11 and L20. Packing descriptions for L1, L3, and L7 have included this change lagen and process wash water have been developed and compared: visual pre- for the past several years. Monolithic silica columns give modern particle-packed cipitation, turbidimetry by UV-visible spectrophotometry, and direct determination column efficiencies and performance but with very much lower backpressures that by ion chromatography (IC). Five exhaustive extraction techniques were assessed allow higher flows to greatly speed analyses without requiring expensive instrumen- for the extraction of sulfate from insoluble collagen at a concentration of 1 g/10 tation upgrades to extend pressure ratings. This greatly helps labs following USP ml: five minutes of extraction in deionized water, two week extraction in water with HPLC methods to more efficiently complete them while still staying within allowable refrigeration (3-7 °C), boiling in water, and salt ion exchange with sodium chloride USP column parameter changes. Robust, reliable methods can be developed hav- or sodium carbonate. Boiling provided the greatest release of sulfate ions from the ing the necessary sensitivity and linearity that meet requirements in selectivity and collagen substrate after about 1 hour. The turbidimetric method exhibits linearity provide cost effectiveness to the lab. It also removes the necessity of redeveloping from 20 to 100 µg/mL (r2=0.9936), precision (≤6.3%), and accuracy (105%). Ion them to in-house methodology and complete revalidations requiring high levels of Chromatography assay exhibits linearity from 5 to 60 µg/mL (r2 = 0.9997), precision resources in time and money. (<6%), and accuracy (106%). 85 Unique Properties of Zirconia Phases for Structurally Similar 89 Improving Precision and Accuracy of Temperature Measurements Compounds and Other Difficult HPLC Separations in Automatic Refractometers Kelly S. Johnson, ZirChrom Separations, 617 Pierce St., Anoka, MN Mark Canestrano, Anton Paar, 10215 Timber Ridge Dr., Ashland, VA 55033, Merlin Bicking, Richard A. Henry 23005 Method development can be challenging for structurally similar compounds and Refractive index measurements are strongly dependent on temperature. In order to complex ionic mixtures. Additionally, validation methods often require a column of achieve high precision in measuring the refractive index, temperatures must be both orthogonal selectivity be used to ensure no impurities are missed. Zirconia based meticulously controlled to a narrow margin of uncertainty, and be determined very phases, with their inherent surface chemistry differences, offer a dramatically differ- accurately, requiring the temperature reading to be true to the value of the sample’s ent selectivity when compared to silica and polymer phases. Here we investigate actual temperature. This paper discusses the effects of innovative methods used two of the most unique zirconia-based phases, a carbon-clad zirconia phase and a to ascertain precision and accuracy of temperature measurements in automatic re- crosslinked polyethyleneimine coated zirconia phase. To demonstrate the differenc- fractometers. es between the zirconia based, carbon-clad ZirChrom®-CARB phase, we highlight a selectivity comparison versus a typical silica bonded phase for 22 non-electrolyte 90 Identification of Micro-Particulates in Pharmaceuticals Using a solutes with varying chemical properties. The analysis of a set of six structurally sim- Novel Imaging Guided Spectroscopy System ilar sulfated steroids will be explored to demonstrate how this orthogonal selectivity Mark Sullivan, Rap.ID Inc., 11 Deer Park Dr., Ste. 201, Monmouth can be successfully applied. The ability of ZirChrom®-CARB to separate closely Junction, NJ 08852, Kathryn Lee, Markus Lankers, Oliver Valet related compounds will be shown in the separation of vitamin D2 from vitamin D3. The detection and identification of foreign particulates in sterile pharmaceutical 14 2015 EAS Abstracts November 2015

products and medical devices is critical for determining the root causes of con- in this study the quantitative values of various inorganic impurities in organic sam- tamination. A successful workflow for completing this type of investigation requires ples were compared by EC and the Background FP method. The correlation coef- an efficient means for: 1) collecting the particulates; 2) measuring particle dimen- ficients between the EC and the Background FP method were more than 0.995 in sions and morphology in the appropriate size range; and 3) identification of the lead, cadmium, arsenic, mercury, ruthenium, rhodium, osmium, cobalt, copper and composition of individual particles with a suitable analysis tool. A combination of molybdenum. And this method achieved detection limits of below 1 μg/g. spectroscopic and imaging analysis is the key enabling technology to accomplish these objectives. In this talk we present pharmaceutical applications of the first com- 95 Molecular Isotopic Engineering (MIE): Industrial Manufacture of mercial instrument designed for particle analysis (500nm to 5mm) equipped with Naproxen of Predetermined Stable-Isotopic Compositions for both Raman (molecular) and laser-induced breakdown spectroscopy (elemental) Novel Intellectual Property Coverage As Well as for Identity and capabilities for the fast and highly automated identification of metals, inorganics, Security Protection organics and biologics. John P. Jasper, Nature’s Fingerprint / Molecular Isotope Technologies LLC., 8 Old Oak Ln., Niantic, CT 06357 91 Container Closure Integrity Testing (CCIT) Method Development Molecular Isotope Technologies LLC (MIT) has developed four patented or pat- for Single-Use Auto-Injectors ent-pending generations of stable-isotopic methods and technologies: (i) product Nikunj Vasoya, Bristol-Myers Squibb, 1 Squibb Dr., New Brunswick, characterization (for both small molecules and biologics), (ii) process characteriza- NJ 08903, Joe Koo, Casey Tyrrel-Pawlowic, Antonio Fernandez, Steve tion, (iii) in-process (continuous) analysis, and now, (iv) molecular isotopic engineer- Klohr ing. Early work in cooperation with the United States Food & Drug Administration Container closure integrity testing (CCIT) is a growing regulatory concern for the on the product characterization of naproxen revealed process manufacturer-level pharmaceutical industry. New Injection-delivery systems, such as single-use au- isotopic provenance of this small analgesic molecule (Wokovich et al., 2005) which to-injectors, are designed for self-administration to enable dexterity-compromised was referred to as “The Manufacturer’s Fingerprint.” Rather than merely accepting patients to self-dose, however, they introduce new challenges to product develop- the random effects of variable sourcing and synthetic process on the stable-isotopic ment, particularly for CCIT. Bristol-Myers Squibb container closure integrity methods compositions of products, we take a proactive approach to purposefully determine utilize fluorescein dye and involve visual inspection of packages under UV Light. the stable-isotopic composition of bio/pharmaceutical products. The main rationale Visual analysis of drug product filled syringes in an auto-injector assembly becomes for MIE is to predetermine the isotopic ranges of products for reasons of product challenging because the fluorescent properties of the auto-injector parts and drug identification and of product security, and also for intellectual property consider- product itself impedes visual detection of the fluorescein dye. The development of a ations. As an example of MIE, we analyzed the products of the isotopic-synthetic re- novel visual aid employing focused light and dye-specific lenses improved our CCIT actions for the last two steps of naproxen synthesis: 2-Bromo-6-methoxynapthalene methodology and enabled us to overcome the packaging component challenges. + Bromopropionate ◊ ±Naproxen ◊ (S)-Naproxen Pre-selection of the stable-isotopic compositions of the starting material, 2-Bromo-6-m25.19 ± 0.04‰ vs VPDB). In 92 Optimizing Dye Ingress for Container Closure Integrity Testing principle, the MIE approach should be readily adapted to existing bio/pharmaceutica Elizabeth C. Moroney, Bristol-Myers Squibb, 1 Squibb Drive, New ethoxynapthalene (viz., δ13C = -29.88 ± 0.04‰, -28.73 ± 0.03‰, -24.01 ± 0.04‰ vs Brunswick, NJ 08903, Casey Tyrrel-Pawlowic, Antonio Fernandez, VPDB) respectively yielded the product of discrete stable-isotopic ranges (-29.75 ± Steven E. Klohr 0.08‰, -29.18 ± 0.08‰, and -l manufacturing units. The manufacturing apparatus Container Closure Integrity Testing (CCIT) is done to demonstrate the capability of a would remain unchanged. This approach could have broad application in securing container system to maintain integrity and sterility over the product lifespan, though drug identity/provenance from manufacturing plant to consumer. it does not demonstrate sterility of the original preparation. As more pharmaceutical products are packaged in a greater variety of container systems, testing techniques 96 Strategic Approaches to Method Development and Analysis for have had to be modified and/or redeveloped. Dye ingress techniques have been Genotoxic Impurities widely used for years owing to familiarity, low capital cost, and relative ease of use. Zhen T. Yang, Wilmington PharmaTech Company, 229A Lake Dr., Historically, methylene blue dye has been the dye of choice used to detect breaches Newark, DE 19702, Ying Chen, Hangchun H. Hu, Chen Zhao, Feili in container systems, though other dyes such as fluorescein may be easier to detect Tang, Carl Behrens, Hongfeng Chen, Harry Li, Yong Chen visually. In this presentation, I summarize the results of experiments carried out to The pharmaceutical industry faces challenges in new drug development as each better understand factors that affect robustness, stability and sensitivity of CCIT dye different active pharmaceutical ingredient (API) process presents its own unique ingress methods. challenges based on the stage of development and commercialization. As genotox- ic impurities (GTIs) in small molecule development present their own risks during 93 Unusually High Recovery in Trace Analysis of Acetohydrazide and the manufacturing process, risk evaluation and control of GTIs are critical for the Hydrazine by Chemical Derivatization due to Sample Matrix Effects assurance of patient safety aligned with today’s regulatory landscape. Wilmington Kaina Jiang, GlaxoSmithKline, 709 Swedeland Rd., PO Box 1539, King PharmaTech (WPT), a United States based fast-growing API manufacturing con- of Prussia, PA 19406, Li Cui tract research organization (CRO) / contract manufacture organization (CMO) since Unusually high recoveries were encountered during chemical derivatization meth- 2003, is specialized in finding practical solutions to the complex challenges of GTIs od development for the determination of acetohydrazide and hydrazine in a drug and known carcinogens analysis involving trace chemical method development and candidate. Systematic studies demonstrated that the high recoveries were caused method validation in both the API and the formulation matrix. Striving to provide by the acidity of the active pharmaceutical ingredient in the sample matrices. Upon the best service, Wilmington PharmaTech has years of GTI handling experience understanding the underlying chemistry of the derivatization reaction, a strategy of and can promptly develop, qualify and validate GTI methods that are custom de- acidifying the derivatization reagent with benzoic acid to minimize the sample matrix signed for our customer’s needs and meet the requirements for regulatory filings. In effect was implemented. As a result, the unusually high recovery method problem this poster presentation, strategies on systematic development of robust analytical was solved and a method for simultaneous determination of acetohydrazide and methods for different type of challenging GTIs and Carcinogens are discussed along hydrazine at 10 ppm was successfully validated in support of setting the overall with the state of the art of technologies such as gas chromatography (GC), GC- project genotoxin control strategy. mass spectrometry (MS), liquid chromatography (LC)-MS, and derivatization with LC-MS, and others. 94 Challenge of Small Sample Analysis for Pharmaceutical Products and Foods Using Theoretical Scattered X-Rays 97 Evaluating Amorphous Content in an Active Pharmaceutical Dan Davis, Shimadzu Scientific Instruments, 7102 Riverwood Dr., Ingredient Using NIR and Raman Spectroscopy Columbia, MD 21046, Hiroaki Furukawa, Naoto Ichimaru, Keijiro Suzuki, Lili Feng, Bristol-Myers Squibb, 1 Squibb Dr., New Brunswick, NJ Shinji Watanabe, Makoto Nishino, Hirotomo Ochi 08816, Boyong Wan The United States Pharmacopeia (USP) has recently established guidelines for It is well known that amorphous active pharmaceutical ingredients (APIs) can have general analysis using X-ray fluorescence (XRF) methodology. XRF is an attractive very different physicochemical properties, stability, bio-availability and process abil- methodology because it does not require chemical pretreatment and allows non-de- ity compared to the crystalline APIs. Thus it is often critical to evaluate and con- structive analyses. Energy dispersive X-ray fluorescence (EDX) can be utilized in trol the amorphous content in API. There can be significant batch to batch varia- the screening process for the control of inorganic impurities in pharmaceutical prod- tion in the amount of amorphous content in API during process development. As ucts, excipients, and active pharmaceutical ingredients (API’s). This study proposes a result, health authorities can request a test to monitor the amorphous content. the use of the scattered X-ray corrected - fundamental parameter (FP) method, While techniques such as X-ray powder diffraction (XRPD), differential scanning hereinafter called “Background FP method”, which uses scattered X-rays for quan- calorimetry (DSC), isothermal microcalorimetry (IMC) and solid state nuclear mag- titation of low ppm level impurity elements in organic samples. The FP method can netic resonance (ssNMR) are considered the primary tools to quantify crystallinity, reduce the need to measure standard samples compared to empirical correction there exist limitations to each technique, especially for quantification of low levels of (EC) method. In order to show the applicability of using the Background FP method, amorphous API. This study highlights the simplicity of using near-infrared (NIR) and 15 2015 EAS Abstracts November 2015

Raman to monitor low level of amorphous content in an API. With limited number pH 4.5, the degradation directionality and the quantity of degradation products were of standard samples, it was demonstrated that NIR spectroscopy can detect as low different. Furthermore, the spatial aggregation propensity (SAP) model was used to as 5% amorphous content in API-1. Back scattered Fourier transform Raman spec- predict the degradation behaviors. However, the stability data was not consistent troscopy and transmission Raman spectroscopy were also compared for monitoring with the predictions. Taken together, this study indicated that neither conformational amorphous content in API-1. stability data nor surface characteristics indicated by SAP modeling alone could fully predict aggregation or fragmentation. A comprehensive understanding of stabilizing 98 Investigation of Compaction Induced Amorphization of Crystalline properties, as well as the key characteristics that influence degradation, are critical API for developing an effective strategy during formulation development for each mAb. Chengbin Huang, Merck, 770 Sumneytown Pike, West Point, PA 19486, Abstracts 101-199 Jerry Klinzing, Jie Ren, Fengyuan Yang, Fengyuan Yang, Anthony Leone, Rubi Burlage, Lei Zhu, Adam Procopio, Yongchao Su 101 Determination of Isoelectric/Isoionic Point of Collagen via Ion Phase transformation during pharmaceutical processing can have major impact on Exchange Resin the quality of pharmaceutical products. The underlying mechanism of crystalline to Bryan M. Vayda, Integra Life Sciences, 313 Enterprise Dr., Plainsboro, amorphous transition has been rarely reported in the literature despite of its import- NJ 08536, John Kemnitzer, Thomas Twardowski, Rachel Katz ant bio-performance and stability implication. Most recently, active pharmaceutical Collagen is a zwitterion, exhibiting an expected isoelectric point (pI) at a pH of about ingredient (API) amorphization has been found in these compaction processes, 4.7 depending upon source, e.g., pig or calf, type I or III, the presence and concen- which may trigger risks of chemical stability, bioavailability and quality. The mecha- trations of salts, and to changes occurring to collagen due to processing. A rapid nism of such undesired conversion is largely unknown. We aim to study model APIs determination of the pI of a commercial, purified, insoluble collagen can be obtained to investigate the amorphization upon compaction. Solid-state nuclear magnetic with the ion exchange resin technique of Janus, et al. In brief, a mixture of Na+ resonance (NMR) is utilized to quantify the amorphous content for this purpose. and Cl- resins in a ratio of 2:1was added at a 1:1 ratio with 1% collagen solution in The quantification of amorphous content indicates posaconazole produces ~ 20% H2O. The collagen/resin mixture was incubated at 40 °C for 2 hours. The pH after crystalline to amorphous conversion after compressing at 400 MPa, which shows extraction was taken to establish the isoelectric point. The isoelectric point of the very sensitive response to compression force. The finite element method (FEM) insoluble collagen was determined at an average of 5.65 with a standard deviation simulation has shown shear stress distribution inside a tablet, which might induce of 0.737. This technique will be compared to the isoelectric point as measured by the amorphization of crystalline drug substance. To test this hypothesis, we have more intensive titration, dialysis and zeta potential techniques.[1] carried out comprehensive investigations on the impact of compaction process, Reference: i.e. axial and shear stress, in the compaction-induced amorphization. A correlation [1] Janus, J.W., Kenchington, A.W., and Ward, A.G. (1951) A rapid method for the between amorphous contents and various compression pressures has been es- determination of the isoelectric point of gelatin using mixed bed deionization. tablished. Moreover, the API-diluent binary system has been employed to evaluate Research 4, 247-24813. the effects of the attributes of excipient materials including weight concentration, type of diluent and lubrication. The preliminary results suggest the dependence of 102 Circular Dichroism of Protein-Dye Complexes as a Characterization both pressure and weight concentration in diluent-API binary system. Our quanti- Tool for Protein Higher Order Structure tative investigations on amorphization of crystalline API provide mathematical and Christopher Sucato, Blue Stream Laboratories, 8 Henshaw St., Woburn, experimental analysis to understand the underlying mechanism of amorphization of MA 01801, Shreyas Laghate, Libo Wang, Mario DiPaola crystalline API during compression. The fluorescent dye, 1-anilinonaphthalene-8-sulphonate (ANS), has a fluorescence quantum yield that increases significantly after binding to the hydrophobic regions 99 Mass Spectrometry Based Proteomics of Oxidative Stress: of proteins. As a result of this effect, ANS has been widely used as a hydropho- Investigating of Amino Acids Modifications by 4-hydroxy-2- bic probe for the study of conformational changes in protein samples, especially nonenal (HNE) Using Lysozyme and BSA as Model Proteins when assessing the structural characteristics of two or more proteins in a compa- Roshanak Aslebagh, Clarkson University, 8 Clarkson Ave., Potsdam, rability study. The change in fluorescent properties of the dye, upon association NY 13699, Costel C. Darie, Steven J. Fliesler, Bruce A. Pfeffer with the hydrophobic regions of protein structural domains, is a key feature of the Oxidative stress has been associated with many metabolic, hereditary, and age-re- ANS molecule. However, the information from extrinsic fluorescence spectra alone lated diseases. One process that occurs in oxidative stress is the oxidation of poly- reveals little information about protein conformational changes at the secondary unsaturated omega-6 fatty acids, which causes the production of 4-hydroxy-2-non- or tertiary structural level. Here we present the results of circular dichroism stud- enal (HNE) as a by-product. HNE is a reactive aldehyde that can modify proteins ies of protein-ANS complexes that indicate the near-UV ellipticity spectral data of by forming covalent adducts, causing alterations in protein function. Although mod- the complexes can be used to gain information that is complementary to both the ification of some amino acids, such as cysteine, histidine and lysine, havebeen ANS-protein fluorescence data, as well as the circular dichroism data on the native described previously, others are possible, but yet to be described. In this study, we protein in the absence of ANS. used lysozyme and BSA for investigation of HNE-modifications. lysozyme and BSA were treated with different concentrations of HNE; treated and untreated samples 103 Label-Free Analysis by HPLC with Charged Aerosol Detection of were then analyzed by electro spray ionization-quadrapole time of flight micro mass Glycans Separated by Charge, Size and Isomeric Structure spectrometer (ESI-QTOF-Micro MS) in positive ionization mode. In parallel, sam- David H. Thomas, Thermo Fisher Scientific, 22 Alpha Rd., Chelmsford, ples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis MA 01824, Ian N. Acworth, Marc Plante, Rainer Bauder, Daniel Kutscher (SDS-PAGE); the Coomassie Blue-stained bands were excised, trypsin-digested, We have developed a quantitative glycan profiling assay that is simple, fast, and and then extracted and purified (C18 Ziptip), followed by nano-liquid chromatog- eliminates the hassle of labeling glycans with a fluorophore. The method is intended raphy tandem mass spectrometry (nanoLC-MS-MS) analysis using a NanoAcquity for characterization and quality control of glycoprotein biotherapeutics. The meth- ultra-pressure liquid chromatography (UPLC) coupled with a QTOF Ultima active od uses a volatile mobile phase fully compatible with mass spectrometry, if further pharmaceutical ingredient mass spectrometer. Data analysis was performed using characterization is desired. Changes in the number, type, composition or linkage ProteinLynx Global Server (PLGS version 2.4) and Mascot database search. Using pattern of glycoprotein glycans may serve as a biomarker of disease or influence the ESI-MS analysis, we found HNE modifications on up to seven amino acid residues efficacy of a biotherapeutic product. For this reason, the ability to correctly identify per molecule of lysozyme; we also used nanoLC-MS-MS analysis to investigate the and measure these glycans quickly and inexpensively is of great practical benefit. sites of modifications. We found that in addition to cysteine, histidine and perhaps ly- This work explores direct detection of native glycans as an alternative to the com- sine residues, arginine residues also were modified, which heretofore has not been mon techniques for glycan analysis that rely on labeling reactions to render glycans detected commonly. Additional modifications are under investigation. detectable. The lack of a detectable chromophore in native glycans is overcome by using UHPLC with charged aerosol detection, which can quantitatively measure any 100 Investigating the pH-Dependent Conformational States and non-volatile compound. N-linked glycans are released from proteins by PNGase-F Stabilities of Therapeutic Monoclonal Antibodies (mAbs) and separated by ultra-high-performance liquid chromatography (UHPLC) on a Songyan Zheng, Bristol-Myers Squibb, 1 Squibb Dr., New Brunswick, new UHPLC platform that integrates the charged aerosol detector into the system NJ 08903, Pedro Smith, Ming Chen, Difei Qiu, Mariana Vlad, Monica for increased performance and ease of use. The mixed mode analytical column Adams employs both weak anion exchange and reversed-phase separation mechanisms This study investigated the degradation behaviors of several therapeutic monoclonal to resolve glycans based on charge, isomerism and size. The native glycans are antibodies (mAbs) and the influence of buffer pH. The results obtained from differen- detected directly without labeling by using charged aerosol detection. Quantitative tial scanning calorimetry (DSC) indicated that the conformational states of all mAbs performance including precision, detection limits and dynamic range is presented. were closely associated with buffer pH and that different mAbs also possessed sim- Figures of merit include sensitivity at the low-nanogram on-column level, dynamic ilar conformational states under the same pH conditions. A further heat-induced range over two orders of magnitude, and peak area precision averaging less than stress study showed that although the mAbs had similar conformational states at three percent relative standard deviation. 16 2015 EAS Abstracts November 2015

104 Label-Free Profiling of O-linked Glycans by UHPLC with Charged serve as initial candidate phosphorylation sites. Synthetic peptide corresponding Aerosol Detection to predicted sites are assayed by matrix-assisted laser desorption/ionization mass David H. Thomas, Thermo Fisher Scientific, 22 Alpha Rd., Chelmsford, spectrometry (MALDI-MS) for kinase-specific in-vitro phosphorylation. These pre- MA 01824, Ian N. Acworth, Rainer Bauder, Marc Plante, Liz Kast liminary hits are further investigated for in-vitro protein at the protein level, followed The goal of this work was to develop a quantitative profiling assay for O-linked by in-gel digestion and MALDI-MS and MS2. Protein-level hits are subsequently glycans released from glycoproteins by reductive beta elimination. While glycan evaluated for biological relevance using site-specific antibodies and mutagenesis. release under reducing conditions is desirable to reduce peeling reactions, the resulting O-linked glycan alditols can’t be derivatized easily with a fluorescent la- 108 Proteomic Investigation of Saliva from People with Smith-Lemli- bel. Charged aerosol detection does not require a fluorophore or chromophore for Opitz Syndrome (SLOS) and Controls sensitive, accurate quantification, and so HPLC-CAD provides a simple, direct ap- Emmalyn J. Dupree, Clarkson University, 10 Clarkson Ave., Potsdam, proach to separate and quantify native glycans. The method uses a volatile mobile NY 13699, Megan Borland, Kelly L. Wormwood, Alisa G. Woods, Costel phase fully compatible with mass spectrometry, if further characterization is desired. C. Darie, Cassia Chapman O-linked glycans are released from proteins by reductive beta elimination. The Saliva samples were analyzed from people with Smith-Lemli-Opitz Syndrome released glycans are separated by ultra-high-performance liquid chromatography (SLOS) and controls using sodium dodecyl sulfate polyacrylamide gel electropho- (UHPLC) on a new UHPLC platform that integrates the charged aerosol detector resis (SDS-PAGE), in-gel trypsin digestion, and nanoliquid chromatography-tandem into the system for increased performance and ease of use. The mixed mode an- mass spectrometry (nanoLC-MS-MS) to investigate the differences between the alytical column employs both weak anion exchange and HILIC separation mecha- proteomes of people with SLOS and controls. The pending results will hopefully nisms to resolve glycans based on charge, size and polarity. The reduced glycans provide more information about the protein changes that occur in SLOS, potentially are detected directly without labeling by using charged aerosol detection. O-linked leading to identification of treatment targets. These results may also shed some light glycan pools released from various proteins were analyzed including those from bo- on the possible link between SLOS and Autism Spectrum Disorder (ASD), as ASD vine fetuin, bovine submaxillary mucin, and IgG. Quantitative performance including is almost always co-morbid with SLOS. This could possibly lead to better diagnosis, precision, detection limits and dynamic range is presented. Figures of merit include treatment and understanding of ASD as well. sensitivity at the low-nanogram on-column level, dynamic range over two orders of magnitude, and peak area precision averaging less than three percent relative 109 LC-MS Analysis of Monoclonal Antibody Structure Utilizing HALO® standard deviation. BioClass Fused-Core™ Particles; Multilevel Analysis for Proteins and Glycovariants 105 Solution Structure of the Microtubule-Targeting COS Domain of Benjamin Libert, Advanced Materials Technology, 3521 Silverside Rd., MID1 Ste. 1-K, Wilmington, DE 19810, William Miles, Stephanie Schuster, Katharine M. Wright, George Washington University, 800 22nd St. NW, Thomas Waeghe, Barry Boyes Washington DC 20052, Haijuan Du, Michael A. Massiah The structural characterization of monoclonal antibodies (mAbs) is a challeng- MID1 is a tripartite motif protein (TRIM18) shown to be important for the proper ing and complex task. Efforts have been directed at optimizing high performance development of the ventral midline during embryogenesis. Microtubule association HALO® BioClass Fused-Core™ silica materials in order to improve the separa- and targeting of MID1 is dictated by a 60-amino acid region termed the COS do- tions component of liquid chromatography mass spectrometry (LC-MS) analysis of main, located at the C-terminal of the coiled coil domain. Here, we report the struc- proteins, mAbs, glycopeptides and released glycans. Recently improved superfi- ture of the COS domain, which adopts a helix-loop-helix structure. This structural cially porous silica packing materials have been applied to high-performance liquid motif is found in many microtubule-associated proteins. Electrostatic mapping of the chromatography (HPLC) – ultra-HPLC characterization of biomolecules including structure reveals a distinct basic patch where the N- and C-terminal ends of the two glycosylation variants from mAbs. HALO BioClass Fused-Core™ materials show helices meet and this may be important for microtubule binding. We also character- considerable utility for analysis of these highly complex molecules, using conditions ized the effect of the Lys370Glu mutation, associated with X-linked Opitz Syndrome that permit excellent separations and analysis via LC-MS. Alternatives to standard (XLOS) and showed this mutation, which is positioned directly in the center of that trifluoroacetic acid (TFA) and formic acid (FA) mobile phase conditions are shown basic path, did not cause unfolding of the COS domain. This observation suggests for intact molecule separations, subunit analyses, and for analysis of tryptic digests. that the disruption in the basic path could affect microtubule binding or binding of other partners. Microtubule-binding assays showed the COS domain in construct 110 Membrane Protein Structural Biology and Solid-State NMR: Past, with the coiled coil domain directly associates with the microtubules. Present and Future Timothy Cross, Florida State University, 1800 E. Paul Dirac Dr., 106 Spectroscopic Characterization of Abatacept, a Protein-Based Tallahassee, FL 32310 Pharmaceutical It is only the native structure of proteins that facilitate the characterization of na- Rose Soskind, Rutgers University, 6 Sycamore Dr., Plainsboro, NJ tive functional mechanisms. Membrane proteins exist in a very heterogeneous 08536, John Wasylyk environment with strong gradients of dielectric constant, electric potentials, water In recent years, there has been a rise in the development of protein-based phar- concentration, hydrophobicity and dynamics, as well as dramatic lateral pressure maceuticals. As a result, there is an increased need in developing techniques to profiles and lipid monolayers with negative or positive curvature. Nuclear magnetic characterize these pharmaceuticals. Fourier transform infrared (FTIR) spectroscopy resonance (NMR) is the only technology that can characterize membrane protein and Raman spectroscopy are fast, non-destructive techniques that have been used structure in a native-like liquid crystalline lipid bilayer environment. These mem- to determine the secondary structure of proteins. This is largely based on the amide brane properties in addition to the amino acid sequence not only dictate membrane functional group vibrations present for proteins. In this study, bovine serum albumin protein structure, dynamics and function, but they shift the whole balance of molec- and Abatacept were stressed at varying conditions, particularly at rising tempera- ular interactions that stabilize the structure. This is especially true for small helical tures, and spectra were taken over time. Spectral results were supported using membrane proteins that represent a majority of bacterial membrane proteins and additional analytical techniques, including dynamic light scattering and differential a large portion of mammalian membrane proteins. Currently, these small helical scanning calorimetry, and size-exclusion chromatography. With exposure to stress, membrane proteins are under-represented even among membrane proteins in the variations were observed in the FTIR and Raman spectra of the protein-based phar- protein data bank (PDB). Here, I describe progress and challenges in the develop- maceuticals, corresponding with changes in protein secondary structure. ment of solid-state NMR spectroscopy as a technology for characterizing membrane protein structure, dynamics and functional mechanisms. I describe the combined 107 VIF-Phosphorylation in HIV Infection use of orientational and distance restraints from Oriented Sample solid-state NMR Pratikkumar N. Rathod, York College- CUNY, Graduate Center-CUNY, and magic angle spinning NMR. I illustrate the development with our early work on 94-20 Guy R. Brewer Blvd., Jamaica, NY 11451, Hsin-Pin Ho, Abbas gramicidin in which we achieved a complete, all atom structure, to the more recent Nazir, Ai-Mei Chen, Emeka Nnaji, Manjeet Kaur, Katrina Murtozaeva, work with membrane proteins where we have achieved backbone structures of the Lois Anti, Xu Yu, Mathias Lichterfeld, Emmanuel Chang M2 proton channel from Influenza A and the cell division protein, CrgA, from Myco- There are several published instances of phosphorylation of various human im- bacterium tuberculosis and then to the future. munodeficiency virus (HIV)-proteins. However, to date, a comprehensive study of HIV-proteins phosphorylation is lacking. This research focuses on studying the 111 NMR Methods for Structural Studies of Membrane Proteins phosphorylation of HIV-proteins in comprehensive manner and evaluating their bio- Stanley J. Opella, University of California-San Diego, 9500 Gilman Dr., logical importance in HIV infection. Amino acid sequences of HIV proteins from 12 La Jolla, CA 92093 different strains are aligned using CLUSTALW to assess sequence conservation of Membrane proteins are important targets for structure determination. One quarter potential phosphorylation sites, across. Phosphorylation prediction tools such as of the proteins in the human genome are membrane proteins, and the majority of NetPhosK, GPS and KinasePhos are used to predict potential phosphorylation sites drug receptors are membrane proteins, especially G-protein coupled receptors (GP- of various HIV proteins. High scoring predictions that are conserved across strains CRs). Despite the importance of both small and large membrane proteins, both 17 2015 EAS Abstracts November 2015

their structure determination and dynamics characterization have lagged because croscopic specimens. Examples of the use and misuse of this technique, from the of technological challenges resulting largely from the phospholipid environment in first documented use of IMSP in a forensic case (the author utilized this instrument which they reside. Progress in the development of new and more powerful nuclear to identify the chemical composition of a minute deposit residing on a pubic hair) magnetic resonance (NMR) methods for determining the structures of membrane through a variety criminal cases, in which important chemical evidence, brought to proteins in phospholipid bilayers is presented. This research takes advantage of light by the infrared spectrum, could have been obtained in no other way. The pre- a panel of membrane proteins that we have developed for this purpose, including sentation concludes with a discussion and example of the complimentary nature of the determination of their backbone structures. Examples include Vpu from HIV- the microanalytical tools (both techniques and instruments) available to the forensic 1, MerF from the bacterial detoxification system, and filamentous bacteriophage microscopist in today’s modern forensic laboratories. coat proteins. Special emphasis is placed on structural studies of the 350-residue chemokine receptor CXCR1 and its complexes with IL-8, G-proteins, and drugs. 115 The Synergism between IR and Raman Microscopy Fran Adar, Horiba Scientific, 3880 Park Ave., Edison, NJ 08820 112 Insights into the Mechanism of Action of Antimicrobial Peptide Infrared (IR) and Raman spectroscopies are the most widely used vibrational tech- Piscidin: One Peptide Family, Multiple Roles in Host Defense niques that have been applied to the intrinsic derivation of chemical and physical Myriam Cotten, Hamilton College, 198 College Hill Rd., Clinton, NY properties of materials. While IR absorption was first performed by William Coblentz 13323 at Cornell University at the beginning of the last century, and CV Raman first re- Antimicrobial peptides are multifunctional molecules that perform vital host defense ported in 1928 the light scattering phenomenon now recognized by his name, it functions to prevent and fight infections. Not only do they directly eradicate bacteria was only in the latter half of the 20th century that an optical microscope was used by targeting their cell membranes or intracellular processes but they can also have as a sampling device for either. The availability of the IR and Raman microscopes immunomodulatory effects. Several members of the piscidin family, which includes immensely increased the areas of investigation where the spectroscopies can be the first antimicrobial peptides to be discovered in mast cells, display antibacterial, used, and consequently the number and scope of applications described in the last antiviral, antifungal, anticancer, and anti-inflammatory properties. In this research, 30 years has grown exponentially. However, when one discusses these microsco- biochemical and biophysical experiments have been carried out to investigate pis- pies, the limitations in spatial resolution always have to be recognized. The spatial cidin’s molecular targets and mechanisms of action in bacterial and cancer cells, resolution of an optical microscope will be determined by the quality of the optics, and their synthetic mimics. Using wide range of tools that include high-resolution and ultimately the working wavelength. Since a Raman spectrum is measured (usu- solid-state nuclear magnetic resonance (NMR), neutron diffraction, oriented circular ally) with a visible laser, and an IR spectrum is measured in the IR (at much longer dichroism, permeabilization assays, biological-activity testing, confocal microscopy, wavelengths), the spatial resolution of the first is about 0.5µm while the second is and molecular dynamics, we contribute to the efforts of mapping at the molecular between 2.5 and 10-20µm (it will be different in different parts of the spectrum). But and atomic levels the landscape of intrinsic structural features and environmental in recent years the diffraction-wavelength barrier has been broken using near field conditions that allow a single peptide to unleash a multiplicity of functions to support technologies that will be described. host defense. 116 A Novel Integrated Instrumentation Platform that Combines Raman 113 NMR Sensitivity Enhancement by Hyperpolarization and Molecular Spectroscopy, Dynamic Light Scattering and Microrheology for Imaging Deriving Unique Insights into the Physicochemical Properties of Eduard Chekmenev, Vanderbilt University, 1161 21st Ave South, Proteins at High Concentration Nashville, TN 37232 Neil Lewis, Malvern Instruments, 7221 Lee Deforest Dr., Columbia, MD Nuclear spin polarization can be enhanced by several orders of magnitude to near- 21046, Wei Qi, Steven Blake, Samiul Amin ly theoretical maximum of unity. This process of dramatic signal enhancement or The combination of dynamic light scattering (DLS), DLS-microrheology and Raman hyperpolarization enables the corresponding gains in nuclear magnetic resonance spectroscopy offers concomitant measurements of protein structure, viscosity and (NMR) sensitivity. The main application for hyperpolarized compounds is their use aggregation. By linking the changes observed in the molecular structure measured as molecular contrast agents, which are administered via injection or inhalation. by Raman spectroscopy with the nanoscale physical properties derived from DLS NMR signal gain of hyperpolarized contrast agents enable detection of metabolism and DLS-microrheology, unique insights into the optimum solvent (formulation) con- and other biological functions in-vivo on the time scale of seconds truly allowing for ditions that stabilize the native state of the protein can be obtained. In particular the real time metabolic imaging. We show efficient hyperpolarization of many biologi- Raman measurements provide information about the secondary and tertiary (intra- cally relevant molecular agents through processes of pairwise addition or exchange molecular) structural changes as well as conformational changes or modifications with parahydrogen molecule providing the source of nuclear spin hyperpolarization. of the disulfide linkages. The DLS and measurements provide direct observations of In particular, 13C-hyperpolarized phospholactate can be obtained via pairwise ad- changes in the hydrodynamic radius or polydispersity (monomer and higher order dition of parahydrogen to phosphoenolpyruvate in seconds, which enables in-vivo oligomers) of the protein resulting from aggregation or unfolding while the DLS-mi- lactate imaging of cancer and brain. Unsaturated nitrogen-containing compounds crorheology records changes in viscosity and other rheological parameters as a can be efficiently 15N-hyperpolarized via the process of chemical exchange on Ir- result of intermolecular interactions which create higher order protein networks and based catalyst at microTesla magnetic field in seconds. These compounds include ultimately gels. These additional physical parameters can be further correlated with 15N-imidazole for pH imaging of cancer, and azidothymidine, 15N-nicotrinamide and other Raman markers that directly measure changes in the hydrogen bonding net- its derivatives for potential HIV and tuberculosis imaging applications. Propane gas work that are the basis for these physical manifestations. In addition a number of can be hyperpolarized by a chemical reaction of pairwise addition of parahydrogen kinetic and thermodynamic parameters can also be obtained through temperature to propene gas, and if low-field NMR is utilized, propane proton hyperpolarization studies and/or the use of deuterium exchange experiments and these data can be is sufficiently long-lived (T1 ~5-6 s) to enable gas imaging, which can be potentially correlated with other measures of the thermodynamics of these systems with tech- utilized for functional lung imaging on conventional LF MRI scanners. LF MRI has an niques such as differential scanning calorimetry (DSC). Specifically we have stud- additional benefit of producing more signal-to-noise ratio (SNR) than high field MRI ied a number of monoclonal antibodies (mAbs), modified mAbs and simple smaller for hyperpolarized compounds. proteins under a variety of solvent conditions. We present data that demonstrates that new insights into the mechanisms and pathways of protein aggregation, as well 114 Some Milestones in Role of Infrared Microspectroscopy as Applied as some of the driving forces associated with changes in viscosity, can be obtained to Microscopic Trace Evidence: A Personal Perspective using this hybrid physicochemical technique. Skip J. Palenik, MicroTrace, 790 Fletcher Dr., Elgin, IL 60123 The infrared microspectrophotometer (IMSP) and scanning electron microscope 117 Uniting Microscopy and Molecular Spectroscopy have become, in recent times, almost as common in trace evidence laboratories as John A. Reffner, John Jay College, City University of New York, Dept. of light microscopes. During the course of a career that began before IMSPs became Sciences, 524 West 59th St., New York, NY 10019 commercially available, the author has had the opportunity not only to observe the When a microscope is used to examine materials, many features provide informa- effect of such powerful instrumentation in the forensic laboratory but also to have tion about the material’s composition, history and providence. One example is the taken an active role in this development as it applies to real-life casework. This pre- microscopical examination of a piece of yarn. Examining the microstructure of indi- sentation begins with an account of discussions with my scientific colleagues about vidual fibers it’s possible to determine if the yarn is composed of a synthetic fiber, the limitations of polarized light microscopy and microchemistry in the mid-1970s in a natural fiber or a mixture of several different fiber classes. Some synthetic fibers our day-to-day microanalytical work and how we eventually attempted to overcome have unique cross-sectional shapes that allow their polymer and manufacturer to be some of these difficulties by cannibalizing the microscope from a Perkin-Elmer dis- determined by microscopical inspection. Not all fibers are distinguishable solely by persive microspectrophotometer that was originally obtained for the laboratory by their morphology. Infrared absorption or Raman spectral analysis is required to con- John Reffner. This was eventually coupled to a Fourier transform infrared (FTIR) firm their molecular composition. Fibers with different morphologies may have the spectrophotometer, allowing us to obtain high quality infrared spectra from truly mi- same molecular composition. Spectroscopically these fibers are similar, microscop-

18 2015 EAS Abstracts November 2015

ically they can be differentiated. Uniting microscopy with spectroscopy is synergistic art systems can acquire a three-dimensional (3-D) image up to 10 times faster than and has real advantages for scientific analysis of physical and biological mater. previously. This paper presents the state of the art in pulsed terahertz systems with Over the past five decades, it has been a privilege to participate in instrumental some application examples in the fields of art conservation, cultural heritage study, development and the integration microscopy with spectroscopy. Uniting microscopy and high-speed imaging. and spectroscopy is not a new concept. In the late 1880’s, Henry Clifton Sorby published essays on the construction and use of the micro-spectroscope. He ap- 123 Battelle Smart Corrosion Detector® Bead: Self-Healing with plied microspectroscopy in his studies of animal and vegetable coloring matter. After Corrosion Detection by Terahertz World War II, electron microprobes and mid-infrared microspectrometer systems Cindy Conner, Battelle, 505 King Ave., Columbus, OH 43201, Ram were developed and showed the feasibility of coupling these technologies. When Lalgudi solid-state electronics and digital computers became available, this union moved Corrosion-related deterioration of metallic structures remains a significant problem forward; making it practical to apply microbeam analysis research and development. across many industries. The development of the Battelle Smart Corrosion Detec- The advances in microspectroscopy for the analysis of molecular composition are tor® bead enables coatings that can both detect and mitigate early-stage corrosion. the main focus of this presentation. Based on Battelle’s Encapsulation by Design technology platform, Smart Corrosion Detector beads are microcapsules comprised of a self-healing agent core with a 118 Detection of Cathinone and Mephedrone in Plasma by LC-MS-MS functionalized shell which reacts with corrosion by-products. The corrosion-trig- Using Standard Addition Quantification Technique gered shell is the key to the self-healing and detection functionality of the Smart Shu-Yuan Cheng, John Jay College, 524 West 59 St., New York, NY Corrosion Detector beads. Combined with terahertz pulsed imaging, a non-destruc- 10019,Theron Ng-A-Qui, Bruce Eng, Jonathan Ho tive method, the onset of corrosion can be detected through the coating system. Cathinones (a.k.a. bath salts) have been listed as illicit drugs since 2011 and are the structural analog of Drug Enforcement Agency (DEA) Schedule I substances. 124 Hybrid Mid-IR OCT System for Imaging and Spectroscopy with They have been banned in numerous countries. Newly synthesized cathinones New High-Power QC Superluminescent Emitters which mimic the effects of illegal drugs of abuse and to bypass the provisions of Deborah M. Varnell, Princeton University, Equad Olden St., Princeton, drug regulations are still available. These products have caught concern among NJ 08544, Mei Chai Zheng, Samantha Lee, Nyan Aung, Ahmed Musse, law enforcement agencies. Acute toxicity and numerous fatalities have been linked Claire Gmachl with the abuse of designer cathinones. Despite the increased availability of designer Optical coherence tomography (OCT) is a fast, high resolution, non-invasive imag- drugs, few studies have focused on the analytical extraction techniques and the ing technique using reflected light from inside a sample to create three-dimensional matrix effect for their detection and quantification in biological samples. Accurately (3-D) images. Currently, many OCT systems are available in the visible or near-in- quantifying the concentration of cathinones in biological samples is important for frared (IR) spectral regions, however, few or none are available using mid-IR light. the regulation of cathinones. This study is to share our work about on developing A hybrid mid-IR OCT system could create a 3-D chemical map of a sample. This is liquid chromatography tandem mass spectrometry (LC-MS-MS) method that utilized possible due to the spectral ‘fingerprints’ many materials have in the mid-IR. A mid- standard addition without internal standard to quantify cathinone and mephedrone IR OCT system has less scattering than the currently available visible or near-IR in plasma. OCT systems due to reduced Rayleigh scattering at long wavelengths. Applications of a mid-IR OCT system include industrial process monitoring, art conservation and 119 Bioanalysis of 2-Hydroxypyridine N-Oxide (HOPO) in Rat Plasma biomedical research and diagnosis. We have designed, assembled and tested a Using LC-MS/MS with a Simple Derivatization mid-IR OCT system capable of simultaneous imaging and chemical analysis. The Guowen Liu, Bristol-Myers Squibb, Rte. 206 & Province Line Rd., system uses time-domain scans and the beam propagates in free space. The axial Princeton, NJ 08543 resolution of the system is 50-μm. The system is able to image complex samples No abstract submitted by the author. made of layers of different materials. It is also able to measure the absorption spec- trum of a sample. This system is made possible by the recent development in our 120 Applications of Tandem Mass Spectrometry in Forensic Toxicology lab of a mid-IR superluminescent emitter which provided, for the first time, a suitable Thomas Rosano, Albany Medical Center Hospital and College, 43 New source for mid-IR OCT. The power achieved by the source is over 100 mW, which Scotland Ave., Albany, NY 12208 should allow for real time imaging. This work was supported in part by MIRTHE No abstract submitted by the author. (NSF-ERC). 121 Advances in High-Performance Mass Spectrometry for Quantitative 125 Some Archaeometric Studies on Metallic Artifacts from Anatolian Applications and Enhanced Productivity Region in 2014 Keeley Murphy, Thermo Fisher Scientific, 355 River Oaks Parkway, San Irmak G. Yuceil, Directorate of Central and Regional Laboratory for Jose, CA 95134 Restoration and Conservation, Topkap Saray I.Avlu Bab Humayun cad. High-throughput screening (HTS) is a valuable part of identifying new leads and No:9 Sultanahmet, Istanbul 73015, Turkey, Tuğçe Pamuk directing drug design in pharmaceutical research. The ability to quickly perform Directorate of Central and Regional Laboratory for Restoration and Conservation large numbers of analyses is critical to the success of any HTS assay. Traditionally in Istanbul is a public institution which works for Ministry of Culture and Tourism the quantitative components of these analyses are performed using fluorescence, of Turkish Republic. The laboratory focuses on technical and material researches, luminescence, absorption, or radiolabel techniques. While these techniques pro- documentation, conservation and restoration procedures of movable and immov- vide for extremely rapid sample analysis they also contain inherent limitations, such able cultural properties. In this context, this paper includes technical examination as the occurrence of false positives, increased time for method development, and of some metallic artifacts by X-ray techniques, describing corrosion morphology (by sometimes costly sample preparation. Mass spectrometry (MS) coupled with liquid Raman spectroscopy and XRD), the creating simulations for deterioration factors chromatography multi-channel technology provides rapid sample analysis as well depends on the soil type which artifacts has been buried for centuries and also treat- as a more selective alternative to traditional methods and without the need for la- ment proposals, conservation/restoration practices. Main topics in this paper are beled substrates. This novel combination of hardware and software provides the gilded metal icons of Don cossacks’ from Hagia Sophia Museum Icon store room. unique capability to rapidly analyze large samples sets while retaining the ability to Their gilding techniques, elemental analysis by powder X-ray diffraction (PXRD), chromatographically separate unwanted components and background interference. microscopic examinations and treatment methods. Additionally, Hagia Sophia’s Through the implementation and integration of this technology, high quality MS data store room conditions and air quality would be open for discussion through some can now be collected at speeds capable of supporting the demands of large scale inductively coupled plasma optical emission spectrometry and ion chromatogra- high throughput screening. phy results of the water which collected dehumidifier machines from store room. A bronze Eros Statue (with puppy) from Roman Period. Its radiographic examinations, 122 Terahertz in Art Culture and Coatings elemental analysis by powder x-ray fluorescence, corrosion analysis (by Raman Irl Duling, Advanced Photonix Inc., 2925 Boardwalk Dr., Ann Arbor, MI spectroscopy and XRD) and simulation of buried environmental conditions. A met- 48104 al manufacturing fashion on very beginning of Iron Age in Anatolia (9-6 BC) from The study and conservation of art and cultural heritage artifacts can be hindered by two civilizations: Phyrig and Urartu. The microscopic and elemental examinations lack of knowledge of the internal structure of the object. Pulsed terahertz systems of their high tin bronzes which have golden appearance and their manufacturing have proven to be useful in determining this internal structure because not only can techniques. they image objects that might be under the surface, they can provide information on the layer structure of the object similar to ultrasound but without the need for a cou- 126 What is Your Cultural Competence Level as a Laboratory Manager? pling medium. The practicality of these systems has improved remarkably over the Ephraim Muchada Govere, The Pennsylvania State University, 457 past 10 years in both sensitivity and speed. The quality of the data and the analysis Agricultural Sciences and Industry Building, University Park, PA 16802 performed can lead to higher resolution of the layered structure, while state-of-the- Increases in minority populations in North America and across Europe, and in- 19 2015 EAS Abstracts November 2015

ter- and intra-population migrations within Africa and Asia coupled by the rapidly 131 Wildlife Forensics: Techniques and Applications increasing globalization of economies and lifestyles are greatly influencing the racial Jane Huffman, East Stroudsburg University, Biology Dept., Moore Hall, and ethnic composition of the laboratory personnel and target markets. It is a matter 200 Prospect St., East Stroudsburg, PA 18301 of arithmetic that in countries like the United States of America and United Kingdom, No abstract submitted by the author. minorities will become the majority and one third of the population, respectively, by 2050. Because the laboratory manager is directly involved in the day-to-day opera- 132 What is RAPID DNA Analysis? Introduction and New Research tions of the laboratory, she/he cannot afford to be culturally incompetent. A culturally Directions incompetent laboratory manager is one who lacks exposure to and understanding of Tracey Dawson Cruz, Virginia Commonwealth University, 1015 Floyd different racial, ethnic, and cultural groups and injects individual biases, prejudices, Ave., Richmond, VA 23284 and acts of discrimination into the decision making process. Are you that kind of a In 2008, the Federal Bureau of Investigation’s (FBI) Rapid DNA initiative partnered manager? If not, at what level are you culturally competent? The starting point to with the Departments of Defense and Homeland Security to lead development becoming an effective cross-cultural manager is to know your cultural competence efforts for point-of-collection DNA analysis. The goals of these agencies were to level. Are you at the Distinguished, Superior, Advanced, (advanced high, mid or produce fully automated instruments that would replace the manual laboratory low), Intermediate (intermediate high, mid or low), or Novice (novice high, mid or processing steps of extraction, amplification, separation and detection, and allele low) cultural competence level? By the end of the lecture, you will know your cultural calling/data interpretation such that CODIS-eligible profiles from (known) reference competence level and that you will know how to develop a plan of action to reach samples could be produced in less than 2 hours. It was envisioned that these instru- the distinguished cultural competence level. Cultural competence has been asso- ments would be made available to both accredited laboratories and booking stations ciated with increased customer and personnel satisfaction, and higher productivity in an effort to eliminate the weeks-to-months processing time currently needed. As and profits. of today, two RAPID instruments are commercially available – RapidHIT 200 by IntegenX and the DNAScan by GE Healthcare. Both systems employ microfluid- 127 Using Technology to Advance your Lab Through the Use of a LIMS ics strategies for fluidic control across microscale chip modules that are designed Solution to individually replace each traditional analytical step of the forensic DNA analysis Kim Charles, Labvantage Solutions, 265 Davidson Ave., Somerset, NJ workflow. While a quality assurance addendum has been released laying a foun- 08873 dation for implementation of these RAPID systems in accredited labs, data from No abstract submitted by the author. neither system has been approved by NDIS for CODIS upload. Further, since exist- ing federal legislation requires CODIS DNA records to be generated by accredited 128 Managing the Sandbox: Coaching Toward Collaboration and laboratories, statutory authorization would be needed before DNA profiles generat- Teamwork ed at booking stations could be searched or stored in CODIS. Current RAPID DNA Richard Durand, Sun Chemical, 631 Central Ave., Carlstadt, NJ 07072 research and development efforts include modification of microanalytical systems Collaboration is a key tool for experiential knowledge transfer and can provide for to accommodate compromised casework samples, which would also require the sustaining tacit knowledge within your laboratory over the passage of time and addition of a quantitation module (per existing QAS requirements). Lastly, research through changes of personnel. Collaboration is not something that that we as lead- efforts to change microfluidic control mechanisms to those that do not require bulky ers can expect will naturally happen in our laboratories. We can definitely encourage pumps and pneumatic valving could greatly reduce instrument footprints and im- and stimulate the process, but we need to understand how perspectives of your prove portability and ruggedization. team members may impact reaching a strong engagement by all the players. Deci- sions on which projects and which team members should be involved needs to be 133 Validation and Implementation of the RapidHIT® 200 System in a carefully considered to gain the technical benefits as well as to maintain team har- Crime Lab mony to sustain the collaborative model going forward. In cases where resources Megan M. Boll, NMS Labs, 2300 Stratford Ave., Willow Grove, PA are limited and a timely response is critical, collaboration can improve the overall 19090, Britton Morin, Christian Westring quality of the outputs of the team. Some examples of how to manage this important Rapid DNA analysis is a popular emerging topic in the field of forensic science. process within the laboratory are shared. Currently, processing of biological evidence for DNA left at crime scenes is a la- bor-intensive task and can take from a week, up to several months to perform. 129 Lab Leadership-Are you a Chemist or a Manager? The introduction of rapid DNA analysis to a laboratory can significantly decrease Jean-Francois Borny, C B & I, 10100 Bay Area Blvd., Pasadena, TX the amount of time needed to perform DNA analysis by automating the process. 77507 IntegenX has developed the RapidHIT™ Human DNA Identification System which In the ever shrinking world of analytical laboratories, one thing is certain: we must all integrates the multi-component process of DNA analysis into a single instrument wear multiple hats. We look like a travelling vendor on the beaches of Mexico or the that generates a DNA profile in just under two hours. This presentation discusses Trocadero in Paris with six or seven hats on at the same time. Which role is more an internal validation conducted for use of the RapidHIT® on casework at NMS important for you? Being a manager or being a chemist? Unfortunately, at times, we Labs and its implementation into the current workflow. The instrument performed get promoted because we do such a good job as a chemist, but we don’t necessarily exceptionally in all of the validation studies including: sensitivity, repeatability, repro- desire to be a manager. The job is ours, now what? Managing a laboratory requires ducibility, mixtures of different contributors, and inhibition. Mock-case type samples a different way of thinking, a different set of skills, and a different culture that can were also analyzed using the RapidHIT®. Results generated were comparable to be overwhelming when we are chemists first. In this presentation, we explore the what is expected from the current DNA analysis methods for most sample types shift necessary to manage a lab, remain sane and hold on to what it means to us to tested. This novel technology can be used in forensic laboratories to help decrease be chemists. Let’s not experience burn out, but let’s strive in what we enjoy doing. sample processing time significantly while maintaining the high standards placed on the forensic DNA community. 130 Wildlife Forensics: Development of In-House Assays for Non- Human Testing with Emphasis on Elephant DNA Testing 134 Quality-by-Control Approaches for Crystallization Systems Using Jillian Fesolovich, University of the Sciences, 600 South 43rd St., Spectroscopy-Based Feedback Control Technologies Philadelphia, PA 19104, Meredith Rohrbaugh Zoltan Nagy, Purdue University, School of Chemical Engineering, West Wildlife forensic science is an emerging sub-field of forensic science with origins Lafayette, IN 47907 dating to the late 1980s. Wildlife forensics bridges law enforcement to the conserva- The advent of process analytical technologies (PAT) more than a decade ago has tion and protection of non-domesticated animals. The loss of biodiversity has alerted brought the applications of advanced control of pharmaceutical manufacturing governments and prompted law enforcement to strengthen legislation regulating processes in the realm of possibility. In this presentation the benefits of modern hunting and trade in wildlife products and derivatives. A novel approach to conser- model-free and model-based feedback control approaches will be exemplified in vation is the development of laboratory tests to identify wildlife species significantly the case of batch and continuous pharmaceutical crystallization systems. Recent affected by illegal hunting and trade throughout the world. Several molecular ap- advances in the monitoring, modeling and control of crystalline product properties, proaches to wildlife forensics exist, such as antibody-based assays, mitochondrial such as purity, size and shape distribution as well as polymorphic form will be cor- and nuclear DNA sequencing, single nucleotide polymorphism (SNP) typing, and roborated with industrially relevant examples. A Crystallization Process Informatics short tandem repeat (STR) typing. The presentation gives an overview of molecular System (CryPRINS) with a composite PAT array will be described, that enables the tests available and their application to wildlife. The talk summarizes the in-house application of a series of novel control approaches, including aspect ratio control development of such assays and highlight current research for the identification of (ARC), competitive purity control (CPC) and active polymorphic feedback control elephant DNA. (APFC). The role of rigorous mathematical modelling in the systematic optimal de- sign, start-up and control of continuous crystallization processes to achieve and maintain the desired controlled stage of operation will be illustrated through ex- amples for both cascade of mixed suspensions mixed product removal (MSMPR) 20 2015 EAS Abstracts November 2015

as well as plug flow crystallization systems. The talk intends to provide motivating varying number of components over a wide range of peak capacities. In addition, the examples of the next stage of innovation in PAT-based pharmaceutical manufactur- probability of a successful separation is compared between traditional high-perfor- ing, illustrating the potential benefits of the new quality-by-control (QbC) framework mance liquid chromatography and SE-LC to emphasize the advantage of the latter in improving product quality and process efficiency while reducing costs and time- approach. Comparisons to experimental results are examined and applications of to-market. the predictive programs are explored for future work. References: 135 Quantitative Transmission Raman Spectroscopy of Bilayered [1] A. Socia and J. P. Foley, J Chromatogr A, 2014, 1324, 36-48. Tablets [2] M. Martin, D. Herman, and G. Guiochon, Anal Chem, 1986, 58, 2200-2207. Gary McGeorge, Bristol-Myers Squibb, 1 Squibb Dr., New Brunswick, NJ 08901, Yan Zhang 139 Modeling HPLC Method Robustness Transmission Raman spectroscopy (TRS) has become an increasingly applied Imre Molnár, Molnár-Institute for Applied Chromatography, technology in the analysis of pharmaceutical tablets for quality control purposes Schneegloeckchenstraße 47, Berlin 10407, Germany and developing formulation and process understanding. One area that has received It is 40 years ago, that Csaba Horváth discovered in 1975 the reason for the suc- only cursory attention to date is that of bilayered tablets that represents an unusu- cess of reversed phase chromatography (RPC) at Yale – changes in the structure ally challenging situation. A bilayered tablet is composed of two discretely unique of water, what he called the “Solvophobic Theory”, explaining the power of gradi- formulations that are sequentially compressed, one after the other to create a lay- ent elution in RPC with aqueous eluents. He wrote a program in Basic about how ered composite tablet. Traditional absorption techniques are unable to quantitatively the dissociation of acids might be modeled by equations in high-performance liquid assess the tablet composition without imposing constraints on several of the tablets chromatography (HPLC). Later in 1985 the development of the HPLC modeling soft- manufacturing variables. This presentation will provide an understanding of the rela- ware DryLab started under the leadership of Lloyd Snyder to improve the efficiency tionship of the active pharmaceutical ingredient (API) content and the transmission in HPLC method development. In 2013 the Nobel Prize for Chemistry was granted Raman spectral response in bilayered pharmaceutical tablets to facilitate develop- to scientists, who were modeling physico-chemical interactions in biochemistry. The ment of quantitative models for the prediction of API content in bilayer tablets. The Molnár-Institute is modeling HPLC since 1985, i.e., since 30 years. The recognition Raman intensity was considered as a function of the Raman photon generation and by the Nobel committee about the importance of modeling is a great motivation for decay in a layer of interest and Raman photon decay from a second layer. The com- the whole Life Science community. Modeling helps to find the best column and the plex spectral response was effectively modeled according to a modified Schrader, best separation conditions in HPLC by calculating a large number of possible chro- Kubela-Munk model where both the Raman photon generation factor and photon matograms. Today we are modeling separation selectivity choices in a multi-variable losses were accounted for. The combined results of these studies yields a compre- design space to be able to find the most robust method for industrial drug production hensive approach for modeling multi-component bilayer tablets. The addition of a at reasonable cost. With a computer generated separation model at hand, we can beam enhancer on the bottom surface allowed for a selective over-enhancement of calculate the influence of various experimental parameters and model the robust- the bottom layer, which helps in the analysis of thin layers or coatings. ness of a separation. This is extremely useful if a method should be used for a long period of time, where one has to take into account day-to-day variations in method 136 Hyperspectral Image Calibrations for Transdermal Delivery Sys- conditions. The talk demonstrates in case studies, how modeling software can work tems together with modern instruments to produce the best separation in as fast as a Carl A. Anderson, Duquesne University Center for Pharmaceutical few hours. Technology, 600 Forbes Ave., Pittsburgh, PA 15282, Benoît Igne, James K. Drennen III 140 Kinetic Model of HPLC Column Re-Equilibration after Gradient Monitoring and controlling the manufacturing transdermal delivery systems poses Elution a number of challenges. A critical aspect is the amount of drug containing coating Michael R. Fletcher, Drexel University, Dept. of Chemistry, 3141 deposited on the backing of the drug product. The key to controlling this quality at- Chestnut St., Philadelphia, PA 19104, Joe P. Foley tribute of the product during development and routine manufacturing is an accurate In traditional high-performance liquid chromatography (HPLC), column re-equilibra- determination of coating thickness during processing. The traditional approach to tion between gradient elution runs is necessary to prepare a column for subsequent this determination is to cut samples out of the web and weigh them. This process experiments. This process replaces the final mobile phase in the column with the is slow (in comparison to the speed of the web) and destructive. Near-infrared hy- mobile phase employed at the start of the run. The replacement of mobile phase perspectral imaging has been used as an effective tool to monitor coating thickness occurs in three regions within the column: 1) between the particles (interparticle); of a web-based transdermal delivery system from development through routine 2) within the pores of the particles (intraparticle); and 3) in the interfacial region manufacturing. This talk focuses on the calibration and implementation of such a between the mobile phase and stationary phase. Column re-equilibration, which we system. In particular, the key contributions to this project from this year’s winner of hypothesize is limited by the rate of diffusion within the pores and in the interfacial the “EAS Award for Outstanding Achievements in Near-Infrared Spectroscopy,” Dr. region, traditionally consumes a large quantity of mobile phase due to the high flow Benoit Igne, are highlighted. rates commonly employed during the re-equilibration process. To evaluate this un- derstanding, the effect of flow rate on the re-equilibration time and solvent consump- 137 The Role of PAT in Continuous Manufacturing tion is studied using a variety of column sizes and stationary-phase composition. In Jennifer Schubert, Vertex, 50 Northern Ave., Boston, MA 02210 addition, the effect of the solvent identity (acetonitrile vs. methanol) and the magni- No abstract submitted by the author. tude of the change in mobile phase composition on the re-equilibration time is also evaluated. The volume of mobile phase needed for re-equilibration is shown to be 138 Development of Computational Approaches for the Prediction of proportional to the flow rate employed during the re-equilibration process, thereby Elution Order and Probability of a Successful Separation in showing that solvent consumption can indeed be reduced by lowering the flow rate Sequential Elution Liquid Chromatography during this process. In addition, the time necessary for re-equilibration at different Erin J. Ennis, Drexel University, Dept. of Chemistry, 3141 Chestnut St., flow rates is shown to be influenced by two terms, one which is flow rate dependent Philadelphia, PA 19104, Joe P. Foley and one which is flow rate independent. Sequential-elution liquid chromatography (SE-LC), through the use of multiple elu- tion modes, can separate analytes by class and within class by employing one or 141 Relative Cost-Effectiveness Modeling to Understand the Value of more selective gradients followed by a universal gradient [1]. During the method Preparative Chiral Separations in Pharmaceutical R&D development process, it is beneficial to be able to predict the elution order and, Keith Galyan, Averica Discovery, 260 Cedar Hill St., Marlborough, MA furthermore, the feasibility of utilizing the selective gradients for a multi-component 01752, Jeffrey Kiplinger, Paul Lefebvre, Emily Showell-Rouse sample of different classes of compounds, such as weak acids, weak bases, and Chiral chromatography has been used to prepare samples of single stereoisomers neutral compounds. Predictive calculations developed in matrix laboratory (MAT- for small to mid-scale efficacy, pharmacology, and toxicity testing. Conventional LAB) simulate SE-LC conditions for the separation of multi-component samples wisdom holds that chromatographic isolation of isomers of drugs becomes less to determine elution order. Additionally, the probability of successful separation is cost-effective as the synthetic scale increases, so efforts to develop a stereospecific explored. In the past, the probability of a successful separation (resolution >= 1.5) synthesis or a “batch resolution” approach are often initiated well before clinical for moderately complex samples could be estimated using theory developed by trials begin. The question of when – at what point in the research and development Martin et al. in which two peaks defined the boundaries of the separation space, (R&D) timeline, or at what scale-up level – chromatographic preparation becomes while the remaining peaks are placed randomly [2]. To avoid the obvious bias in a cost burden is not often quantitatively evaluated. The choice often depends on that approach, we employ a stochastic approach utilizing MATLAB that allows all of balancing the costs of developing a non-chromatographic process (which will be the peaks to be placed randomly in the separation space. This stochastic approach wasted if the compound does not progress to clinical stage) against the cost-effec- was used to predict the probability of a complete separation for samples with a tiveness of chromatography. Traditionally chromatographic efficiency is measured

21 2015 EAS Abstracts November 2015

through loading studies, which provide an absolute productivity figure of merit in the resultant prediction errors are evaluated. Determining a “good” tuning param- units such as KKD (kg feedstock/kg stationary phase/day). We suggest that an alter- eter value with this one model evaluation measure is sometimes difficult. Including nate approach, based on evaluating the relative efficiency of feedstocks produced additional measures of model quality allows selection of a better balanced model. by different chemistry options, offers a better assessment of cost-effectiveness. For To demonstrate the utility of the fusion processes for outlier detection and model se- example, partial enrichment of a desired isomer, changes in the impurity profile of a lection, a near-infrared (NIR) spectral data set is evaluated using PLS and RR. The mixture, or protection/deprotection strategies can dramatically influence separation fusion processes are extended to selecting tuning parameters for multiple model efficiency. Planning to compare production efficiency of different feeds is a rapid penalties (constraints). Presented are NIR examples for calibration model updating process, and offers more information than a final loading study on a synthetic prod- and smoothing. uct. Developing a chemistry strategy based on this feedback optimizes R&D pro- gram support during drug candidate profiling and de-risking, and facilitates cost-ef- 145 The “Corporate Approach” in Chemometrics: Using a Hierarchy in fective decision making prior to and during good manufacturing practice production. Classification of Complex Data Steven D. Brown, University of Delaware, Dept. of Chemistry, 163 The 142 Sequential Elution Liquid Chromatography-Mass Spectrometry Green, Newark, DE 19716 Using a Wide-Range pH Gradient Classification is among the oldest methodology in chemometrics, but surprisingly, Catherine Kita, Drexel University, Dept. of Chemistry, 3141 Chestnut St., no effort has been made to take advantage of a source of information present in Philadelphia, PA 19104, Joe P. Foley classifications where many classes are present. By considering the relationships Sequential elution liquid chromatography (SE-LC) is a novel approach for the liquid among classes - e.g., a family tree or a corporate chart – it may be possible to chromatographic separation of samples comprised of two of more classes of ana- include additional information. This presentation considers structure in classes and lytes via the use of multiple selective elution modes. By employing a wide-range pH examines when there may be a benefit to explicitly indicating that structure in the gradient in the mobile phase, a sample composed of weak acids and weak bases classification, and how that might be accomplished. Some comparisons are made can be separated by class and within class (i.e., each analyte from another). Pre- with conventional “flat” classification. vious work showed a higher probability of a successful separation utilizing SE-LC compared to conventional high-performance liquid chromatography (HPLC), due to 146 Mythbusters - Chemometrics Edition the increased peak capacity and decreased separation disorder of the former.[1] Christopher Brown, 908 Devices, 309 Willow Hill Court, Los Gatos, CA In this work, a mass-spectrometry-compatible pH gradient based on a wide-range 95032 buffer system is employed for the sequential-elution mediated separation of ion- Chemometric knowledge and application continues its march into mainstream an- izable compounds by liquid chromatography-mass spectrometry. The compounds alytical chemistry and emerging fields such as ‘omics. As with all maturing tech- examined thus far include small organic molecules and amino acids. Experimental nologies, practitioners now run the gamut from topical tool-users to deep domain emphasis is on the optimization of chromatographic figures of merit and on the experts. Folklore emerges, passed down from generation to generation and practi- viability of the proposed gradient for the analyte detection by mass spectrometry. tioner to practitioner. Some folklore is well-founded, but not all, and as in all scien- Reference: tific endeavors separating fact from fiction can be a productive exercise. I’ll review [1] A. Socia and J. P. Foley, J Chromatography A, 2014, 1324, 36-48. some common chemometric myths, their presumed origins and, with an eye to the underlying theory of these methods, comment on their likely status as fact/fiction. 143 Automating Multivariate Curve Resolution for Success David M. Haaland, Spectral Resolutions, 809 Richmond Dr. SE, 147 On the Evolution of Data-Driven to Error-Driven Multivariate Albuquerque, NM 87106, Howland D.T. Jones, David M. Melgaard, Analysis Jerilyn A. Timlin Peter D. Wentzell, Dalhousie University, Dept. of Chemistry, PO Box Multivariate curve resolution (MCR) is a widely used approach for identifying spec- 15000, Halifax, NS B3H4R2, Canada tral pure components and their relative concentrations from hyperspectral images. The emergence of chemometrics as a substantial field of analytical chemistry in the Using data from a confocal hyperspectral fluorescence imaging microscope, we 1970s represented a profound expansion in chemical measurement science from show that a combination of proper experimental design and thorough characteri- approaches that were primarily model-driven to those that included data-driven par- zation of the hyperspectral imaging system can greatly improve the accuracy with adigms. This fundamental philosophical change has not always been warmly em- which the MCR algorithm unmixes hyperspectral image data and increases the in- braced, but has proven itself time and again in addressing complex problems, and terpretability of the results. A full understanding of each hyperspectral imaging sys- changed the face of analytical chemistry. Thousands of methods and applications tem’s noise characteristics along with identifying when to use spatial compression have been described over the years, with the majority targeted at the primary areas and the introduction of automated spectral and spatial region of interest selection of multivariate regression/calibration, exploratory data analysis/classification, and can aid in automating the MCR analysis to reduce user input and minimize potential modeling/curve resolution. However, most of these approaches have been based variability in results. Examples of successful strategies for experimental design and on statistical tools that make implicit assumptions about the measurement error data analytical treatment approaches that improve MCR automation and success structures, specifically that the measurement noise is independent and identically are presented. In particular, the results of MCR analysis applied to hyperspectral distributed with a normal distribution (iid normal). In fact, most multivariate chemical fluorescence microscope images of rat basophilic leukemic (RBL) cells with multiple measurements violate this assumption and have complex error structures that in- colored quantum dots used to tag proteins on the surface of the cells will be used to clude non-uniform variance (heteroscedasticity) and correlated errors. This has led demonstrate the challenges the analyses of these images represent and illustrate to data analysis methods that are often suboptimal, the use of data preprocessing the success of the automated multivariate curve resolution approach. *Sandia Na- methods that can seem arbitrary, and inadequate diagnostics for prediction. The tional Laboratories is a multi-program laboratory managed and operated by Sandia recognition of these limitations is also giving rise to further evolution of multivariate Corporation, a wholly owned subsidiary of Lockheed Martin Corporation, for the methods in chemistry (and other fields) based on a better understanding of mea- U.S. Department of Energy’s National Nuclear Security Administration under con- surement errors and the limitations they impose. The term “error-driven” is used tract DE-AC04-94AL85000. here to describe multivariate analysis tools that use measurement error information along with the data to improve analytical results. In this presentation, the current 144 Advancing Multivariate Calibration with Fusion of Multiple state and limitations of some of these methods are described. Measures of Model Quality John Kalivas, Idaho State University, 921 South 8th Ave., Stop 8023, 148 Technical Entrepreneurship: The Value of a Good Idea Pocatello, ID 83209, Brett Brownfield, Alister Temcate Arturo E. Osorio, Rutgers University Business School, 1 Washington Outlier detection is imperative before forming a multivariate calibration model. Stan- Pk., Newark, NJ 07102 dard outlier measures are typically used individually and sometimes in a graphical Neat idea or valuable business proposition? Technical entrepreneurship integrates binary combination. Regardless, diverse measures often identify different samples specialized scientific and/or technological skills with business abilities to develop a as outliers, e.g., a spectrum shape measure versus a spectrum magnitude mea- value proposition that directly solves a concrete problem in exchange for an econom- sure. Additionally, many outlier detection methods require selection of a model (such ic gain. The economic value of the proposition is the direct result of the urgency to as the number of eigenvectors for Mahalanobis distance). Presented are new outlier solve a problem, its recurrence, the size of the affected population, the uniqueness measures and fusion processes that allow multiple outlier measures (and multiple of the solution, and the solution’s implementation costs. Well-tuned entrepreneurial eigenvectors) to be used simultaneously to form a consensus decision for a particu- skills serve to identify a competitive advantage within the value proposition and as- lar sample. Simultaneous evaluation of any number of outlier measures is possible. sess the potential differences between launching a new venture versus transferring Another use for fusion of quality measures is to select tuning parameters for multi- the solution to a third party in exchange for an economic gain. This session presents variate calibration methods such as partial least squares (PLS) or ridge regression a succinct overview of the major elements to take into account when assessing the (RR). A tuning parameter value is often determined through cross! -validation and significance of a new idea with the purpose to determine its potential economic value.

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149 Production and Operations Management for Engineering current thinking about the value of viewing analytical methods from a lifecycle per- Enterprises spective, and incorporating quality-by-design concepts. We address establishing Yao Zhao, Rutgers University Business School, 1 Washington Pk., the requirements in an analytical target profile, developing valuable method under- Newark, NJ 07102 standing that goes beyond the traditional robustness experiments and establishing Production and operations management is the processes and activities that create a control strategy, based on risk assessment, to assure the analytical method per- value in the form of goods and services by transforming inputs into outputs. For forms as expected over its entire lifespan. engineering and manufacturing firms, it typically consists of the following parts: new product development, demand forecasting, production planning and scheduling, 154 Method Validation through the Phases of Drug Development distribution, logistics & customer services, inventory management, manufacturing Kevin Dobmeier, Merck, 770 Sumneytown Pike, West Point, PA 19486 process design & quality control, and sourcing, purchasing and supplier manage- As drugs move through development, the focus of the clinical and product quality ment. Through a relevant example, we explain how science-based production and evaluations surrounding them evolves. In a similar manner, the analytical meth- operations management can improve the productivity, reduce waste and increase ods used must adapt to the changing goals of the program. Applying a risk-based, customer satisfaction. staged approach to method validation allows efficient allocation of time and resourc- es through product and method development. Phase-based validation criteria pro- 150 Data Driven Marketing Innovations in Pharmaceutical and vide a means of ensuring that appropriate attention is paid to the key deliverables Healthcare Industries of a method. The use of standard procedures to define the process and the criteria Lei Wang, Rutgers University Business School, 1 Washington Pk., involved can help standardize results and keep non-value added tasks, including Newark, NJ 07102 excessive documentation and approval, at an appropriate minimum. An example A big-data revolution is under way in pharmaceutical and healthcare. There are of a phase-based strategy for validation of small-molecule drug product methods is unprecedented opportunities to collect and use data innovatively to improve health presented. Generalized practices, suitable for rapid and efficient method validation outcomes and business performance. We review various new data sources that at each stage are discussed. In addition, case studies are provided to illustrate have become available in recent years including: open data, mobile data, and social strategies tailored to changing product and method needs. media data. We also discuss how to integrate these new data sources with tradition- ally available data to better understand patient behavior, predict health outcomes 155 Lifecycle Management for Method Validation of Biologics from a and perform value analysis to improve business efficiency. Compendial Perspective Anita Szajek, United States Pharmacopeia, 12601 Twinbrook Pkwy., 151 Leading Innovation: Getting Everyone on the Same Page! Rockville, MD 20878 David Dobrzykowski, Rutgers University Business School, 1 Washington Management of analytical method validation for biologics presents unique challeng- Pk., Newark, NJ 07102 es throughout the product life cycle. Examples of these challenges are second and Managing innovation is a major challenge for organizations. Most efforts to bring third order structure, aggregation, bioassay, immunogenicity and traceability to In- innovations from the “benchtop to business” fail. So, how can professionals improve ternational Standards. For different biologics, critical quality attributes and the regu- their probability of success? A recent consulting report identified four imperatives latory requirements may vary. Recently, the United States Pharmacopeia and Food for companies seeking success in translating innovative ideas into positive results and Drug Administration published guidances on Analytical Lifecycle Management for the organization. Companies must: 1) break down internal silos, 2) excel in both and Analytical Method Validation, respectively. This presentation highlights exist- speed and cost, 3) commit to a more customer-centric approach, and 4) tap into a ing compendial methods and tools available for biologic drug products, approaches larger idea pool. These imperatives center on improving information processing and for validation of non-compendial methods and considerations for analytical method collaboration, both within the company as well as with external supply chain part- comparability. A discussion on the role of International Standards and USP refer- ners. The question is, “how?” This session highlights research from a series of stud- ence standards for biologics are also be presented. ies into how companies translate their desire for innovation into tangible business results. Research from a variety of organizational settings is discussed ranging from 156 Investigating Viral Membrane Protein Structure and Function by global manufacturers to healthcare providers. Participants will leave the session Solid-State NMR with new ideas regarding how to better acquire external information and process it Mei Hong, Massachusetts Institute of Technology, Dept. of Chemistry, internally to deliver on organizational goals for innovation. In addition, participants 77 Mass Ave., Cambridge, MA 02139 will leave with a tangible tool (the VMOST model) which will position them for suc- Many proteins generate membrane curvature for function. Examples include viral cess in leading their next project by better aligning the actions of team members. fusion proteins and proteins that mediate virus budding and release. How protein structure underlies membrane-curvature generation is so far poorly understood. We 152 Lifecycle Management of Compendial Monographs through are investigating the conformation, dynamics, and lipid interactions of two curva- Modernization ture-inducing membrane proteins: the fusion protein of the paramyxovirus PIV5, Leonel A. Santos, United States Pharmacopeia, 12601 Twinbrook which catalyzes virus-cell membrane fusion, and the influenza M2 protein, which Pkwy., Rockville, MD 20878 mediates membrane scission during virus release. 13C and 15N chemical shifts indi- Drug substances and drug products monographs contain compendial methods that cate that the fusion peptide (FP) and transmembrane domain (TMD) of the PIV5 fu- are used to evaluate the identity, strength, and purity of the articles. Modernization sion protein adopt dramatically different conformations in different lipid membranes. of compendial monographs should be performed to ensure that the analytical pro- 31P nuclear magnetic resonance (NMR) line shapes and correlation spectra reveal cedures continue to ensure the quality of the products. Changes during the lifecy- that these conformations differ in their abilities to induce membrane curvature and cle of products such as new or multi sourced starting materials, revised production dehydration, which are necessary for stabilizing fusion intermediates. We charac- processes, new drug product formulation, etc. will warrant the need to evaluate terize the nature of the membrane curvature by solid-state NMR and small-angle the compendial monographs according to its intended use. New technologies or X-ray scattering, and propose possible FP and TM conformations involved in the methodologies should be evaluated to its applicability in support of a public stan- hemifusion intermediate and post-fusion states. To investigate how the influenza dard. Historical monograph modernization is presented as well as future direction of M2 protein generates membrane curvature for virus budding, we developed novel revising the compendia to keep them up-to-date. Application to the modernization of relaxation and correlation approaches involving oriented bicelles under static and over the counter monographs is discussed. off-magic-angle-spinning conditions. The data show that an amphipathic helix in M2 causes high membrane curvature and M2 is localized in this high-curvature 153 Analytical Method Lifecycle: Recent Updates from USP and FDA domain. These NMR approaches can be applied to other curvature-inducing mem- Greg Martin, Complectors Consulting, 804 Bauman Circle, Pottstown, brane proteins. PA 19465 Development and validation of analytical methods has long been an important topic 157 Expediting the Cures: The Role of Solid-State NMR for the pharmaceutical industry. Recently there has been discussion about taking George B. Crull, Bristol-Myers Squibb, Drug Product Science & these topics to the next level, similar to the use of quality-by-design concepts for Technology, PO Box 191, New Brunswick, NJ 08903 manufacturing processes. In fact, the United States Pharmacopeia (USP) and the When one considers the tools required to select and manufacture a pharmaceuti- United States Food and Drug Administration (FDA) have been very interested in cal several come to mind, e.g., powder X-ray diffraction (PXRD), high-performance pursuing this, and have published some of their thinking. USP has formed an Expert liquid chromatography (HPLC), or dissolution, but this presentation highlights the Panel, which published a stimuli article, “Lifecycle Management of Analytical Proce- importance of solid-state nuclear magnetic resonance (ssNMR) in: the selection of dures: Method Development, Procedure Performance Qualification, and Procedure form, selection of state (crystalline vs. amorphous), the control of polymorphs, the Performance Verification”. In 2014, FDA issued a Guidance for Industry: Analytical application to formulated materials, and support for stability studies and marketed Procedures and Methods Validation for Drugs and Biologics, which superseded the materials. Specific examples using ssNMR of H-2, C-13, F-19, N-15 and P-31 are draft guidance issued in 2000. This talk addresses how these articles represent presented. Regulatory and patent agencies are requesting better characterization of 23 2015 EAS Abstracts November 2015

the specific polymorph and conformation of salt form prior to approval. These details 161 Contribution of THz-Time Domain Imaging to Multi-Layered Artifact are frequently only available from ssNMR studies due to the low drug loading and Inspection complex formulations typical of new pharmaceuticals. Because NMR is inherently Corinna Ludovica Koch Dandolo, Technical University of Denmark, quantitative it is frequently applied as the primary method to support secondary Ørsteds Plads, Kgs. Lyngby 2800, Denmark, Peter Uhd Jepsen techniques, e.g., infrared, near-infrared or Raman with a detection limit of 1%. A Imaging with terahertz (THz) waves offers the capability of seeing through tradition- complete understanding of the number of bound waters, the locations and the mo- ally opaque materials, with a spatial resolution comparable to that of the naked eye. bility is now possible using deuterium ssNMR in conjunction with C-13 cross polar- The use of ultrashort THz pulses allows for depth-resolved imaging by time-of-flight ization magic angle spinning (CP-MAS). Amorphous pharmaceuticals and amor- analysis of reflected signals from a target. Therefore, THz imaging has proven its phous solid dispersions are of growing importance as many compounds have low versatility as a new imaging technology for inspection and analysis of historical as solubility. ssNMR can probe the nature(s) of the active pharmaceutical ingredient well as contemporary art, offering detailed information about the internal structure in the polymer as a function of drug to polymer ratio, production conditions or the of such objects, complementary to information available by other methods such impact of stress. No other tool contributes to as many aspects of the pharmaceutical as x-ray radiography and infrared imaging. Here we present comprehensive, fully development process as ssNMR. depth-resolved imaging and analysis of a medieval wall painting, easel paintings and different kinds of panel paintings and lacquerwares. The results have been 158 Recent Development of Magnetic Resonance: Instrumentation, compared with those obtained by other standard imaging techniques. The informa- Physics and Applications tion provided valuable information about the construction, plastering, painting and Yiqiao Song, Schlumberger, 1 Hampshire St., Cambridge, MA 02139 gilding techniques, and expanded the knowledge about the response of different In recent years, nuclear magnetic resonance/magnetic resonance imaging (NMR/ pigments and underdrawings. We are able to image the optical stratigraphy and MRI) has become an important technique for characterization of a variety of porous the single layers composing the art pieces, and to visualize and localize inclusions, media including inorganic materials and biological tissues. In particular, NMR/MRI restoration materials and defects. For this purpose, an effective numerical method measurement of relaxation and diffusion properties has found applications in pe- to separate single THz pulse reflections of interest from the entire signal across troleum exploration, material sciences, medical imaging, and other fields. This talk the image has been developed, which is adapted for uneven surfaces typically en- outlines the physics of the relaxation and diffusion processes in porous media and countered in practical applications of the technique. For the first time, real hidden the recent MR development including multi-echo techniques, compressed sensing, paintings have been imaged by THz-TDI in both a neoclassical easel painting and a two-dimensional methods for diffusion and relaxation, in particular to study complex 17th-century panel painting, with an unexpected richness of details. diffusion dynamics in porous media. I also discuss several applications of these techniques in the study of polymer degradation, molecular composition, porosity 162 Pulsed Terahertz Spectroscopy and Imaging: A Useful Tool in Art in sedimentary rocks, food productions and biological tissues. New ideas of NMR Conservation instrument are presented to include miniaturization of NMR electronics into a silicon Enrique Castro Camus, Center of Investigations in Opitca A.C., Loma chip, and a fully broadband front-end electronics without using the conventional del Bosque 115, Lomas del Campestre, Leon 37150, Mexico, Alma resonant circuit. M. Gomez-Sepulveda, Arturo I. Hernandez-Serrano, Kirsti Krugener, Michael Schwerdtfeger, Stefan F. Busch, Amin Soltani, Martin Koch, 159 Application of Solid State NMR in the Pharmaceutical Industry Wolfgang Viol Dirk Stueber, Merck, 126 E. Lincoln Ave., Rahway, NJ 07065 The band of the electromagnetic spectrum located between the microwaves and the Solid state nuclear magnetic resonance spectroscopy (ssNMR) has emerged as an mid-infrared remained relatively unexplored until about 30 years ago owing to the essential technique for studying pharmaceutical solids. The selection of the optimal lack of suitable sources and detectors of this particular radiation. The introduction polymorphic form of the active pharmaceutical ingredient (API) and the develop- of the technique known as time-domain spectroscopy allowed scientists access to ment of an appropriate formulation of the best API form are often complicated by a this peculiar portion of the spectrum. Since then numerous applications in physics, complex phase behavior of the API. API molecules often exhibit extensive polymor- chemistry, biology and industry have emerged. In the last decade there has been phism and the tendency to form solvates and hydrates. In addition, the interaction of also a small and active community that has focused on using terahertz radiation the desired API lead form with excipients in formulations during processing or during in the study of art work. In this talk I will be presenting some of our recent results long-term storage may lead to form change and amorphization. In many cases, the using terahertz spectroscopy and imaging for the study of sculptural/architectural physical formulation process itself causes API lead form changes or amorphization. artwork as well as oleo on canvas paintings. The cases of study include deteriorat- Consequently, API and formulated samples studied in early drug development often ed stone fragments of two medieval constructions in Germany, namely the Castle contain complex mixtures composed of the desired API lead form in the presence of Celle and the Cathedral of St. Peter of Trier in which internal fractures and the of other API polymorphs, solvates, amorphous forms, and excipients. The ability to restoration of one of them was assessed. In terms of oleo paintings, two pieces of characterize and quantify the components in these complex mixtures in the pres- the altarpiece of the parish of Purisima del Rincon in Mexico originally painted in the ence of each other made ssNMR an indispensable analytical tool in the pharma- mid XVIII century and repainted in the early XX century were characterized, finding ceutical industry. the hidden early images. 160 THz Characterization of Corroded Archaeological Bronzes 163 THz Waveform Techniques for Nondestructive Evaluation in Dense Ilaria Cacciari, IFAC-CNR, Via Madonna del Piano 10, Sesto Fiorentino Materials (FI) 50019, Italy David F. Plusquellic, National Institute of Standards and Technology, 325 The corrosion of alloyed archaeological artefacts produces the formation of various Broadway Ave., Boulder, CO 80305, Shin G. Chou, Paul E. Stutzman, chemical compounds such as carbonated, oxides, sulphates or sulphides on the Shuangzhen Wang, Sung Kim, Robert D. McMichael, Virgil Provenzano, metal surface. Its accurate characterization is constrained by the presence of these Jack Surek, William F. Egelhoff, Edward J. Garboczi corrosion layers. Archaeological finds are commonly examined using non-destruc- Terahertz (THz) radiation is well suited for transmission through many low density tive techniques, that determine the composition of the superficial corrosion layers materials such as paper, plastic and wood but the high extinction from scattering formed on the surface of the finding but not the composition of the underlying ma- in high density materials such as deck concrete makes detection more challeng- terial. Both the heterogeneity of the alloy and the surface patina are responsible of ing. Here, we discuss progress towards detection of common iron oxide corrosion the difficulties in recovering the shape and the surface details of an ancient metal products (α-hematite and α-goethite) through cement materials and coatings on object. The ability to locate its former surface inside the corrosion product is still surfaces. The method relies on detection of absorption resonances associated with a controversial issue, because ready-made techniques as possible alternative to antiferromagnetic resonance transitions (AFM) in the 80 GHz to 1.2 THz range. Nar- THz are still lacking. As far as this author knows, neither detailed examinations of row absorption resonances alleviate the need for pre-corrosion baseline scans that the metal nor comparative studies to assess its conservation state have yet been have plagued non-resonant-based microwave techniques. We demonstrate the use reported. In this work, a number of laboratory prepared metal samples and original of room-temperature high-power solid-state sources and heterodyne receivers for corroded artefacts are characterized using THz spectroscopy. Advanced concepts corrosion product detection through high density materials and discuss advantages are introduced to describe the impact of the metal surface on the THz signal that is realized from the strong temperature dependence of the AFM resonance frequency. reflected from the corrosion layers. THz measurements have been combined with other well-established techniques, such as three-dimensional microscopy and laser 164 LC-MS for the Development of Translational Biomarkers for induced plasma spectroscopy. This provides a complete overview on the character- Environmental Exposures and Genetic Diseases ization of corrosion layers of archaeologic bronze. Clementina Mesaros, University of Pennsylvania, 421 Curie Blvd., Philadelphia, PA 19104, Andrew J. Worth, Ian A. Blair Discovery and validation of translational disease biomarkers remains a challenge in biomedical research. More specifically, novel approaches to biomarker charac- terization are needed to further our understanding of pathogenic factors leading to 24 2015 EAS Abstracts November 2015

disease states. Here, we have employed liquid chromatography mass spectrometry 167 Bioanalytical Challenges and Strategies for Developing a Highly (LC-MS) as a tool for identifying biomarkers applicable to asbestos exposure, as Sensitive LC-MS-MS Method to Quantify Total and Free levels of a well as the genetic disease Friedreich’s ataxia (FRDA). Asbestos is a broad class Soluble Target, Interferon-gamma-inducible Protein-10 at of magnesium silicate particles known to cause mesothelioma by mechanisms in- Picomolar Levels in Human Serum volving oxidative damage. The exceptionally long latency periods of adverse health Hongwei Zhang, Bristol-Myers Squibb, 311 Pennington-Rocky Hill effects resulting from asbestos exposure makes biomarker discovery a critical as- Rd., Pennington, NJ 08534, Qing Xiao, Baomin Xin, Wendy Trigona, pect to identifying at-risk individuals. We performed untargeted lipidomic analysis Adrienne Tymiak, Ashok Dongre, Timothy V. Olah on serum samples from asbestos exposed individuals and mesothelioma patients As translational medicine integrates into many different activities along the dis- to identify serum markers which may hold utility in defining unknown biochemical covery pipeline, biomarker analyses are being increasingly explored and applied consequences resulting from asbestos inhalation. In addition, we have adapted throughout the process in the areas of target validation, assessment of drug effica- targeted metabolic assays involving isotopic tracing to identify unknown biochem- cy, prediction of drug-related toxicity, disease diagnosis and progression monitoring, ical aberrations in FRDA. FRDA is a genetic disease thought to progress in part and adaptive clinical trial designs. Consequently, the demand for the quantitation due to mitochondrial dysfunction. Utilizing isolated platelets as a surrogate tissue of endogenous targets as biomarkers has significantly increased. The chemokine, with translational applications we have identified a distinguishing metabolic profile interferon-gamma-inducible protein-10 (IP-10), is a soluble target for the treatment in FRDA patients in which reliance on fatty acid metabolism is increased to com- of autoimmune diseases. Therapeutic anti-IP-10 mAbs bind specifically to IP-10 pensate for decreased glucose utilization. Importantly, our metabolic findings are and neutralizes its various biological activities. Measurement of both free and total likely to prove useful as a biochemical output of disease state as they correlate with target ligand levels following dosing has been increasingly used in drug develop- known genetic markers of disease severity. Taken together our results show the util- ment to guide decisions and to assess exposure-response relationships in support ity of metabolomics and novel cellular systems as a useful component to identifying of efficacy and safety evaluations and dose selection. In this work, a liquid chroma- novel biomarkers on environmental exposures and genetic diseases. tography tandem mass spectrometry (LC-MS-MS) based method was developed for quantitation of target ligand IP-10 levels after dosing with a therapeutic anti-IP-10 165 A Promising Alternative to SWATH for Biomarker Discovery: An monoclonal antibody. Given that the target ligand, especially in the free form, is Ion-Current Based Approach for Large-Scale, Accurate and present at very low levels (pM), the biggest challenge for LC-MS analysis was insuf- Extensive Proteomic Quantification with Extremely Low Level of ficient sensitivity of the method. We present method development strategies and the Missing Data optimization process that achieved a highly sensitive LC-MS based assay. These Jun Qu, University of Buffalo 701 Ellicott, Buffalo, NY 14203 strategies have also be applied to other small protein therapeutics and targets. Despite the tremendous advances in quantitative proteomics, achieving BOTH high proteomic coverage and low missing data remains highly challenging. Data inde- 168 Utilizing Computational Predictions to Aid in the Design of pendent acquisition may be limited in proteomic coverage. We describe a novel ion- Chromatographic and Electrophoretic Separations current-based MS1 strategy for accurate and extensive proteomic quantification, Donna Blackney, Drexel University, Dept. of Chemistry, 3141 Chestnut with extremely-low levels of missing value and the capacity of quantifying numerous St., Philadelphia, PA 19104, Erin Ennis, Joe P. Foley technical/biological replicates. Robust sample treatment and liquid chromatography In high-performance liquid chromatography (HPLC) and capillary zone electropho- mass spectrometry (LC-MS) approaches were established to enable reproducible resis (CZE), simulation software has been successfully used to aid in the design of analysis; quantitative features were acquired in a data-independent manner with experiments. However, many of the programs available for public use are expen- high-field Oribtrap and a locally-developed informatics package (Ion Star). The sive, complicated to use, and/or lack features needed for specific research endeav- extensive and reproducible preparation and LC separation, and sensitive Orbitrap ors. This work describes software implementations developed in our lab to aid in Fusion analysis laid solid foundation for reliable and comprehensive proteomic specific problems encountered in sequential-elution liquid chromatography (SE-LC) quantification. Additionally, high-resolution MS1 (e.g., 120,000FWHM) detection and dual-opposite injection capillary zone electrophoresis (DOI-CZE). SE-LC is a and optimal peak alignment were found critical to achieve reliable matching and low separation technique in which two or more elution modes are utilized in series for missing data. High quantitative precision (CV<10%) and accuracy (error<15%) were the separation of two or more groups of compounds. In DOI-CZE, sample is injected achieved for a benchmark biomarker system, with only 0.024% of proteins showed at both ends of the capillary under suppressed electroosmotic flow. A careful design missing value in any of the 16 replicates. Moreover, >70% of true positives and of experimental conditions is required in order for each separation technique to be >98% true negatives were correctly identified. We applied this strategy in two bio- successful. Software was designed using a combination of programs such as Mi- marker applications requiring the analysis of a large number of biological replicates. crosoft Excel, Mathlab, and Python to predict the retention time and elution order First, investigation of the effects of three chemotherapy regimens on pancreatic of analytes with minimal experimental data. Simulated chromatograms and electro- cancer cells, which quantified ~5000 unique proteins in all 45 samples without any pherograms that highlight various aspects of the predictive software are presented. missing data. Second, we investigated the temporal changes in lung proteomes at 11 time points post influenza virus infection (44 animals), which quantified ~3800 169 Trace Level Impurity Analysis in Project Manufacturing proteins without any missing data. Both studies provided novel insights into the Andrew P. Kennedy, DuPont Crop Protection, 1090 Elkton Rd., Newark, mechanisms of disease or therapy. DE 19711 Method development for identification of trace level impurities (< 5 ppm) in technical 166 Biochemical and Biological Characterization of IL-25 Enriched grade and formulated products is complicated by numerous factors, including sensi- from Primary Human T-Cells tivity, interferences, individual analyte chemistry, and stability. Furthermore, analysis Darryl Davis, Jansen R&D, 1444 McKean Rd., Spring House, PA 19477, at manufacturing sites imposes additional constraints, such as, critical analysis time, Yazen Jmeian efficiency, and capacity. Two situations are discussed that highlight the value of a IL-25 is a cytokine known to be a component of a defense response to foreign chal- collaborative relationship between research and development and manufacturing to lenges in the respiratory tract and in the gut. However, the complete role of IL-25, address these requirements. A reactive and unstable process impurity previously re- and cellular sources of IL-25 is not very well known. While expression of IL-25 in gut quired a complicated multi-step derivatization procedure to enable analysis through and respiratory epithelial cells to support defense responses has been reported, we liquid chromatography mass spectrometry (LC-MS); however, this derivatization sought to identify additional cells that express IL-25. Specifically, we focused on IL- produced inaccurate and inconsistent results due to formation of interfering com- 25, specifically circulating immune cells as a source of natively expressed IL-25 for ponents and overall lack of robustness. Therefore, a gas chromatography (GC)-MS validation of in-vitro assays and for possible development of therapeutic agents. We method was developed which eliminated the need for the derivatization process, collected blood samples from normal volunteers and assayed cells for intracellular without compromising analysis time and detection limits. The new method was suc- IL-25 by flow cytometry. Results showed that multiple cell lineages expressed IL-25. cessfully transferred to a manufacturing site where it is utilized to ensure the final We then sought to confirm these findings by expanding cells in culture to generate product meets impurity specifications. In another situation, a LC-ultraviolet (UV) sufficient IL-25 to purify and test in functional assays. We were able to expand T method was initially developed for a pH sensitive impurity that required a lengthy cells in culture to yield sufficient amounts of IL-25. We confirmed the identity of IL-25 gradient to resolve the component of interest from the structurally similar active by mass spectrometry and three different cell based assays. The results confirmed ingredient. Recent acquisition of mass spec instrumentation at the manufacturing the presence of IL-25 in activated T cells and indicated that the material is highly site allowed for development of an improved method that resulted in an 87% reduc- active. These results demonstrate for the first time that IL-25 protein is expressed by tion in analysis time and a 100 fold increase in limit of quantitation. The significant T cells and that endogenous IL-25 may be purified and maintains activity. Expanded improvement to the limit of quantitation allowed the manufacturing site to meet new T cells may be a viable source of human IL-25 that may be used to investigate the specification guidelines for this impurity. role of IL-25 in immune responses and how to possibly target IL-25 to attenuate inflammatory disorders.

25 2015 EAS Abstracts November 2015

170 Trace Analysis of Dioxins and Dioxin-Like PCBs Utilizing GC-MS- and is accordingly potentially mutagenic. Therefore, a sensitive analytical meth- MS with a New Sensitive Source od for quantifying NCS at trace levels to support drug development is imperative Jessica Westland, Agilent Technologies, 2850 Centerville Rd., in terms of implementing a control strategy. The high reactivity of NCS and con- Wilmington, DE 19808 comitant presence of extremely high concentrations of API, which constitutes the The European Union (EU) has instituted regulations governing the levels of poly- sample matrix in this case, requires a robust chemical derivatization strategy for chlorodibenzo-p-dioxins (PCDDs), polychloro-dibenzofurans (PCDFs), dioxin-like quantitatively trapping trace levels of NCS. We report herein a method employing (DL) and non-dioxin-like (NDL) PCBs in food and feed. In a new amendment to EU dibenzoylmethane (DBM) as the derivatization reagent. The resulting reaction prod- legislations No. 589/2014 and No. 709/2014, the use of gas chromatography tan- uct, chlorodibenzoylmethane (Cl-DBM), is detected as a surrogate of NCS using dem mass spectrometry (GC-MS-MS) systems has been accepted as confirmatory coordination ion-spray mass spectrometry and monitoring the potassium ion adduct technique for checking compliance with maximum levels (ML). This presentation [M+K+]. During the method development, however, highly variable recovery (poor displays individual analytical criteria; compare the 7000C MS-MS and the new 7010 method accuracy) was observed presumably due to the sample matrix effect. This MS-MS which utilizes a new more sensitive source; and discuss the new GC-MS- presentation discusses the details of this novel NCS quantitation methodology and MS Analyzer system, which includes automated calculations (blank subtraction and the underlying sample matrix effect that was determined to be the source of the limit of quantitation) and a customized report (including specified dioxins, furans, di- poor recovery. oxin-like polychlorinated biphenyls (PCBs), and non-dioxin like PCBs). The system, based on the 7890 GC, uses the multimode inlet (for temperature programmable 175 Pitfalls and Tips for Accurate Quantification of Genotoxic sample introduction), and retention time locking (RTL) for robust and reliable per- Pharmaceutical Impurities formance. The presentation shows the increased benefit of utilizing the new 7010 Li Cui, GlaxoSmithKline, 709 Swedeland Rd., King of Prussia, PA MS-MS for trace analysis of PCDD/Fs and PCBs in food and feed. 19406, Kaina Jiang, Kevin Facchine According to the International Conference on Harmonization (ICH) M7 guidance and 171 2-D-LC: Practical Considerations and Applications for the benefit of patient safety, genotoxic pharmaceutical impurities need to be con- Marcelo Filgueira, The Dow Chemical Company, 400 Arcola Rd., trolled in drug substances usually at parts-per-million (ppm) levels. Developing and Collegeville, PA 19426 validating robust quantitative methods at such low levels presents major analytical On-line comprehensive two-dimensional liquid chromatography (2-D-LC) has been challenges. In this presentation, we will discuss several case studies with respect around for many years, but recent advancements in commercial instrumentation, to the method development challenges encountered and the corresponding trouble- software and practical understanding now allowed this powerful technique to be shooting strategies to identify and address those issues. Resultantly, several meth- used routinely in industrial laboratories for the solution of practical analytical prob- ods for accurate quantification of trace-level genotoxic impurities in various phar- lems. In this presentation we discuss the key aspects that can make the difference maceutical matrices were successfully developed. We share those learnings here in real world applications. and hope to thereby spark further discussions among the trace analysis community.

172 ICH M7 Guideline Implementation Perspectives 176 The Use of Confocal Raman Microscopy in Samples of Forensic Warren Ku, Boehringer Ingelheim, 175 Briar Ridge Rd., Ridgefield, CT Interest 06470 Jennifer M. Leonard, City University of New York Graduate Center, 365 The International Conference on Harmonization (ICH) M7 guideline for assess- 5th Ave., New York, NY 10016 ment and control of DNA reactive (mutagenic) impurities in pharmaceuticals to limit Raman spectroscopy is often employed due to its ability to give a “fingerprint”, or potential carcinogenic risk reached Step 4 (Implementation) in June and Step 5 molecularly specific information. It is characterized by high spectral resolution and is (Adoption) in September of 2014. An M7(R1) Addendum reached Step 2 (Consul- quick, non-contact and involves minimal preparation, making it a popular technique tation) in June 2015, which will provide useful information regarding the acceptable for chemists and criminalists alike. Normal Raman (NR) is often classified as having limits of known mutagenic/carcinogenic impurities and supporting monographs to a low intensity and fluorescence interferences at high energy excitations that often promote consistency of application. The guideline outlines: 1) the scope, general overwhelm a spectrum. However, surfaced enhanced Raman spectroscopy (SERS) principles including considerations for marketed products; 2) what impurities need may be a solution to these issues. SERS also involves enhancement of the Raman to be assessed; 3) evaluating mutagenic potential through quantitative structure-ac- signal in the order of 106-1014. Normal Raman spectroscopy or surface enhanced tivity relationship (QSAR) and Ames testing; 4) acceptable intakes (use of the TTC, Raman spectroscopy is made a more powerful tool with the addition of confocal compound specific intakes, incorporation of less than lifetime exposures); 5) flexible microscopy and this results in additional output and sensitivity. The light is focused options to control mutagenic impurities; and 6) a staged approach to documentation so that a spectrum can be recorded on a particular part of a sample and then moved in regulatory filings. Although implementation of M7 is encouraged after its publi- and recorded again. In addition, mapping and imaging experiments are made possi- cation, because of the complexity of the guideline, its full application (with limited ble. This study involves obtaining representative data sets in inhomogeneous sam- exceptions) is not expected until January 2016 for clinical development filings and ples of forensic interest. By using confocal microscopy to focus on varying parts of July 2017 for new marketing applications that do not include clinical efficacy and a single sample, multiple spectra were obtained and analyzed to see if significant safety data (e.g., new dosage forms, generics) or applicable post-approval changes differences exist throughout one sample with both NR and SERS techniques. In ad- (e.g., new synthetic route). This presentation provides an overview the main M7 and dition, the power of depth profiling was explored to aid in judging the inhomogeneity M7(R1) guideline elements and some implementation perspectives and challenges of a sample and identifying contaminants or different substances at different depths. encountered over its first year. The samples involved in this study include standards and mixtures of organic gun- shot residue constituents, explosives, and ink samples. 173 Diverse and Sensitivity Enhanced GTI Analysis Applications by HILIC-MS 177 Assessing the Utility of the Light Mineral Fraction of Soils for Mohan Kanthasamy, Bristol Myers Squibb, 1 Squibb Dr., New Brunswick, Forensic Applications NJ 08903 Jack Hietpas, Federal Bureau of Investigation – Oak Ridge Institute Control of genotoxic impurities (GTIs) plays a critical role in drug development. Al- for Science and Education, 2501 Investigation Parkway, Quantico, VA though liquid chromatography (LC) coupled with mass spectrometry (MS) has been 22135, JoAnn Buscaglia, Garrett McMahon, Libby Stern applied to quantify GTIs with low ppm sensitivity, it is often challenging to achieve Soils are complex mixtures of botanical fragments, anthropogenic particles, pollen sub-ppm to ppm sensitivity for polar GTI analytes in hydrophobic solvent as the and spores, and minerals, among a vast number of other detrital particles. The sample matrix. To overcome these challenges, a general LC-MS method was devel- mineral assemblages in soils are commonly utilized to make associations between oped using hydrophilic interaction liquid chromatography (HILIC) separation mode. people, objects, and locations. For forensic and provenance studies, the light min- Due to the high organic content of the mobile phase in HILIC mode, the sensitivity of eral fraction (ρ<2.65 g/cm3) of soils is not considered to be as discriminating as the the MS detection and the retain ability of highly polar GTIs are more feasible com- heavy mineral fraction (ρ>2.65 g/cm3). However, the light minerals typically account pared to reverse phase mode. A presentation of this general HILIC-LCMS method for 90-99% of the fine-to-coarse (> 50 um) mineral fraction and are, therefore, more is given along with its successful application to quantify a variety of GTIs and GTI likely to be present in sufficient quantity in the very small (<<1g) sample sizes com- precursors in a number of different sample matrixes. monly encountered in casework. The goal of our research was to investigate metrics to exploit potentially valuable information from the more abundant light mineral frac- 174 Recovery Issues in Trace Level N-chlorosuccinimide (NCS) tion of soils. To this end, eleven surface soil samples were collected from regions Analysis by Chemical Derivatization: Sample Matrix Effect with distinct bedrock/ surface geology, ecoregion, and physiography. We investigat- Lianming Wu, GlaxoSmithKline, 709 Swedeland Rd., King of Prussia, ed the two most common minerals in the light fraction of soils and sediments (quartz PA 19406, Hong Cai, David Q. Liu and feldspar); we assessed the utility of quartz surface textures (Morgan et al. 2010) N-Chlorosuccinimide (NCS) is a commonly used chlorinating agent in the synthesis and major element composition of the feldspar minerals for source discrimination. of active pharmaceutical ingredients (APIs). NCS has been tested Ames positive Interpretation of quartz surface textures can be used to constrain environmental and 26 2015 EAS Abstracts November 2015

petrogenetic origins (Krinsley and Doornkamp 1973). The major element composi- the desired oxidized product of MK-8133 and ~37% of MK-6096, respectively. NMR tion of feldspar is highly variable, and has been successfully used to determine the analysis showed that the metabolites formed were, within the limits of detection, provenance of ancient sediments (Pittman, 1970). The results from this research 100% regio- and stereo-chemically pure. Second, EC was applied for the reductive may provide new metrics and information for domestic criminal investigations and cleavage of disulfide bonds in proteins and peptides. Typically, chemical reduction intelligence purposes. and alkylation followed by desalting is used prior to protein/peptide sequencing by liquid chromatography tandem mass spectrometry (LC-MS/MS). This process is 178 Characteristics and Impact Dynamics of Frangible Ammunition performed off-line and, is resource intensive. We explored whether EC-MS can be Peter Diaczuk, John Jay College, Dept. of Sciences, 524 West 59th St., applied as an on-line tool for disulfide cleavage, followed by LC-MS/MS analysis New York, NY 10019, Jack Hietpas, Xiao Shan Law for modified insulin molecules. Our results showed that disulfide bonds of MK-2640 Frangible bullets are designed to minimize the dangers from ricochet by breaking were reduced instantly at a 50 µL/min flow rate with -1.5 V reduction potential using up or fragmenting upon impact with hard unyielding substrates. The energy of these 1% formic acid as buffer. A chain and B chain products were observed by MS, and smaller post-impact fragments or powder is so small that they cannot travel very far a 40% reduction efficiency was achieved. Proof of concept was achieved, demon- from the initial impact site. To perform this way, these bullets are made of various strating that ultra-pressure LC-EC-MS can be used for Merck modified insulin com- formulations of powdered metals held together by adhesives or resins. In contrast pounds. This methodology has great potential to be applied to in-vitro and in-vivo to the formulations that are chemically bonded together, at least one manufacturer samples to reduce cycle time for pharmacokinetic/pharmacodynamic analysis and has developed a line of frangible ammunition that instead incorporates a jacket to for the identification of metabolites of insulin molecules. encase the frangible core. Because of this novel design, the manufacturer claims that their bullets will also behave on soft organic targets as they behave on hard 182 Mass Spectrometry Analysis of NXS/T Glycosylation Sites in unyielding materials, i.e., by breaking up into small pieces. Using high-speed pho- Recombinant Glycoproteins tography, the impact dynamics of selected brands of frangible bullets are compared Costel C. Darie, Clarkson University, 8 Clarkson Ave., Potsdam, NY to each other and to traditional full metal jacketed bullets to determine their perfor- 13699, Izabela Sokolowska, Armand Ngounou Wetie, Alisa G. Woods mance characteristics common yielding and unyielding materials. Post-impact bullet Full structural characterization of chimeric recombinant proteins that are used as or jacket fragments were recovered and examined microscopically to determine if therapeutics includes analysis of post-translational modifications (PTMs) such as stria from passage down the firearm’s barrel were present and if so, were the stria disulfide bridge assignment or investigation of glycosylation sites. These PTMs are useful for comparison purposes to determine common origin. essential to understanding the complete three-dimensional structure or the confor- mation of these proteins, as well as their solubility or stability. An important feature 179 Forensic Microscopy in Civil Investigations of IgG-based chimeric proteins is the NXS/T N-linked glycosylation site from the Dale K. Purcell, SSCI, A Division of Albany Molecular Research Inc., Fc part of the IgG, a site that may be critical in determining the solubility or stabil- 3065 Kent Ave., West Lafayette, IN 47906 ity of the chimeric protein. Here, we employed a targeted proteomics approach to The recognition, isolation, and identification of specific microscopic particulate con- investigate whether introduction of new N-linked glycosylation sites into a chimeric stituent materials for comparative or inferential source attribution constitute the ba- recombinant protein influences the glycosylation of existing glycosylation sites. Spe- sis for forensic microscopy and microanalysis investigations. Initial examination by cifically, we over-expressed the chimeric construct containing the Fc region of the light microscopy (stereomicroscopy, polarized light and thermal microscopy) is es- IgG fused to the exons 7-8 of mouse ZP3 (IgG-Fc-ZP3E7) and purified the protein sential for determining the morphological and physical attributes of microscopic par- product. We used an IgG-heavy chain (IgG-HC) control. We analyzed glycosylation ticulates such as shape, size, color, agglomeration, refractive indices, and melting of chimeric and control protein by trypsin-AspN double digest by nanoliquid chro- point among others, and to provide investigative clues as to which additional meth- matography-tandem mass spectrometry (nanoLC-MS/MS) using data dependent odologies may be appropriate for more definitive identification or association. Mo- analysis (DDA) and information dependent analysis (IDA or DDA with inclusion list). lecular structural analyses using Fourier transform infrared (FTIR) microspectros- Our findings suggest that manipulation of N-glycosylation of recombinant proteins copy or Raman microspectroscopy are useful in particulate identification. Variable may aid development of new approaches for controlling solubility and stability of pressure scanning electron microscopy with energy dispersive x-ray spectroscopy recombinant proteins. (SEM-EDS) is useful in classifying a large variety of particulates. Comparative and inferential source attribution are discussed using specific examples and illustrations. 183 Raman Spectral Fingerprinting for Biologics Counterfeit Drug Detection 180 Antibody-Like Biorecognition Sites for Proteins from Surface Anna Luczak, Bristol-Myers Squibb, 1 Squibb Dr., New Brunswick, NJ Imprinting on Nanoparticles 08903, Jeremy Peters, Varsha Ganesh, Ravi Kalyanaraman Snehasis Bhakta, University of Connecticut, 55 North Eagleville Rd., The World Health Organization (WHO) estimates that less than 1% of pharmaceu- Storrs CT 06269, Mohammad Saiful Islam Seraji, Steven L. Suib, James tical products sold in developed countries, and 10% to 15% or more in developing F. Rusling countries are believed to be counterfeits. Although most of the counterfeits reported Natural antibodies have found widespread usage in important areas including bio- are drugs that contain small molecular entities as active ingredients, recent trends analysis, cancer therapy and directed drug delivery, but are expensive and can have also include biologics based drugs and the battle against counterfeit biologics is poor stability. In this report, we describe synthesis of antibody-like binding sites growing. In 2009, Pharmaceutical Security Institute (PSI) reported that 3.5% of all by molecular imprinting on silica nanoparticles (SiNP) using a combination of four counterfeit drugs were biologics, compared to 1.2% in 2006. With the recent United organosilane monomers with amino acid-like side groups providing hydrophobic, States Food & Drug Administration’s biosimilar approval and the Avastin counterfeit hydrophylic and H-bonding interactions with target proteins. This provided good sta- example reported in the United States, it is becoming increasingly important that bility, selectivity and specificity of the artificial antibodies (AA) sites in a relatively low pharmaceutical companies find ways to rapidly screen and detect biological coun- cost synthesis. We made SiNP AAs for human serum albumin and glucose oxidase, terfeits. This talk features a novel technique of using drop coat deposition (DCD) for and testing binding stability, selectivity and specificity against several other proteins the drug sample combined with con-focal Raman spectroscopy for biologics drug using adsorption isotherms and surface plasmon resonance (SPR). In addition, the fingerprinting. Specific examples will include monoclonal antibody and fusion pro- Langmuir-Freundlich adsorption model was used to obtain apparent binding con- tein drugs and the spectral analysis focused on the determination of protein sec- stants (KLF) from binding isotherms of HSA (6.7x104) and GOx (4.7x104) to their ondary structure. respective artificial antibodies. These value were 4-300 fold larger than for a series of non-template proteins. SPR studies of AA binding with proteins attached to a 184 Emerging Analytical Diagnostic Tools to Seeing the Unseen gold sensor confirmed good specificity and faster binding for the target proteins Norberto A. Guzman, Princeton Biochemicals, PO Box 7102, Princeton, compared to non-target proteins. NJ 08543, Daniel E. Guzman Over the years analytical chemistry and immunology have contributed significantly 181 Electrochemistry/Mass Spectrometry (EC-MS) – A Tool for to the field of clinical diagnosis by introducing quantitative techniques that can de- Metabolite Synthesis and Online Disulfide Cleavage for Protein tect crucial and distinct chemical, biochemical and cellular biomarkers present in and Peptide Analysis biosamples. Currently, two-dimensional hybrid analytical technologies are emerg- Huifang Yao, Merck, 126 E. Lincoln Ave., Rahway, NJ 07065, Yong Liu ing as powerful tools for the quantification and characterization of several proteins Recent advances in on-line coupling of electrochemistry (EC) with electrospray simultaneously, including those with subtle structural changes such as variants, iso- mass spectrometry (EC-MS) have successfully extended the use of EC into drug forms, peptide fragments, and post-translational modifications. One such technique metabolism investigations. First, we report the use of an electrochemical cell to to perform this challenging task is immunoaffinity capillary electrophoresis (IACE), synthesize major human circulating metabolites of MK-8133 and MK-6096 in mg which combines the use of antibodies, and/or other affinity ligands, as highly selec- quantities in a totally aqueous environment. Importantly, this advance provides a tive capture agents with the high resolving power of capillary electrophoresis. Since green chemistry alternative to traditional methods. A potential of +1.8 V and 20 mM affinity ligands can be polyreactives, binding and capturing more than one mole- sodium phosphphate or 1.0% formic acid as buffer, resulted in a ~50% conversion to cule, they may generate false positive results when tested under mono-dimensional 27 2015 EAS Abstracts November 2015

techniques, such as enzyme-linked immunosorbent assay (ELISA). IACE, on the 188 Rapid Clean-Up Method for Monitoring the Biomarker of other hand, is a bi-dimensional technique that captures, concentrates, separates Dimethylformamide in Blood by LC-MS-MS and identifies each component of a sample, when coupled to one or more detectors Matthew Cleeve, Kinesis Ltd., 9 Orion Court, Ambuscade Rd., simultaneously, without the presence of false positive data. This disruptive tech- Colmworth Business Park, St Neots PE19 8YX, United Kingdom nique capable of preconcentrate on-line and enhance sensitivity of analytes present Dimethylformamide (DMF) is an organic solvent widely used in the chemical and in simple or complex matrices may change the way of testing biomarkers to obtain pharmaceutical industries. Its use in organic synthesis laboratories is common- accurate diagnosis of diseases, hopefully before symptoms of a disease are man- place. Occupational contact of DMF through respiratory and skin absorption might ifested. In this presentation, we discuss the design of a miniaturized multi-dimen- cause damage to cellular DNA of liver and kidney. 3-Methyl-5-isopropylhydantoin sional IACE apparatus, with potential of being used as a point-of-care instrument, (MVH), a metabolite of DMF, was selected as the biomarker to monitor occupa- and capable of improved sensitivity, specificity and throughput. tional exposure to DMF. This work details a rapid and reliable sample preparation method to, following sample pre-treatment, extract 3-Methyl-5-Isopropylhydanto- 185 Hydrophilic Interaction Chromatography (HILIC) and Enzymatic/ in (MVH) from whole blood. The method uses a TELOS 96-well Plate to remove Spectrometric Methods for the Determination of Uric Acid and interferences and allow direct liquid chromatography tandem mass spectrometry Creatinine in Human Biofluids (LC-MS-MS) analysis of MVH. The 96-well plate utilizes an anion exchange reten- Si Zhou, University of Massachusetts Dartmouth, 285 Old Westport Rd., tion mechanism to remove the breakdown components of haemoglobin. As part of North Dartmouth MA 02747, Xiaofei Lu, Yuegang Zuo the method, 3-Methy-5-isobutylhydantion (MIH) was used as internal standard. The Abnormal concentrations of uric acid (UA) and creatinine (Cr) in plasma and urine work investigated dosage levels from 10 to 100mg/kg. The treated samples were are associated with various diseases and are routinely examined in clinical and analyzed using a C18 reversed-phase column and LC-MS-MS without additional biomedical laboratories using enzymatic/photometric techniques. However, many concentration. The TELOS 96-well 25mg Plate offers a suitable sample preparation endogenous and exogenous compounds interfere with these photometrical mea- method to remove matrix components of haemoglobin which may otherwise inter- surements. In this study, a hydrophilic interaction chromatography (HILIC) [1] was fere with the analytical determination of MVH by LC-MS-MS. Used in conjunction developed for the simultaneous determination of uric acid and creatinine in human with LC-MS-MS, the use of a 96-well plate and 7 minute analytical run time provides fluids. The retention of two analytes (UA and Cr) and the internal standard on the col- a high-throughput solution for monitoring exposure to DMF. umn was found to increase with increasing of both pH (range from 3.75 to 5.75) and the percentage of acetonitrile (ACN) in mobile phase. The developed HILIC method 189 Quantification and Profiling of Twenty Three Different Bile Acids has proved fast, selective, and accurate when compared with commonly used en- Kyle Buckley, Environmental and Health Occupational Sciences zymatic/photometrical methods. The enzymatic/spectrometric methods compared Institute, Rutgers University, 170 Frelinghuysen Rd., Piscataway, NJ were based on different reaction principles for these two analytes. The product of 08854, Ill Yang, Grace Guo, Le Zhan the each reaction was quantitatively analyzed by UV-Vis spectrometry. For uric acid, Quantifying bile acids is important as they can be biomarkers of digestive the recovery for HILIC method (85.5-100.4%) is higher than that for spectrometric disease including a severe complication of long term parenteral nutrition (TPN); method (71.5-91.0%), the concentration determined by HILIC is homologous high- TPN-associated cholestasis (TPN-AC). In order to characterize the changes er than that by colorimetric method; for creatinine, the recovery for HILIC method in homeostasis, bile acid profiles are required. Bile acids present significant (83.7-113.6%) is higher than that for colorimetric method (80.8-106.8%), while the challenges both because the concentration varies dramatically for organ to organ concentration determined by HILIC is lower than that by colorimetric method. Inter- as well as sample to sample. We have developed assays for quantifying bile ference is more significant in colorimetric method than in HILIC method. acids in various bio-fluids serum and plasma and tissue including liver using our Reference: existing instrumentation. The acids and their oxidized metabolites challenge the [1] Zuo, Y. Ed., High-Performance Liquid Chromatography (HPLC): Principles, Pro- chromatographic selectivity as most are difficult to resolve and the concentrations cedures and Practices. Nova Science Publishers, Inc., New York (2014). in serum are often at or near the methods detection limits. Concentrations in organ extracts are thousands of times greater and can differ by 2-3 orders of magnitude 186 Incorporation of Biomarkers Separation Research Projects into between acids. The sensitivity will be critical both because of sample type and the Analytical Chemistry Curriculum need to detect relatively small changes. This poster describes the high-performance Yuegang Zuo, University of Massachusetts Dartmouth, 285 Old liquid chromatography tandem mass spectrometry (HPLC-MS-MS) (ITMS) method Westport Rd., Dept. of Chemistry and Biochemistry, North Dartmouth, for analysis in multiple sample types including liver, gall bladder, Ileum, and serum. MA 02747 Detection limits ranged from .1ng/ml to 100ug/ml depending on sample type Analytical chemistry plays an important role in almost every science and engineer- for the 23 bile acids and metabolites included in the current method. Data from ing disciplines, and it becomes a critical part in addressing and resolving broader the optimization of extraction protocol and recoveries for each analyte are also societal concerns. Thus, today’s analytical chemistry education must be more rel- presented. evant to modern analytical laboratory practices. Although traditional undergradu- ate chemistry curricula provide a solid foundation in the fundamental principles of 190 Analytical Method that can Distinguish O vs. N Glucuronides analytical chemistry, they do not formally value practical skills that enable students Rahul Talekar, Merck, 33 Ave. Louis Pasteur, Boston, MA 02115, Adam to adapt and be successful in today’s rapidly changing and competitive analytical Beard, Yong Liu, Karen Owens, Zheng Tan, Roy Helmy workplace. To bridge the current gap between “real work” experiences and universi- Glucuronidation (addition of glucuronic acid) is often involved in xenobiotic metab- ty training in analytical chemistry, the author has incorporated real world biomarkers olism of drugs. Glucuronidation of parent drug containing OH, NH or NH2 groups, separation research projects [1] into analytical chemistry curriculum to prepare our would produce O-glucuronides, N-glucuronides respectively with same mass. In ad- students academically for what the real world wants from them. The incorporated dition, loss of glucuronide is a very favored process in both in source fragmentation biomarkers research projects challenged students to think creatively and improved or MS-MS based on collision induced dissociation, which make it really challenging their skills in communication, teamwork, and problem-solving. The details of this to differentiate N or O glucuronide. There is a need for an analytical method that can innovative curriculum project are given at the presentation. Reference: [1] Y. Zuo, ambiguously determine O vs. N glucuronides. This poster highlights our efforts to 2014, High-Performance Liquid Chromatography (HPLC): Principles, Procedures develop a rapid and efficient analytical method to identify whether the parent drug and Practices. Nova Science Publishers, Inc., New York. has O vs. N glucuronide. 187 Separation of Vitamin D2 and D3 for Clinical Application 191 Label-Free Detection and Differentiation of Pathogenic Bacteria Mark Woodruff, Fortis Technologies, Clayhill Business Park, 45 Using Synthetic Antimicrobial Peptides Coalbrookdale Rd., Neston CH64 3UG, United Kingdom, Ken Butchart Xiaobo Liu, Clarkson University, 8 Clarkson Ave., PO Box 5810, In this poster we discuss the separation of Vitamin D2 and D3, two crucial vitamins Potsdam, NY 13699, Dawei Xu, He Dong, Silvana Andreescu, Mouna either ingested as supplements or synthesized naturally by the skin. They allow the Marrakchi adsorption of several minerals calcium, phosphate, iron, magnesium and zinc in the The demand for rapid and sensitive detection of bacteria is growing significantly human body, which help prevent the disease osteomalacia and rickets, a weakening in biological, biomedical and clinical fields. Due to their shorter detection time and of the bones due to defective bone mineralization. The separation chromatograph- higher sensitivity over the conventional methods, impedance sensors modified ically is important before detection of these compounds as they have the same with biorecognition molecules have been used for detection and quantification of molecular weight, meaning that mass spectrometry detection cannot be relied on to bacteria. In this study, the design and development of an antimicrobial peptides separate them alone. We highlight a rapid highly sensitive method in which a simple (AMP)-based impedance sensor for the detection and quantification of bacteria is polar-endcapped column and mobile phase combination separates the two forms of discussed. The sensor showed different impedimetric responses to various bacterial Vitamin D, allowing high qualitative and quantitative results to be obtained. strains, which may relate to different affinities of the synthesized peptides for various strains. The detection limit of the sensor is 10^2 CFU/mL for the four strains. Scan- ning electron microscopy (SEM) was used to further examine the binding mecha- 28 2015 EAS Abstracts November 2015

nism and the morphology of the AMP modified substrate surface. The relationship the subcutaneous site after sample injection, can cause a significant decrease in between the AMP immobilization process as well as orientation of the peptides and solubility of the sample, resulting in precipitation. The data presented includes an the sensor performances are discussed. This study demonstrates the AMP-based example of an insulin formulation prepared at pH 4. After injection, the pH rises detection strategy provides a robust platform for bacteria detection with excellent through the pI of insulin, causing a drop in solubility and precipitation. At pH’s above applicability in other fields. the pI, insulin re-dissolved. This behavior was recorded in the light transmission and pH data. Non-specific interactions between the extracellular matrix components and 192 Two-Dimensional Analysis of Protein Therapeutics and Amino Acid the protein formulation, may have a detrimental impact on drug absorption. Using Excipients with Combined UV and Charged Aerosol Detection the Sirius Scissor, post-injection stability and diffusional properties of a drug can Bruce Bailey, Thermo Fisher Scientific, 22 Alpha Rd., Chelmsford, MA be monitored and used to aid formulation development and optimize bioavailability. 01824, Marc Plante, Ian Acworth Therapeutic proteins (antibodies and vaccines) vary considerably due to the nature 195 Optimized High-Throughput Mixed-Mode SPE Method for the and dose of the protein molecule. Vaccine formulations differ from therapeutic anti- Analysis of Arachidonic Acid in Plasma by LC-MS-MS body formulations since they often include an adjuvant for immune-enhancement. Matthew Cleeve, Kinesis Ltd., 9 Orion Court, Ambuscade Rd., Protein therapeutics can aggregate during storage depending on pH, temperature, Colmworth Business Park, St Neots PE19 8YX, United Kingdom and protein concentration. Stabilization of protein formulations can be enhanced A high-throughput sample preparation method was developed using a mixed-mode through the addition of surfactants, sugars and amino acids excipients. Eight specif- solid phase extraction 96-well plate for the determination of free arachidonic acid ic amino acids commonly used as excipients in protein therapeutic formulations due in plasma by liquid chromatography tandem mass spectrometry (LC-MS-MS). Ara- to their physicochemical properties include arginine, aspartic acid, glutamic acid, chidonic acid is a long chain polyunsaturated fatty acid. There is a high content of lysine, proline, glycine, histidine, and methionine. The chromatographic separation free arachidonic acid in the human body which usually comes from dietary animal of therapeutic protein and amino acid excipients is performed using a two-dimen- sources, including meat and eggs. Arachidonic acid and its metabolites have strong sional (2-D) approach. Proteins are separated using an Accucore 150 C4 column biological activity and can regulate a variety of physiological processes, such as the using water: acetonitrile gradient with each solvent containing trifluoroacetic acid regulation of lipid and glucose, prevention of cardiovascular disease and the chemo- as an ion pairing agent followed by detection via diode array detector. A heart cut prevention of cancer cells. It is therefore important to determine the concentrations containing the polar amino acids from the sample injection is transferred to a second of free arachidonic acid in human plasma. In addition to mixed-mode solid-phase column via a switching valve. The separation of underivatized amino acid excipi- extraction (SPE), protein precipitation, liquid-liquid extraction (LLE) and non-polar ents and several ions is complete within 20 minutes using hydrophilic interaction SPE were studied for removing both proteins and phospholipid interferences from liquid chromatography mode on the Acclaim Trinity P1 column with charged aerosol plasma. The matrix effect of both phospholipids and proteins on the recovery of ara- detection. The mixed mode Acclaim Trinity P1 column provides cation, anion and chidonic acid was investigated and the final method was applied for the determina- reversed phase separation characteristics so gradient conditions can be adjusted by tion of arachidonic acid in human blood samples. The calibration curve ranged from selecting appropriate ionic buffer strength, pH and level of organic solvents. A mock 10 to 2500 ng/ml with good linearity. Analyte recoveries ranged from 99.4 to 103.2% protein formulation containing a mixture of surfactant, amino acids, ions and protein with RSDs less than 6%. A limit of detection is 3ng/ml was achieved. The proposed is analyzed to demonstrate the successful capability of the described 2-D method. clean-up method is simple, accurate, precise and a high throughput determination of arachidonic acid in plasma samples. In contrast to protein precipitation, liquid-liquid 193 Charging YOYO-1 on Capillary Wall for Online DNA Intercalation extraction and non-polar SPE, the mixed-mode method offers improved elimination and Integrating this Approach with Multiplex PCR and Bare Narrow of matrix effects from phospholipids and proteins for the analysis of arachidonic acid Capillary–Hydrodynamic Chromatography for On-Line DNA in plasma by LC-MS-MS. Analysis Huang Chen, University of Oklahoma, 101 Stephenson Pkwy., Norman, 196 Investigations of Potential Aptamer Interactions Between G4- OK 73019, Zaifang Zhu, Joann Juan Lu, Shaorong Liu forming Oncogene Promoter Sequences and Tumor Proteins Multiplex polymerase chain reaction (PCR) has been widely utilized for high-through- Christina M. Albanese, Rensselaer Polytechnic Institute, 110 8th St., put pathogen identification. Often, a dye is used to intercalate the amplified DNA Troy, NY 12180, Suttipong Suttapitugsakul, Linda B. McGown fragments, and identifications of the pathogens are carried out by DNA melting The ever-increasing demand for targeted biological, diagnostic, and therapeutic curve analysis or gel electrophoresis. Integrating DNA amplification and identifica- tools has necessitated further study and development of effective affinity reagents, tion is a logic path toward maximizing the benefit of multiplex PCR. Although PCR such as aptamers. We have previously proposed a novel method for aptamer dis- and gel electrophoresis have been integrated, replenishing the gels after each run is covery focusing on the genomically prevalent G-quadruplex (G4) motif as a potential tedious and time-consuming. In this technical note, we develop an approach to ad- basis for aptamer capability. Our approach investigates whether G-rich sequences dress this issue. We perform multiplex PCR inside a capillary, transfer the amplified from human oncogene promoter regions can behave as aptamers by specifically fragments to a bare narrow capillary, and measure their lengths online using bare binding proteins extracted from tumor cells. Prior success with the erb-b2 recep- narrow capillary–hydrodynamic chromatography (BaNC-HDC), a new technique re- tor tyrosine kinase 2 (ERBB2) breast cancer promoter region has encouraged the cently developed in our laboratory for free-solution DNA separation. To intercalate expansion of our study to include other G-rich oncogene promoters, including the the DNA with YOYO-1 (a fluorescent dye) for BaNC-HDC, we flush the capillary col- c-myb, c-myc, Rb, and VEGF promoter regions. In order to further investigate the umn with a YOYO-1 solution; positively charged YOYO-1 is adsorbed (or charged) specificity of our DNA-protein interactions, we have begun comparative studies us- onto the negatively charged capillary wall. As DNA molecules are driven down the ing proteins extracted from breast tumors and adjacent normal, non-cancerous tis- column for separation, they react with the YOYO-1 stored on the capillary wall and sue of a single donor. Preliminary matrix-assisted laser desporption/ionization mass are online-intercalated with the dye. With a single YOYO-1 charging, the column spectrometry (MALDI MS) results from experiments performed using sequences can be used for more than 40 runs, although the fluorescence signal intensities from c-myb and c-myc promoter regions have indicated protein capture by both of the DNA peaks decrease gradually. Although the dye-DNA intercalation occurs promoters from the primary tumor but not from the adjacent normal tissue. Here, during the separation, it does not affect the retention times, separation efficiencies, we focus on sequences from the Rb and VEGF promoter regions. Protein capture or resolutions. is screened using MALDI MS. Captured proteins of interest are then analyzed using liquid chromatography MS-MS with Mascot scoring and Western blot interrogation. 194 A New In-Vitro Method to Estimate Formulation Performance of In this way, we aim to discover affinity interactions that will lead to new aptamers Subcutaneously Administered Biopharmaceuticals and that may also have biological significance that may aid in cancer diagnosis and Jon J. Mole, Sirius Analytical Inc., 100 Cummings Center, Beverly, MA treatment. 01915, Karl Box, Hanne M. Kinnunen, Randall J. Mrsny Subcutaneously administered biopharmaceuticals often have low or largely vari- 197 Sedimentation Velocity Analytical Ultracentrifugation as a Method able bioavailability. The chemical, physical and physiological environment of the for Quantifying the Amount of Empty Virion Particles in Adeno- subcutaneous tissue may play a role in determining the extent of biotherapeutic Associated Virus Preparations absorption. A novel instrument for predicting how the characteristics of the subcu- Christopher Sucato, Blue Stream Laboratories, 8 Henshaw St., Woburn, taneous tissue may influence the post-injection stability and diffusional properties MA 01801, Libo Wang, Mario DiPaola has been developed. Sirius Scissor enables identification of any instabilities or un- Adeno-associated virus (AAV) is a small, benign virus which infects humans, and is wanted interactions that may hinder absorption from the injection site in a tractable a promising delivery system for gene therapy applications. While AAV vector sys- manner, thus allowing for rational formulation design. A sample is injected into a tems have been used in many clinical trials worldwide, and promising results have membrane cassette containing an extracellular matrix, where light transmission and been obtained for a number of diseases, certain aspects of the efficiency of vector pH are monitored. Any decrease in transmission indicates precipitation, which may transduction remain to be well characterized. During the production of recombinant be related to pH. Diffusion of sample from the cassette into a carbonate buffer may AAV vectors for gene therapy, empty virions are present after the packaging pro- also be monitored by analysis of collected aliquots. The pH change that occurs in cess, which can result in vector lots with mixtures of full and empty virions at variable 29 2015 EAS Abstracts November 2015

ratios. Further, it has been shown that the presence of empty virions correlates with ranges of 2.16-2.82 and 2.70-4.62 ng/L, respectively; and no 4-cumylphenol was suppression of transduction in human tissue. In this presentation we show that ana- observed. The detailed kinetic study on the photodegradation of BPA and its ana- lytical ultracentrifugation (AUC) is a powerful analytical assay for the quantitation of logues are reported at the presentation. structural heterogeneity in recombinant AAV preparations, allowing for the charac- Reference: terization of empty-to-full virion ratios. The results of sedimentation velocity AUC are [1] Y. Zuo and Z. Zhu (2014) Simultaneous identification and quantification of presented on a number of AAV sample types encompassing the range from empty 4-cumylphenol, 2,4-bis-(dimethylbenzyl)phenol and bisphenol A in prawn Mac- (null) AAVs to fully packaged, and the resulting size distributions are evaluated in the robrachium rosenbergii. Chemosphere 107, 447-453. calculation of empty-to-full ratios. 201 Nano-Impact Electrochemistry for the Routine Characterization of 198 Influence of the “Wire” on the Stability of Enhanced Serum Amine the Chemical Reactivity and Surface Properties of Metal and Metal Oxidase Used in Biosensing Oxide Nanoparticles Mihaela D. Leonida, Fairleigh Dickinson University, SONS, 1000 River Anahita Karimi, Clarkson University, 8 Clarkson Ave., Potsdam, NY Rd., Teaneck, NJ 07666, Eugene Kang, Mikel Romero, Ish Kumar 13699, Daniel Andeescu, Silvana Andreescu Serum amine oxidase (SAO) known to have cardioprotective effect is a potential The electrochemical study of single particle impacts with an electrode is a rapidly therapeutic agent for heart patients. Amine oxidases were also studied for use as developing field which provides extensive and unique capabilities for the detec- sensing agents in biosensors for clinical laboratory and food industry applications. tion and characterization of nanoparticles. The method has demonstrated signifi- SAO (oxidoreductase) has limited stability. This study targets SAO stabilization cant promise for studying nanoparticle properties including nanoparticle coating, by modifying its structure using a green reagent, a room temperature ionic liquid size and shape and catalytic activity. In this presentation, we describe the devel- (RTIL). Since enzymes are more stable in the presence of their substrates, SAO opment of an electroanalytical collision technique to characterize the fundamental was suspended in a RTIL in the presence of pyridoxal phosphate (molecule similar surface properties, functionalization and redox reactivity of metal and metal oxide to its prosthetic group). After removal by dialysis of the denaturing RTIL, SAO refold- nanoparticles by nano-impact electrochemistry. We demonstrate the potential of this ed entrapping some of the pyridoxal phosphate present in the denaturing mixture. method: 1) as a screening tool of particle reactivity; 2) for study of the adsorption/ Other modifiers were used in parallel procedures: copper ions (SAO is a copper desorption of environmental contaminants with single particle resolution; and 3) to enzyme), lipoic acid (LA) - potent antioxidant and intrinsic part of some redox en- extract mechanistic information predictive of the chemical reactivity of nanoparticles zymes, and binary combinations thereof. Activity assays were done to assess the for various applications. We discuss the potential of this approach to complement effect of the modification (“wiring”) on the SAO activity and compare the influence of or replace costly characterization techniques and enable routine characterization of the nature of the modifier. Periodically assays were done to assess the effect of the nanoparticles and their reactivity. modification on enzyme stability and on its antioxidant effect. “Wiring” was beneficial for the modified enzymes (ME) affording enhanced stability compared to starting 202 Simultaneous Direct Analysis of Glyphosate and Aminomethyl- SAO and enhanced kinetics of electron transfer. Biosensors with ME as sensing phosphonic Acid in Surface Water Using UPLC-MS in Selected Ion element were built in which the modifiers served as mediating, enzyme-friendly spe- Recording Mode cies. The biosensors were evaluated for catalytic effect, linearity in amine concen- Anthony A. Provatas, University of Connecticut, CESE, 3107 Horsebarn tration, and stability. Hill Rd., Unit 4210, Storrs, CT 06269, Naman N. Buch, Steven L. Kolakowski, James D. Stuart, Christopher R. Perkins 199 Antibiotic Effects of Essential Oils: Clove and Oregano Oils, on Glyphosate, a nonselective, broad-range weed killer, is one of the most widely used Cariogenic Bacteria as Studied by Microbiological and NMR herbicides in applications for vegetation controls due to its low toxicity. Although Techniques glyphosate has a very low toxicity, it can be harmful to aquatic life in surface water Fernando Commodari, Caldwell University, 120 Bloomfield Ave., at higher concentrations. Glyphosate and aminomethylphosphonic acid (AMPA), its Caldwell, NJ 07006, Agnes T. Berki, Marli F. Pimenta, Dohee Han, main metabolite, are very polar and have a high solubility in water. Methods that Christine L. Cordi, Matthew J. Pampin, Alex Paleski, Rira Lee are usually used to analyze glyphosate are high-pressure liquid chromatography Our goal is to design oral hygiene products that protect the resident bacteria of (HPLC)- fluorescence detection, gas chromatography-mass spectrometry (GC-MS) the oral biota, while inhibiting the growth of cariogenic and gingivitis bacteria. Mi- or HPLC-MS-MS. Due to glyphosate’s high polarity, these current methods require crobiomes of the mouth, from healthy individuals, Escherichia coli and Streptococ- a derivatization step which may slow down the process of analysis. Additionally, If cus sanguinis, bacteria of the healthy oral flora, and Streptococcus salivarius, a HPLC is selected as the analytical technique, it is very challenging to retain under- cariogenic bacterium, were tested. These bacteria were subjected to Kirby-Bauer ivatized glyphosate utilizing reverse-phase chromatography. A validated analytical antibiotic susceptibility testing using the oils and controls without oils. Clove and methodology has been developed and is presented for the direct analysis (without oregano oils inhibited the growth of all tested bacteria with more extensive inhibition derivatization) of glyphosate and AMPA in surface water, utilizing ultra-performance of the cariogenic bacteria, suggesting that these oils could be used to inhibit bacte- liquid chromatography (UPLC)-MS, a UPLC amide column and single ion record- rial growth in the oral cavity. Eugenol is known to be active in clove oil, but work is ing (SIR). The presented analytical approach is significantly faster compared to the to be done in identifying any minor components in this oil and the major and minor existing methods and eliminates any complications associated with analyte deri- components in oregano oil. High resolution (14.1 Tesla) 1H and 13C one-dimensional vatization. and two-dimensional nuclear magnetic resonance (NMR) findings are presented to identify and assign the active component molecules responsible for anti-microbial 203 A New TO-17 Tube for the Investigation of Volatiles and Semi- activity in the essential oil mixtures, using a cryoprobe. Volatiles: Are Targets in Soil Gas being Missed? Abstracts 200-299 Tom Mancuso, PerkinElmer, 710 Bridgeport Ave., Shelton, CT 06484, Bill Hahn, Lee Marotta 200 Examination of Bisphenol A Analogues and their Photodegradation Through rigorous investigation, a new sampling tube and method have been devel- in Natural Water oped to monitor 1,3-butadiene, benzene, toluene, ethyl benzene, xylenes (BTEX) Yuegang Zuo, University of Massachusetts-Dartmouth, 285 Old and the 16 Environmental Protection Agency (EPA) regulated polynuclear aromat- Westport Rd., North Dartmouth, MA 02747, Avis L. Francis, Mohammed ic hydrocarbons (PAHs) in one air sample. The current soil vapor intrusion tube Alshanqiti Alshanqiti, Joseph Michael for measuring analytes in soil gas covers the analyte range from chloromethane Bisphenol A (BPA) is widely used in plastic and other industrial consumer prod- through pyrene. This new tube will enable the determination of analytes whose ucts. Release of bisphenol A and its analogues into the aquatic environment during boiling point range is greater than pyrene extending to Dibenz(a,h)anthracene and manufacture, use and disposal has been a great scientific and public concern due nC40. This research provides “side-by-side” experiments using the current soil va- to their toxicity and endocrine disrupting effect on aquatic wildlife and even human por intrusion (SVI) tube and the new XRO-40 tube to determine if analytes above the beings. More recent studies have shown that these alkylphenols may be bioac- boiling point of phenanthrene are not being recovered in soil gas by the current tube. cumulated in crustacean tissues and other aquatic organisms from polluted water Three sampling sites were investigated in these experiments. Method parameters, and affect the molting processes and survival of American lobster, crab, shrimp and sampling procedures, analytical parameters and reporting limits, and comparison of other wild lives in natural water. [1] Thus, it is important to examine BPA analogues site data of this new tube and method are discussed. and their photodegradation in natural waters. In this study, we have developed gas chromatography-flame ionization detection (GC-FID) and GC-mass spectrometry 204 Benchmarking Real-Time SIFT-MS Analysis of VOCs Against methods for the determination of bisphenol A and its analogues in fresh and coast- Regulatory GC-MS Analysis al marine waters. Bisphenol A, 2,4-bis-(dimethylbenzyl)phenol and 4-cumylphenol Barry J. Prince, Syft Technologies Limited, 3 Craft Pl., Christchurch were found in the concentration ranges of 2.07–2.55, 2.07–2.43, and 0.217-0.223 7691, New Zealand, Vaughan S. Langford, Murray J. McEwan, Daniel ng/L, respectively, in a fresh lake water. In New Bedford Harbor water samples, B. Milligan bisphenol A and 2,4-bis-(dimethybenzyl)phenol were detected in the concentration GC-MS is one of the most widely accepted and used methods for analyzing mix- 30 2015 EAS Abstracts November 2015

tures of volatile organic compounds (VOCs). Although GC-MS is the method of desorption, mass spectrometry and gas chromatography. Preliminary results show choice for most applications, there are difficulties associated with monitoring low appreciable conversion is possible at room temperature and pressure, thus at the levels of VOCs from gaseous samples. Many analyses require adsorption of the bench scale, energy intensive experiments can be avoided. Future work should VOC matrix on to a substrate for pre-concentration which is followed later by a involve mechanistic analysis as well as catalyst modification to increase light ab- thermal desorption step before entering the GC-MS. New real-time methods offer sorption efficiency and conversion. simpler operating procedures and much shorter analysis times than those offered by GC-MS. We present the results of a comparison between a GC-MS study and 208 Quantification of Pesticides in Fruit and Vegetable Samples in the emerging technology of selected ion flow tube mass spectrometry (SIFT-MS). Southwestern Pennsylvania The SIFT-MS instrument was calibrated for 25 compounds from three certified gas Kaelyb Suchevits, California University of PA, 250 University Ave., standards using a serial dilution method in Tedlar sampling bags. Calibration was California, PA 15419 undertaken over a concentration range of 5 ppbv to 1 ppmv (limited at the bottom In this project, fresh fruits and vegetables including apples, peaches, nectarines, end by the bag and at the top end by the standard) and observed to be linear for all and celery grown in a farm in Washington, Pennsylvania were collected and ana- 53 ion-molecule reactions used to quantify the compounds (R2 ≥ 0.997 for all reac- lyzed for pesticide residues using a gas chromatography mass spectrometer. The tions). Four ppbv-range mixtures of 17 compounds were prepared in 6-L passivated fruit and vegetable samples were extracted using the QuEChERS (quick, easy, stainless steel canisters for comparative analysis by GC-MS (using the USEPA TO- cheap, effective, rugged, and safe) method for sample concentration and purifica- 15 method) and SIFT-MS. Good agreement (within 30%) was observed for 14 of the tion. Prior to the extraction, an internal standard of triphenyl phosphate was spiked 25 compounds. Several environmental samples were also compared. Particularly to all samples. About 60 pesticide compounds were identified in the samples by good comparative data were obtained for the aromatic compounds benzene, tolu- authentic multi-residue standard mixtures. A recovery study of all 60 pesticide com- ene, and the sum of ethylbenzene and the three xylenes. pounds was conducted on randomly selected samples (2 per fruit and vegetable). Quantification of each of the 60 pesticide compounds was performed with their rela- 205 Preparation Characterization and Application of H3PO4 Activated tive response to triphenyl phosphate and three quantification m/z ions. Maize Tassel for Remediation of Eutrophic Phosphorus Adebayo Am Shofolahan, Sefako Makgatho Health Sciences University, 209 Methodology for Environmental Assessment of Silver Nanotoxicity PO Box 234, Pretoria 0002, South Africa, Nana Nm Agyei Eduard Dumitrescu, Clarkson University, 8 Clarkson Ave., Box 5810, Technologies for phosphate removal from contaminated waters, such as chemical Potsdam, NY 13699, Xiaobo Liu, Kenneth Wallace, Silvana Andreescu precipitation with lime, are expensive. In this study the feasibility of utilizing low- The last years have seen an increasing use of nanomaterials in industry and ac- cost activated maize tassel for the adsorptive removal of phosphate was assessed. ademic research. This translates to a greater risk of circulation and accumulation Raw maize tassel powder was impregnated with H3PO4 and activated at 600 °C in the environment, as nanomaterials are eliminated through wastewater from do- under an inert atmosphere of N2. The 2:1 (w/w H3PO4: tassel) impregnation prod- mestic and industrial sites. As a result, their potential toxic effects target primarily uct was characterized by BET and scanning electron microscope (SEM) and used the aquatic species. Therefore, methodologies for the assessment of nanotoxicity for adsorption studies. The activation process resulted in an increase in specific in relevant biological models are required. In this work, embryonic zebrafish (Danio surface area and porosity. Batch experiments were performed to study the removal rerio) is used to evaluate the environmental impact of silver nanoparticles (Ag NPs) of phosphate from simulated samples; the optimal parameters were found to be: in aquatic environment. The lethal effects of these NPs and their size dependency contact time of 90 min, pH 7 and adsorbent dosage of 1.5 g per 100 mL solution. were assessed using viability assays through the 5-day embryogenesis of zebrafish The kinetics data were fitted to a pseudo-second order model (R2 > 0.99). The embryos. For comparison purposes, the toxicity of released Ag+ ions was also de- adsorption data were fitted to the Langmuir isotherm model (R2 > 0.99), yielding an termined. A flow injection system (FIA) was used to determine the dissolution profile estimated adsorption capacity of 15.31 mg phosphate per g adsorbent. The activat- of the used Ag NPs in experimental conditions. The alteration of the intestinal nitric ed product was successfully applied for the remediation of phosphate in selected oxide (NO) level as a result of NPs action was studied using a modified-carbon fiber sewage samples. microelectrode. The effects of Ag NPs on the intestine and other tissue of zebraf- ish embryos were studied using histology assay. RNA in-situ probe for inducible 206 Ambient Air Monitoring: What are the Right Tools for the Job? nitric oxide synthase (NOS2) was synthesized and used to determine the elevated Chris Hall, Markes International, 11126 Kenwood Rd., Ste. D, Cincinnati, expression of NOS2. The dose-dependent toxic effects observed for AgNPs and OH 45242, Nicola Watson, Caroline Widdowson soluble ions in zebrafish embryos are discussed. Many techniques are available for the sampling and measurement of volatile organ- ic compounds (VOC) in ambient air, but which is the best to use? Canister samples 210 Withdrawn by the author. are the current gold standard, stipulated by the Environmental Protection Agency (EPA) for TO15 for analysis of air toxics and other volatile organic compounds at 211 Hydrophilic Interaction HPLC Method for the Determination of various concentrations, but as the list of target compounds continues to grow, canis- Ascorbic Acid in Citrus Fruits and Pharmaceutical Formulations ters are being pushed to their performance limit. Another option is the use of sorbent Ruiting Zuo, University of Michigan, 500 S. State St., Ann Arbor, MI tubes. They are very versatile and depending on the sampling situation various 48109, Yiwei Deng, Yuegang Zuo, Si Zhou techniques can be used to retain VOCs on the media; diffusive/passive (EPA Meth- Ascorbic acid (AA), also known as vitamin C, is a natural antioxidant and plays od 325) and pumped/active (EPA TO-17) sampling. On-line analysis for the very an important role in various physiological processes such as biosynthesis of col- volatile, ozone precursors and reactive sulfur compounds is becoming increasingly lagen, norepinephrine, peptide hormones, and tyrosine, intestinal absorption of popular around the US and worldwide. There is no one correct method but rather iron, defense against cellular oxidation, and prevention of cardiovascular disease a range of sampling techniques that can be implemented for varying situations. and cancer. Since humans cannot synthesize AA themselves, they must take this This poster covers the various options available for sampling a variety of VOCs in essential nutrient from foods and beverages. Many analytical methods including a range of situations/conditions, and discuss the advantages and disadvantages of spectrophotometry, voltammetry, gas chromatography, capillary electrophoresis, each technique. In addition to the sampling we also look at the latest developments and high-performance liquid chromatography (HPLC) have been developed for the in analysis and detection of VOCs. determination of AA in fruit juices. Due to the complex matrices with numerous non- specific interferences for the AA determination in fruits by other techniques, HPLC 207 Carbon Dioxide (CO2) Conversion into C1 Chemicals Using has gained popularity. As AA is very polar small molecule, it is difficult to be retained N-Based Photocatalytic Systems in conventional reverse-phase (RP)-HPLC and separated from void volume. A rapid Sherry-Ann Tim Kee, New Jersey Institute of Technology, 1 Rutgers Dr., and sensitive hydrophilic interaction chromatography (HILIC) method is reported Newark, NJ 07103, Xianqin Wang here for the determination of AA in citrus fruits and pharmaceutical formulations. Global warming is currently one of mankind’s biggest challenges. A key player in the Fresh fruits were hand-squeezed, pharmaceutical pills were dissolved in the HILIC global warming issue is the primary greenhouse gas, carbon dioxide (CO2). Carbon mobile phase, then centrifuged, filtered, diluted and separated on a Waters Spher- capture and conversion is an initiative to capture carbon dioxide at point sources isoro S5NH2 column fitted with a C18 guard column using an isocratic elution pro- and convert it into useful products such as formic acid and methanol. Our aim is to gram consisting of a 50% acetonitrile and 50% aqueous phosphate buffer solution. produce a carbon neutral fuel through biomimicry using conventional chemicals and The UV detection was made at the wavelength of 205 nm. AA had a good retention solar energy photocatalysis. This provides the opportunity to transform CO2 from on the amino column and was well separated from other components in citrus juices an environmental threat to an economic resource while also facilitating the closing and pharmaceutical matrices. The developed HILIC technique has been also suc- of the carbon cycle. CO2 will serve as a feedstock to fuels that will replace current cessfully applied to determine AA in human and other animal liquid samples. fossil fuel (gasoline, crude oil and coal) consumption without changing the fossil fuel economy. In this study, we are investigating a N-based photocatalytic system to convert CO2 to C1 chemicals. Various N-based photocatalysts were screened and analyzed using characterization techniques such as temperature programmed 31 2015 EAS Abstracts November 2015

212 Solid-Phase Micro Extraction of Tea Flavor Components liquid chromatography columns with different stationary phase chemistries. The re- Anne Jurek, EST Analytical, 503 Commercial Dr., Fairfield, OH 45014, sults of these analyses were evaluated for optimum resolution and selectivity, and Mike Moses, Kelly Cravenor the conclusions are presented. Tea flavors can vary from spicy to flowery to fruity and any combination thereof. Moreover, the flavor profile of tea can depend on where the tea leaves are grown, 216 The Use of High Definition TD-GC-TOF-MS for Challenging the brewing time and temperature, the processing of the tea leaves, and the type of Analyses in the Food Industry leaf used. Using head space solid-phase micro extraction sampling in conjunction Nicola Watson, Markes International, Ste. 130, 2355 Gold Meadow Wy., with gas chromatography/mass spectrometry for separation and analysis, assorted Gold River, CA 95670, Laura McGregor, Charles Haws, Chris Hall teas are examined for their varied flavor components. Aroma profiles of food contain a wide variety of components at a range of concentra- tions. Detection and identification of important keynote compounds with a low odor 213 Examination of the Adulteration, Counterfeiting and Contamination threshold and those responsible for off-odors is a challenging prospect. The use of Spices, Botanical Products, and Supplements by ICP-OES and of high definition gas chromatography coupled with time-of-flight mass spectrom- ICP-MS etry (GC-TOF-MS) combined with thermal desorption (TD) offers an ideal solution Patricia L. Atkins, SPEX CertiPrep, 203 Norcross Rd., Metuchen, NJ for routine analysis. Low detection limits and fast acquisition speeds allow trace 08840, Huifang Lang components, including adulterants and odor taints, to be identified even within the The consumption of botanical products has increased over the past two decades most challenging of matrices. TD brings the added advantage of retaining part of as consumers trend to what are perceived to be natural and high quality botani- the sample for re-analysis, allowing confident and secure repeat analysis. Addi- cal products. The primary regions of spice and tea production around them have tionally, this presentation explores the use of soft electron ionization by Select-eV often been cited as having less stringent safety and quality standards in regards technology. Select-eV provides hassle free soft ionization, with no requirement for to consumer products. Products from these regions have been noted to contain source-switching and no inherent loss in sensitivity. The ability to provide enhanced a variety of adulterants and contaminants. Common spices and botanicals in the molecular ions whilst retaining structurally-significant fragment ions provides com- United States (black pepper, red pepper, cinnamon, mustard seed, cumin, and tur- plementary spectra for enhanced confidence in compound identification. An over- meric) in various forms (i.e., spices, teas, condiments and supplements) were pur- view of this novel TD-GC-TOF-MS system is presented using specific case studies chased at dollar stores, farmer’s markets, chain stores, and on-line vitamin outlets. on aroma profiling of food products. Products selected covered the range of preparations including organic products. Cryogenic grinding and microwave digestion were employed in sample processing. 217 Withdrawn by the author. Physical and chemical screening methods were used to detect gross adulteration and counterfeiting. Inductively coupled plasma optical emission spectrometry (ICP- 218 Profiling of Aromas Components in Wine with GC-MS-MS with Full OES) was used to determine the macroelement components (Si, Na, Mg, Fe, and K) Spectrum Information that indicated possible adulteration or contamination. High percent levels of bulking Tom Mancuso, PerkinElmer, 710 Bridgeport Ave., Shelton, NJ 06484, agents including silica and sodium were often found in low cost spice and botanical Sharanya Reddy, Bill Hahn samples indicating potential adulteration. ICP-mass spectrometry was used to de- There are many volatile compounds in wine that help define the complex sensory termine the presence and level of heavy metal contamination and adulteration. Most effects of wine. These compounds contribute to aroma character of wines such as of the spice groups studied had many examples of high heavy metals content at the fruity, vegetal, butterscotch, leathery etc. Characterization of the aroma products in ppm level including very high lead levels which could be indicative of adulteration by wine can help with quality control of the product. We analyzed red, white and rosé lead chromate or lead oxides. wines using a hybrid gas chromatography tandem mass spectrometry (GC-MS-MS) instrument in scan mode. The peaks detected were processed with software tools 214 Cleanup of Fatty Food Matrices Using Zirconia-Based SPE and matched against National Institute of Standard and Technology library to iden- Sorbents tify analytes. The data was then exported to TIBCO Spotfire® software for princi- Jennifer Claus, Supelco, Division of Sigma-Aldrich, 595 North Harrison pal component analysis and unique identifiers that differentiated the wines were Rd., Bellefonte, PA 16823, Katherine K. Stenerson, Michael Halpenny, mapped out. Olga I. Shimelis, Emily Barrey, Patrick Myers, Michael Ye A major obstacle for food chemists continues to be the analysis of compounds in 219 Analysis of Target Pesticides in Essential Oils Using a Novel GC- fatty food samples. Interfering lipids can contaminate liquid chromatography (LC) MS-MS System and gas chromatography (GC) systems, produce elevated detection limits, and ul- Thomas Dillon, Perkin Elmer, 710 Bridgeport Ave., Shelton, CT 06484, timately decrease instrument and column lifetime. Because traditional solid-phase Sharanya Reddy, Samuel Tolley extraction (SPE) cleanup techniques often provide insufficient fatty matrix removal Essential or botanical oils are used as flavors and fragrances in aromatherapy and for analysis, a new approach using zirconia-based sorbents has been developed alternative medicine. The presence of pesticide residues in these essential oils is for selective lipid removal. Zirconia-based sorbents employ Lewis acid/base inter- of growing concern because of the widespread commercial usage of these oils. actions as well as hydrophobic interactions to selectively retain unwanted lipid in- Analysis of pesticides using a single quadrupole mass spectrometer in the com- terferences. To further enhance fatty compound removal, these sorbents may be plex matrix of botanical oil normally requires extensive sample clean up prior to combined with traditional phases such as C18 and/or Florisil in SPE and/or disper- analysis. We present a study of the analysis of target pesticides spiked in lemon sive SPE (quick, easy, cheap, effective, rugged, and safe) formats. These innovative oil using a novel hybrid gas chromatography tandem mass spectrometry (GC-MS- sorbents have been shown to remove more fatty matrix interferences than tradition- MS) system, with no additional sample clean up other than a simple dilution and an al cleanup sorbents, including PSA/C18 and silica. A comparison of zirconia-based on-line Swafer backflush system to remove high-boiling matrix components. Unlike sorbents to traditional cleanup sorbents for fat removal in various fatty food matrices a traditional triple quadrupole system, this novel GC-MS-MS system has the ability is demonstrated. Background removal, analyte recovery, and reproducibility of the to measure all product ions all the time with no loss in sensitivity. Besides method different cleanup techniques are compared in this presentation. simplification, monitoring for multiple product ions improves confidence in results. The other big advantage of full product spectrum acquisition is realized when the 215 Study of Chromatographic Selectivity for Analysis of Multiple unanticipated interference of a matrix ion with a target product ion can be eliminat- Mycotoxins by LC-MS-MS ed in post run processing by simply selecting and quantifying an interference-free Olga I. Shimelis, Supelco, Division of Sigma-Aldrich, 595 North Harrison product ion without having to re-acquire the calibration and sample chromatograms. Rd., Bellefonte, PA 16823, Emily R. Barrey, David S. Bell, Michael Ye, This saves both time and expense. The pesticides detection limits were well within Jennifer Claus USP regulatory limits. Mycotoxins are toxic secondary metabolites produced by fungi, which can exist in food as a result of fungal infection of crops. Their strong resistance to decomposi- 220 Vitamin B6 Fluorescence Response Enhanced by Post-Column tion and digestion cause mycotoxins to remain in the food chain. The analysis of Photochemical UV Irradiation mycotoxins in food and animal feed has been a challenge mainly due to the com- Henry Joshua, Aura Industries, 545 8th Ave., New York, NY 10018 plexity of food matrices and desired low detection limits. In recent years, significant A simple, reliable, sensitive and selective high-performance liquid chromatographic advances in the analytical techniques were applied to the detection of mycotoxins. method for the determination of vitamin B6 has been developed using post-column There has been an increasing need for a method to detect multiple mycotoxins with photolytic derivatization with 245 nanometer UV light. The method allows for the a single sample preparation and analysis method. Previously, research concentrat- confirmation of analyte identity by the simple expedient of performing the analysis ed on a liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for with and without irradiation. The simplicity of the apparatus (no moving parts) and multi-mycotoxin analysis as mass spectrometry provides appropriate selectivity and technique provides significant advantages over methods using post-column chem- sensitivity for detection. This study investigated the selectivity for over 15 common ical derivatization. mycotoxins on a variety of solid-core and fully porous sub 2-µm high-performance 32 2015 EAS Abstracts November 2015

221 Time Domain NMR Study of Triglyceride Oils and Soybean Seeds 225 NMR Structure of a Cranberry Xyloglucan with Anti-Escherichia Kaitlyn I. Doolittle, E. I. DuPont de Nemours & Co., PO Box 8352, coli Adhesion Activity DuPont Experimental Station 500/1206B, Wilmington, DE 19803, John Gary D. Strahan, United States Department of Agriculture, Eastern D. Everard, Weilan Pan, Kevin L. Stecca, Elizabeth F. McCord Regional Research Center, 600 E. Mermaid Ln., Wyndmoor, PA 19038, We have used both high and low field nuclear magnetic resonance (NMR) to as- Alberto Nunez, Hoa K. Chau, Andre K. White, Christina Khoo, Arland sign the time domain NMR signals in refined bleached and deodorized (RBD, tri- T. Hotchkiss glyceride) oil samples and soybean seeds. After inverse Laplace transform (ILT), the Consumption of cranberry juice is often used as a treatment for urinary tract infec- time domain data from the seeds contained many different characteristic signals. tions. The oligosaccharides found in residues from cranberry juice production have Variable humidity and water mixing experiments were used to assign water relat- been found to inhibit the adhesion of Escherichia coli to epithelial cells.[Hotchkiss, et ed peaks. Comparison with high field NMR data allowed assignment of oil related al., J. Agric. Food Chem., 2015, 63(23), pp 5622–5633.] We herein report on the use peaks. The seed data was similar to that of the RBD oil. of two-dimensional nuclear magnetic resonance (2-D-NMR) to determine the im- portant structural characteristics and conformations of the components in a fraction 222 Development of a Paper-Based Screening Method and Mass of this residue. Carbohydrate analysis and mass spectrometry indicated that it likely Spectral Library for Adulterated Milk Samples by Matrix-Assisted contained a branched arabino-xyloglucan composed of three hexose and four pen- Laser Desorption Ionization Mass Spectrometry (MALDI MS) tose sugars. Using 2-D-NMR the resonances of the sugar atoms were assigned and Jacquelyn Cali, Kean University, 1000 Morris Ave., Union, NJ 07083, their important through-bond correlations determined, establishing that the structure Michelle V. Joyce, Kevin Rhoads contained an S-type xyloglucan structure with the linkages: α-L-Araf (1→2) α-D-Xylp Milk is a primary nutritional source across the globe; however, quality control of milk (1→6) β-D-Glcp. These xyloglucan oligosaccharides represent a novel, bioactive in many developing countries is lacking due to unregulated supply chains. Farm- component in cranberry have a novel SSG/GSS structure, where “G” and “S” refer ers sell milk to collection stations, where it is commonly diluted and tainted with to the single-letter designations used to describe xyloglucan sequences (Reference: chemicals in order to increase volume, extend shelf life, and boost profit. Some of S.C. Fry, et al.,. Physiol. Plant. 1993, 89, 1−3). the most common adulterants include sugar, detergents, starches, and preserva- tives. Milk contamination is an international problem, and India, in particular, is a 226 Headspace GC-MS Analysis of Flavanoids from Cocoa Beans to country in which 68% of its milk is adulterated.[1] Our research focuses on the use Chocolate of matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) to Katarina Mladenovic, Kean University, 1000 Morris Ave., Union, NJ analyze cow’s milk samples for expected large proteins and common potential small 07083, Dil Ramanathan, Tom Mancuso molecule contaminants that have been found in milk in India. Using MALDI-MS over With an industry rapidly approaching $100 billion, chocolate has become a staple in three mass ranges, a screening process for cow’s milk was developed. Pure milk the lives of many people throughout the world. Recent research has also shown that was analyzed to characterize protein composition, while milk spiked with contami- chocolate taken in moderation can prevent colon cancer. This study focuses on the nants was analyzed to detect adulterants and study potential protein alterations. A chemical profiling of ingredients in each step of the chocolate making process. By paper-based screening method was developed to address issues with transporta- qualitatively and quantitatively understanding the chemical components, we hope tion and degradation of milk samples, as well as to reduce cost of analysis. By spot- to make the best quality chocolates that provide many benefits for consumers. All ting the milk on chromatography paper spots adhered to the MALDI target, proteins experiments were performed using the Perkin-Elmer gas chromatography (GC) and small molecules in milk were detected and showed mass spectral profiles anal- Clarus 680 and mass spectrometer (MS) Clarus SQ 8T. For comparative purposes, ogous to those generated from milk spotted directly onto the MALDI target. Using samples will be introduced to the GC-MS via headspace and an autosampler. Sev- the mass spectra collected from the analyses of both untainted and spiked milk on eral methods will be compared to develop the best GC-MS method, and once that paper, a library is being created to screen samples for known milk proteins and to method is established, studies will be performed to profile the compounds during detect the presence of contaminants. each step of the chocolate making process. The profiling results will be compared Reference: with the National Institute of Standards and Technology and United States National [1] Food Safety and Standard Authority of India. Executive Summary on National Library of Medicine’s PubChem database to identify the flavonoids and other import- Survey on Milk Adulteration. 2012. ant chemicals. Preliminary data with the methods that were developed have shown promising results. Both roasted and unroasted samples contained tetramethyl pyr- 223 Headspace Gas Chromatography Mass Spectrometry Analysis of azine, a compound known to give off strong flavor and aroma in cocoa and coffee Leachable Compounds in Baby Bottles and Formula beans. The tests have also found that unroasted cocoa beans had more compounds Jacquelyn Cali, Kean University, 1000 Morris Ave., Union, NJ 07083, that are responsible for flavor and aroma than the roasted cocoa beans. Once the Tom Mancuso, Dil Ramanathan study is completed, the results can be used to obtain a final product which will guar- Bottle-feeding infants dates back to the 15th century, but mass production of poly- antee batch-to-batch consistency and a higher quality chocolate. propylene bottles in the 1950s increased its popularity. In the early 2000s, suspicion arose that chemicals within the plastic, including bisphenol A (BPA), have the ability 227 The Use of High Resolution Accurate Mass GC-MS for Metabolomics to leach into foods and cause harmful effects. In 2012, the United States Food and Workflows Drug Administration banned the use of BPA in baby bottles due to its ability to leach Rafael Acosta, Thermo Fisher Scientific, 2215 Grand Avenue Pkwy., into the formula. Though the use of BPA was banned, concerns whether or not the Austin, TX 78728 products are truly free of chemicals that can be harmful to infants remain. In this Typically high resolution accurate mass liquid chromatography mass spectrome- study, a mother’s process of bottle sterilization, formula preparation, refrigeration try (LC-MS) in combination with unit mass gas chromatography mass spectrome- storage, and heating using a bottle warmer were simulated. The bottles used in this try (GC-MS) has been used for metabolomics workflows. With the introduction of study were store brand, and were tested with both brand name and store brand for- Orbitrap technology 10 years ago and the recent introduction of the new Thermo mulas. Samples of the plastic and formula were taken from bottles after being stored Scientific™ Q Exactive™ GC mass spectrometer, it is now possible to use the pow- over several different time intervals and were analyzed using a Perkin-Elmer GC erful information obtained with an Orbitrap based analyzer, namely high resolution Clarus 680-MS Clarus SQ 8T with headspace capabilities. Headspace gas chro- spectra and excellent mass accuracy, combined with the high selectivity of gas chro- matography mass spectrometry (GC-MS) analysis detected volatile and aromatic matography to generate greater confidence in analytical results. With up to 100,000 compounds that leached from the plastic. Chemicals found to have leached from resolving power (m/z 272) and sub part per million mass accuracy, the Q Exac- the bottles were profiled, and preliminary results are promising. tive GC system makes identifying unknown metabolites less labor intensive. The large linear dynamic range and triple-quadrupole equivalent sensitivity ensures that 224 Fast and Precise Flavor and Fragrance Analysis in Whisky low concentration analytes are correctly identified and quantitated, and having no Shyam Verma, Sigma-Aldrich, 595 N. Harrison Rd., Bellefonte, PA spectra saturation eliminates the need for re-injection of valuable and limited sam- 16823, Rudolf Koehling, Eva Katharina Richter ple quantities. Combining these aforementioned features with a new powerful and Many whisky lovers rave about the peaty and smoky taste of certain whiskies. This revolutionary deconvolution algorithm provides the ultimate assurance in unknown taste originates primarily from phenolic compounds in peat smoke. Typically, aro- identification in a metabolomics workflow. matic compounds are easily detected by high-performance liquid chromatography ultra-violet (HPLCUV). The UV absorbance of a whisky sample becomes a measure 228 Characterization and Determination of Irganox 1076 and 1010 in of the “peatiness” of a whisky’s taste, reflecting only the sum of all compounds. In Polyethylene Using Thermal Desorption and Reactive Pyrolysis – this article, some challenges in the analysis of these complex mixtures that one GC-MS may initially regard as an ideal sample matrix for direct injection and standard re- Roger Tank, Frontier Laboratories USA, 3952 Tallgrass East Ct., versed-phase liquid chromatography are discussed. We report results of LC-UV Holland, MI 49424, David Randle, Itsuko Iwai, Akihiko Hosaka, Ichi and LC-mass spectrometry methods for detecting these complex and contributing Watanabe, Michael Soli, Terry Ramus aromatic compounds. Two of the more commonly used antioxidants, Irganox 1076 and 1010, are phenolic 33 2015 EAS Abstracts November 2015

primary antioxidants used to provide long-term thermal stability to polyolefins such 232 Expansion of Micellar Liquid Chromatography: From Transdermal as polyethylene. Analysis of 1076 and 1010 is difficult using gas chromatography Permeability Prediction to Bile Salts Studies (GC) because each is highly resistant to conventional extraction and has a very low Dina S. Shokry, University of Huddersfield, Queensgate, West vapor pressure. This report details a GC- mass spectrometry (MS)-based analytical Yorkshire, Huddersfield, HD1 3DH, United Kingdom, Laura J. Waters, method for the qualitative and quantitative determination of Irganox 1076 and 1010 Gareth Parkes in polyethylene. 1076 is thermally desorbed from polyethylene at 320 ˚C. Both 1076 Micellar liquid chromatography (MLC) has been used in a variety of applications, and 1010 have an ester linkage which can be thermally hydrolyzed and methylated one of the most recent of these is the prediction of partition coefficient values (log using tetramethylammonium hydroxide (TMAH). Factors affecting the accuracy and P) for pharmaceutical compounds and then its incorporation in a skin permeability precision of each technique will be discussed. Calibration is performed using stan- model as an attempt to predict drug permeation through human skin. This work is dard addition which eliminates the need for in-matrix (additive in polymer) primary an attempt to expand the applications of MLC for studying bile salts and their effect standards and takes in account both instrument changes and matrix interference. on the retention behavior of drugs. Degassed surfactant-based mobile phase was The precision of the method for both compounds is on the order of 5% relative stan- pumped through the chromatographic system with a reversed-phase cyanopropyl dard deviation and the %error is <10%. column. Samples of 0.2 mM drugs were analyzed at a wavelength appropriate for each drug producing a peak indicating the retention of the solute within the column 229 Determination and Quantification of Perchlorate in a Fruit Matrix as a function of time. Data were recorded and then analyzed to obtain capacity Using Accelerated Solvent Extraction and IC-MS factors. Results successfully showed the binding behavior of the drugs to bile salts. Kyle B. Renfrew, Thermo Fisher Scientific, 2215 Grand Avenue Pkwy., Also through plotting the inverse of the calculated capacity factors against the differ- Austin, TX 78728, Amanda Hartman, Nicholas Santiago ent bile salt concentrations used, a linear relationship was obtained and the partition Perchlorate is an oxidizing agent used in munitions and firework manufacturing that coefficients of used drugs were calculated from the slope and intercept relationship. has been identified as an environmental contaminant of water, soil, and food crops. Through statistical methods the results were used to predict absorption of the used Sufficiently high doses of perchlorate can inhibit iodide absorption in the thyroid, drugs. Application of MLC was a successful method for the determination of partition leading to metabolic disruption with effects on fetal and infant development. Con- coefficients of the used drugs and consequently for the prediction of their absorption sequently, the United States Environmental Protection Agency and Food and Drug through statistical methods. Administration have developed chromatographic methods for its routine monitoring in water and food crops. Unlike its analysis in drinking water, which requires minimal 233 Analysis of Residual Solvents Using a Headspace Syringe sample preparation, perchlorate determination in fruit and vegetable matrices pres- Autosampler ents considerable challenges due to high levels of interfering matrix ions; these are Anne Jurek, EST Analytical, 503 Commercial Dr., Fairfield, OH 45014, particularly problematic when quantifying at typical parts-per-billion (ppb) concen- Mike Moses, Kelly Cravenor trations. In addition, food samples require laborious extraction procedures that limit During the synthesis of some pharmaceuticals it is sometimes necessary to use throughput and reduce precision due to variable handling. The use of accelerated solvents in order to increase yield or purity of the product. After the pharmaceu- solvent extraction (ASE) and ion chromatography coupled with mass spectrometry tical is produced, the solvent(s) are removed to the greatest extent possible. The (IC-MS) presents a complete workflow solution that minimizes sample preparation products are then tested for any residual solvents in order to limit patient exposure. and maximizes sensitivity for perchlorate in foods. Mass detection reduces the need Residual solvents are separated into classes according to the risk to patient health. for post-extraction clean-up procedures through added selectivity relative to con- Class 1 solvents are to be avoided at all costs as they are known to be human ductivity detection. Moreover, ASE increases sample-to-sample consistency and lab carcinogens or have adverse effects on the environment. Class 2 solvents have productivity. Altogether, these data highlight a simplified method for reliable monitor- less severe toxicities but should be avoided because of the potential of adverse ing of ppb-level perchlorate in fruits and vegetables. effects. Finally, Class 3 solvents have low toxicity and can be used when needed. Thus, pharmaceutical manufacturers are required to test for residual solvents in 230 Analysis of Underivatized Steroids Using Cold EI GC-MS order to ensure that any residual solvents in the product are below established ex- Adam Patkin, Perkin Elmer, 710 Bridgeport Ave., Shelton, CT 06484, posure limits. United States Pharmacopeia (USP) general chapter <467> describes Tom Mancuso a static headspace gas chromatography procedure for the determination of residual Many laboratories use gas chromatography-mass spectrometry (GC-MS) and GC- solvents. This application demonstrates the USP <467> procedure using the FLEX MS-MS for the analysis of steroids and hormones to detect and monitor health dis- auto sampler and the gas tight syringe static headspace sampling option. orders. While liquid chromatography (LC)-MS-MS performed with triple quadrupole instruments is being used more frequently, it suffers from being a target analysis 234 Simultaneous Determination of Kolliphor HS15 and Miglyol 812 in technique. Only the target analytes are detected, and it is not possible to retrospec- Microemulsion Formulation by Ultra-High Performance Liquid tively query the data for unexpected steroids, precursors, or derivatives in novel me- Chromatography Coupled with Nano Quantity Analyte Detector tabolomes. GC-MS is non-selective, providing a comprehensive view of all steroids (UPLC-NQAD) in the metabolome for non-targeted steroid profiling. GC-MS also allows for spectral Honggen Zhang, Merck, 124 E. Lincoln Ave., Rahway, NJ 07065, searching of unknown compounds against large libraries of reference spectra. LC Zhenyu Wang, Oscar Liu introduction has the advantage that derivatization is generally not required, whereas Kolliphor HS15 is a potent non-ionic solubilizer and emulsifying agent. Miglyol 812 GC typically requires it because of thermolability at the high analyte boiling points. is a penetration solvent. Both surfactants are often formulated into microemulsion The technique of cold electron ionization (EI) GC-MS allows the introduction of high formulations. A novel method for simultaneous determination of kolliphor HS15 and flow rates of carrier gas into the MS interface. Using relatively short GC columns, miglyol 812 was developed by using an ultra-high-performance liquid chromatog- the high flow rates reduce the temperature required to elute the analytes and min- raphy nano quantity analyte detector (UHPLC-NQAD) instrument. All components imize decomposition. The cold EI axial flow-through ion source and supersonic jet in kolliphor HS15 and miglyol 812 were well separated by an Acquity BEH C18 expansion reduces ion source decomposition compared with conventional closed column. Mobile phase A was a 0.1% trifluoroacetic acid in water and mobile phase EI source designs by eliminating analyte contact with hot source walls. Cold EI also B was acetonitrile. A gradient elution (60% to 100% mobile phase B) within 8 min enhances the molecular ion intensities, improving technique specificity. When used and held for 5 min was employed. The flow rate was 0.7 mL/min and injection vol- in a novel q-time-of-flight configuration the enhanced molecular ion also increases ume was 10 µL. The method was characterized for specificity, linearity, precision, MS-MS quantitative sensitivity, selectivity, and information content. accuracy and quantification limits (QL). Good linearity (r > 0.990) was obtained in range of 27.6-1381.1 μg/mL for kolliphor HS15 and 0.8-221.9 μg/mL for miglyol 231 Analysis of FAMEs Using Cold EI GC-MS for Enhanced Molecular 812. Overall recoveries were 98.0-102.4% (RSD =1.5%) for kolliphor HS15 and Ion Selectivity 90.7-106.6% (RSD=2.0%) for miglyol 812. QL were determined as 27.65 μg/mL for Adam Patkin, Perkin Elmer, 710 Bridgeport Ave., Shelton, CT 06484, kolliphor HS15 and 0.8 μg/mL for miglyol 812. No interferences were found in the re- Tom Mancuso tention time of major components in kolliphor HS15 and miglyol 812. The proposed Characterization of fatty acid methy esters (FAMEs) is used in several important fields method has been applied in analysis of microemulsion sample. ranging from biofuel analysis to fat content in foods and blood. They are generally derivatized from free fatty acids and mono-, di-, and triglycerides. FAMEs may be sat- 235 Trace-Level Aliphatic Amines in Cationic Pharmaceutical urated, mono- or polyunsaturated, linear or branched, and of variable chain lengths. Ingredients Electron ionization gas chromatography-mass spectrometry (EI GC-MS) is often Stuart J. Procter, Metrohm USA, 6555 Pelican Creek Circle, Riverview, used to characterize FAMEs, but may fail to produce a useful molecular ion for short, FL 33578, Harihara Subramanian, Andrea Wille unsaturated, or branched chains, making identification more difficult. Contrastingly, Low-molecular-weight amines find widespread use in raw materials or intermediate cold electron ionization GC-MS (Cold EI GC-MS) can substantially increase the mo- products in the manufacturing of numerous chemicals, pharmaceuticals, polymers, lecular ion intensity of these compounds, while retaining the EI fragmentation pattern pesticides, rubber, dyes, adhesives, solvents and corrosion inhibitors. Their moni- for spectral library searching without modification to established GC methodologies. toring is crucial as most of them are toxic and irritate the skin, mucous membranes 34 2015 EAS Abstracts November 2015

and respiratory tract. Moreover, secondary amines can react with nitrite forming car- ent compound, produces degradants that can be classified into four types. Selection cinogenic nitrosamines. The analytical challenge confronted by the presented work of the appropriate deuterium label can greatly simplify degradant tracking. A com- describes how to detect sub-ppm concentrations of low-molecular weight amines in mon moiety among the four degradant types is the benzimidazole ring. Deuterium the presence of strongly retained cationic drugs using ion chromatography (IC). An labeling on the benzimidazole ring provides a stable label that reflects all four degra- upstream inline coupled-column matrix elimination (CCME) technique was utilized dant types. A prototype dosage form containing omeprazole and deuterium labeled to eliminate cationic drug from the analytical flow path. In contrast to direct-injection omeprazole was stressed at 60 °C / 60% relative humidity for 1 week. LC-MS anal- IC, where the late elution of strongly retained drugs requires eluents with added ysis revealed known and unknown degradants that were uniquely identified by their acetonitrile. CCME significantly shortens the analysis time and improves amine deuterium label. Further characterization was carried out using preparative HPLC sensitivity and selectivity. From the determination of monomethylamine in Nebivolol and nuclear magnetic resonance spectroscopy to structurally confirm two unknown hydrochloride presented, the CCME technique shows to be a promising tool for degradants of omeprazole: 1-(5-methoxy-1H-benzo[d]imidazol-2-yl)-2,3,5-trimeth- detecting further low-molecular-weight amines in a wide range of drugs products. ylpyridin-4(1H)-one and 5-methoxy-2-(methylthio)-1H-benzo[d]imidazole.

236 Quantification of Genotoxic Impurities Benzyl Bromide and Benzyl 239 LC-MS Method Development for the Identification of Route Specific Chloride in Pharmaceutical Intermediates and API by GC-MS MDMA Impurities Jesse Martinez, Abbvie Inc., 1401 Sheridan Rd., North Chicago, IL Rebecca F. Dunn, Arcadia University, 885 N. Easton Rd., Apt. 3A6, 60064 Glenside, PA 19038, Heather L. Harris, Warren Korn, Karen S. Scott Benzyl chloride (BnCl) and benzyl bromide (BnBr) are potential impurities in either This presentation demonstrates the simple dry extraction techniques possible with an intermediate or API. If present they must be eliminated or reduced to acceptable liquid chromatography mass spectrometry (LC-MS) instrumentation for impurity pro- levels in subsequent processing to ensure patient safety since both compounds filing of a popular club drug. MDMA, 3,4-methylenedioxymethamphetamine, isa are genotoxic. For this study, they must be controlled to not more than 2 ppm at an Schedule 1 hallucinogenic stimulant commonly found in “Ecstasy” tablets or referred intermediate stage. At these trace levels, high-performance liquid chromatography to as “Molly.” By analyzing the organic by-products, or impurities, in MDMA tablets, ultraviolet or gas chromatography flame ionization detector (GC-FID) does not have it is possible to identify the synthetic route used to prepare the sample. The aim of the appropriate sensitivity to be viable analytical techniques. An analytical method this research was to develop a simplified method for the extraction and identification has been developed employing GC-mass spectrometry (MS) for the determination of 11 previously identified route specific MDMA impurities, in the presence of MDMA of BnCl and BnBr at these trace levels, in a pharmaceutical matrix. The GC-MS and caffeine. By only identifying the impurities selected, this method is able to focus method offers sensitive detection; capable of detecting both BnBr and BnCl at 0.004 on the impurities, typically found at low concentrations, which differentiate between µg/mL (4 picogram) levels which corresponds to 2 ppm relative to a 2 mg/mL sample two popular synthesis routes. Three dry extraction methods using methanol, 0.05 concentration. The GC-MS method was qualified by determining linearity, accuracy, N hydrochloric acid in methanol, and 0.1% trifluoroacetic acid in methanol, respec- practical quantitation limit, and injection precision. For both compounds the method tively, were utilized for the extraction of the compounds of interest from tablets. is linear in the range of 4 pg to 40 pg, accuracy (spike and recover) is in the range The compounds of interest were extracted from four different common excipients: of 99% to 106% at the 4 pg level, and an injection precision of less than 5% (RSD) cornstarch, d-lactose, d-sorbitol, and microcrystalline cellulose. Percent area ratios at 4 pg. In summary, a GC-MS method has been developed for trace detection and were used to determine the percent recovery of each method and these results were quantitation of BnBr and BnCl in a pharmaceutical matrix. compared to the percent recoveries obtained from a previously optimized liquid-liq- uid extraction method. Of the three dry extraction methods, the methanolic HCl had 237 Practical Utilization of a Lab Mobile Direct Analysis in Real-Time the highest average percent recovery from cornstarch at 52% while the liquid-liquid (DART) Ambient Ionization Mass Spectrometer in Quality Control extraction had 58%. When the percent recoveries of MDMA and caffeine were ex- and Product Authenticity Screening cluded from the average, the data showed the methanolic HCl dry extraction to be Brian D. Musselman, IonSense, 999 Broadway, Ste. 404, Saugus, MA the optimum extraction method. 01906, Joseph Lapointe, Robert Goguen, Joseph Tice, Emily Dunn, Taylor Feraco 240 Headspace Grade Solvents for Trace Analysis Integration of a lab mobile direct ionization in real-time (DART®) ionization mass Subhra Bhattacharya, Thermo Fisher Scientific, One Reagent Ln., Fair spectrometer systems is described. The adaptation of a state-of-the-art Waters Ac- Lawn, NJ 07410, Eric Oliver, Stephen C. Roemer quity® QDa detector to enabling ambient ionization with a commercial liquid chro- Headspace analysis is a common practice in pharmaceuticals to detect residual matography mass spectrometry (LC-MS) is facilitated by development of a custom solvents. Per United States Pharmacopoeia (USP), residual solvents in pharma- VAPUR® ion transport interface, new pulsed DART gas control to facilitate low gas ceuticals are defined as organic volatile chemicals that are used or produced in the consumption, and an industrial strength cart to permit rapid deployment of the sys- manufacturing of drug substances or excipients. Since the residual solvents are dif- tem between laboratory and production sites. As DART does not require utilization ficult to remove completely from the drug products, the amount of residual solvents of solvents for sample preparation or ionization the system can be deployed safely should be evaluated by headspace gas chromatography (GC) analysis. Based on without requirement for transport of those solvents. Using this instrument we doc- their toxicity effects, residual solvents are classified as class 1 (most toxic), class 2 ument analysis of a variety of raw materials and finished pharmaceutical products and class 3. In headspace GC analysis, the pharmaceutical compound of interest as well as forced degradation studies. Uniquely, as the temperature of the DART is often dissolved in a high boiling solvent to assess the amount of volatile compo- gas can be modulated routinely, we describe a thermal profile method for product nents. Although water is the most common solvent for this type of analysis, other characterization. In the thermal profile experiment the desorption gas temperature solvents are of frequently used when solubility of the pharmaceutical compound is is modulated to produce mass spectra at four different temperatures. The combi- an issue. We have evaluated five different solvents for headspace analysis. The nation of those mass spectra provides a unique chemical fingerprint, the so called solvents are water, dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), total product mass spectrum (TPMS), which we will demonstrate has utility for use N,N-dimethylacetamide (DMAC) and 1-methyl-2-pyrrolidinone (NMP). The purpose in rapid quality assessment, detection of contaminants in finished goods and raw of our study is to qualify these high purity solvents for residual solvent analysis materials, and detection of non-specific threats encountered in cases of economic from pharmaceuticals. Four different pain medications were purchased from Wal- adulteration. greens and residual solvent analysis performed using DMSO and water. Our results showed that all five solvents are free of trace level impurities and qualified to detect 238 Structure Elucidation of Omeprazole Degradants in an Over the volatile organic components at trace level from pharmaceutical compounds. Counter Formulation Cassandra J. Schmitt, Pfizer Consumer Healthcare, 1211 Sherwood 241 On-Line Supercritical Fluid Extraction-Supercritical Fluid Ave., Richmond, VA 23220, Keith A. Rippel Chromatography: A Novel Approach in Cleaning Validation for Omeprazole is a proton pump inhibitor currently available in over the counter (OTC) Pharmaceutical Manufacturing dosage forms. The demand for an OTC dosage form of omeprazole creates chal- William Hedgepeth, Shimadzu Scientific Instruments, 7100 Riverwood lenges in formulation to minimize degradation while maintaining bioavailability. The Dr., Columbia, MD 21046, Ken Tanaka market for an OTC drug necessitates a more consumer-friendly dosage form, which Cleaning validation is necessary to establish the quality and safety of pharma- often results in a more complex formulation. These more complex dosage forms re- ceutical drug products. Although swabbing is the preferable method to validate quire degradant characterization methods that are specific and uniquely tag degra- cleaning, it has some problems. The TOC analyzer is not applicable to hydropho- dants for identification. Applying “stable isotope labeling technique” (SILT) provides bic compounds because ethanol is needed for swabbing. Sample concentration is a unique way to track degradants within a complex dosage form by liquid chroma- also sometimes required before high-performance liquid chromatography (HPLC). tography mass spectrometry (LC-MS). Commercially available deuterium labeled Thus, we developed a novel sample pretreatment and analysis method for cleaning omeprazole was used in this study to track omeprazole degradants. Degradant validation using supercritical fluid for both extraction and analysis. A commercial- characterization of omeprazole is complicated by the structural rearrangement be- ly available detergent containing alkylbenzene sulfonates was used as a standard tween the benzimidazole and pyridine ring. This rearrangement, along with the par- sample. For the test of sample extraction, the sample was dropped onto a swab 35 2015 EAS Abstracts November 2015

(ITW Texwipe, USA). The Nexera UC system (Shimadzu Corporation, Japan) was 245 Developing Novel Spectroscopic Imaging Methods for Functional used for both the screening of the method using supercritical fluid chromatography and Hybrid Materials Analysis (SFC) and the supercritical fluid sample extraction (SFE) followed by SFC directly Joel F. Destino, University at Buffalo, Dept. of Chemistry, 359 Natural (SFE-SFC). Shim-pack UC series columns were screened and used for subsequent Sciences Complex, Buffalo, NY 14260, Andrew K. Craft, Zachary R. analysis. The analytical method for SFC was developed by screening four columns Jones, Frank V. Bright and gradient conditions. The performance of the method was measured by evalu- Designing high performance functional materials often requires a deep understand- ating the linearity and the reproducibility. The method provides great linearity for a ing of the fundamental relationships between physical and chemical properties. broader range of the detergent concentration and great reproducibility of the reten- For measurements such as these, bulk sampling methods are typically sufficient; tion time, the area and the height of the peak. The performance of the method was however, for the development of thin films and coatings, it is usually necessary measured by evaluating the recovery rate, the linearity and the reproducibility. The to investigate these relationships across a surface, demanding advanced imaging evaluation results showed that on-line SFE-SFC can provide reliable data for the techniques. Over the past few years, research from our group has focused on the cleaning validation for pharmaceutical manufacturing. The benefit of on-line SFE- development and application of novel spectroscopic imaging methods to elucidate SFC analysis is not only the data reliability but also the convenience and safety the relationship between various chemical and physical properties of advanced and gained by skipping a manual extraction process. hybrid functional materials across length scales. This poster includes various re- search projects that best represent this work. The first project presented includes 242 Using Headspace and Liquid Autosampler Gas Chromatography the application of statistical methods with colocalized Raman and scanning probe Mass Spectrometry to Profile Costa Rican Forest Herbs for microscopy to study a ternary hybrid xerogel thin film with antifouling properties. The Pharmacological Activity second project shows the application of the same techniques for the analysis of 2D Danielle J. Antonucci, Kean University, 1000 Morris Ave., Union, NJ MoS2 nanomaterials. The third presents a fluorescence based method to rapidly de- 07083, Mirna Giron, Alyssa Bellomo, Jui Chaugule, Tom Mancuso, Dil termine surface pH and chemical segregation in hybrid thin film structures. Finally, Ramanathan future work on the development of additional spectroscopic imaging methods from Herbal remedies have been used for centuries to treat all sorts of illnesses in many our lab is presented. parts of the world. They are a cheaper alternative to prescription drugs and are more convenient to obtain since they can be homegrown or found commercially 246 Identification and Classification of Botanical Forensic Evidence in the form of tablets, capsules, powders, teas, extracts, and fresh or dried plants. Using Direct Analysis in Real Time High-Resolution Mass The objective of this study was to analyze and profile the medicinal plants used by Spectrometry Costa Rican locals in the Maquenque National Wildlife Refuge in order to find out Ashton D. Lesiak, University at Albany-SUNY, Dept. of Chemistry, 1400 which compounds in the plants had remedial and pharmacological properties. The Washington Ave., Albany, NY 12208, Justine E. Giffen, Robert B. Cody, plants studied in this experiment included (but were not limited to) Santa Maria, A. John Dane, Rabi A. Musah Guava, Avocado, Mano de Tigre, Aceituno, Gavilana, Vismia ferruginea, and Nance. With increasing frequency, the drugs that are being submitted to crime labs for anal- Bark and leaf samples were analyzed using a Perkin-Elmer GC Clarus 680 and MS ysis are no longer restricted to well-characterized substances such as cocaine and Clarus SQ 8T. Two different methods of sample introduction were used: headspace heroin, but often include a variety of plant-based products of abuse. Conventional and liquid autosampler. Headspace gas chromatography mass spectrometry (GC- methods of drug analysis cannot be efficiently applied to these new psychoactive MS) specializes in the detection of aromatic and volatile compounds, while the liquid substances, as the complex matrices of which they are comprised are difficult to autosampler GC-MS is used to detect semi-volatile compounds. With the results analyze, and there are few if any standard operating protocols currently used by obtained from this study, we are able to profile the individual compounds within the forensic laboratories to identify emerging mind-altering botanicals. While traditional plants for their pharmacological activity. methodologies can provide identification of these products, the method develop- ment and validation needed for each new drug on the market makes the application 243 An Innovative Approach for the Quantitation of Captopril in of these techniques impractical. We demonstrate that high-throughput direct anal- Industrial Hygiene Monitoring ysis in real time high-resolution mass spectrometry (DART-HRMS) can be used to Sheetal Patel, Bristol-Myers Squibb, 1 Squibb Dr., New Brunswick, NJ rapidly identify plant-based drugs of abuse in complex matrices, including Kratom, 08844, Hugh Yao Datura spp. Salvia divinorum, and Kava, among others. This was accomplished Bristol-Myers Squibb Company (BMS) ensures its employees’ health and work envi- based on their unique but reproducible chemical fingerprint profiles, without the ronment safety by the exposure control of potent chemicals to prevent occupational need for complex and time-consuming sample preparation steps. Moreover, the illness associated with the chemical agent(s). Monitoring of exposure is performed psychoactive compounds contained within the plant matrices are easily detected. by the collection and analysis of industrial hygiene air and surface samples. Cap- Through comparison of the in-source collision-induced dissociation spectra of bo- topril is an angiotensin converting enzyme (ACE) inhibitor and has an occupational tanical unknowns to those of authentic standards, the species identity of the plant exposure limit (OEL) of 100 µg/m3. Air samples from the manufacturing facilities can be confirmed. Furthermore, the output of these experiments can be subjected are routinely taken for exposure monitoring. During air sampling and subsequent to statistical analysis methods to enable species-level classification and class dis- storage, Captopril tends to convert to a dimer, Captopril disulfide, due to the forma- tinction between multiple types of plant-based drugs of abuse with defined levels of tion of disulfide bond as a result of its exposure to air. This creates challenges for confidence, an approach which is currently lacking in forensic reporting. analytical quantitation. Captopril and Captopril disulfide reference standards had to be used for calibration. In addition, the interconversion of Captopril and Capto- 247 Developing Magnetic Resonance Imaging (MRI) Techniques for In- pril disulfide also occurs in uncontrolled solutions, including the standard solutions, Situ Physiochemical Tomography of Heterogeneous Reactions which makes the quantitation difficult. In this presentation, an innovative approach is Nanette N. Jarenwattananon, University of California-Los Angeles, 607 demonstrated with a reducing reagent, Tris (2-carboxyethyl) phosphine HCl (TCEP), Charles E. Young Dr. East, Los Angeles, CA 90095, Stefan Glöggler, being added to solutions to convert the disulfide dimer to Captopril monomer as Trenton Otto, Louis Bouchard well as to keep Captopril from being converted back to the dimer. As a result, the More than 85% of all chemical industry products are generated via catalytic reac- quantitation can be successfully done using one calibration curve (Captopril) with tions, the majority of which are heterogeneous reactions occurring at a gas-solid improved robustness. Various parameters for the conversion reaction have been interface. As a result, considerable resources are devoted to improving catalytic studied, including reaction time, temperature, molar ratio, solution stability, etc. The efficiency and conditions. However, optimizing catalytic reactor design requires method has been validated successfully for precision, accuracy, linearity, specificity, knowledge of the coupling between heat transfer, fluid dynamics, entropy, enthalpy, detection and quantitation limits, and robustness. and reaction kinetics, which is difficult to model accurately without experimental ob- servations. These computations must be calibrated against experimental observa- 244 Practical Advanced Analytical Techniques for Mutagenic Impurity tions of temperature, pressure, velocity, and chemical sensing. Magnetic resonance Quantitation imaging (MRI) is a promising candidate for imaging catalytic reactions because it Timothy M. Nowak, Merck, 126 E. Lincoln Ave., Rahway, NJ 07065 can “see” through opaque media. The long-term goal of our research is the devel- Mutagenic impurities (MIs) are chemical compounds that can potentially cause DNA opment of novel MRI methods for imaging in-situ chemical reactions. While MRI can damage, even when present at low levels. To limit the potential risk of these reactive provide the detailed inner views of a reactor, we also require a theoretical framework compounds, the MIs need to be controlled to trace levels. Due to the reactivity of to extract the aforementioned thermodynamic parameters from the reaction. We are some of these impurities, trace-level control and quantitation of these compounds in developing MRI methods to visualize non-equilibrium thermodynamics in-situ and raw materials, process intermediates, and active pharmaceutical ingredients have gather three-dimensional chemical sensing, transport, and thermodynamic parame- proposed challenges to pharmaceutical scientists. Since mutagenic impurities can ters at all points inside the reaction chamber, allowing us to report on the state of the have a wide-range of chemical and physical properties, different analytical tech- catalyst and the reactor. By using MRI, the reacting flow remains unperturbed, and niques are needed to accurately quantitate the amount of MIs present in a phar- we can map spatial distributions of velocities, concentrations, and temperatures. maceutical substrate. In this presentation, a few examples of MI quantitation using From these maps, we extract “internal views” of pressure, heat capacity, thermal various analytical techniques are discussed. 36 2015 EAS Abstracts November 2015

conductivity, kinetics, reaction enthalpy, and entropy production, which provide a interfacial capacitance, the three compounds all exhibited similar catalytic activity, quantitative basis for reactor optimization. Thus far, we have demonstrated microm- suggesting that the activity per accessible active site was comparable between the eter-scale images of catalytic conversion, enhanced signal by hyperpolarization and three samples. This disagrees with recent results on metallurgical samples, raising mapped temperatures of reacting gases. the question of whether powdered materials behave similarly to bulk alloys and/ or whether capacitance measurements are suitable indicators of electrochemically 248 Design and Development of a Portable Aptasensor for Toxicity active surface area. Monitoring of Field Samples Gonca Bulbul, Clarkson University, Dept. of Chemistry and Biomolecular 251 Electromodification of Carbon Nanotubes with Prussian Blue by Science, 8 Clarkson Ave, Potsdam, NY 13699, Akhtar Hayat, Silvana Cyclic Voltammetry Andreescu Thomas O’Connor, Vassar College, Box 2937, 124 Raymond Ave., The adoption of nanoparticle based analytical technologies has gained increasing Poughkeepsie, NY, 12603, Stuart L. Belli, Christopher Smart, Holger acceptance in various fields of chemical analysis in the environmental, clinical, Moustakas food and biomedical sectors. Different types of methodologies which employ elec- Carbon nanotubes (CNTs) grown from acetylene gas by chemical vapor deposi- trochemical, fluorimetric, piezo-electric output signals are currently developed for tion (CVD) on a conducting nickel-electroplated gold substrate have been shown to the detection of various molecules. In this respect, colorimetric assays are very undergo covalent modification with Prussian Blue (PB), a metal-hexacyanoferrate promising since they enable rapid visual detection on the spot without any need for (MHCF) compound. PB was bound to CNTs by a novel electrochemical technique elaborate laboratory equipment. Additionally, they provide specific advantages such that utilizes cyclic voltammetry, providing a straightforward as well as versatile meth- as portability, ease-to-operate, and low cost which make them very attractive from od to functionalize CNTs; thereby expanding upon their unique properties. Current an application view of point. Nanoceria particles have gained significant interest due efforts are focused on altering the composition of the conducting substrates and to their catalytic and free radical scavenging properties. Here, we report a newly exploration of compounds that can also be covalently attached to CNTs by this discovered phenomenon for quantifying molecular recognition based on the revers- method. ible assembly of single-stranded DNA (ssDNA) aptamers on redox active nanoce- ria particles. The method involves target tunable electrostatic and steric repulsion 252 A Genome-Inspired Reverse Selection Pathway towards Aptamer phenomena of the ssDNA to the surface of nanoceria which changes its spectral Discovery and functional catalytic properties upon binding of the target analyte. As a proof of Suttipong Suttapitugsakul, Rensselaer Polytechnic Institute, Cogswell concept, the proposed strategy was employed to construct an aptaswitch for the Laboratory, 110 8th St., Troy, NY 12180, Christina M. Albanese, Linda colorimetric sensing of OchratoxinA (OTA), providing a detection limit of 0.15 nM B. McGown OTA. This approach is generally applicable for sensitive and specific detection of a Aptamers have become an increasingly mainstream alternative to antibodies as wide spectrum of analytes, since any aptamer–target binding event can in principle affinity reagents and have potential applications in protein analysis, analytical and be translated to conformational transition and be detected based on this strategy. biosensor devices, and medical diagnostics. The conventional method for aptamer discovery, systematic evolution of ligands by exponential (SELEX), aims to discover 249 Reducing Feature Spreading in Contact Pin-Printed Organosilane new aptamers by incubating a selected target protein with pools of oligonucleotides. Arrays on Porous Silicon Despite its success, this process has many drawbacks, including underrepresen- Sitora Khodjaniyazova, SUNY-Buffalo, Dept. of Chemistry, 507 Natural tation of G-quadruplex sequences. G-quadruplex structures have been proven to Sciences Complex, Buffalo, NY 14260, Sidney G. Coombs, Frank V. be an important aptamer motif and are prevalent throughout the human genome, Bright particularly oncogene promoter regions, suggesting their biological significance. Porous silicon (pSi) exhibits strong, red-orange photoluminescence (PL) at room In this study, we have employed a reverse selection method developed in our lab temperature. pSi can be used in sensing, optoelectronics, and lab-on-chip devices that incubates a single G-rich oligonucleotide based on human oncogene promoter because pSi surface is easily modified and its PL is analyte responsive. However, sequences with pools of proteins from human cancer cells. Previous success has pSi PL is unstable because as-prepared, H-passivated pSi (ap-pSi) is easily oxi- been achieved in our lab using the ERBB2 breast cancer promoter. Now, we expand dized under ambient conditions. The goal of our research is to stabilize the PL and our study to include other potential aptamer sequences, such as the c-myc and simultaneously impart chemical functionality to the pSi surface through silanization c-myb promoter regions. In our overall approach, these sequences are attached with an organically modified silane. Traditionally, the ap-pSi surface is pretreated to to streptavidinated magnetic beads and protein capture is performed in-vitro us- create oxidized porous silicon (ox-pSi) before silanization. Our previous research ing nuclear extracts from the human cancer cell lines BT-474, K-562, and MCF-7. showed, through combined PL and infrared (IR) imaging studies, that ox-pSi si- Captured proteins are collected and initially screened using matrix-assisted laser lanization can be achieved through direct contact pin-printing (CPP) of organosilane desporption/ionization-time of flight mass spectrometry. These protein mixtures are arrays. In the case of the popular organosilane 3-aminopropyltriethoxysilane (APT- then separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis ES), silanization of ox-pSi results in rapid feature formation (< 1 min) with significant and identified by liquid chromatography with tandem mass spectrometry. Protein feature spreading, up to 5x the pin diameter. This extensive feature spreading pres- identities are confirmed for the selective capture by Western blot experiments and ents a major barrier to the creation of high-density contact pin-printed microarrays the potential biological significance of the interactions is determined using chromatin on pSi. To address feature spreading, we explore CPP on ap-pSi as a strategy to immunoprecipitation (ChIP). This presentation focuses on Western blot interroga- mitigate feature spreading. In this presentation we present the discovery of in-situ tion of potential protein candidates. oxidation of ap-pSi by CPP APTES and its impact on feature spreading and com- position. 253 Affinity Methods in Laboratory Medicine: Novel Tools for Clinical Analyses 250 Electrochemical Hydrogen Evolution from Ni and Ni–Mo William Clarke, Johns Hopkins University School of Medicine, 1800 Composites Orleans St., Sheikh Zayed Tower B-1020F, Baltimore, MD 21784 Peter M. Csernica, Cornell University, 124 Westbourne Ln., Apt. 1241, Affinity-based separation methods are an emerging and exciting area of research Ithaca, NY 14850, James R. McKone, Francis J. DiSalvo, Héctor D. in analytical chemistry. Methods based on affinity interactions are widely used in a Abruña variety of research areas including environmental analysis, proteomics, pharmaceu- Electrolysis of water is an attractive method for producing clean hydrogen for storing tical analysis, and clinical studies. This lecture discusses the use of affinity-based and transporting renewable energy. Although hydrogen production by this method methods for clinical analyses, ranging from basic chemical affinity methods (e.g., is most effective using precious metal catalysts, non-noble metals are attractive HbA1c and boronate) to complex hybrid methods involving antibody-based ex- alternatives because of their reduced cost. Among the many bimetallic materials traction and mass spectrometry (e.g., immunoextraction-liquid chromatography tan- that have been investigated as catalysts for the hydrogen evolution reaction (HER), dem mass spectrometry and parathyroid hormone). Opportunities and challenges nickel–molybdenum (Ni–Mo) compounds have been shown to be particularly active. of affinity-based separations in clinical laboratories are also discussed, as well as Importantly, their high activity suggests synergy between the two metals, as the potential future applications in clinical analyses. bimetallic compounds are substantially more active than either of their constituents. Previous studies of Ni–Mo HER catalysts have focused primarily on disordered al- 254 Natural Product Screening Using Affinity Chromatography loys, which have a molybdenum content that can be difficult to control and is re- Ruin Moaddel, National Institute on Aging, NIH, 8C232 BRC, 251 stricted to be < 25%. Here, an established method for producing the alloy was mod- Bayview Blvd., Baltimore, MD 21224 ified to synthesize ordered Ni–Mo intermetallics that incorporate very nearly 50% No abstract submitted by the author. molybdenum. The HER activity of the intermetallic powder was tested alongside a disordered alloy and pure Ni sample under identical conditions. When normalized to mass loading, the alloy was by far the most active. However, when normalized to

37 2015 EAS Abstracts November 2015

255 Affinity Chromatography as a Probe of Cellular Pharmacology ear in these biogenic liquids, MCR can extract useful informative data about the Current Practice and Future Directions changes in some amino acids and the protein product. Here MCR clearly showed Irving W. Wainer, Mitchell Woods Pharmaceuticals, 4 Corporate Dr., Ste. the increase of a unique dityrosine emission from the product glycoprotein with pro- 287, Shelton, CT 06484 cess time. Accurate quantitative predictive regression models for end glycoprotein In biological systems, molecular recognition at pharmacologically active sites is yield using EEM were possible with relative errors of <5%. This approach can be based upon highly selective interactions between biopolymers such as proteins used for process management at early stage by predicting final process outcome (targets) and other proteins or small molecules (ligands). One approach to the study at the early, small-scale (100-200L) stage of fed-batch manufacturing processes. of small molecule–protein interactions is cellular membrane affinity chromatography The incorporation of extra dimensions to MDF measurements such as anisotropy, (CMAC) where cellular membrane fragments are immobilized within a chromato- provides an alternative approach to macromolecular component and fluorophore graphic column. Frontal affinity chromatography (FAC) techniques are used to as- resolution based on rotational diffusion and/or molecular size. We show how protein sess binding interactions between test compounds and receptors, transporters, etc. can be quantified in model cell culture media (yeast hydrolysate) and how the meth- contained within the immobilized membranes. The CMAC technology was initially odology can be extended to the structural analysis of multi-fluorophoric proteins. developed using cellular membrane fragments and tissue homogenates. Recently, This anisotropy resolved multi-dimensional emission spectroscopy (ARMES) meth- the method has been expanded to include nuclear and mitochondrial membranes. od for example could clearly resolve different tyrosine fluorophore populations and The results obtained on the nuclear and mitochondrial membrane affinity columns room temperature phosphorescence from albumin protein emission. are discussed as will the application of this approach to the identification of new drug candidates and to elucidation of the associated molecular mechanisms. 259 Insightful Analytical Data from Upstream Bioprocesses with a Real-time In-Situ Monitor 256 Frontiers in Affinity-Based Separations: Exploring New and Unique Mark Arnold, University of Iowa, Center for Biocatalysis and Tools for the Rapid Analysis of Clinical, Pharmaceutical and Bioprocessing, Iowa City, IA 52242 Environmental Samples Near-infrared spectroscopy is an established process analytical technology (PAT) David S. Hage, University of Nebraska-Lincoln, Dept. of Chemistry, 704 for monitoring and controlling the production and formulation of small molecule Hamilton Hall, Lincoln, NE 68588 therapeutics. Despite considerable effort by the analytical community, near infrared The use of biologically-related binding agents, such as antibodies and receptors, spectroscopic sensing has not been successfully implemented as a PAT monitoring has been of great interest for decades as a means for the selective isolation and technology for bioprocesses, where real-time measurements of cellular nutrients analysis of chemicals and biochemical in complex mixtures. Affinity chromatogra- and metabolites are necessary to optimize upstream processes used in the produc- phy and high-performance affinity chromatography are powerful separation meth- tion of biotherapeutics. This presentation will focus on advances associated with ods that make use of these same types of binding agents as stationary phases. monitoring cellular nutrients and metabolites with in situ near infrared spectroscopy. This presentation provides a brief overview of affinity-based separation methods Selectivity will be discussed as it relates to the robustness of analytical measure- and discusses recent developments in the creation and bioanalytical applications ments with noninvasive near infrared spectroscopy. A novel near infrared monitor of high-performance affinity chromatography. This includes a discussion of how developed by ASL Analytical, Inc. will be introduced and its ability to monitor glycerol high-performance liquid chromatography (HPLC)-based affinity columns can be and methanol in real-time during Pichia fermentations as well as glucose and lactate used in chromatographic immunoassays, rapid affinity extractions, multi-dimension- during CHO cell cultivations will be demonstrated. Lastly, the utility of continuous al systems, and flow-based biosensors for targets such as drugs, hormones, pro- analytical information provided by this monitor will be discussed in the context of teins, and environmental agents. The use of high-performance affinity columns and extracting valuable kinetic information related to the upstream bioprocess. microcolumns for characterizing the thermodynamics and kinetics of biological inter- actions are also considered, as well as the use of affinity-based separations in the 260 Using IR Absorption and Raman Spectroscopy for Characterization high-throughput screening of drug-protein interactions and personalized medicine. of Biomass Hydrolysis Sergey Mozharov, University of Washington, 1013 NE 40th St., Seattle, 257 Identification of Model Parameters in Cell Culture Bioreactor by WA 98105, Brian Marquardt, Charles Branham Raman Spectroscopy via Calibration-Free Way Mid-infrared (IR) absorption and Raman spectroscopy are complimentary tech- Nicolas Spegazzini, Massachusetts Institute of Technology, 77 niques that have been used for characterization of biomass conversion processes Massachusetts Ave., Cambridge, MA 02139​ for many years. Chemical and physical complexity of lignocellulosic biomass as An outstanding challenge in photonics and bioprocess research is to devise a well as chemical interactions involving its constituents make it difficult to find the method for continuous, non-invasive monitoring of analytes, which constitutes a most suitable strategy of data analysis. Multivariate data analysis techniques are significant component of quality-by-design (QbD) and critical process parameters commonly used to analyze vibrational spectra. They often rely on a sequence of monitoring (CPPs). Vibrational spectroscopy potentially provides a powerful tool for pre-processing steps, some of which can be ambiguous and ineffective in sepa- simultaneous, quantitative and label-free measurement of multiple analytes, due to rating relevant and irrelevant variance. In this report, the use of mid-IR and Raman its intrinsic chemical and biochemical specificity. Here, we propose a novel calibra- spectroscopy for characterization of a large set of biomass hydrolysis reactions is tion framework that enables spectroscopy-based estimation of analyte information presented. Gradually increasing complexity of the samples, it is shown how over- without necessitating extensive a priori concentration information. In a nutshell, a looking instrumental, optical and physical factors can significantly influence mul- kinetic model of the investigated process provides a guide to the ‘‘missing’’ concen- tivariate prediction models. Factoring process understanding in the model is nec- tration portion of the ill-posed problem of concentration estimation. We employ this essary to avoid these mistakes. Using IR and Raman Spectroscopy concurrently new method in bioprocess modeling. Biological drugs or another type of cell culture with a combination of theory-driven and data-driven multivariate techniques was are produced in a bioreactor by cells growing, a tank designed to maintain carefully shown to be an effective approach to advance process understanding, substantially calibrated conditions. The result can be a bit different every time you run a reactor. A improve reliability of measurements by cross-validating the results in real time, learn bioreactor control provides special challenges due to significant process variability, how to overcome limitations of each instrument and identify their failure points. the complexity and nonlinearity of biological systems. Based on in process analyti- cal technology monitoring it was possible by Raman spectroscopy obtain in-situ in a 261 Survivor-Selected Ion Monitoring (Survivor-SIM): Combining High bioreactor quantitative determination of bioanalyte in a batch process. Mass Accuracy, Resolution, and Chemical Noise Elimination to Augment the Quantitative Performance of a Q Exactive Orbitrap 258 The Use of Multi-Dimensional Fluorescence Spectroscopy for the Eugene Ciccimaro, Bristol-Myers Squibb, Rte. 206 and Province Quantitative Analysis of Liquid Media: From Hydrolysates to Line Rd., Princeton, NJ 08543, Asoka Ranasinghe, Carrie Xu, Joelle Protein Solutions Onorato, Kimberly Snow, Dieter Drexler, Celia Darienzo, Timothy Olah Alan Ryder, Nanoscale Biophotonics Laboratory, School of Chemistry, Due to observed collision induced dissociation (CID) fragmentation inefficiency, de- National University of Ireland Galway, Galway, Ireland, Boyan Li, Radu veloping sensitive liquid chromatography tandem mass spectrometry (LC-MS-MS) Groza assays for CID resistant compounds is especially challenging. As an alternative to Multi-dimensional fluorescence (MDF) spectroscopies like excitation-emission ma- traditional LC-MS-MS, we present here a methodology that preserves the intact trix (EEM) and total synchronous fluorescence scan (TSFS) offer unique possibil- analyte ion for quantification by selectively filtering ions while reducing chemical ities for the quantitative and qualitative analysis of proteins in complex biogenic noise. Utilizing a quadrupole-Orbitrap MS (Q Exactive), the target ion is selectively liquids. Conventional EEM spectroscopy was used for the quantitative predictive isolated while interfering matrix components undergo MS-MS fragmentation by CID, analysis of glycoprotein production in a CHO cell fed-batch process. EEM spectra allowing noise-free detection of the analyte’s surviving molecular ion. In this man- of complex solutions are very sensitive to compositional change and this can be ner, CID affords additional selectivity during high resolution accurate mass analysis monitored using multivariate curve resolution (MCR). Despite the fact that the rela- by elimination of isobaric interferences, a fundamentally different concept than the tionship between spectral change and fluorophore concentrations are highly non-lin- traditional approach of monitoring a target analyte’s unique fragment following CID.

38 2015 EAS Abstracts November 2015

Survivor-SIM expands the targeted quantitative options of the Q Exactive, which allel reaction monitoring (PRM) modes. Both instruments showed excellent linearity include selected ion monitoring (SIM) and parallel reaction monitoring (PRM). We and repeatability; however the high-resolution PRM mode (at 10 ppm product ion highlight the utility of the survivor-SIM analysis using cyclic peptides as a case study. mass accuracy) demonstrated the highest selectivity. Therefore, the high-resolution mass spectrometry is preferred for analyses of these important estrogen metabo- 262 Ultra-Sensitive and Accurate Analysis of Biotherapeutics Using lites in serum samples from postmenopausal women and older men. Supported by Low-Flow LC-MS NIH grant P30ES013508. Jun Qu, SUNY- Buffalo, 701 Ellicott, Buffalo, NY 14203 Though representing a promising alternative to ligand-binding-assays for quantifi- 265 The Role of Millisecond Conformational Motions in Enzyme cation of biotherapeutics, LC-MS-based methods face challenges associated with Function and Allostery sensitivity and accuracy. To cope with these, we developed a suite of strategies Patrick Loria, Yale University, 225 Prospect St., PO Box 208107, New based on low-flow LC-MS. The workflow is comprised of: 1) an orthogonal array Haven, CT 06520 optimization (OAO) for high-throughput and reliable method development, which Nuclear Magnetic Resonance (NMR) relaxation dispersion experiments are used to greatly facilitates the selection of the most sensitive and stable signature peptides characterize millisecond and microsecond motions and the role of these motions in that ensures reliable; 2) optimal blood removal and extraction approaches for tissue regulation of protein tyrosine phosphatases (PTPs) and in allostery in the enzyme analysis; 3) surfactant aided on-pellet digestion (SOD) sample treatment method for imidazole glycerol phosphate synthase (IGPS). In a pair of mechanistically indistin- highly efficient and reproducible sample cleanup and digestion with high-through- guishable PTPs with catalytic rates that vary by 50-fold these experiments indicate put (45min); 4) a ultra-sensitive, robust and high-throughput trapping micro-LC- that catalytic activity is regulated by the rate of active site loop closure. Millisecond MS technique for routine analysis of biotherapetuics with extremely low limits of motions also appear to play a role in the allosteric activation of IGPS. Here, the quantification (LOQs). 5) a dual-mechanism, antibody-free enrichment method to extent of millisecond motions correlates closely with the effectiveness of allosteric enrich signature peptides from highly complex matrices in a high-throughput man- ligands to activate catalysis in IGPS, a V-type allosteric enzyme. ner; 6) hybrid calibration method for accurate quantification without the need of iso- tope-coded protein internal standard. Several paradigms using these methods are 266 Use of 19F NMR to Probe Conformational Heterogeneity and presented, including applications in ultra-sensitive pharmacokinetic (PK) analysis Dynamics of Exchange in Functional RNA Molecules and tissue PK. Nancy L. Greenbaum, Hunter College-CUNY, 695 Park Ave., New York, NY 10065, Caijie Zhao 263 Application of Differential Mobility Spectrometry-Triple Quadrupole Functional ribonucleic acid (RNA) molecules are often very plastic and undergo Mass Spectrometry to Sensitive and Selective Bioanalysis of changes in base pairing patterns, thus forming alternative secondary and tertiary Peptides not Suited for MRM Analysis conformations. This property is critical for multiple roles of some noncoding RNA Mingshe Zhu, Bristol-Myers Squibb, Rte. 206 & Province Line Rd., molecules in gene expression. In order to understand mechanisms of RNA-medi- Princeton, NJ 08543, Eugene Ciccimaro, Jr., Naiyu Zheng, Yuan-qing ated activity, it is important to probe the kinetics and thermodynamics associated Xia with conformational rearrangement of functional RNA molecules. Solution nuclear Triple quadruple-based multiple reaction monitoring (MRM) is the method of choice magnetic resonance (NMR) provides excellent tools for analysis of conformational for quantitative bioanalysis of intact peptides or signature peptides from digested heterogeneity and dynamic exchange, the timescale of which (ms to sec) is within proteins in biological matrix. However, some cyclic peptides, such as human sun- the NMR detection range. However, technical challenges associated with spectral flower trypsin inhibitor (SFTI), cannot generate useful product ions under collision overlap of uniformly 13C or 15N labeled RNA molecules or the selective labeling of induced dissociation (CID) for sensitive or selective MRM analysis. Herein, we individual groups to be tracked may limit monitoring of these nuclei. By comparison, present a new workflow that combined differential mobility spectrometry (DMS) with the ease of incorporating single 19F-labeled nucleotides by commercial chemical multiple ion monitoring (MIM) for bioanalysis of peptides that have poor CID frag- synthesis and large spectral dispersion and sensitivity to chemical environment of mentation efficiency. SFTI was spiked into blank rat plasma (0.125 to 2,000 ng/mL), 19F presents unique advantages. In this work, we measure the rates associated followed by quantitation using AB SCIEX QTRAP® 6500 equipped with IonDrive™ with spontaneous interconversion between major conformers in folded RNA se- source and SelexION™ DMS technology. A liquid chromatography (LC)-DMS-MIM quences by use of a 19F-19F EXSY NMR experiment on RNA samples into which method was developed using double charged precursor ion of m/z 757 in Q1 and single 5-19F-pyrimidine labels are incorporated. We first utilize this approach to de- Q3, respectively. An optimized composition voltage was obtained at 9.5 V at a fixed termine kinetic exchange rates between conformers in a model bi-stable RNA stem separation voltage at 3500 V. A lower limit of quantitation (LLOQ) of LC-DMS-MIM loop, and then to probe the dynamic nature of rearrangements between multiple for SFTI in plasma was 0.125 ng/mL, 40 folds better than that analyzed by LC-MIM conformers in a larger RNA construct representing the U2-U6 snRNA complex of (DMS-off mode). The LC-DMS-MIM baseline was significantly lower than that of the human spliceosome. In the case of the U2-U6 snRNA complex, such a rear- LC-MIM. This suggested that the improvement of the signal-to-noise was mainly rangement in the context of the intact spliceosome may have critical implications contributed by the reduction of noise levels by DMS. In addition, use of higher CE in splicing activity. in Q2 was able to reduce baseline further, resulted in a two-fold improvement of the sensitivity. Therefore, LC-DMS-MIM workflow is a very valuable option to com- 267 Hybrid Approaches for Protein Structure Determination Combining plement LC-MRM and LC-HRMS approaches in bioanalysis of peptides molecules Computational Modeling with Sparse NMR Restraints and/or signature peptides. Gaetano Montelione, Rutgers University, CABM 679 Hoes Ln., Piscataway, NJ 08854, Yeufeng Tang, Yuanpeng J. Huang, Thomas 264 Ultrasensitive Quantification of Serum Estrogens Using Pre- Hopf, Debra Marks, Chris Sander ionized Derivatives and LC-MS Accurate protein structure determination by nuclear magnetic resonance (NMR) is Ian A. Blair, University of Pennsylvania, Penn SRP Center, 854 BRB II/ routine for smaller proteins, but more challenging for many larger proteins, for which III, 421 Curie Blvd., Philadelphia, PA 19104, Qingqing Wang, Clementina experimental data is often incomplete and ambiguous. The massive increase in evo- Mesaros, Nathaniel W. Snyder lutionary sequence information coupled with maximum likelihood covariance anal- Quantification of multiple estrogens and their metabolites in serum or plasma re- ysis now provides a rich complementary source of structural constraints. Exploiting quire the use of stable isotope dilution methodology in combination with ultra-per- this synergy, we have developed a hybrid approach that uses evolutionary couplings formance liquid chromatography – selected reaction monitoring mass spectrometry (EC) from the sequence record together with sparse NMR data to determine accu- (UPLC-SRM-MS). Extremely high sensitivity, which can be obtained by the use rate three-dimensional (3-D) protein structures. We demonstrate this hybrid “EC- of pre-ionized derivatives, makes it possible to determine the low levels present NMR” method by determining accurate structures of eight proteins ranging in size in samples from postmenopausal women and older men. An ultrasensitive stable from 64 to 370 amino-acid residues (6 to 41 kDa). For small proteins and domains isotope dilution liquid chromatography (LC)-SRM/MS method has been developed up to 150 residues (< ~15 kDa) with extensive sequence information, EC-NMR is a and validated for multiplexed quantitative analysis of six unconjugated serum es- powerful and efficient new approach for protein structure determination. For larger trogens and their hydrolyzed conjugates as N-methyl pyridinium-3-sulfonyl (NMPS) proteins ECs can be combined with sparse NMR data to provide structures that are derivatives using a triple quadrupole mass spectrometer (Thermo TSQ Vantage). more accurate and complete than those obtained using such NMR data alone. We This method requires only 0.1 mL of human serum, yet detected 1 to 10 fg on also discuss other hybrid methods combining computational modeling with NMR column for the six estrogens and their hydrolyzed conjugates. However, interfer- data to determine 3-D structures of larger proteins and enzymes. These advances ence was still observed in the channels used to monitor the catechol estrogens significantly expand the range of proteins for which accurate structures can be de- - 4-hydroxy-estradiol (4-OH-E2) and 2-OH-E2. Therefore, assay performance for termined using NMR spectroscopy. serum samples from postmenopausal women on the triple quadrupole instrument has been compared with analyses of the same samples conducted on a Thermo Q-Exactive Plus hybrid quadrupole/Orbitrap high-resolution mass spectrometer. The Q-Exactive was operated in the full scan/single ion monitoring (SIM) and par- 39 2015 EAS Abstracts November 2015

268 FBLD Yields Orally Active Brain Penetrant Inhibitors for BACE1 272 Quantitative Single-Molecule Imaging of DNA Hybridization and PDE10A Eric M. Peterson, University of Utah, Dept. of Chemistry, 315 South Daniel F. Wyss, Merck, 2015 Galloping Hill Rd., Kenilworth, NJ 07960 1400 East, Salt Lake City, UT 84112, Joel Harris, Michael W. Manhart, Fragment-based lead discovery (FBLD) has become one of the preferred lead dis- Frances Morris covery methods in the pharmaceutical industry. Starting with low molecular weight, Quantitative imaging of individual fluorescently-labeled molecules is a powerful and ligand efficient fragments promise to deliver chemical series with superior physi- extremely sensitive method to characterize equilibria and dynamics of bio-recog- cochemical properties compared to those derived from traditional high throughput nition events at liquid-solid interfaces. Total-internal-reflection excitation combined screening (HTS) approaches. This may be particularly important when a small mol- with high-efficiency imaging of fluorescence allows surface-bound molecular pop- ecule needs to reach a drug target in the brain to elicit its disease-modifying activ- ulations to be quantified in-situ by counting individual molecules. We haveap- ity. This presentation describes two such examples where highly structure-driven plied this methodology to characterize oligonucleotide hybridization, the chemistry FBLD approaches yielded orally active, brain penetrant inhibitors. In the first exam- of which is integral to the design and understanding of modern chip-based DNA ple, FBLD was essential to the discovery of the non-planar cyclic amidine class of screening. DNA hybridization is challenging to measure by single-molecule tech- BACE1 (β-secretase) inhibitors which show tremendous promise in Alzheimer’s dis- niques because of nonspecific adsorption, which is typically mitigated by fluores- ease (AD) and which have produced the most advanced inhibitors in clinical devel- cence resonance energy transfer (FRET) methods that compromise detection effi- opment as potential disease-modifying treatments for AD. In the second example, ciency. In this work, we describe a scheme for immobilizing probe single-stranded FBLD recently led to a series of highly potent and selective phosphodiesterase 10A DNA at a glass interface passivated against nonspecific adsorption with anionic (PDE10A) inhibitors. Scaffold modification then yielded a compound that displayed blocking groups, yielding surfaces where complementary binding dominates the an overall excellent in vivo profile across various anti-psychotic preclinical efficacy population of fluorescently-labeled target DNA on the surface. Surface populations models. With multiple compounds from several organizations progressing in the of complementary target DNA are nearly three orders of magnitude greater than clinic, the potential of PDE10A inhibitors for the treatment of schizophrenia should those of a scrambled sequence. The surface density of immobilized probe DNA soon be known. can be controlled by the probe concentration used in the immobilization reaction. At low surface densities, association and dissociation kinetics are measured under 269 New Nano Tools for “Follow-that-Molecule” in Single Live Cells equilibrium conditions where individual probe molecules can be resolved so that Nancy Xu, Old Dominion University, Dept. of Chemistry and hybridization and dissociation rates can be measured directly from single-molecule Biochemistry, Norfolk, VA 23529, Tao Huang, Pavan K. Cherukuri, trajectories. At higher probe capture site densities, the substrates are more efficient Preeyaporn Songkiatisak at capturing target strands from solution, making them excellent single-stranded Binding of a few ligand molecules with its receptors on single live cells can initiate DNA sensors with very low detection limits. dynamic cascades of cellular signaling pathways, alter cellular functions and cause diseases. Such signaling pathways can take minutes to hours. Currently, fluores- 273 Low-Cost Multispectral Imaging for Art and Archaeology cence microscopy using fluorescence imaging probes is the primary workhorse Antonino Cosentino, Cultural Heritage Science Open Source, Piazza for live cell imaging. Unfortunately, fluorescence probes (fluorophor, fluorescence Cantarella 11, Aci Sant’Antonio 95025, Italy protein, QD) suffer intrinsic photobleaching, making them unable to continuously We present a low-cost multispectral imaging system for art and archaeology real- capture the dynamic events of single live cells over hours. Photobleaching also ized thanks to the first ever crowdfunding project in art conservation science which makes quantitative analysis over time difficult. We have developed photostable sin- has seen the participation of 43 donors from 16 countries. The system uses a set of gle nanoparticle imaging probes, single molecule nanoparticle optical biosensors bandpass filters, a digital single-lens reflex camera and free software for editing the (SMNOBS), and far-field photostable-optical-nanoscopy (PHOTON). We have used multispectral images. This system allows to map pigments in polychrome artifacts these new nano tools to follow the same single molecules in single live cells over and to obtain their reconstructed reflectance spectra. Scientific examination and hours, watch them initiating the dynamic cascades of signaling pathways of sin- documentation of art is expensive. Typically larger museums have budgets suffi- gle live cells in real-time at single-molecule and nanometer resolutions. We have cient for scientific departments equipped with cutting-edge technologies while small showed that these new tools can overcome the drawbacks of fluorescence micros- to medium sized cultural institutions have very limited access to the same science copy for quantitatively imaging of single molecules and studying signaling pathways and technology. The most important and recognizable works of art from prestigious of single cells with superior temporal and spatial resolutions. These new tools can museums get extensive scientific studies, unfeasible for the vast majority ofcul- be used to determine cellular functions of individual proteins in single live cells to tural heritage objects, existing in local communities since they lack comparable fi- address fundamental questions in cell biology, identify new protein biomarkers and nancial resources. Cultural Heritage Science Open Source (CHSOS) initiative was rare subsets of tumor cells for disease diagnosis, as well as, characterize underlying launched in 2012 and it aims to develop and disseminate affordable and sustainable molecular mechanisms of toxicity of environmental pollutants. The detailed experi- methodologies for art examination that can reach a much larger audience of cultural mental design and the updated results are presented. The work is supported in part institutions. CHSOS pursues its mission in three significant ways: its popular blog, by NSF (CBET 0507036 & 1450936) and NIH (R01 GM0764401). publications and training programs. So far, CHSOS has developed and dissemi- nated low-cost technical solutions for panoramic infrared reflectography, technical 270 Microwell Arrays for Measuring Single Molecules photography, reflectance transformation imaging and reflectance spectroscopy. David Walt, Tufts University, Dept. of Chemistry, 62 Talbot Ave., Medford, MA 02155, Barrett Duan 274 Revealing the Hidden Writing of a 15th Century Palimpsest Using Presently available methods for detecting biomolecules are primarily based on mea- Hyperspectral Imaging Analyzed by Principal Component Analysis suring “analog” signals—the higher or lower the concentration, the higher or lower and Generalized Least Squares Weighting the signal, respectively. Digital measurements, based on counting single molecules, Donald Dahlberg, Lebanon Valley College, 101 N. College Ave., PO enable extremely high sensitivity because low background signals can be readily Box 1400, Annville, PA 17003, Neal Gallagher, Meghan Wilson, Fenella distinguished from the high digital signals making for a much lower limit of detection. France We have developed a method that allows us to measure the concentration of pro- Hyperspectral imaging of a 15th century palimpsest was undertaken at the Library teins more than a thousand times lower than enzyme-linked immunosorbent assays of Congress with the Preservation Research and Testing Division (PRTD) standard- (ELISAs). The method also allows us to observe the behavior of individual enzyme ized spectral imaging system comprised of illumination panels and camera with molecules and nanoparticles. The application of the technology to new diagnostic integrated software and hardware. This system contains a 39 MegaPixel Mono- tests in the fields of oncology and infectious disease is described. A discussion of chrome E6 Camera using a Kodak CCD sensor (7216 × 5412 pixel array with linear how the technology is being commercialized is also presented. dimension of 6.8 microns). Integrated light emitting diodes (LED) illumination panels containing 13 wavebands from the ultraviolet (UV-365nm), through the visible (VIS) 271 Single-Molecule Photoelectrocatalysis and into the infrared (IR-940nm) regions of the visible and non-visible spectrum, Peng Chen, Cornell University, Dept. of Chemistry, Baker Laboratory, were captured in reflected illumination mode. Four additional images were taken Ithaca, NY 14853 with very low angle illumination at 470nm and 910nm. The resulting 17 grayscale This talk presents our recent results in using single-molecule fluorescence micros- TIFF images were combined and analyzed using Solo+MIA software by Eigenvector copy to image photoelectrochemical reactions on single semiconductor nanostruc- Research. Multivariate image analysis by principal component analysis (PCA) was tures. We separately image hole and electron induced reactions, driven by light dominated by the shadows created by the wrinkles and texture in the palimpsest and electrochemical potential, and map the reactions at single reaction temporal and revealed only faint images of the original writing. Generalized least squares resolution and nanometer spatial resolution. We also correlate the surface reactivity weighting (GLSW) is a pretreatment method that deweights variables unrelated to with the overall performance of each nanostructure in photoelectrochemical splitting the property of interest. In GLSW a region of the image not containing writing in se- of water. lected and a PCA model is constructed of this region. Any variation within the select- ed region is considered clutter and variables important to the clutter are deweighted

40 2015 EAS Abstracts November 2015

before the final PCA analysis of the whole document. GLSW-PCA was much more ing in greater confidence in the analytical results. While GC-MS-MS is the laboratory successful in suppressing the wrinkle and texture shadows and revealing the origi- workhorse for the analysis of volatile residues, it can only find compounds if they are nal cursive writing of the palimpsest. Examination of the GLSW deweighted variable included in the method. A screening method capable of looking for hundreds of pes- loadings revealed that the lower wavelengths were most useful in revealing the ticides without the need to purchase standards or calibrate would complement the original writing. targeted GC-MS-MS approach. This talk describes a new pesticide screening meth- od using a high resolution accurate mass GC- quadrupole time-of-flight (Q-TOF) 275 Spectral Imaging: Capturing and Illuminating Cultural History - with a new exact mass pesticide database/library. This screening method has been Scholarly and Scientific Information applied to QuEChERS (quick, easy, cheap, effective, rugged, and safe) extracts of Fenella G. France, Library of Congress, 101 Independence Ave. SE, organic fruits and vegetables spiked at the 10 and 100 ppb level. More than 97% of Washington DC 20540 the spiked pesticides were found at both spiking levels. The refinement and development of non-invasive imaging technologies: multi- and hyper-spectral imaging, provide a depth of information from cultural heritage objects 278 An Improved QuEChERS Method for LC-MS/MS Determination of that has previously remained hidden, providing new layers of scholarly and pres- Multiresidue Mycotoxins in Grains ervation data by capturing images of heritage materials in distinct wavebands of Michael Young, Waters, 34 Maple St., Milford, MA 01757, Kim Van Tran, the visible and non-visible spectrum. Advancing these capabilities has enabled ob- Dimple Shah scure and ambiguous historic facts/records to be liberated allowing new revelations Recently a method was published describing a modified QuEChERS (quick, easy, and interpretations of previously assumed information from founding fathers to be cheap, effective, rugged, and safe) method for liquid chromatography tandem mass amended or confirmed. Image processing of the large datasets generated allow for spectrometry determination of 14 mycotoxins in rice (Koesukwiwa et al., 2014). This a wealth of information to be garnered. Two main processing techniques utilized are year, an evaluation of that method was presented for mycotoxins analysis in whole principal component analysis (PCA) and spectral curve analysis. These techniques grain wheat, maize and rice flours (Young et al., NACRW 2105). Good recovery was respectively generate pseudo color images to separate materials that appear similar observed in all three matrices for most compounds including aflatoxins, fumonisins, in the visible region, and analyzing spectral curves from the same pixel location ochratoxin, T2-Toxin and HT-2 toxin. However, the recommended dispersive sol- throughout the range of wavebands derives a unique spectral curve for the specific id-phase extraction (dSPE) provided cleanup suitable only for analysis using highly materials – colorants, inks or substrates. These imaging technologies have been sensitive instrumentation. An alternative cleanup strategy was then investigated expanded to gather a range of preservation information including the characteriza- using a novel reversed-phase SPE cartridge in the pass-thru cleanup mode. This tion of colorants non-invasively, and comparative ink analysis. A further advantage simple SPE protocol effectively removed the fat and phospholipid (lecithin) from is the ability to undertake predictive assessment of the impact of environment and the QuEChERS extract. The more effective cleanup allows for more reproducible treatments – both historic and modern. Baseline imaging and tracking changes over evaporation and reconstitution of the samples and therefore makes the QuEChERS time allows for more objective decisions to be made for exhibition and storage of method more suitable for use with less sensitive instruments. heritage materials, and the ability to ensure the effectiveness of conservation treat- ments for stabilization. Imaging technologies have advanced the capacity to better 279 Heavy Metals in Seafood: Let’s have a Risk/Benefit Conversation preserve, understand degradation, and capture scholarly information in cultural her- Marc E. Engel, Florida Dept. of Agriculture and Consumer Services, itage materials. 3125 Connor Blvd., #9, Tallahassee, FL 32399, Donald M. Axelrad Cadmium, lead and mercury are commonly found in seafood products, and are 276 It Takes a (technological) Village: A Marriage of Traditional and known human health hazards. Consequently, canned, fresh, and frozen seafood Modern Conservation Methodologies to Reveal Invisible 18th were analyzed for these metals and the results reviewed to identify products that Century Spanish Colonial Frescoes Found on the Sacristy Walls in may be of concern regarding human health. Shrimp, oysters and clams had very the Alamo low levels of mercury. However canned oysters from Asia had relatively high levels Pamela Rosser, The Alamo, 300 Alamo Plaza, San Antonio, TX 78205, of cadmium and lead; on average cadmium levels (n=28) were 1.24 µg/g and lead Dennis A. Baltuskonis, Michelle M. Bushey (n=25) 0.61 µg/g. Four of the canned oyster samples were smoked oysters and A combination of modern scientific, coupled with more traditional conservation these had on average cadmium levels of 2.54 µg/g and lead levels of 1.4 µg/g. The methodologies, has been successfully employed to discover 18th century mission smoked oyster sample with the highest levels of cadmium and lead had 7.07 µg/g artwork. This abstract is on the implications of recent technical and scientific discov- and 3.72 µg/g respectively. Considering the long half-life for cadmium, its classifica- eries regarding the interpretation of early 1700’s Spanish frescoes and how three tion as a potential carcinogen and the sensitivity of children to lead, these levels are conservation specialties joined together to make the invisible- once again visible. of concern for human health. Results confirm that the higher the trophic level of a fin- Traditional conservation cleaning techniques first uncovered the Spanish colonial fish species the higher its mercury levels. Mercury levels in shark, and swordfish are era frescoes on the interior walls of the Sacristy in the Alamo in 2000. The original typically above the United States Food and Drug Administration guidance level. Fin- designs covered the entire surface of each wall. Distinct patterns have been discov- fish are a good source of low fat protein and salmon for example is a good source of ered that encompass the entire room. Additional design elements found suggest omega-3 fatty acids, which are important to fetal and children’s neurodevelopment that each wall was once elaborately painted according to a pre-ordained master and adult heart health. It is important to consider the health benefits of fish con- plan that included the use of stencil patterns and multi-colors of a highly symbolic sumption while also considering mercury exposure. Based on omega-3 fatty acids nature. Two major technological advancements, multi-spectral imaging (MSI) and and mercury concentration, canned white tuna is a “healthier” choice than grouper. X-ray fluorescence (XRF) analysis were then enlisted to gain further insights into the nature of the designs. The ability to capture digital images in distinct, wavebands 280 The Analysis of Pesticide Residues in Foods Using Liquid of the electromagnetic spectrum, (MSI) proved indispensable in elucidating fresco Chromatography and High Resolution Mass Spectrometry design nuances, e.g., the use of “pouncing”, and even the existence of significant Brian Eitzer, The Connecticut Agricultural Experiment Station, 123 design features which could only be visualized under UV illumination. These discov- Huntington St., New Haven, CT 06511, Walter Krol, Christina Robb eries when further combined with XRF analysis verified the existence of the design One of the challenges in the analysis of pesticide residues in foods is the large num- and its composition including the sophisticated use of pigments of red ochre, earth ber of residues that are potentially present. It is therefore prudent to use analytical green, copper green, black, white, copper leaf, and yellow ochre, the presence of techniques that can identify and quantify many different residues within a single which suggest the source of their manufacture in both the Old and New worlds. analytical run. One such technique that is becoming more common is the use of a QuEChERS (quick, easy, cheap, effective, rugged, and safe) based extraction of the 277 Pesticide Residue Analysis Using GC-MS-MS for Target sample followed by high-performance liquid chromatography (HPLC) / high-resolu- Compounds and GC-Q-TOF for Screening tion mass spectrometry (HRMS). One advantage of HRMS is that it does not require Philip L. Wylie, Agilent Technologies, 2850 Centerville Rd., Wilmington, specific tuning or conditions for each pesticide residue, instead a small number of DE 19808 scan functions can be used to unambiguously identify and quantify hundreds of Gas chromatography tandem mass spectrometry (GC-MS-MS) is a highly selective different residues. In our laboratory we are using an Agilent 1200 HPLC linked to a technique with more than adequate sensitivity to meet regulatory requirements for Thermo Exactive Orbitrap mass spectrometer to conduct these analyses. Pesticide chemical residue limits in foods. But, a new GC triple quadrupole (QQQ) design residues are identified and quantified in a full scan at a mass resolution of 50,000, increases the ion generation in the EI mode by about 30-fold, resulting in a 10-fold while the residues are confirmed using one of two all ion fragmentation (AIF) scans increase in sensitivity. So, how can this increased sensitivity be used to improve using higher-energy collisional dissociation (HCD). Typically pesticide residues can results in the chemical residue laboratory? First of all, one can make smaller sample be identified by using the exact mass of their chemical formula (plus an adduct to injections and still have improved sensitivity for the analysis. For example, one could create an ion). Examples of the use of this technique in our laboratory are shown inject 0.5 µL instead of 2 µL resulting in longer column and liner lifetime and less during this talk. frequent source cleaning. Short dwell times can be used allowing one to expand the number of compounds analyzed or to add more qualifier ions to the method, result- 41 2015 EAS Abstracts November 2015

281 Is Your Method Detecting the Right Analyte? Methanol Created 284 Vacuum Chromatography from Carbamate Compound during Headspace GC Analysis Kuriakose T. Joseph, Coconut Associates, 755 Magee Ave., Leih-Shan Yeung, Merck, 126 East Lincoln Ave., Rahway, NJ 07065 Philadelphia, PA 19111 Merck Compound A is a pharmaceutical compound with low solubility in intestinal A new technique named vacuum chromatography (VC) is introduced for the sepa- fluid. In order to improve the bioavailability, the compound was spray-dried with ration of organic compounds in a mixture. It is very similar to gas chromatography amorphous polymer. In the design of experiment (DOE) study, methanol was ob- (GC) and could also be called phase-less chromatography or time of flight chro- served at 0.03% to 0.24% w/w. Methanol is a class 3 solvent with International matography. A capillary column is used with no stationary phase, neither solid nor Conference on Harmonization (ICH) control limit of 0.3%w/w, but methanol was liquid. Also no mobile phase, neither liquid nor gas is used. A vacuum is applied at not a process related solvent during the spray-drying process. The observed lev- the outlet to pull the compounds through the column in vapor state. The lighter mol- el of methanol in the spray-dried intermediate was approaching the ICH limit and ecules travel through the column faster than heavier molecules and elute primarily therefore triggered a thorough investigation to determine the root cause. Possible in the reverse order of molecular weight as peaks in a chromatogram. However, the sources of methanol include excipients, drug substance, spray-dry solvents, analyt- vapor pressure (boiling point), structure, functional group, etc. have high influence in ical method bias, and system contamination. Upon confirmative testing, the project the order of elution. Compounds with low boiling points elute earlier and some iso- team has excluded methanol contamination from the raw material or the process mers appear as well separated peaks. Also relative elution order changes between as the potential culprit. The investigation uncovered that Compound A degraded columns of different dimensions and as the oven temperature programming condi- during the headspace gas chromatography (GC) analysis and released methanol tions are varied, however, reproducibility seems to be good under same conditions. as a side product. The residual solvent test method deploys a headspace GC with Compounds elute at relatively lower temperatures than GC and so advantages of headspace oven temperature of 105 ˚C. Pharmaceutical compounds containing vacuum distillation are applicable to VC also. As no stationary phase is needed, the carbamate group such as Compound A could degrade to yield methanol under ba- columns are cheap and VC could be employed to very high temperatures, especially sic condition and high temperature by hydrolytic reaction R-NHCO2CH3 + OH- → without column bleeding (theoretically) and could be cleaned by baking out or rins- R-NH2 + CH3OH + CO2. This presentation describes our research in confirming our ing with solvents. The possibilities of using metal columns also exist. Overall, VC hypothesis by identifying Compound A hydrolytic degradates post GC analysis. The could be amenable to far more compounds than GC. Split injection is accomplished degradation kinetics at different headspace oven temperature was also explored to using vacuum line with switch valve and regulator. evaluate the impact of temperature on methanol level. The optimized test method with lower headspace oven temperature and acidified sample diluent minimized the 285 A Model Study of Pseudo-Absolute Quantitative Analysis Using methanol generation as a side product and served the purpose of detecting the true Gas Chromatography - Vacuum Ultraviolet Spectroscopy process related solvents. Ling Bai, The University of Texas-Arlington, 700 Planetarium Place, Box 19065, Arlington, TX 76019, Kevin A. Schug, Jonathan Smuts, Phillip 282 Comparison of Quantitative Approaches for GC Methods to Walsh Characterize Solvate Content of Pharmaceutical Substances In addition to conventional methods of quantification (internal and external standard), Daisy Soares, Bristol-Myers Squibb, 1 Squibb Dr., New Brunswick, NJ, gas chromatography-vacuum ultraviolet (VUV) spectroscopy also has the potential Yan Zha for pseudo-absolute quantitation of analytes based on pre-recorded cross sections During the development of drug candidates, various types of solid-state active phar- without the need for traditional calibration. The pseudo-absolute method has been maceutical ingredients are evaluated, including hydrates, co-crystals, and solvates. used in this research where the sample loss and recovery sources associated with Solvates are defined as crystals having a solvent molecule as part of its crystal- sample introduction into the instrument can be evaluated experimentally. The VUV line structure. The selected solvate for a particular compound intends to improve detector records full-range absorbance spectra for each scan (125-240nm).The ab- physiochemical stability, process scalability, and biological properties. Solvates can sorbance spectra are integrated either over the full, or partial, wavelength regions to be analyzed using several different analytical techniques, but gas chromatography generate a two-dimensional chromatogram. The chromatogram is then integrated (GC) is likely the most widely employed technique to quantitate solvate content. This to determine a peak area. The concentrations of eluted solutes are proportional to presentation will describe the development of a robust GC method for characterizing the areas or the height under the recorded peaks integrated in gas chromatography a pharmaceutical substance containing a fairly high level of N-N-dimethylacetamide system. Standard samples of benzene and natural gas have been used to assess (~ 8%). Quantitation using either internal or external calibration standards was sys- error or sample loss for the analysis of liquid and gaseous samples. Results indicate tematically evaluated. Critical method parameters such as dissolution solvent, cali- that column installation, split ratio accuracy, sampling times for splitless analysis, bration standards, linear response range and injection precision as well as sample detector scan rate, and make-up gas flow can all contribute factors to error introduc- preparation procedures were effectively optimized to achieve a well-defined, robust tion and sample loss. The capability of VUV detector provides an excellent means operating range. It was found that an introduction of an internal calibration standard for carrying out system performance checks and solving challenging quantitative offered many advantages and provided higher method precision than use of ex- analytical problems. ternal standards. The established methodology shows high method precision and overall method robustness, as well as demonstrated greater suitability for the use in 286 Evaluating Deconvolution Capabilities of Vacuum Ultraviolet routine quality control analysis. Detection for Gas Chromatography of Co-Eluting Isomers of Dimethylnaphthalene 283 An Evaluation of Ionic Liquid Capillary Columns for the FAME Jamie L. Schenk, University of Texas-Arlington, 700 Planetarium Pl., Isomer Analysis Box 19065, Arlington, TX 76019 Len Sidisky, Supelco, Division of Sigma-Aldrich, 595 North Harrison One issue with most gas chromatography (GC) detectors is their inability to decon- Rd., Bellefonte, PA 16823, Greg A. Baney, Jamie L. Desorcie, Dan L. volute coeluting analytes. Electron ionization mass spectra of coeluting analytes Shollenberger, Gustavo Serrano can become highly complex and misleading. Dimethylnapthalenes (DMNs) are a Analyses of fatty acid methyl esters (FAMEs) are continuing to gain importance as class of compounds that are virtually indistinguishable by GC-mass spectrometry more research is focusing on their biomedical impacts. This includes the analysis (MS) because of significant coelution and identical mass spectra. The effectiveness of saturated and polyunsaturated FAMEs along with the positional geometric (cis of a vacuum ultraviolet (VUV) detector paired with a GC to deconvolve mixtures of and trans) FAME isomers. Traditionally, FAME analyses have been performed using DMN is demonstrated. Various mixture combinations and different ratios of concen- silicone polymer or polyethylene glycol based stationary phases that yield typical trations were used to test the accuracy, efficiency, and sensitivity of the detector. All elution patterns. Analysts performing the task of analyzing the fatty acid composition DMNs were analyzed together to see which coeluted. The compounds that coeluted of food have a wide variety of capillary column selectivity’s available for resolving were mixed in ratios of 75/25, 50/50, and 25/75 and deconvolved using the GC- the fatty acids as FAMEs depending upon the information they require from their VUV software. All compounds were successfully convoluted with little error between analyses. Nonpolar methylsilicone columns provide a boiling point separation of the calculated values and theoretical values. The most challenging two isomers to the FAME isomers with limited resolution of polyunsaturated isomers. Polar poly- deconvolute were taken and mixed into smaller ratios ranging from 85/15 to 99/1. ethylene glycol (PEG) columns resolve the isomers by degree of unsaturation with These were ran and decolulved with an error between the calculated value and the minimal overlap of the carbon chain lengths. The highly polar cyanosilicone columns theoretical values also being negligible. Further characterization of the state-of-the- will resolve cis and trans isomers along with possibly providing positional geometric art in theoretically computed VUV spectra was also investigated and compared with isomer separations depending upon the column type. New classes of stationary experimental measurements. GC-VUV was shown to be successful for deconvolv- phases based (SLB) on ionic liquid (IL) technology have been developed and have ing all DMN isomers introduced as standards, as well as from fuel samples. demonstrated to provide unique elution patterns for FAME isomers compared to the traditional silicone or polyethylene glycol based stationary phases. The two new phases are SLB-IL60 with a PEG like selectivity and the SLB-IL111 with highly polar selectivity. We compare and contrast the selectivity of the ionic liquid phases with polymeric based phases for various FAME samples. 42 2015 EAS Abstracts November 2015

287 Image Directed ID of Sub-Visible Particles in Protein Based (PDE’s) of certain elemental impurities when setting specifications, provided they Therapeutics. Classification According USP<787> of Intrinsic, can prove that the species present in their product is more or less toxic than the spe- Inherent and Extrinsic Particulate Matter on the Sub-Visible Level cies considered when setting the PDE values in International Conference on Har- Olga Laskina, rap.ID Inc., 11 Deer Park Dr., Ste. 201, Monmouth monization (ICH) Q3D. The speciation studied for this presentation covers arsenic, Junction, NJ 08852, Kathryn A. Lee, Markus Lankers, Oliver Valet mercury, and chromium. Speciation experiments were performed using high-per- Protein aggregation is a key quality attribute of bio-therapeutics. Aggregates hold formance liquid chromatography inductively coupled plasma mass spectrometry the potential for adversely impacting production and patients. The newly released (HPLC-ICP-MS) consisting of a NexION 350 ICP-MS coupled to Altus HPLC and United States Pharmacopeia (USP) <787> “Subvisible Particulate Matter in Ther- Waters Empower 3 software. apeutic Protein Injections” defines particle types: Truly foreign particles are “extrin- sic”; particles from the production environment or primary packaging are “intrinsic” 291 Demystifying USP <232>/<233>: Sample Preparation Using and formulation particles are “inherent”. In the visible inspection process inherent Microwave Digestion particles must be distinguished from the other two. USP<787> furthermore states Johan Nortje, Milestone, 25 Controls Dr., Shelton, CT 06484 that membrane microscopy is not the preferred method and is suited only for other New regulations for the analysis of trace metals in pharmaceutical ingredients have than inherent particles. USP<788> and USP<787> confirm this, stating MM gridded generated new challenges for the pre-analytical preparation on the wide range filter paper cannot isolate fragile or translucent particles, nor can they sufficiently of samples that would undergo testing. For United States Pharmacopeia (USP) be visualized in conventional microscopes. We show a method that allows the iso- <232>/<233>, the demand for a clear process is evident. To obtain high quality lation of particles on a gold membrane with subsequent enumeration of particles multi-element inductively coupled plasma optical emission spectrometry mass by membrane microscopy utilizing a new illumination technique. Besides dark-field spectrometry (ICP-OES/MS) data, several techniques have been used without a illumination, a UV light induces the auto-fluorescence of tryptophan and allows spe- solution for all samples. Conventional closed vessel microwave digestion has been cific counting of inherent particles. In a second step, Raman spectroscopy can ob- recognized as the most effective technique for the digestion of the widest range tain fingerprint spectra that allow material identification and differentiation between of sample types in metals analysis. Unfortunately, pressure and temperature con- extrinsic, intrinsic, and inherent particles. This method gives chemical composition straints using traditional techniques have complicated the picture: multiple steps, of hundreds of particles per analysis and allows root cause investigation of con- incomplete digestions and specific methods for troubling elements. Single reaction taminants to avoid further contamination. With a novel sampling method we have chamber (SRC) microwave technology is a new technique with the capability of di- overcome the limitation of the lower count number from the membrane method com- gesting several different types of samples simultaneously at temperatures up to 300 pared to micro flow imaging (MFI). Image directed spectroscopy is used to obtain degrees Celsius and pressures up to 199 bar. Utilizing this new microwave design Raman spectra of the spherical silicone droplets specifically from a biopharmaceu- with user feedback, an optimized primer on critical factors in preparation choices as tical formulation between two quartz slides. well as a single method solution utilizing SRC technology and ICP-MS multi-element analysis are presented. 288 Raman, IR, and LIB Spectroscopic Particle Identification for Improvement of Pharmaceutical Production 292 USP 232/233 Heavy Metals Testing by WD-XRF: A Simplified Lin Bui, rap.ID Inc., 11 Deer Park Dr., Ste. 201, Monmouth Junction, NJ Approach 08852, Kathryn Lee, Markus Lankers, Oliver Valet Glenn Williams, Rigaku Americas Corp., 9009 New Trails Dr., The Analyses including identification, counting, and sizing of un-wanted particles in Woodlands, TX 77381, Thanh Nguyen pharmaceutical products can be very beneficial for product quality and process im- The United States Pharmacopeia (USP) has issued guidelines for heavy metals/in- provement because it can lead to elimination of specific particle sources and higher organic impurities. This is detailed in General Chapters 232 and 233. USP 233 spe- quality products. Particles can be from a variety of sources including products, vials, cifically describes analytical testing by inductively coupled plasma (ICP) techniques. syringes, IV bags, on stoppers, production equipment, swabs, scoops, and filters. Wavelength dispersive X-ray fluorescence spectrometry (WD-XRF) is an analytical Fast and accurate methods of analysis for even micro particles include Raman, technique that is accepted by USP as an alternative for heavy metals testing if laser induced breakdown (LIB), and infrared (IR) spectroscopies. Materials may be sensitivity and validation requirements are met. High end WD-XRF instrumentation combinations of things, and it is important to understand all of the components to is capable of analyzing inorganic impurities to sub part-per-million levels in most get a better idea of the source of the material. Due to the different physics behind pharmaceutical matrices. XRF is a non-destructive, direct analysis technique that each technique, each different spectroscopic technique may provide only a partial requires minimal sample preparation and no extraction or digestion steps. Sever- answer, and thus the complete information may be obtained by using more than al real pharmaceutical examples on methods analyzing Class 1 (As, Pb, Cd, and one technique. In addition, spectral interpretation is very important to finding the Hg) and Class 2A (Ni, Co, and V) are shown and discussed. In addition, it is well complete answer. Analyses of particles using the different techniques as well how to known that mercury standards have stability issues due to its high volatility. Several interpret data better is discussed. additives designed to stabilize mercury have been investigated. Stability data for mercury in the presence of several different matrices and additives are presented 289 Handheld LIBS Instrument Based on High Repetition Rate Laser and discussed. Qun Li, B&W Tek, 19 Shea Wy., Ste. 301, Newark, DE 19713, Katherine Bakeev, Jing Li, Sean Wang 293 Ambient Ionization Mass Spectrometry In this paper, we present a new type of handheld laser-induced breakdown spec- R. Graham Cooks, Purdue University, 560 Oval Dr., West Lafayette, IN troscopy (LIBS) instrument. A micro diode-pumped passive Q-switched solid-state 47907, Michael Wleklinski, Yafeng Li, Ryan Bain, Anyin Li, Xin Yan laser with high repetition rate of well above 1 kHz in comparison to 1-10 Hz as This presentation deals with low voltage forms of ambient ionization mass spec- used in a traditional LIBS instrument is employed to produce a train of laser pulses. trometry, specifically with 0 volt paper spray ionization. Comparisons are made with The laser beam is further fast scanned over a pre-defined area, hence generating conventional kV forms of the experiment and a model of droplet ionization is develop several thousands of micro-plasmas per second at different locations. Synchro- for the case in which voltage is not applied. The analytical properties and especially nized miniature charge-coupled device (CCD) array spectrometer modules collect the enhanced sensitivity to surface active species are discussed and compared the LIBS signal and generate LIBS spectra. By adjusting the integration time of the with Enke’s early treatment of surface active compounds in electrospray ionization. spectrometer to cover a plurality of periods of the laser pulse train, the spectrometer Reactive forms of ionization are also discussed and accelerated derivatization in the integrates the LIBS signal produced by this plurality of laser pulses. Hence the inten- (initially) uncharged droplets is followed. New methods of sampling small volumes sity of the obtained LIBS spectrum can be greatly improved to increase the signal- of solution and performing 0 volt spray ionization are presented as are arrays of to-noise ratio (SNR). This unique feature of the high repetition rate laser based LIBS emitters in this experiment. system allows it to measure elements at trace levels, hence reducing the limit of detection (LOD). The increased signal intensity also lessens the sensitivity require- 294 Real-Time Analysis of US EPA Method TO-14A Compounds Using ment for the optical spectrometer. In addition, the energy of the individual laser pulse Selected Ion Flow Tube Mass Spectrometry (SIFT-MS) can be reduced in comparison to traditional LIBS system to obtain the same signal Barry Prince, Syft Technologies, 3 Craft Pl., Christchurch 8024, New level, making the laser pulse less invasive to the sample. The typical measurement Zealand, Vaughan S. Langford, Daniel B. Milligan, Murray J. McEwan time is within one second. Several examples of real world applications including Selected ion flow tube mass spectrometry (SIFT-MS) is a real-time analytical tech- quantitative LIBS analysis are presented. nique that offers potential for rapid screening of volatile organic compounds (VOCs) to ultra-trace levels in air.[1] Quantitation limits in the low part-per-trillion range (by 290 Applying HPLC-ICP-MS Speciation in Support of ICH Q3D Risk volume; pptv) are achieved without sample preparation or pre-concentration and re- Assessments sults compare well with those obtained at an accredited laboratory using the United Jonathan L. Sims, PerkinElmer, Chalfont Rd., Seer Green HP92FX, States Environmental Protection Agency (US EPA) TO-15 Compendium Method.[2] United Kingdom, Kenneth Neubauer This paper presents the results obtained during development of a rapid analytical Manufacturers of drug products can vary from the stated permitted daily exposures method that targets the compounds in the US EPA TO-14A Compendium Method. 43 2015 EAS Abstracts November 2015

A Syft Technologies Voice200Ultra SIFT-MS instrument was used in this study. The 297 Formation of Molecular Anions from Primary Aliphatic Amides and quantitation limits specified by the TO-14A method are achieved within a few sec- Oximes under Helium-Plasma Ionization (HePI) Mass Spectrometric onds for each analyte. SIFT-MS also offers significantly enhanced dynamic and Conditions linearity ranges compared to the canister-gas chromatography/mass spectrometry Isra Hassan, Stevens Institute of Technology, 560 Belgrove Dr., Kearny, method. Direct mass spectrometry techniques, such as SIFT-MS, cannot resolve all NJ 07032, Athula Attygalle isomers as required by the TO-14A method. However, this study has demonstrated Electron capture is the process in which an external electron is incorporated into the that SIFT-MS provides a rapid screening tool that saves busy laboratories signifi- orbital of a molecule. It is well known that nitro compounds and halogenated com- cant money through pre-screening of incoming samples prior to regulatory analysis. pounds undergo electron capture. We can predict whether a compound will undergo These results also form the basis of deployment of SIFT-MS instrumentation as electron capture by calculating its electron affinity; a large and positive electron real-time analyzers for TO-14A method compounds, such as in ambient or fence- affinity value (such as 1.006 eV for nitrobenzene) indicates that a certain compound line monitoring scenarios. is likely to capture an electron in gas phase. Molecular anions were detected using References: helium-plasma ionization mass spectrometry (HePI-MS) on a Waters Quattro Ultima [1] Prince, B.J., Milligan, D.B., & McEwan, M.J. (2010). Rapid Commun. Mass Spec- mass spectrometer. We report that oximes derived from aliphatic aldehydes as well trom., 24, 1763-1769. as several primary amides undergo electron capture. In negative mode, the HePI [2] Langford, V.S., Graves, I., & McEwan, M.J. (2014). Rapid Commun. Mass Spec- mass spectra reveal a peak at m/z M-. for the molecular anion. In addition, a peak trom., 28, 10-18. at m/z [M + 32]-. is observed for the superoxide radical anion. We hypothesize that the superoxide radical anion is also involved in the electron capture process. Theo- 295 Application of Direct Analysis in Real Time Mass Spectrometry retical calculations of the electron affinities were also made. (DART-MS) to Investigations of Plant Root Emissions of Organosulfur Compounds into the Environment 298 Improving Precision and Accuracy of Temperature Measurements Rabi Ann Musah, SUNY-Albany, Dept. of Chemistry, 1400 Washington in Automatic Refractometers Ave., Albany, NY 12222, Ashton D. Lesiak, Max J. Maron, Kristen Mark Canestrano, Anton Paar, 10215 Timber Ridge Dr., Ashland, VA Fowble, Michael C. Long, Robert B. Cody, David Edwards, A. John Dane 23005 The leaves of some plants are known to produce volatile organosulfur compounds Refractive index measurements are strongly dependent on temperature. In order to (VOSCs) that confer upon the plants agricultural, horticultural and/or medicinal im- achieve high precision in measuring the refractive index, temperatures must be both portance. Previous studies of these volatiles have relied heavily on gas chromatog- meticulously controlled to a narrow margin of uncertainty, and be determined very raphy-mass spectrometry (GC-MS) techniques for their characterization. However, accurately, requiring the temperature reading to be true to the value of the sample’s VOSCs are prone to decomposition as a result of the harshness of the conditions actual temperature. This paper discusses the effects of innovative methods used generally used for GC analysis, which often lead to artifact formation. Using plants to ascertain precision and accuracy of temperature measurements in automatic re- of the Mimosaceae genus as models, we demonstrate the utility and advantages of fractometers. direct analysis in real-time (DART)-MS methodology for the analysis and identifica- tion of plant derived biogenic sulfur. High resolution DART-time-of-flight (TOF)-MS 299 Application of a Unique Microwave Digestion Technology Coupled analysis revealed that the roots, in contrast to the aerial parts of these plants, emit- with ICP-MS for the Determination of Key Elements in Dietary ted a cocktail of small molecules into the environment, including SO2, methylsulfinic Supplements acid, pyruvic acid, lactic acid, ethanesulfinic acid, propane sulfinic acid, mercap- Reynhardt Klopper, Anton Paar USA, 10215 Timber Ridge Dr., Ashland, toaniline, S-propyl propane 1-thiosulfinate, and thioformaldehyde, an elusive and VA 23005, Paul Dodson highly unstable compound never before reported to be emitted by a plant. None of Dietary supplements are regulated by the United State Food and Drug Adminis- these compounds were observed in GC-MS analyses even under mild conditions. tration (FDA) and can be found in various forms, including tablets and powders, The composite of small-molecule species detected by DART-MS provided an un- consisting of vitamins, minerals, herbs or botanicals. Under the Dietary Supplement precedented glimpse of the in-situ root emissions profile that was unobscured by Health and Education Act of 1994 (DSHEA), dietary supplement manufacturers are the presence of the intracellular molecules normally observed in mass spectrometric responsible for ensuring the safety of products before market release. Elements analysis of tissue extracts or macerates. The detection of these molecules led to the such as Zn, Mg and Se are added for nutritional purposes, and need to be deter- cryo-scanning electron microscopy facilitated discovery of novel root structures that mined for labeling confirmation. The presence of certain toxic elements, e.g., Pb, may be important in the emission of root volatiles. Hg is highly regulated and may not exceed set concentration levels. A number of analytical techniques are used for dietary supplement testing, the most common 296 A Two-Fold Approach Towards Improved Pu/U Chronometry in being inductively coupled plasma mass spectrometry (ICP-MS) coupled with micro- Thermal Ionization Mass Spectrometry wave sample digestion. In this study we illustrate how a new and unique microwave Floyd E. Stanley, Los Alamos National Laboratory, 30 Bikini Atoll Rd., digestion technology allows for the efficient and fast processing of dietary supple- Los Alamos, NM 87545, Benjamin L. Byerly, Khalil J. Spencer ments. The compact and lightweight Multiwave GO microwave system features the Chronometry, or “age-dating”, is a critical signature in forensic efforts designed to patent pending directed multimode cavity (DMC) technology, which enables focused determine the history of an interdicted nuclear material. This technique exploits application of microwave energy to a compact cavity system, while allowing for the the radioactive decay of certain nuclides (e.g., 240Pu → 236U via alpha decay) simultaneous processing of up to 12 samples in 18 minutes, yielding clear solutions to determine the time of a material’s last processing. While several factors may of multi-gram samples. impact findings, such as imperfect cleanup, multiple re-processing, or open system behavior, determination of a material’s model age is widely accepted as valuable 300 Monolithic Silicas in High-Performance Liquid Chromatography: information in identifying potential actors in its creation and diversion from legiti- The Alternative to Conventional Packed-Particle Columns mate control. Thermal ionization mass spectrometry (TIMS) has traditionally been Egidijus Machtejevas, Merck KGaA, Frankfurter Str. 250, Darmstadt the gold standard in measuring nuclear material isotopic relationships for both Pu 64293, Germany and U systems. However, many materials of interest in nuclear chronometry con- In contrast to conventional packed-particle columns, monolithic silica columns are tain extreme isotope relationships, with abundances spanning nine or more orders made of a single continuous-bed rod of high purity porous silica that is then bond- of magnitude, and may be of sufficiently recent production that progeny in-growth ed with C18, C8 and the other modifications that chromatographers have known is minimal (part-per-billion). These unique concerns require new techniques and and trusted to solve their separation needs through the history of high-performance quality control (QC) strategies to ensure highest confidence and legally defensi- liquid chromatography (HPLC). For the first time, the physics of the HPLC column ble chronometric measurements for plutonium-bearing materials from a variety of have been completely reengineered, providing dramatically reduced backpres- sources. In the present work, we investigate the combined usage of novel, mixed-ar- sures due to the open flow-through design, while maintaining the high resolution ray total evaporation measurement techniques and QC comparators mimicking U and performance that chromatographers expect today. Monolithic columns remove in-growth in Pu matrices to provide concordant model ages for Pu-U chronometers backpressure as the primary consideration in method development and give back (e.g., 240Pu/236U, 239Pu/235U, 238Pu/234U) and improved measurement uncer- the flexibility of choices in flow rates for much higher throughput, column lengths tainties. These strategies were ultimately applied to real-world Pu materials and for superior resolution, and solvent choices for optimum selectivities that smaller provided both excellent agreement between primary chronometers (Dev. ~ 0.1%) and smaller sized packed-particle columns have slowly taken away over the de- and improved uncertainties (~0.2%, k=2). cades. Because they have no individual particles to shift or break, column perfor- mance is very consistent over much longer lifetimes, making them ideal for method transfer into production quality control or multiple sites. Their high permeability also makes them very forgiving of shortcuts and timesaving in sample preparation as well as easier to aggressively flush out to re-equilibrate. This presentation guides

44 2015 EAS Abstracts November 2015

you through the world of monolithic silica materials. Benefits are demonstrated with (EC) method. In order to show the applicability of using the Background FP method, many application examples including pharmaceutical and bioanalysis separations in this study the quantitative values of various inorganic impurities in organic sam- (proteomics, peptidomics, etc.), calibration curves, recovery calculations, and meth- ples were compared by EC and the Background FP method. The correlation coef- od robustness overviews. ficients between the EC and the Background FP method were more than 0.995 in lead, cadmium, arsenic, mercury, ruthenium, rhodium, osmium, cobalt, copper and 301 Ambient Air Monitoring: What are the Right Tools for the Job? molybdenum. And this method achieved detection limits of below 1 μg/g. Chris Hall, Markes International, 11126 Kenwood Rd., Ste. D, Cincinnati, OH 45242, Nicola Watson, Caroline Widdowson 305 The Use of High Resolution Accurate Mass GC-MS for Metabolomics Many techniques are available for the sampling and measurement of volatile organ- Workflows ic compounds (VOC) in ambient air, but which is the best to use? Canister samples Rafael Acosta, Thermo Fisher Scientific, 2215 Grand Avenue Pkwy., are the current gold standard, stipulated by the Environmental Protection Agency Austin, TX 78728 (EPA) for TO15 for analysis of air toxics and other volatile organic compounds at Typically high resolution accurate mass liquid chromatography mass spectrome- various concentrations, but as the list of target compounds continues to grow, canis- try (LC-MS) in combination with unit mass gas chromatography mass spectrome- ters are being pushed to their performance limit. Another option is the use of sorbent try (GC-MS) has been used for metabolomics workflows. With the introduction of tubes. They are very versatile and depending on the sampling situation various Orbitrap technology 10 years ago and the recent introduction of the new Thermo techniques can be used to retain VOCs on the media; diffusive/passive (EPA Meth- Scientific™ Q Exactive™ GC mass spectrometer, it is now possible to use the pow- od 325) and pumped/active (EPA TO-17) sampling. On-line analysis for the very erful information obtained with an Orbitrap based analyzer, namely high resolution volatile, ozone precursors and reactive sulfur compounds is becoming increasingly spectra and excellent mass accuracy, combined with the high selectivity of gas chro- popular around the US and worldwide. There is no one correct method but rather matography to generate greater confidence in analytical results. With up to 100,000 a range of sampling techniques that can be implemented for varying situations. resolving power (m/z 272) and sub part per million mass accuracy, the Q Exac- This poster covers the various options available for sampling a variety of VOCs in tive GC system makes identifying unknown metabolites less labor intensive. The a range of situations/conditions, and discuss the advantages and disadvantages of large linear dynamic range and triple-quadrupole equivalent sensitivity ensures that each technique. In addition to the sampling we also look at the latest developments low concentration analytes are correctly identified and quantitated, and having no in analysis and detection of VOCs. spectra saturation eliminates the need for re-injection of valuable and limited sam- ple quantities. Combining these aforementioned features with a new powerful and 302 Enhanced Characterization of Allergens in Cosmetics by GC×GC– revolutionary deconvolution algorithm provides the ultimate assurance in unknown TOF MS with Soft Electron Ionization identification in a metabolomics workflow. Chris Hall, Markes International, 11126-D Kenwood Rd., Cincinnati, OH 45242, Charles Haws, Laura McGregor, Steve Smith 306 Label-Free Analysis by HPLC with Charged Aerosol Detection of In 2003, an EU Directive restricting the use of allergenic compounds in fragrances Glycans Separated by Charge, Size and Isomeric Structure was released. The Directive named a total of 27 allergens, stating that they should David H. Thomas, Thermo Fisher Scientific, 22 Alpha Rd., Chelmsford, be labelled if present at >100 ppm in ‘wash-off’ products (such as shower gels), or MA 01824, Ian N. Acworth, Marc Plante, Rainer Bauder, Daniel Kutscher >10 ppm in ‘leave-on’ products (such as perfumes). Compliance with this directive We have developed a quantitative glycan profiling assay that is simple, fast, and therefore requires that these compounds are identified and quantified accurately. eliminates the hassle of labeling glycans with a fluorophore. The method is intended Two-dimensional gas chromatography with time-of-flight mass spectrometry for characterization and quality control of glycoprotein biotherapeutics. The meth- (GC×GC–TOF MS) is well-suited for the analysis of allergens within complex od uses a volatile mobile phase fully compatible with mass spectrometry, if further cosmetics extracts. The enhanced separation provided by the coupling of two characterization is desired. Changes in the number, type, composition or linkage columns of different stationary phase minimizes tedious sample preparation steps, pattern of glycoprotein glycans may serve as a biomarker of disease or influence the which can also introduce error into the analytical process. Despite the superior efficacy of a biotherapeutic product. For this reason, the ability to correctly identify separation of GC×GC, the identification of individual compounds in complex and measure these glycans quickly and inexpensively is of great practical benefit. samples remains challenging when multiple compounds in a chemical class have This work explores direct detection of native glycans as an alternative to the com- similar spectra, or weak molecular ions. This problem can be addressed by the use mon techniques for glycan analysis that rely on labeling reactions to render glycans of soft ionization to reduce the degree of ion fragmentation, but this approach has detectable. The lack of a detectable chromophore in native glycans is overcome by been cumbersome to implement until now. This poster presents the use of novel using UHPLC with charged aerosol detection, which can quantitatively measure any ion-source technology to obtain complementary, simplified soft EI spectra for full non-volatile compound. N-linked glycans are released from proteins by PNGase-F characterization of complex fragrances in a single analytical sequence. and separated by ultra-high-performance liquid chromatography (UHPLC) on a new UHPLC platform that integrates the charged aerosol detector into the system 303 Are you Compliant to the New USP Guidelines for UV/VIS? for increased performance and ease of use. The mixed mode analytical column Birgit Pils, Mettler Toledo, Sonnenbergstrasse 74, Schwerzenbach employs both weak anion exchange and reversed-phase separation mechanisms 8603, Switzerland, Gustavo Sierra to resolve glycans based on charge, isomerism and size. The native glycans are In ultraviolet–visible (UV-VIS) spectroscopy, regular performance verification is es- detected directly without labeling by using charged aerosol detection. Quantitative sential to ensure accurate and reliable instrument performance. Widely accepted performance including precision, detection limits and dynamic range is presented. guidelines for performance verification of spectrophotometers are described in the Figures of merit include sensitivity at the low-nanogram on-column level, dynamic United States Pharmacopeia (USP). The recommended tests include the check range over two orders of magnitude, and peak area precision averaging less than of photometric accuracy and repeatability, wavelength accuracy and repeatability, three percent relative standard deviation. instrument resolution as well as stray light measurement. Recently, the USP intro- duced a new chapter on ultra-violet visible spectroscopy and adapted the test for 307 Label-Free Profiling of O-linked Glycans by UHPLC with Charged stray light. Here, we compare the methods for measuring stray light according to Aerosol Detection the current and previous version of the USP and highlight the advantages of the David H. Thomas, Thermo Fisher Scientific, 22 Alpha Rd., Chelmsford, new test. MA 01824, Ian N. Acworth, Rainer Bauder, Marc Plante, Liz Kast The goal of this work was to develop a quantitative profiling assay for O-linked 304 Challenge of Small Sample Analysis for Pharmaceutical Products glycans released from glycoproteins by reductive beta elimination. While glycan and Foods Using Theoretical Scattered X-Rays release under reducing conditions is desirable to reduce peeling reactions, the Dan Davis, Shimadzu Scientific Instruments, 7102 Riverwood Dr., resulting O-linked glycan alditols can’t be derivatized easily with a fluorescent la- Columbia, MD 21046, Hiroaki Furukawa, Naoto Ichimaru, Keijiro Suzuki, bel. Charged aerosol detection does not require a fluorophore or chromophore for Shinji Watanabe, Makoto Nishino, Hirotomo Ochi sensitive, accurate quantification, and so HPLC-CAD provides a simple, direct ap- The United States Pharmacopeia (USP) has recently established guidelines for proach to separate and quantify native glycans. The method uses a volatile mobile general analysis using X-ray fluorescence (XRF) methodology. XRF is an attractive phase fully compatible with mass spectrometry, if further characterization is desired. methodology because it does not require chemical pretreatment and allows non-de- O-linked glycans are released from proteins by reductive beta elimination. The structive analyses. Energy dispersive X-ray fluorescence (EDX) can be utilized in released glycans are separated by ultra-high-performance liquid chromatography the screening process for the control of inorganic impurities in pharmaceutical prod- (UHPLC) on a new UHPLC platform that integrates the charged aerosol detector ucts, excipients, and active pharmaceutical ingredients (API’s). This study proposes into the system for increased performance and ease of use. The mixed mode an- the use of the scattered X-ray corrected - fundamental parameter (FP) method, alytical column employs both weak anion exchange and HILIC separation mecha- hereinafter called “Background FP method”, which uses scattered X-rays for quan- nisms to resolve glycans based on charge, size and polarity. The reduced glycans titation of low ppm level impurity elements in organic samples. The FP method can are detected directly without labeling by using charged aerosol detection. O-linked reduce the need to measure standard samples compared to empirical correction glycan pools released from various proteins were analyzed including those from bo- 45 2015 EAS Abstracts November 2015

vine fetuin, bovine submaxillary mucin, and IgG. Quantitative performance including tribute of the product during development and routine manufacturing is an accurate precision, detection limits and dynamic range is presented. Figures of merit include determination of coating thickness during processing. The traditional approach to sensitivity at the low-nanogram on-column level, dynamic range over two orders this determination is to cut samples out of the web and weigh them. This process of magnitude, and peak area precision averaging less than three percent relative is slow (in comparison to the speed of the web) and destructive. Near-infrared hy- standard deviation. perspectral imaging has been used as an effective tool to monitor coating thickness of a web-based transdermal delivery system from development through routine 308 Absolute Molar Mass and Size in UHPLC manufacturing. This talk focuses on the calibration and implementation of such a Michelle Chen, Wyatt Technology, 6300 Hollister Ave., Santa Barbara, system. In particular, the key contributions to this project from this year’s winner of CA 93117, Sophia Kenrick, Eric Seymour, Bob Collins, Aym Berges the “EAS Award for Outstanding Achievements in Near-Infrared Spectroscopy,” Dr. Multi-angle light scattering (MALS) is well-established as a means of determining Benoit Igne, are highlighted. molar masses and sizes of macromolecules and nanoparticles, downstream of size-exclusion chromatography or gel-permeation chromatography (SEC/GPC). In 312 Equivalence among NIR Makes and Models; What Benoît Started combination with a refractive index detector for universal concentration measure- Charles Hurburgh, Iowa State University, 3167 NSRIC, Ames, IA 50011, ment, MALS provides these measurements from first principles, independently Samantha McGinnis, Glen Rippke of retention time and molecular reference standards. SEC-MALS overcomes the Equivalent measuring units will give results with no more variance across a system limitations of traditional SEC/GPC analysis presented by the assumption that the than is created by using copies of the same make and model. United States Drug analyte is identical to the reference molecules in terms of conformation, density Administration (USDA) has supported Iowa State University to determine if makes and non-ideal column interactions. Ultra-high performance liquid chromatography and models of near-infrared transmittance (NIRT) beyond the one presently used (UHPLC) offers several benefits over standard HPLC: short run times, reduced con- in the USDA Official System can be considered equivalent, and if so, under what sumption of sample and mobile phase, and improved resolution. However, MALS conditions of calibration and maintenance. A test and variance model involved three instruments designed for HPLC applications are not suitable for UHPLC. In order to makes of NIRT that had previously passed the state-operated National Type Eval- migrate the benefits of MALS analysis to UHPLC, it has been necessary to develop uation Program accuracy test. Samples of wheat, barley, corn and soybeans were a new MALS detector and a new refractive index detector with greatly reduced in- tested 3 times each in 5 copies of the three models. The initial analysis was done terdetector dispersion. We present data from the first complete UHPLC-SEC-MALS with the calibrations and instrument setup as received. For wheat protein, the stan- system that robustly analyzes proteins, aggregates and fragments with unprece- dard deviation across the 15 units on a randomly chosen sample was 0.15 % points dented resolution compared to standard SEC-MALS. A small, poorly understood of protein (12% moisture basis). The standard deviations across copies were 0.12, shoulder on the main peak in a bovine serum albumin sample is clearly identified as 0.06 and 0.10 % points, respectively. With present calibration and standardization a protein fragment with molar mass of 57 kDa. Fragments of a 142 kDa monoclonal practices, equivalence was not met for the wheat samples. Changes to these prac- antibody are resolved, quantified and identified with molar masses of 93, 61 and tices have a good possibility to create equivalence. The three makes had standard 27 kDa, corresponding to heavy-heavy, heavy-light and light chains, respectively. deviations across the three replicates (per sample) of less than 0.05 % points, indi- These results are achieved with just micrograms or nanograms of protein. cating that hardware consistency was quite good. Data for the other grains will be presented in November. This project was a direct continuation of Dr. Igne’s disserta- 309 Highly Efficient Purification of Enantiomers Using Polysaccharide tion, intra and inter-brand calibration transfer for near-infrared spectrometers, which Type Chiral Stationary Phases and Recycle Purification Technology demonstrated on a lab scale that equivalence can be met under specific conditions. Ernest Sobkow, YMC America, 941 Marcon Blvd., Allentown, PA 18109, Keiko Kihara, Hideo Gabari, Takashi Sato, Saoko Nozawa, Noriko Shoji, 313 Successful Calibration Transfer: Technical, Business and Noritaka Kuroda, Takatomo Takai Regulatory Considerations The need for chiral separations in small molecule pharmaceutical development is Gary McGeorge, Bristol-Myers Squibb, 1 Squibb Dr., New Brunswick, NJ important. Selective, fast, and efficient analytical and preparative separations are 08901, Carl Anderson, Benoît Igne, Robert W. Bondi, Doug Steinbach, an essential part of the drug development tool kit. Key hurdles to be overcome in James Drennen III, Yan Zhang preparative separations involve improving the productivity of a given separation and Multivariate models applied to spectroscopic data presents an unusual situation for the difficulty in cost effectiveness of the purification. We have recently developed the analytical chemist in that there is no calibrator sample during the run sequence. various coated and immobilized chiral stationary phases with polysaccharide deriv- All quantitative assessment is derived from the application of a historical calibration atives. The coated phases give great resolution and the immobilized phases offer model and new samples projected onto the model. Consequently the validity of the a wider range of solvent compatibility. A separation method developed at analytical historical model should always be assessed during roll-out of the application. Of par- scale can be easily and linearly scaled up to purification from milligrams to gram ticular concern is that of calibration transfer whereby the model is applied at a new scale (and beyond) by using a preparative scale column and the liquid chromatog- manufacturing site that may present new sources of variation that the original model raphy (LC)-Forte/R preparative LC system. In this poster we show that the efficien- did not observe. A successful calibration transfer method for quantitative analysis of cy of purification is improved by applying recycle chromatography. This recycling pharmaceutical products represents one that is robust against instrument changes method is widely applicable to cases where ideal resolution is not achieved at the as well as raw material variability, changes in production scale and site, and any method screening stage. other foreseeable sources of variation that may deteriorate model performance. In this presentation we provide an example of a model tablet product where acetamin- 310 SFC Analytical Method Development for Vitamin D3 and Related ophen represents the active and the active assay was predicted. Models were de- Compounds veloped using both transmission Raman and near-infrared spectroscopic modalities Ernest Sobkow, YMC America, 941 Marcon Blvd., Allentown, PA 18109, and the relative performance upon calibration transfer to different instruments was Junko Kawabata, Roland Spaegele, Toshikazu Adachi, Noritaka Kuroda assessed. From a regulatory perspective it is becoming clear that health authori- By utilizing the advantages of supercritical fluid chromatography (SFC) such as ties are laying down expectations on how to manage calibration transfer for such high permeability and diffusibility, we could generally achieve higher resolution by methods and we will add some perspective to the difficulties of meeting the various SFC analysis in a shorter run time than by high-performance liquid chromatography regulatory expectations. (HPLC). Thus a combination of achiral columns and SFC is one of the effective strategies for reducing analysis cycle time of natural products such as fat-soluble 314 When the Sample Makes (or Breaks) the Technology vitamins and terpenoids. It has currently been recognized that some normal-phase Benoît Igne, Glaxo-SmithKline, 709 Swedeland Rd., King of Prussia, HPLC methods to assay Vitamin D3 and three related compounds (Pre-Cholecal- PA 19406 ciferol, 5,6-trans-Cholecalciferol, Tachysterol3) with various impurities in nutritional Near-infrared spectroscopy has seen a tremendous development since the 1970s, products are insufficient because analysis times of them are slightly longer and when the first commercial spectrometers became available. Applications of the tech- integration of them into a single method is difficult. In this poster, we describe the nology have been reported in many fields, from space exploration to biomedical development of a robust and efficient SFC method to solve these problems. research. Agriculture has significantly benefited from the information rich, low ab- sorbing, near-infrared signal in the laboratory and in the field, and manufacturing 311 Hyperspectral Image Calibrations for Transdermal Delivery processes (chemical, food, petrochemical, pharmaceutical, etc.) employ the tech- Systems nology for in-line, real-time, analyses. By the physico-chemical nature of its signal, Carl A. Anderson, Duquesne University Center for Pharmaceutical near-infrared spectroscopy probes both the chemical and the physical information Technology, 600 Forbes Ave., Pittsburgh, PA 15282, Benoît Igne, James of samples (the sample matrix). This is one of the properties that makes it so widely K. Drennen III applicable, but also complicates its uses. By the means of case studies spanning Monitoring and controlling the manufacturing transdermal delivery systems poses the fields of agriculture and pharmaceutics, the tremendous power of near-infrared a number of challenges. A critical aspect is the amount of drug containing coating spectroscopy will be explored. Central to this discussion is the quantity of informa- deposited on the backing of the drug product. The key to controlling this quality at- tion available about the sample in every single near-infrared spectrum. However, 46 2015 EAS Abstracts November 2015

the cost of this information rich data is the complications it brings to the life-cycle energy implants is still the exclusive domain of SIMS. However, SIMS can also be of analytical methods (maintenance and transfer). The relationship between accu- used to characterize thin films, interfaces, and contamination in thick layers (both racy/precision and robustness are explored in these examples. Finally, a reflection conducting and insulating). This presentation will highlight multiple successful uses on where near-infrared technology is heading and the issues that practitioners are of SIMS for the characterization of semiconductor device materials as well as some facing are provided. of the limitations of the technique with regard to artifacts associated with sputtering solid surfaces. 315 Molecular Analysis at the Nanoscale Emile A. Schweikert, Texas A&M University, Dept. of Chemistry, College 318 Trapped Ion Mobility Mass Spectrometry and the Preservation of Station, TX 77843 Sample Features The importance of characterizing nanosize particles and domains is widely recog- Melvin A. Park, Bruker Daltonics, 40 Manning Rd., Billerica, MA 01821, nized. Yet, measurement techniques for molecular assemblies in dimensions below Mark E. Ridgeway, Joshua Silveira 50 nm are lagging. Our approach to advance nanoanalysis is based on second- As analytical chemists we endeavor to create descriptions of our samples; i.e., mo- ary ion mass spectrometry (SIMS), with novel methodology and instrumentation. lecular composition, relative abundances of species, spatial distribution, etc. One Nanodomains or individual nanoparticles are bombarded with a sequence of indi- of the issues associated with analytical instrumentation and related methods is the vidual nano-projectiles (e.g., C_60, Au_400). At impact energies of ~ 1 keV/atom, possible disturbance or outright destruction of those features in our sample that we they generate notable secondary ion, SI, emission from a surface volume of 10-15 are trying to measure. Historically, this has been the case with mass spectrometry nm in diameter and up to 10 nm in depth. The ejecta from each impact are mass (MS). The combination of electrospray ionization (ESI) and matrix assisted laser analyzed and recorded individually. Thus, the co-emitted ions originate from mol- desorption ionization (MALDI) with MS led to a revolution in its application to bio- ecules co-located within < 15 nm. The nature of the ejecta may differ from that logical samples because these techniques preserved molecular information through observed from bulk of similar composition. Isolated nano-objects in dimensions the ionization step. This has led to detailed descriptions of biochemical systems in below 50 nm constitute finite systems where the energy imparted by the projectile terms of mass. More recently, the community has been attempting to hybridize, in cannot be dissipated as in a semi-infinite solid. As a consequence, sputter, ion and an effective way, ion mobility analyzers (IMS) with MS. The addition of IMS to MS electron yields are size-dependent. The presentation will describe the custom-built promises to improve specificity and detection limits as well as provide additional instrumentation and the methodology for data acquisition and analysis. The charac- information – collision cross section – about sample species. As part of this effort, terization of nanoscale domains and particles is illustrated with several examples: we’ve developed a novel ion mobility spectrometer – Trapped Ion Mobility Spec- evolution of catalytic sites with temperature, changes in the chemical environment trometry (TIMS) – and corresponding theory. The central idea behind TIMS is to of gold nanoparticles varying by as few as ~ 100 atoms, compositional and structur- mobility analyze ions in a moving, rather than stationary, gas stream. This results in al make-up of nanodomains. Work supported by the National Science Foundation a compact, “high” performance, but easily hybridized analyzer. Grant CHE-1308312. 319 Two-Dimensional Liquid Chromatography (LC) Strategies Coupled 316 Micrometer-Scale Ion Traps from Micro-Mass Spectrometry to with Mass Spectrometry for Efficiency, High Resolution Quantum Information Processing: Why Instrumentation Still Characterization of Therapeutic Monoclonal Antibodies Matters Dwight Stoll, Gustavus Adolphus College, 800 West College Ave., St. Matthew Blain, Sandia National Laboratory, PO Box 5800, Albuquerque, Peter, MN 56082, David Harmes, John Danforth, Elsa Wagner-Rousset, NM 87185 Alain Beck, Szabolcs Fekete, Davy Guillarme Microfabricated ion traps offer important advantages for both micro-mass-spectrom- The characterization of therapeutic monoclonal antibodies (mAbs) is a tremendous etry and quantum information science. If properly engineered for the experimen- challenge to state-of-the-art separation technologies. Historically, these assess- tal objectives, these micro-devices offer the ability to extend the performance of ments have been made using different orthogonal separation techniques (e.g., their macroscopic equivalents, and can even allow new concepts to be explored in size-exclusion LC, ion exchange LC, reversed-phase LC capillary electrophoresis), both classical and quantum ion physics and chemistry. Some technical aspects of with either direct or indirect detection using mass spectrometry (MS). Two-dimen- the realization of micrometer-scale traps are presented. One early example to be sional liquid chromatography (2-D-LC) provides rich opportunities to increase both described is the design, simulation, and construction of a large array of microme- the efficiency of the characterization, and the value of the information gained in the ter-sized cyclindrical ion traps for application as a micro-mass-analyzer [1]. As a process. In this presentation, we describe results of the characterization of a ther- second example, the ability to fabricate complex and arbitrarily arranged two-di- apeutic mAb by first partially digesting the antibody and separating the fragments mensional (2-D) trap electrode geometries and arrays has been recognized to be by online selective two-dimensional liquid chromatography coupled to time-of-flight critical for a number of trapped ion quantum information experiments (both pro- mass spectrometry (sLCxLC-TOF-MS). We use cation-exchange (CEX) chromatog- posed and realized). In particular, surface electrode ion traps have enabled the ion raphy in the first dimension, and reversed-phase LC in the second dimension – this trap CCD (charge coupled device) architecture [2], whereby individual ions can be approach provides, in a single analysis, information about the charge variants of moved between different trapping regions optimized for ion loading and qubit ini- each fragment, and it’s mass. The selective comprehensive approach to two-dimen- tialization, entanglement, storage, and read-out. The ability to design and fabricate sional separation provides a powerful compromise between analytical throughput, surface electrode trap junctions for the spatial re-ordering of single ions [3], preci- and very high resolution separation, because it enables the second dimension sep- sion through-chip holes for ion loading and photon collection/delivery, and arbitrarily aration of multiple fractions per mAb fragment eluting from the first dimension CEX shaped trap chips for increased optical access to the ions, are critical for construct- separation. We will demonstrate that sLCxLC reveals the presence of mAb variants ing the required trap features and ultimately rendering a highly evolved ion trap having the same mass, but different 2-D retention times, that would otherwise be chip technology. This “micro-systems” approach to the instrumentation of microme- missed using other separation technique. We will show comparisons of the sLCxLC ter-scale ion traps relies on microfabrication techniques to render devices optimized separations to fully comprehensive 2-D-LC (LCxLC), heartcutting 2-D-LC (LC-LC), for the experimental application. Sandia National Laboratories is a multi-program and 1-D-LC separations of comparable analysis time. laboratory managed and operated by Sandia Corporation, a wholly owned subsid- iary of Lockheed Martin Corporation, for the U.S. Department of Energy’s National 320 Investigation of One- and Two-Dimensional Approaches for the Nuclear Security Administration under contract DE-AC04-94AL85000. Chromatographic Separation of Complex Mixtures of Closely References: Related Species [1] M.G. Blain, et al., Int. J. Mass Spec. 236 (2004) 91. Chris Welch, Merck, RY 818 B224, Rahway, NJ 07065 [2] D. Kielpinski, et al., Nature, 417 (2002) 709. The rapid and efficient chromatographic separation of closely related species (en- [3] D L Moehring, et al., New Journal of Physics 13 (2011) 075018. antiomers, diastereomers, regioisomers, positional isomers, etc.) is of considerable interest in our laboratories. In this presentation we survey recent results in the use 317 Characterization of Semiconductor Materials by Secondary Ion of single dimension chromatography as well as off-line and on-line two-dimensional Mass Spectrometry high-performance liquid chromatography (2-D HPLC) methods for rapid resolution Joe Bennett, National Institute of Standards and Technology, 100 of difficult mixtures of closely related species. Bureau Dr., Gaithersburg, MD 20899 Secondary ion mass spectrometry (SIMS) has proven to be an essential tool for 321 Heart-Cutting Two-Dimensional Ultra-High-Pressure Liquid the qualitative and quantitative analysis of a variety of solid materials. Under ap- Chromatography for Process Development: Asymmetric Reaction propriate operating conditions SIMS is uniquely positioned to provide quantitative Monitoring analyses with high dynamic range (ppb to % levels) and high precision (<1% RSD) Shengli Ma, Boehringer Ingelheim, 900 Ridgebury Rd., Ridgefield, CT at in-depth resolutions of <1 nm. These characteristics make SIMS ideally suited to 06877, Nelu Grinberg address the analytical challenges encountered during the manufacturing of semi- This contribution presents the first application of two-dimensional, ultra-high-pres- conductor devices. Extremely sensitive, highly reproducible depth profiling of low sure liquid chromatography (2-D-UHPLC) for monitoring asymmetric reactions in 47 2015 EAS Abstracts November 2015

process development. Several asymmetric transformations were studied to illus- 325 Kinetics of Surface-Assisted Silica Nucleation on Model Biological trate the operation of the instrument and evaluate the performance of 2-D-UHPLC. Interfaces Two-dimensional UHPLC is particularly advantageous because it allows a simulta- Adam F. Wallace, University of Delaware, 101D Penny Hall, Dept. of neous analysis of the reaction conversion and its enantiomeric excess. By employ- Geological Sciences, Newark, DE 19716 ing UHPLC the analysis time can be reduced significantly, and the achiral−chiral Nature has evolved the ability to manufacture functional nanomaterials with such 2-D coupling approach allows for direct injection of the reaction mixture. This study sophistication that their structural complexity and materials properties often exceed demonstrates the utility of 2-D-UHPLC in asymmetric transformations for drug de- those of materials synthesized by state-of-the-art techniques. Therefore, there is velopment. much potential that may be realized by investigating the fundamental physical and chemical processes that drive the nucleation, growth, and assembly of biomineral- 322 Microdosing Device and Drug Formulation Compatibility Study by ized tissues. Additionally, because the trace element and isotopic compositions of 2-DLC-MS biominerals reflect to some extent the conditions in which they initially formed, these Lulu Dai, Genentech, 1 DNA Way, South San Francisco, CA 94080, materials comprise one of the primary records of life and environmental change on Kelly Zhang planet Earth. The proper interpretation of this record requires a microscopic un- Compatibility of a parenteral drug microdose formulation with the intravenous (IV) derstanding of biomineral nucleation and growth mechanisms. Specialized proteins infusion devices was studied in support of a bioavailability clinical trial by micro-trac- (silaffins, silacidins, silicateins) and long-chain polyamines have been found in as- er method. It was discovered that a polyethylene glycol (PEG) based microdose sociation with siliceous diatom frustules and sponge spicules and are thought to solution was prone to fast degradation in contact with the IV tubing and generated initiate and control silica deposition within highly regulated internal environments; a new impurity. However, it was not possible to identify the impurity by convention- however, the mechanisms through which these species initiate silica nucleation al liquid chromatography mass spectrometry (LC-MS) due to strong and complex in vivo remain poorly understood. In this work, an in-situ, atomic force microsco- matrix interference from PEG. On-line two-dimensional (2-D) LC-MS was utilized in py-based experimental approach is developed to directly measure the kinetics of attempt to solve the problem. In the 2-D chromatography scheme, a reverse phase silica nucleation on model biosubstrates under chemical conditions that mimic natu- method was used in the first dimension to separate the active pharmaceutical in- ral biosilica deposition environments. Relative contributions of thermodynamic and gredient (API), the impurity and partial of PEG component. The impurity and the kinetic drivers to surface nucleation are quantified by use of amine-, carboxyl-, and co-eluted PEG portion were sent to the 2nd dimension by heart-cutting and were hybrid amine/carboxyl−terminated surfaces as surrogates for charged moieties on subsequently separated in the 2nd dimension, thus the matrix interference from PEG silica-mineralizing organic matrices. was minimized and the MS information of the impurity was obtained. The 2-DLC-MS information allowed successful identification of the degradation product. The study 326 Surface Analysis and Correlation to Coating’s Adhesion to Metal through 2-DLC-MS suggests that the incompatibility was caused by oxidation of the Xiaochun Zhang, Ashland, 500 Hecules Rd., Wilmington, DE 19808, API with reactive peroxide impurities in PEG and the peroxide reactants were likely Mackenzie Williams, Venkataram Krishnan, Bruce Fillipo formed through free-radical initiated oxidation of PEG. The study demonstrated that Architectural coatings include paints, sealers, and specialty coatings for building 2-DLC is an enabling tool in cleaning out the strong matrix interference, resolving and construction applications. They are designed to provide a protective and/or complex co-elution and overcoming mass suppression by the matrix. It indicates decorative layer on the surface in both indoor and outdoor applications. One of the that microdose formulation is especially vulnerable to incompatibility issues due to important performance attributes of water based architectural coating is its adhesion low drug amount and high exposure to reactants in the excipients and devices. strength to most substrate surfaces including metal. Multiple surface analysis tools such as X-ray photoelectron spectroscopy (XPS) and contact angle measurement 323 Surface and Interface Nanostructures of Oxide Catalysts for Solar were employed in this study to understand the mechanism of coating’s adhesion to Water Splitting metal surfaces. Combined with adhesion application test, specific interaction be- Bruce E. Koel, Princeton University, Dept. of Chemistry and Biochemistry, tween coating film and metal surface was investigated and surfactant’s impact at A311 Engineering Quadrangle, Olden St., Princeton, NJ 08544 this sub-micron scale interface was explored. Improved utilization of solar energy is a key challenge for addressing our energy needs. In this quest, fabrication and control of the nanostructure at surfaces and 327 Analyzing Cannabis for Physiological Purposes interfaces plays a central role in development of efficient conversion schemes for Jeffrey C. Raber, The Werc Shop, 2585 Nina St., Pasadena, CA 91107 producing fuels from sunlight. We report here on our studies of the influence of Cannabis sativa L. is currently cultivated via a multitude of diverse methods involv- surface modifications on surface and interface nanostructures of oxide catalysts for ing numerous varietals. A significant percentage of the currently cultivated varietals solar water splitting. We consider in these materials: 1) the role of surface dopants, produce predominantly tetrahydrocannabinolic acid (THCA) as the major cannabi- co-catalysts, and composite oxide components; and 2) the role of different morphol- noid. Recently, testing laboratories have seen an increase in cultivating activities ogies and crystal facets. Characterization of these materials utilized surface-sensi- involving high cannabidiolic acid (CBDA) varietals. All cannabis consumption is tive electron and ion spectroscopies, along with atomic imaging using electron and sought by the consumer to elicit a specific physiological response. For patients who scanning probe microscopy, and electrochemical water oxidation measurements. are critically ill, or ailing with chronic debilitating conditions, selection of the right Model catalysts of metal-doped and oxide single-crystal surfaces were formed cannabis cultivar can be exceptionally important. Selection of a preferred cannabis by vapor deposition under controlled conditions. In addition, we investigated the flower product has traditionally been performed based on olfactory inspection and facet-dependent activity and stability of Fe2O3 and Co3O4 nanocrystals for water visual appearance. With the introduction, and increased requirement by regulations, oxidation using the well-defined morphologies of nanomaterials synthesized using of independent testing laboratories within the supply chain consumers can also use hydrothermal methods. analytical data to further inform themselves about their selections. The importance of broad based analytical methods to fully profile cannabis flowers, including both 324 The Role of Surface Defect Sites of Transition Metal Oxide Nanopar- cannabinoid and terpene components of interest, is described and discussed. The ticles in Clean Energy Production importance of product purity and some of the analytical testing required to ensure Xianqin Wang, New Jersey Institute of Technology, Dept. of Chemical, unadulterated cannabis is available to end consumers are also highlighted. Biological and Pharmaceutical Engineering, 323 MLK Blvd., Newark, NJ, 07102 328 Improved Gas Chromatographic (GC) Quantification of Acidic and Directing matter and energy now is to synthesize new forms of matter with tailored Neutral Cannabinoids properties that will operate at the theoretical limits and will enable catalysts that are Amanda Rigdon, Restek, 110 Benner Circle, Bellefonte, PA 16823, Jack 100% specific and produce no unwanted byproducts. Fundamental understanding Cochran, Joan Serdar of the active sites and how to control the stability of the active sites are key to tackle Quantification of cannabinoids is required for labeling purposes in most states the challenge. Low coordination defect sites on transition metals, such as steps and where medical and/or recreational cannabis is legally dispensed. Accurate quanti- kinks, as well as open “rough” crystal faces that make low coordination metal sites fication of cannabinoid content is important for dosage purposes and assurance of available have been uniquely active for breaking H–H, C–H, C–C, C=O, O=O and product uniformity, and is required in most states where cannabis is available. The N_N bonds. Oxide–metal interfaces provide highly active sites for reactions of C–H most straightforward way to accurately quantify both acidic and neutral cannabi- and C=O bonds. Thus the capability of controlling and tuning the properties of the noids is liquid chromatography (LC), however some laboratories do not have access transition metal or metal oxide in the catalysts determines whether we can address to LC instrumentation and are limited to GC techniques. Neutral cannabinoids may the main challenge. A fundamental understanding of structure-function relationships be quantified using GC, but acidic cannabinoids cannot be separately quantified for catalysts is vital for the design of new catalytic materials. To accomplish this, new via GC because they are decarboxylated in the hot injection port, resulting in the and efficient methods of in-situ time-resolved characterization and rapid throughput acidic and neutral cannabinoids being reported as a sum. Another drawback of GC testing of catalytic properties are crucial. In this talk, the functions of transition met- cannabinoid quantification is the phenomenon of irreproducible decarboxylation, al oxides in tackling the energy and environment challenges are reviewed. Their which results in inaccurate reporting. A fast and simple derivatization method using structure-function relationships in hydrogen production and biofuel upgrading are BSTFA + 1% TMCS was developed to address both of the drawbacks associated discussed in detail. 48 2015 EAS Abstracts November 2015

with GC cannabinoid testing. This method was tested for linearity and stability us- 333 Kinetic and Mechanistic Studies of Enantioselective Synthesis of ing different concentrations of acidic and neutral cannabinoids, as well as, different Hemiaminals via a Pd-Catalyzed C-N Coupling plant matrix concentrations. Because some of the derivatization sites are sterically Yining Ji, Merck, 126 E. Lincoln Ave., Rahway, NJ 07065, Qinghao hindered, mass spectrometry was employed to ensure derivatization went to com- Chen, Robert Thomas Williamson, Edward Sherer, Andrew Brunskill, pletion during the course of the experiment. Results show that the derivatization Alan Hyde, Kevin M. Belyk, Hongming Li, Jingjun Yin, Louis-Charles process was completed in all samples. This presentation explores the phenomenon Campeau, Kevin R. Campos, Michael Williams, Ian Davies, Rebecca of incomplete decarboxylation, introduce a fast derivatization method for cannabi- T. Ruck noids in plant matrix, and explore the feasibility of using this method in alternative A novel Pd-catalyzed asymmetric synthesis of chiral N,O-acetals through C-N cou- matrices such as food. pling has been developed and is described. Detailed kinetic and mechanistic stud- ies have been carried out to both understand and aid in reaction development. By 329 Multi-Residue Pesticide Analysis in a Highly Resinous Natural monitoring the reaction by 1H nuclear magnetic resonance (NMR) spectroscopy, we Product Matrix; Sample Preparation Strategies for Ultra-High- were able to determine the reaction driving forces, assess the catalyst stability, and Performance LC-MS and SFC-MS Analysis identify and probe key catalyst poisons. Furthermore, the use of NMR spectroscopy Michael S. Young, Waters Corporation, 5 Technology Dr., Milford, MA and computational modeling toward establishing the stereodetermining step of the 01757, Kim Tran catalytic cycle is discussed. Additionally, we were able to identify and track the ap- Recent advances in sample preparation, such as the QuEChERS (quick, easy, pearance and disappearance of transient catalytic species aided by the combination cheap, effective, rugged, and safe) methods, have simplified and streamlined pesti- of both 1H and 31P NMR spectroscopy. These studies revealed the existence of a cide analysis for many commodities. Although effective for many types of samples, competitive pathway leading to the formation of a less active precatalyst. These there are significant challenges when this technique is applied to highly resinous findings indicate the need to design better precatalysts that may contribute to the commodities such as cannabis. Unlike many fruits and vegetables, cannabis con- reduction of the catalyst loading, leading overall to a more robust process. tains a multitude of resin constituents similar in physicochemical properties to many pesticides. Because these interfering resin constituents are so similar in solubili- 334 Utilization of Low-Field NMR for the Characterization of Chemical ty and polarity to the target analytes, standard methods of extraction and cleanup Reactions used for other dried commodities may be ineffective for pesticide analysis in canna- Brian Marquez, Mestre Lab, 207A Yantic St., Norwich, CT 06360 bis. In this presentation options are discussed for optimized sample preparation of No abstract submitted by the author. cannabis for tandem ultra-performance liquid chromatography mass spectrometry (UPLC-MS), supercritical fluid chromatography-MS, and gas chromatography-MS 335 Recent Advances in In-Vitro Testing Methods for Inhaled Drug pesticide analysis. Topics include modified QuEChERS methodologies and other Products alternative protocols for initial extraction of pesticides from cannabis. Topics also Mark Copley, Copley Scientific Limited, Colwick Quays Business Park, include strategies using dispersive solid-phase extraction (dSPE) and pass-through Nottingham NG4 2JY, United Kingdom SPE for improved cleanup of the extracts. Traditional in-vitro test methods for inhaled drug products are derived from quality control (QC) tests developed over a number of years by pharmaceutical companies 330 Applications of Supercritical CO2 for the Analysis and Preparation and subsequently absorbed into pharmacopeial chapters. As a result, these tests, of Cannabis as a Natural Therapeutic which are designed to assess the critical quality attributes (CQAs) of delivered dose Christopher Hudalla, ProVerde Labs, 420 Fortune Blvd., Milford, MA uniformity (DDU) and aerodynamic particle size distribution (APSD) are designed 01757 to be simple, reproducible and above all discriminatory. However, in a research The cannabis industry has been thriving for many years, with the use of cannabis and development environment they lack clinical realism and achieving good In-vitro tracing back thousands of years. However, the illicit status of the plant throughout in-vivo correlations is challenging at best. In a contemporary QC environment they much of the world has stifled commercialization and research, forcing most activities may be considered laborious and the metrics used to assess product quality out- underground, often times with high risk and minimal accountability. As a result of dated. In a market dominated by generic development the combination of a need this, technological advances in science and analytical instrumentation have found to demonstrate bioequivalence and improve product understanding through qual- little to no application to this diverse field. Recent advances throughout the world in ity-by-design (QbD) has driven the recent evolution in in-vitro test methods. This legislation, regulation and public acceptance have opened the door for legitimacy talk discusses recent advances in more clinically relevant methodologies as well as of this industry. This provides the new opportunity for the use of the latest advances techniques such as abbreviated impactor measurements (AIM) and efficient data in scientific instrumentation and methodologies to be applied to different aspects of analysis (EDA) to improve the relevance of data generated. this industry, including ensuring consumer safety, basic research, optimization of cultivation practices, and the design and development of marijuana infused products 336 Experimental Observation of Powder Dispersion Mechanism (MIPs). Here we present the application of supercritical fluid technologies to the Xiang Kou, Novartis, Building 8 Halei Rd. 898, Shanghai 201203, China, analysis, extraction and purification of cannabinoids for the preparation of cannabis Steven T. Wereley based therapeutics. Supercritical fluid extraction (SFE) is used in combination with Dry powder inhalation (DPI) has been used for drug delivery due to its many ad- supercritical fluid chromatography (SFC), both analytical and preparative, to facili- vantages. Improvement of the delivery performance has been a continuous effort tate cultivation and production, following the process from seed to sale. to meet higher expectations. One approach is to understand the fundamentals of powder dispersion mechanism. The investigation reported here is to study powder 331 Using NMR with Other PAT Instruments for Reaction dispersion of carrier based DPI formulations by visualization of the aerosolization Characterization and Monitoring process. The dry powder aerosolization process was visualized in a transparent Andreas Kaerner, Eli Lilly, Lilly Corporate Center, Building 87, model Rotahaler® chamber using high speed camera and the collected images Indianapolis, IN 46285 were analyzed by micro particle image velocimetry (µPIV) technique. Influencing No abstract submitted by the author. parameters, including particle size, surface morphology, ratio of fines, are varied and investigated. It is found fine particles (1-5µm) formed agglomerates when it was 332 Chemical Reaction Development Using NMR dispensed alone. These agglomerates were difficult to dispered. Particles larger David Foley, Pfizer, 445 Eastern Point Rd., Groton, CT 06340 than 20µm tend to exist singly even when high percentage of fines was added. On-line sampling using nuclear magnetic resonance (NMR) provides in-depth, re- Particles dispersed by drag force, particle-particle collision, particle-grid impact and al-time kinetic and structural information on complex reaction mixtures containing particle fragmentation during administration of DPI were directly seen. A revised multiple components in solution. The use of online NMR technology in the devel- ratio of aerodynamic response time (τA) to the mean time between collisions (τC) opment of organic reaction processes provides information rich data which can be was found to be 6.8 suggesting the impact and collision were of strong interest for used to gain a deeper understanding of the process under investigation. This tech- the particle dispersion. The collision and impact motions were beneficial for parti- nique has been employed in process development at Pfizer for a number of years, cle-particle dispersion to occur since collision and impact augmented the relative predominantly at the development stage of pharmaceutical research. NMR working motion between particles. Without particle-particle and particle-powder mass colli- together with other process analytical technologies provides an opportunity to build sions, the powder would remain at the bottom of the chamber due to the influence robust chemical processes in development that can be scaled and transferred to of gravity. These dispersion mechanisms are due to the turbulence created by the manufacturing environments with confidence. inhaler device configuration.

49 2015 EAS Abstracts November 2015

337 Computational Performance Analysis of Electronic Cigarettes as knowledge in order to assess, exploit, and decipher practical real-world problems. Nicotine-Delivery Devices, This is accomplished by examining real-world case studies, problem analysis, and Yu Feng, North Carolina State University, Engineering Bldg. 3, Campus prototyping studies. Box 7910, 911 Oval Dr., Raleigh, NC 27695, Clement Kleinstreuer Numerous inhalable aerosols consist of multiple nano-to-micro-scale solid or liq- 340 3-D Scanning: Rapid Analytics uid particles with dissolved or embedded compounds, as well as associated va- Richman Siansimbi, DigitialScan3D, 8703 N. Crawford St., Portland, OR pors. In general, of interest are the transport and conversion phenomena leading 97203 to local particle/droplet/vapor depositions and possible species-mass transfer into No abstract submitted by the author. systemic regions. Examples include aerosol inhalation from fuel handling, welding operation and smoking, as well as oral drug administration for direct delivery to 341 Continuous Liquid Interface Production for Layerless Fabrication pre-determined lung sites. In this study the focus is on hygroscopic growth of na- Rima Janusziewicz, University of North Carolina-Chapel Hill, 131 South no-size multi-component droplets and droplet-vapor interactions during transport Rd., Caudill Labs, Rm. 234, Chapel Hill, NC 27599, John Tumbleston, with subsequent deposition in a human upper lung-airway model. For that purpose Adam Quintanilla, Sue Mecham, Joseph M. DeSimone a comprehensive, accurate and efficient computational fluid-particle dynamics The fabrication process of additive manufacturing (AM), or more commonly, (CF-PD) model has been developed which is capable of simultaneously analyz- three-dimensional (3-D) printing, was first realized in the 1980’s with the introduc- ing multi-component droplet-vapor interactions with evaporation and condensation tion of the Stereolithography Apparatus (SLA). Since then, many iterations of the effects for different sets of inhalation conditions. Selecting inhaled electronic ciga- selective addition of material in a layerwise manner have been developed to encom- rette (EC) smoke as an application, the experimentally validated simulation results pass a range of materials and properties. In spite of this growth, the AM field has include detailed transport, deposition and absorption data for different constituents been restricted to the realm of rapid prototyping (RP). This stagnation in the field (i.e., water, propylene glycol, glycerol and nicotine) in both vapor and liquid forms can be largely attributed to the inherent flaws of layer-by-layer printing; in particular for an idealized human upper lung airway geometry, i.e., from mouth to generation anisotropic mechanical properties with respect to print orientation. This anisotropic 3. Results indicate that liquid-vapor phase change impacts significantly the depo- characteristic arises from the discrete nature of the AM process and results in lay- sition concentrations of aerosols via inertial impaction, secondary flows, Brownian ers of material with little chemical interaction, visibly indicated by the stair-stepping motion, and the vapor-specific absorption rates. Parametric sensitivity analyses effect. Continuous liquid interface production (CLIP) is an alternative approach to were performed to evaluate the influence of different inhalation flow waveforms on AM that capitalizes on the fundamental principle of oxygen inhibited photopolymer- EC-aerosol transport, interaction, and deposition. The CF-PD model can be readily ization to generate a constant liquid-interface of uncured resin between the growing extended to provide predictive simulation results for the design and operation of part and the exposure window. This interface eliminates the necessity of an itera- other pulmonary aerosol-delivery devices. tive layer-by-layer process allowing for continuous production. Herein we report the advantages of continuous production, specifically the fabrication of layerless parts. 338 Probing the Dynamics of Electrodynamicly, or Optically, Trapped These advantages include the production of parts containing large overhangs with- Pharmaceutical Aerosol out the use of supports, the reduction of the stair-stepping effect without compro- Allen Haddrell, University of Bristol, Tyndall Ave., Bristol BS8 1TH, mising printing time and the fabrication of parts with isotropic mechanical properties. United Kingdom, Jonathan Reid, Lea Ann Dailey, Darragh Murnane A consensus forum of industrial, academic and regulatory experts have identified a 342 3-D Technology poor understanding of the relationships between physicochemical characteristics of Jonathan Teeple, Cimquest, 3434 US-22 #130, Branchburg, NJ 08876 drug formulations and their performance in the humid environment of the respiratory No abstract submitted by the author. tract as one key barrier to progress in inhalation therapeutics. Thus, quantifying the properties of pharmaceutical aerosol that govern hygroscopic growth will yield the 343 2D-LC-SFC: Extending Capabilities of Multi-Dimensional potential for the rational design of new formulations for drug delivery to the lung Separations (DDL). Described here is a comprehensive approach to study the complex relation- Meenakshi Goel, Genentech, 1 DNA Way, South San Francisco, CA ship between aerosol inhalation and human health utilizing cutting-edge single drop- 94080, Mohammad Al-Sayah, C.J. Venkatramani, Guannan Li let analysis technique (the comparative kinetic electrodynamic balance (CKEDB)) Two-dimensional (2-D) chromatography is becoming more popular in the phar- coupled with a novel whole lung model capable of determining total and regional maceutical world due to its higher resolving power compared to one-dimensional doses in the lung. The CKEDB has the ability to trap and confine a droplet dis- chromatography. The high resolving power is pronounced especially when using pensed only <100 ms before. The dispensed droplets can originate from aqueous or “orthogonal” separation modes in the two dimensions. In this work we report the organic solutions, or even from metered dose inhalers (MDI) starting formulations. design of the first on-line two-dimensional liquid chromatography-supercritical fluid Once trapped the absolute radius of the droplet can be measured at a frequency chromatography (2D-LC-SFC) system with an interface that allows the coupling of of 100 Hz with size accuracy better than ±50 nm, mapping out the kinetics of water reversed-phase liquid chromatography (RPLC) with SFC to achieve simultaneous transport and the time-dependence in aerosol size during inhalation. The CKEDB achiral and chiral analysis of pharmaceutical compounds. SFC, a normal phase can readily measure the radial growth factor of an aerosol up to very near saturation separation technique, was chosen in the second dimension due to its versatility, (>99.5% RH), something that cannot be accomplished via common conventional higher efficiency, and faster analysis times. The peaks of interest from thefirst techniques, and critical to predicting aerosol hygroscopic behavior in the lung and RPLC dimension column were effectively focused as sharp concentration pulses on the inevitable dose. This unique perspective offers a significant opportunity to gain small volume C-18 trapping column’s and then injected onto the second dimension insights into the treatment of lung and systemic diseases. SFC columns through an eight port/dual position switching valve interface. The first dimension RPLC separation provides the achiral purity result (impurities and related 339 3-D Technology: A Journey through the Limits of Current Design substances), and the second dimension SFC separation provides the chiral purity and Implementation result (enantiomeric excess). This truly orthogonal system could have a huge impact Alex Baranowski, Bristol-Myers Squibb, 1 Squibb Dr., New Brunswick, on API process development as the sample preparation, chromatographic analysis NJ 08901, George Currier times, and data analysis times would be reduced to allow high-throughput analysis. Three-dimensional (3-D) technology in the context of 3-D printing and 3-D scanning, The interface design, proof of concept, effect of focusing columns, and comparative encompasses a massive design space with numerous scientific possibilities to ex- studies to conventional SFC are discussed. The high-throughput capabilities of this plore. This science lies at the nexus of mathematics, computer science, chemistry, fully automated 2D-LC-SFC system are also demonstrated through various case physics, and almost all natural sciences in between. With that in mind one may studies. ask, “What can 3-D technology offer me?” In order to address this question one must first journey through the current capabilities and limitations of each technology. 344 Analytical Development to Support Low Titer Investigation of a One technique, additive manufacturing, better known as 3-D printing, is a mecha- Biologics Drug Substance nized process by which a variety of materials are usually deposited by layers under Su Pan, Bristol Myers Squibb, 1 Squibb Dr., New Brunswick, NJ 08903, computer controls or through a predefined path. This paves the way for complex Yan Zha, Colleen Alexander, Yueer Shi precision parts manufacturing which was previously impossible or required a full Recently we encountered unexpected low titer results during production of a genet- computerized numerical controlled (CNC) machine shop. 3-D scanning, a compli- ically engineered fusion protein drug. One of the critical targets for this immediate mentary technique to 3-D printing, was instrumental in reverse engineering and investigation was the chemically defined media for Chinese hamster ovary cell line rapid analytics. 3-D scanning is deployed by acquiring shape and appearance of (CD-CHO); this basic nutrition concentrate contains amino acids and vitamins for real-world objects in three dimensional space; a technique which can be exploited in cell growth. This presentation will outline our approaches to rapidly develop quan- almost nearly industry. This presentation will provide an overview of 3-D technology titative methods for the determination of two amino acids - L-asparagine (L-ASN) and its corresponding processes. The ultimate goal of the presentation is to afford and L-aspartic Acid (L-ASP) and two vitamins - thiamine and folic acid in several the audience with enough knowledge to dive deeper into 3-D workspaces and gain media. A robust LC/MS method was developed for analyzing L-ASP and L-ASN in 50 2015 EAS Abstracts November 2015

a complex mixture of 16 amino acids using a mixed-mode column. This direct mea- as a result of extended re-equilibration. Retention mechanisms in HILIC consist of surement of amino acids by mass spectrometry is sensitive, rapid and reproducible a complex mixture of partition, polar and ionic interactions. As ionic interactions are compared to conventional derivatization methods. Another LC/UV/MS method was often dominant in HILIC mode, the charge state of the analyte and surface are of developed for folic acid and thiamine analysis using a novel reversed-phase column importance. Trace ubiquitous ions such as sodium and potassium are often over- containing embedded charged groups on which polar acidic and basic compounds looked that may compete for surface ionic sites and thus impact establishment of can be retained and resolved. Batch analysis of the 9-month “aged” media showed equilibration. Herein we report a novel approach to investigate the dynamics of such an increase of L-ASP concentration and decrease of L-ASN concentration. This ions under HILIC conditions through the use of mass spectrometric (MS) detection can be attributed to a conversion of L-ASN to L-ASP due to acidic hydrolysis and and introduction of a phthalate probe to the mobile phase. The phthalate probe is consistent with the solution stability data established by the vendor. On the other allows one to track the dynamics of surface ions during the equilibration process. hand, the concentration of folic acid is found to be significantly lower than the target Several HILIC stationary phases were evaluated under a variety of buffer concen- value with a more than 3- fold reduction at time of the investigation. trations, buffer types, organic solvents and pH values. It is envisioned that such in- formation will assist in furthering our understanding of HILIC retention mechanisms 345 Using Mobile Phase Modifiers to Improve Reversed Phase and Size and provide necessary knowledge to develop robust and reproducible methods in Exclusion Separations of Biomolecules this valuable mode of chromatography. Stacy Shollenberger, Supelco, Division of Sigma-Aldrich, 595 North Harrison Rd., Bellefonte, PA 16823, Hillel Brandes, Lauren Swiger, 348 Automating Method Development and Prep Purification in Counter David S. Bell Current Chromatography (CCC) Non-specific protein adsorption onto a high-performance liquid chromatography Gary W. Yanik, PDR-Separations, 3 Old Meadow Wy., Palm Beach (HPLC) column has been shown to affect protein recovery, peak shape and res- Gardens, FL 33418 olution. Therefore in order to get complete recovery and improve chromatographic Counter current chromatography (CCC) offers advantages for some separations/ performance, it is necessary to select a mobile phase that reduces interaction be- purifications, as compared to high-performance liquid chromatography – supercrit- tween the protein and the accessible surface area in the column. This talk focuses ical fluid chromatography (HPLC/SFC). But CCC method development has been on the role that the addition of various co-solvents to the mobile phase plays in too labor-intensive for most prep purification groups supporting medicinal chemistry suppressing protein adsorption in size exclusion (SEC) as well as reversed phase (many short-run jobs each year). We developed a universal hardware/software solu- (RP) high-performance liquid chromatography (HPLC). The effect of organic sol- tion (AutoCCC) and recommended procedure for automatically screening methods vents on retention and adsorption of protein standards in SEC was determined and and purifying compounds. CCC can operate routinely along-side HPLC/SFC using the mechanism of this co-solvent effect will be described. In general, the addition the same software, user interface, and procedure to screen methods via large se- of a co-solvent suppressed protein adsorption, the degree of which varied based quences and then scale-up for automated peak collection. on the mechanism of suppression. We extended this concept to RP separations of monoclonal antibodies (mAb), specifically focusing on the addition of low-levels 349 Dissolution Modeling of a Controlled Release Osmotic Tablet of aliphatic primary alcohols to the mobile phase. Additionally, as we and others Using Terahertz Pulsed Imaging (TPI) have shown that temperature plays a pivotal role in mAb recovery, we studied the Brian P. Regler, Merck, 126 E. Lincoln Ave., Rahway, NJ 07065, Donna impact of adding 1-5% of several aliphatic primary alcohols on the chromatography Carroll, Gerard M. Bredael, David Harris, James DiNunzio, Khek-Khiang of mAbs as a function of temperature. We found that adding certain, but not all, Chia, Gerard Klinzing, Alessia Portieri, Philip Taday aliphatic primary alcohols dramatically reduced the temperature required to achieve Push-pull osmotic tablets are currently being developed for controlled release of optimum recovery and chromatography of mAbs. This data will benefit analysts as- actives. Zero order release is achieved through the use of a bilayer tablet coated sessing optimum conditions for the separation of proteins or mAbs during develop- with a semipermeable membrane. One layer of the tablet contains an osmogen ment or biomarker research. that serves as an engine for water flux into the tablet. Swelling of the internal tablet core drives the discharge of active through a port in the apex of the tablet’s cap. In 346 Size Exclusion Chromatography of Reversible Peptide Aggregates development studies conducted to date, the coating thickness has the greatest influ- Paul L. Walsh, Merck, 126 E. Lincoln Ave., Rahway, NJ 07065, Jameson ence on release rate of active from the osmotic tablet, with an inversely proportional Bothe, Claudia Neri, Suzanne D’Addio, Daniel Yin, Yun Mao relationship to coating thickness. Secondary product characteristics, such as the Size exclusion chromatography (SEC) is the best analytical tool available for the tablet’s internal structure, show less of an influence on release behavior compared assessment of the physical instability of large molecule parenteral pharmaceutical to coating thickness. Terahertz pulsed imaging (TPI) uses short pulses of terahertz formulations, including peptides, proteins, and even monoclonal antibodies. While radiation (2-120 cm-1) to penetrate typical pharmaceutical matrixes. The time delay orthogonal techniques exist, such as turbidity, dynamic light scattering (DLS), and of reflections of these pulses from materials of different refractive indexes in the analytical ultracentrifugation (AUC), SEC is the only method which can be translat- sample allows for the measurement of thickness of various materials. Using TPI, the ed from early development to filing due to its scalability, small volumes needed for coating thickness of osmotic tablets may be measured in a rapid, non-destructive analysis, acceptance by regulatory agencies, and ability to be coupled to multi-angle manner across an entire tablet. The coating thickness data obtained using TPI was light scattering (MALS), and even in-line DLS. SEC detects higher order molecular found to be in line with other analysis techniques, such as X-ray computed tomogra- weight species (aggregates) of peptide and protein formulations by allowing ag- phy and scanning electron microscope. The release rate of active from tablets coat- gregates to pass through the column while the smaller molecular weight species ed to different thicknesses was measured and a correlation was developed based are retained in the pores of the stationary phase particles. While fibrils and other on statistical models to estimate the release rate as a function of coating thickness. irreversible aggregates can be detected easily using typical SEC analytical columns and methods, reversible aggregation, sometimes due to interactions of the active 350 Withdrawn by the author. ingredient with excipients, are not as easily detected. Reversible aggregation does not have the same risks as irreversible aggregation, as it may not have an effect 351 Withdrawn by the author. on bioperformance, and is less likely to cause an immunogenic response. Here, we present the development of an SEC method which is able to detect the reversible 352 Withdrawn by the author. aggregation of a peptide formulation by utilizing the vehicle of the formulation as Development of Suitable Dissolution Methods to Assess the the mobile phase, and utilizing a semi-preparative column. The reversibility of the 353 Formulation of BMS-986001/Lamivudine/Efavirenz Fixed-Dosed aggregation of the formulation was observed through heating the formulation from Regimen Tablets its storage temperature (5 ˚C) to 25 ˚C, and eventually to 37 ˚C. Adriene Malsbury, Bristol-Myers Squibb, 1 Squibb Dr., New Brunswick, NJ 08903, Zongyun Huang, Frank Tomasella, Duohai Pan 347 Fundamental Investigations of Equilibration Dynamics in Hydrophilic Interaction Liquid Chromatography (HILIC) A fixed-dosed regimen (FDR) tablet formulation was under development for three David S. Bell, Supelco, Division of Sigma-Aldrich, 595 North Harrison anti-HIV drugs: BMS-986001, lamivudine and efavirenz. During formulation devel- Rd., Bellefonte, PA 16823, Craig Aurand, Richard A. Henry opment, it was observed that BMS-986001 and efavirenz co-crystallized during Although hydrophilic interaction liquid chromatography (HILIC) is known to provide granulation and in accelerated storage conditions, which was expected to hinder valuable retention and selectivity of polar compounds, it is often avoided due to their bioavailability. An exploratory dissolution method was developed to monitor issues surrounding robustness, repeatability and long equilibration times. In this co-crystal formation using 0.1 N HCl medium since the co-crystal is insoluble in study, systematic investigations of possible contributions to reproducibility in HILIC that medium. A reduction in the dissolution profile provided evidence of co-crystal are reported. The impact of gradients and subsequent equilibration procedures on formation in the formulation. With this method, 5-16% co-crystallization was ob- retention times were investigated using several HILIC stationary phases. Surpris- served and such results were consistent with those obtained by near-infrared (NIR). ingly, retention and selectivity were repeatable even after short equilibration times; A dissolution method accommodating all three drugs was developed using United however, retention and, at times, selectivity and peak shape continued to change States Pharmacopeia (USP) Apparatus 2 (paddles) at 75 rpm in 1000 mL of 2% 51 2015 EAS Abstracts November 2015

SDS aqueous medium, which provided a good dissolution profile for efavirenz and half-lifes for crystal growth can be fitted using a first order decay to the precipitation rapid dissolution for BMS-986001 and lamivudine. To quantify the three drugs si- vs. concentration curves. Monitoring of the solution concentration profiles with time multaneously, a gradient reversed-phase chromatographic method was developed showed that induction times to nucleation and subsequent precipitation (crystalli- to separate and elute nucleoside reverse transcriptase inhibitor (NRTI), lamivudine sation) rates were dependent on the supersaturation levels achieved. Precipitation and efavirenz at reasonable run time. This poster describes the development work rate half-lifes between 200 and 1300 seconds were observed for papaverine. of these two dissolution methods in detail. 357 In-Use and Multi-dose Stability of Taxol Injection: Overcoming the 354 Determination of Surcide P Anti-Microbial Preservative in Carbon Analytical Challenges Associated with Determination of Active Black Process by Light Absorption Spectrophotometry Using Ingredient Stability Schiff’s Reagent as Colorimetric Agent Nalini Anand, Bristol-Myers Squibb, 1 Squibb Dr., New Brunswick, NJ Peter P. Yeh, MacDermid, Inc., 245 Freight St., Waterbury, CT 06702 08907, Peter Tattersall, Thomas Haby Surcide P, containing hexahydro-1,3,5-tris(2-hydroxyethyl)-s-triazine as the active The objective of this work was to support registrational submissions with compat- ingredient, is a broad-spectrum biocide used as an anti-microbial preservative in ibility/in-use and multi-dose stability data for Taxol Injection, manufactured with a variety of house-hold, industrial, and institutional products. It is effective at low semi-synthetic paclitaxel drug substance. An in-use stability study was required concentrations ranging from 0.05 to 0.2% and is active in both acidic and alkaline to demonstrate the compatibility and stability of diluted paclitaxel in infusion solu- environments. In the Carbon Black process, Surcide P is used as a preservative to tions throughout the intravenous administration period, whereas multi-dose stability suppress bacteria growth. A simple analytical method has been developed in our was necessary to confirm chemical and physical stability of Taxol Injection during laboratory for determining the Surcide P concentration in the Carbon Black process multi-dose administration. For the in-use stability study, Taxol Injection (6 mg/mL) solution. In this analytical procedure, the process solution is first acidified to pre- was diluted with 0.9% sodium chloride, 5% dextrose injection or 5% dextrose and cipitate the carbon suspended in an aqueous dispersant. To the process solution 0.9% sodium chloride injection to paclitaxel concentrations of 0.3 and 1.2 mg/mL, is added Schiff’s reagent which reacts with Surcide P and develops a magenta bracketing the concentration range used clinically. Every effort was made to test all solution color. After filtration, the light absorption spectrum of the sample solution in samples as close as possible to real time. For the multi-dose stability study, 70% the visible region is scanned and the solution absorbance at the 554-nm wavelength of the contents of each Taxol Injection vial were removed by needle and syringe, is measured. The Surcide P concentration in the process solution is determined and tested at the initial time of puncture and after a period of 28 days as the worst by comparing the solution absorbance with those of the standard solutions from a case storage condition. To assess any physical and chemical changes, qualified calibration plot. This analytical procedure is used for Carbon Black process control. (registered) methods were used to perform all required tests. We discuss several In this paper, details of the analytical procedure and the analytical results obtained challenges encountered during these special stability studies, sample preparation are presented and discussed. and subsequent analysis. The challenges include overcoming air bubbles created during sample handling, the sample drawing using syringe, liquid sample weighing, 355 Investigation of an Oxidative Degradation Mechanism in a Tablet the effect of preparation technique of the mobile phase preparation on the sensitive Formulation of the active peak , unconventional procedure in potency (mg/vial) and challenges in Matthew A. Janson, Genentech, 465 E. Grand Ave., South San using an older technology gas chromatography packed column for solvent analysis. Francisco, CA 94080, Karthik Nagapudi, Mohammad Al-Sayah, Michael Hayes, Yanzhou Liu 358 The Completed Dissolution of Pharmaceuticals Using and Omitting Accelerated stability studies are typically conducted to evaluate and propose the HF pharmaceutical drug shelf-life assignment. During early phase prototype tablet sta- Bob Lockerman, CEM Corp., 3100 Smith Farm Rd., Matthews, NC bility studies, a new unknown degradation product was observed under accelerated 28104, Daniel Iversen 40 °C/75%RH stability studies. The identity and hence the mechanism of formation The completed dissolution of pharmaceuticals required by the upcoming United was needed to enhance drug product formulation development. Liquid chromatog- States Pharmacopeia (USP) method 232/233 utilizes hydrogen fluoride (HF). Many raphy mass spectrometry (LC-MS) analysis indicated the addition of two hydroxyl labs shy away from the use of HF due to its safety and handling procedures. This groups to the drug substance molecule, thus proposing an oxidative degradation study will compare a complete dissolution and a leach of the target analytes by pathway. The position of the hydroxyl groups couldn’t be proposed by LC-MSn, using and omitting HF. The types of products to be tested will range from gel caps, necessitating the need for the degradation product isolation and further identification safety caps, and pressed pills. The results are compared and presented. by nuclear magnetic resonance (NMR) analysis. This work shows various oxidative forced degradation attempts to help understand the degradation mechanism. An 359 In-Vitro Evaluation of Humidity Effects on the Performance of interesting reverse concentration dependent behavior was observed. Different sam- pMDI-VHC Combinations – A Full-Factorial DOE Analysis ple treatment procedures were investigated to generate significant amounts ( >10%) Xiaofei Liu, United States Food and Drug Administration, Center for of the degradation product. The solution, enriched with the degradation product, Drug Evaluation and Research/Division of Pharmaceutical Analysis, 645 was purified by preparative LC followed by a chiral supercritical fluid chromatogra- S. Newstead Ave., St. Louis, MO 63110, Diem Ngo, Changning Guo, phy (SFC) step. NMR analysis proposed the addition of two hydroxyl groups onto Badrul Chowdhury, Peter Starke, Susan Limb, Prasad Peri, Richard the conjugated six member ring of the drug substance which yielded stereoisomers. Lostritto, Alan Schroeder, William Doub Finally, polysorbate, one of the excipients used in the formulation, was determined To assist coordination of pressurized metered-dose inhalers (pMDIs) actuation to be the root cause of the formation of this degradation product. Polysorbate con- and patient inhalation, valved holding chambers (VHCs) are often prescribed with tains residual peroxides which triggered the formation of this degradation product. pMDIs. The pMDI-VHC combination can collect the larger particles that would The tablet formulation was then modified to replace polysorbate with a different otherwise be deposited in the oropharynx allowing only the finer particles to en- surfactant, sodium dodecyl sulfate, to prevent this oxidative degradation. ter the lungs. The United States Pharmacopeia (USP) chapter <1602> (in-process revision), together with USP <601> [1], provides guidelines to evaluate the per- 356 Approaches for Measuring Intestinal Precipitation Rates of Oral formance of pMDI-VHC combinations. However, testing under less than perfect Drugs patient-use conditions (e.g., a high humidity environment) was not discussed in Jon J. Mole, Sirius Analytical Inc., 100 Cummings Center, Beverly, MA the proposed compendial methods. This poster analyzes the pMDI-VHC APSD 01915, Karl Box, Robert Taylor, John Comer, Rebeca Ruiz (aerodynamic particle size distribution) data obtained using different levels of hu- Weak bases are susceptible to precipitation when travelling through the gastro-in- midity and various patient-use scenarios. Results are presented for a pMDI tested testinal tract following a pH increase. Effective oral absorption may depend on the alone and with two different VHCs in environments of 35±5% or 85±5% relative precipitation behavior of the drugs. In this study we evaluate a method for studying humidity, with inhalation delay times of 0 or 5 seconds, and with controlled elec- supersaturation and precipitation rates. The pH-shift method was used to create su- trostatic charge (~0 or ~20 kV/in) on the surface of the VHC. A 3×23 full-factorial persaturated solutions of basic drugs by dissolving the drug in ionised form and then design on experiment (DOE) analysis was conducted to evaluate the humidity ef- titrating with strong base to create a supersaturated solution of the neutral form. The fects on a set of performance characteristics (responses). The results show the concentration of drug in solution is followed using the principles of mass and charge importance of the humidity factor in influencing pMDI-VHC performance. The hu- balance. In this way, induction times and precipitation rates during the crystalliza- midity factor’s interaction with other factors is also discussed. A better understand- tion process can be measured. Results of papaverine using the pH-shift method ing of environmental condition effects on the performance of pMDI-VHC combina- were obtained; “time zero” denotes the start of data collection after reaching the tions will inform better assessment of the safe and effective use of pMDI products target pH. After time zero, pH values are more or less constant until the “Induction with VHC add-on devices, and enhance patient/health care provider’s inhalation time”, which denotes the onset of precipitation. After this time, crystals grow and the therapy adherence and manufacturer’s ability to provide patient-friendly products. neutral species concentration reduces until it reaches the intrinsic solubility. The su- Reference: persaturation ratio may be calculated from the concentration in solution at time zero [1] USP PF 41(5) In-Process Revision <1602> Spacers and Valved Holding Cham- divided by the intrinsic solubility. After induction, precipitation rates and precipitation bers Used Withinhalation Aerosols--Characterization Tests 52 2015 EAS Abstracts November 2015

360 Karl Fischer Oven Temperature Ramp: A One-Step Method chiral column variability. The developed chiral method was able to separate Enan- Development Tool tiomer and Diastereomer from the analyte. The method robustness was demon- Sofia Silva, Hovione FarmaCiencia SA, Sete Casas, Loures 2674-506, strated across different column batches and through design of experiment studies Portugal, Jorge Moreira, Pedro Serôdio, Constança Cacela on method parameters. The method was successfully validated and transferred to Water content determination is one of the most important techniques in the chemical manufacture site to support the chemical process to manufacture API. characterization of different materials during the drug product development lifecycle. During the traditional method development of Karl Fischer (KF) oven techniques, 365 Increasing Stability of a Core-Shell Particle the sample has to be prepared and measured several times and at different con- Mark Woodruff, Fortis Technologies, Clayhill Business Park, 45 ditions (e.g., different temperatures), which can be labor-intensive and lead to very Coalbrookdale Rd., Neston CH64 3UG, United Kingdom, Ken Butchart long development times. In this study, an innovative and simple KF oven method Much has been made of the ability of core-shell particles to improve speed of anal- development strategy, in which a temperature ramp is applied to optimize the con- ysis and increase sensitivity in high-performance liquid chromatography (HPLC). ditions needed to determine the total amount of water in the sample, is presented. In this poster we look at the use of a core-shell column that can withstand high This approach is particularly of great advantage for products in which the water con- pH conditions. Up until now core-shell columns have had a very limited pH range, tent is very low and/or when the sample temperature behavior is unknown. By using which means that the use of pH to alter selectivity, and in particular retain polar basic this technique, a temperature ramp is set (usually from 50 ºC to 250 ºC), where the analytes with good peak shape has been limited. Now with a new surface grafted temperature is being continuously recorded, along with the amount of water release technology the core-shell particle pH range has been extended. We can look at the and the drift, as a function of time. The heating rate must be selected in a way that entire pH range and show how this technology works, allowing method development can guarantee that the temperature inside the sample is actually achieved, which options not previously available. Utilizing pH from 1-12 should allow us to screen typically translates into a heating rate of 2 ºC/min. In this way, it is possible to cal- acidic, basic and neutral compounds in order to obtain the optimum pH in which to culate the temperature curve and the optimum conditions that will release the total run our LC method. We discuss how this affects our robustness and reproducibility amount of water present in the sample, without decomposition. This approach was in method development. used in several products for an easy and fast method development, saving time and resources during the early stages of method and product development. Together 366 A Novel Diphenyl Stationary Phase for Metabolite Profiling with the strategy presentation, several case studies are given. Mark Woodruff, Fortis Technologies, Clayhill Business Park, 45 Coalbrookdale Rd., Neston CH64 3UG, United Kingdom, Ken Butchart 361 Differentiation of Sugars by Gas Chromatography - Vacuum In chromatography selectivity is often hard to achieve in a simple manner, complex Ultraviolet Spectroscopy samples often require a complex mobile phase system to be produced that is not Jamie L. Schenk, University of Texas-Arlington, 700 Planetarium Pl., seen as productive, reproducible and easily transferable between sites. We discuss Box 19065, Arlington, TX 76019 the use of a novel di-phenyl bonded phase chemistry which allows the use of simple Carbohydrates are key essentials in all life forms but they have always been chal- mobile phase systems even for complex sample types, such as positional isomers, lenging to analyze. Identification and determination of configuration using gas chro- loss of functional groups and metabolites which are closely related. We discuss the matography-mass spectrometry (GC-MS) and other separation methods are fraught use of this unique stationary phase in terms of its physical and chemical character- with difficulties in determining differences in alpha/beta anomeric position, ring con- istics and stability, particularly in mass spectrometry (MS) where the unique bonding figuration, and the overall stereochemistry of the sugars. The vacuum ultraviolet function allows for a stable baseline to be achieved. No “MS-bleed” is achieved detector for gas chromatography (GC-VUV) collects full absorption spectra from and this in turn allows sensitivity and resolution to be optimized. We compare to 120-240 nm, where all analytes absorb and have unique absorption spectra. In this both traditional C18 chemistry and also to other phenol functionalities in terms of study, permethylated sugars were prepared for GC analysis. GC-VUV was used to the extra selectivity and extra stability that is available. Applications highlighting distinguish epimers, ring configurations, and alpha/beta conformations for various the unique selectivity that can be achieved with simple mobile phases and unique different pentoses and hexoses. di-phenyl functionality are shown, examples of positional isomers, metabolites and other closely related species are referenced. Which mobile phases are best and 362 Withdrawn by the author. how simplicity is retained due to the unique nature of the stationary phase. 363 Development of a Mixed-Mode HPLC Method to Simultaneously 367 Fast Radio-Pharmaceutical Analysis by UPLC-MS and Beta-RAM Separate Neutral and Ionic Pharmaceutical Antimicrobial Detector Preservatives Van Truong, Merck, 126 E. Lincoln Ave, Rahway, NJ 07065, Roy Helmy, Parul S. Kadakia, Merck, 126 E. Lincoln Ave., Rahway, NJ 07065, David Waterhouse Zhenyu Wang, Oscar Liu The objective of this study was to develop fast screening methods for the radio- Preservatives are widely used in pharmaceutical formulations, especially aqueous chemical purity analysis of radio-pharmaceutical compounds using Waters ul- and suspension dosage forms to ensure the medicine is microorganism free. Com- tra-performance liquid chromatography mass spectrometry (UPLC-MS) system. mon pharmaceutical preservatives contain various physicochemical forms, e.g., The challenges of using fast LC systems with the standard radiochemical (RAD) neutral, anionic or cationic. The goal of this study was to develop a simple high-per- detector were peak broadening and loss of radio resolution and sensitivity due to formance liquid chromatography (HPLC) method to simultaneously separate and large system dead volume and reduced residence time in the RAD detector. In quantify these preservatives. Four commonly used preservatives in pharmaceuti- this study, the IN/US Beta-RAM Model 5 RAD detector, which was configured to cal formulation were selected for method development. Use of a silica-based, tri- solve those problems, was evaluated. The Aquity UPLC with four-column switching ple-mode column (anion-exchange, cation-exchange, and reversed-phase mech- coupling with a signal quality detector and a Beta-RAM Model 5 detector enables anism) on a conventional HPLC-UV system provided best selectivity and great simultaneous analysis of radiochemical purity and mass spectrometry of radiola- versatility to separate ionic and neutral compounds. The effect of separation tem- beled compounds on four orthogonal methods. The stationary phases evaluated perature, buffer strength, pH, and organic strength on retention mechanism and were Poroshell EC 120 C18 1.7-µm, Kinetex Phenyl Hexyl 1.7-µm, BEH C18 1.7- selectivity were investigated during method development. The optimized method µm, and Biphenyl Pinacle 1.9-µm. The method conditions and parameters, such as showed ability to analyze all four presented preservatives in a short run time of 25 column dimension, UPLC flow rate, flow cell volume, liquid scintillation mixing ratio, minutes. Validation of the developed method indicated excellent accuracy, linearity, radiochemical detector dwell time, mobile phase pH conditions and organic modifi- and precision. This method also demonstrated its suitability of usage in preservative ers were optimized to achieve highest efficiency, resolution and sensitivity. The fast analysis when employed for screening pharmaceutical formulation. The method can screening methods with 3 minutes gradient offered different selectivity and impurity further be applied to incorporate more pharmaceutical preservatives as needed. profiles; therefore, enhanced confidence in purity analysis. The radiochemical purity and LC-MS analysis can be performed on four orthogonal methods in the same pe- 364 Develop a Robust Chiral Method riod as one traditional Merck’s fit for purpose (FFP) method which uses a standard Jun Chen, GlaxoSmithKline, 709 Swedeland Rd., King of Prussia, PA column dimension of fused-cored C18 with 15 minute gradient. 19406, Shawn Yang, Ted Chen Active pharmaceutical ingredients (API) often contain one of more chiral centers. 368 Overview of the Normal Phase and HILIC Modes of Chromatography However, the enantiomers and diastereomers of the API are considered impurities in the Analysis of Polar Analytes that need to be controlled under acceptable levels. The control of chiral impurities Crystal Benner, Tosoh Bioscience, 3604 Horizon Dr., Ste. 100, King of (undesired enantiomers and diastereomers) in the API is achieved by testing the Prussia, PA 19604, Atis Chakrabarti API and/or its precursors using chiral methods. Fit for purpose and robust chiral Reversed-phase chromatography (RPC) is the most widely used mode of retention methods are critical to control the chiral impurities. This presentation gives an ex- in high-performance liquid chromatography (HPLC). Very polar compounds are of- ample on how to develop a robust chiral high-performance liquid chromatography ten not sufficiently retained in low percent organic, or even in 100% aqueous mobile method through the understanding the chemical properties of the analyte and the phase in RPC mode. By using an amide or amino bonded phase column, polar com- 53 2015 EAS Abstracts November 2015

pounds can be retained by a normal phase or hydrophilic interaction chromatogra- phase separation technique, and molecular dynamic simulations were used to link phy. Although non-polar organic mobile phases and a silica stationary phase were solution-phase structures to gas-phase structures. Capillary electrophoresis (CE) used traditionally in normal phase LC, today most normal phase separations are allows for separation of charged species in solution, leading to the use of CE for performed with aqueous-organic mobile phases and a more polar-bonded station- direct experimental observation of solution-phase structures. Using CE with ultra- ary phase. This mode of HPLC is now commonly referred to as HILIC, hydrophilic violet-visible detection, we have observed intermediates in the transition between interaction chromatography. The order of elution in normal phase / HILIC is opposite PPI and PPII for polyproline-13 using a similar experimental protocol to the IMS that found in RPC for the same mixture of compounds. Here in this presentation experiment. Preliminary data analysis indicates that the occurrence of observed we discuss about the applications using an amide bonded phase and amino-alkyl intermediates is consistent with ion mobility spectrometer (IMS) data, and the re- bonded phase for the separations of glycan, saccharides, oligosaccharides, polyols, action time-scale observed by CE parallels that for IMS. These observations show pyridylaminated oligosaccharides, water-soluble vitamins, nucleic acid fragments. CE is capable of providing the experimental evidence necessary to bridge the gap This discussion also focuses on the effect of particle size on resolution and analysis between solution and gas-phase structures, and that ion mobility is able to provide time, retention and selectivity. information on solution-phase structures. Reference: 369 Analysis of Volatiles from PVC Electrical Tapes by Direct Thermal [1] Shi et al., JACS, 136, 12702–12711 (2014). Extraction-Gas Chromatography/Mass Spectrometry Emily E. Prisaznik, Cedar Crest College, 100 College Dr., Allentown, PA 372 Supercritical Fluid Chromatography in Support of Pharmaceutical 18104, Thomas A. Brettell Development - A Study of Scale-Up from Analytical to Preparative Black polyvinyl chloride (PVC) electrical tape is often used in the construction of im- Scale with Isocratic Conditions provised explosive devices (IEDs) as a means of securing its components. For this Mirlinda Biba, Merck, PO Box 2000, Rahway, NJ 07065, Jinchu Liu, reason, direct thermal extraction - gas chromatography/mass spectrometry (DTE- Lindsey Jacobs, Judy Morris, Ingrid Mergelsberg GC-MS) was explored as an analysis technique for black electrical tapes. DTE-GC- Supercritical fluid chromatography (SFC) has been the preferred technology at Mer- MS was used to examine black electrical PVC tape samples purchased from various ck for routine enantiopurity analysis and preparative scale purifications in support commercial sources. DTE was performed using an SIS AutoDesorb™ system (Sci- of drug discovery and development for many years. The use of pressurized car- entific Instrument Services, Inc., Ringoes, NJ). Trace amounts (<40 mg) of samples bon dioxide, as opposed to organic solvents, leads to faster, more efficient, and were thermally desorbed at 150 °C with a purge and flow of helium gas for 1 minute. more sustainable separations compared to high-performance liquid chromatogra- Desorption time for all samples was set to 10 minutes with an initial column trap phy (HPLC). However, the advantages afforded by SFC are coupled with various temperature at 30 °C to focus the volatiles onto the gas chromatographic column. unknowns when scaling to preparative scale. Due to the compressible nature of the Gas chromatography (GC) was performed using a 5890 Agilent gas chromatograph. carbon dioxide mobile phase, scaling from analytical to preparative scale in SFC is A Zebron™ ZB-5HT capillary column (30 m + 5 m Guardian x .25 mm i.d. x .10 µm) considerably complicated and not widely understood. In these research studies, we (Phenomenex, Torrance, CA) was used for all analysis. A helium carrier gas with a investigated scaling from analytical SFC with 3-μm particle size chiral columns to flow rate of 1.0 mL/min was used with a 10:1 split. The injector temperature was set preparative SFC with 5-μm chiral columns, using isocratic conditions. The systemat- to 225 °C. The initial column temperature was held at 30 °C for 2 minutes, followed ic scaling evaluation using different chiral stationary phases showed that the mobile by its first ramp to 150 °C increasing 10 °C/minute. The second ramp was to 240 °C phase is not very compressible at the operating conditions with 20% methanol used increasing 2 °C/minute, finishing with a final ramp to 280 °C increasing 10 °C/min- as modifier. The results obtained from this systematic SFC scaling study illustrate ute. The oven was held at 280 °C for five minutes. A post run program returned the the practical application and scaling rule from analytical to preparative SFC. oven temperature to 30 °C and lasted 10 minutes. Mass spectrometry was carried out on a 5973 Agilent Mass Selective Detector with a scan range of 40-500 m/z and 373 Acrylic Oligomer Quantification by Size Exclusion Chromatography auxiliary temperature of 250 °C. Martin Nosowitz, Arkema, 900 First Ave., King of Prussia, PA 19301 Size exclusion chromatography can in some cases be adapted to quantify small 370 Absolute Molar Mass and Size in UHPLC molecules that have been incorporated in polymers. Detector selection is a key step Michelle Chen, Wyatt Technology, 6300 Hollister Ave., Santa Barbara, in assembling a method. The poster details the development of a method that allows CA 93117, Sophia Kenrick, Eric Seymour, Bob Collins, Aym Berges quantification of methyl methacrylate oligomers present in poly-methyl methacrylate Multi-angle light scattering (MALS) is well-established as a means of determining (PMMA) samples. molar masses and sizes of macromolecules and nanoparticles, downstream of size-exclusion chromatography or gel-permeation chromatography (SEC/GPC). In 374 Recent Hardware Advances in Supercritical Fluid Chromatography, combination with a refractive index detector for universal concentration measure- Extend the Use of SFC into New Application Areas ment, MALS provides these measurements from first principles, independently Jennifer Van Anda, Agilent Technologies, 2850 Centerville Rd., of retention time and molecular reference standards. SEC-MALS overcomes the Wilmington, DE 19808, Rick Wikfors limitations of traditional SEC/GPC analysis presented by the assumption that the Recent advances in hardware have extended the use of supercritical fluid chro- analyte is identical to the reference molecules in terms of conformation, density matography (SFC) into new application areas and enhanced the application areas and non-ideal column interactions. Ultra-high performance liquid chromatography currently using SFC. The development of a low dispersion back pressure regulator (UHPLC) offers several benefits over standard HPLC: short run times, reduced con- allows full flow to a mass spectrometer without excessive peak broadening. A new sumption of sample and mobile phase, and improved resolution. However, MALS splitter for interfacing SFC flow streams to a mass selective detector or evaporative instruments designed for HPLC applications are not suitable for UHPLC. In order to light scattering detector increases robustness and reliability while allowing generally migrate the benefits of MALS analysis to UHPLC, it has been necessary to develop constant flow across a gradient is described. The capability of a solvent less injec- a new MALS detector and a new refractive index detector with greatly reduced in- tion (SPE) has been developed using a G4227A Flexible cube hosting two switching ter-detector dispersion. We present data from the first complete UHPLC-SEC-MALS valves. An example of the solvent less injection of sulfonamides is shown. The use system that robustly analyzes proteins, aggregates and fragments with unprece- of a valving configuration to use individual columns or columns in series will be il- dented resolution compared to standard SEC-MALS. A small, poorly understood lustrated. SFC is particularly amenable to columns in series due to the low viscosity shoulder on the main peak in a bovine serum albumin sample is clearly identified as of CO2. The column coupling can modify selectivity and increase the number of a protein fragment with molar mass of 57 kDa. Fragments of a 142 kDa monoclonal theoretical plates enhancing separations. antibody are resolved, quantified and identified with molar masses of 93, 61 and 27 kDa, corresponding to heavy-heavy, heavy-light and light chains, respectively. 375 Enhanced Characterization of Allergens in Cosmetics by GC×GC– These results are achieved with just micrograms or nanograms of protein. TOF MS with Soft Electron Ionization Chris Hall, Markes International, 11126-D Kenwood Rd., Cincinnati, OH 371 Observing Intermediates of Peptide Folding Using Capillary 45242, Charles Haws, Laura McGregor, Steve Smith Electrophoresis In 2003, an European Union Directive restricting the use of allergenic compounds John D. Barr, Moravian College, Dept. of Chemistry, 1200 Main St., in fragrances was released. The Directive named a total of 27 allergens, stating Bethlehem, PA 18018, Alison E. Holliday, Liuqing Shi, David E. Clemmer that they should be labelled if present at >100 ppm in ‘wash-off’ products (such as Polyproline takes an all-cis helical structure (PPI) in solutions of aliphatic alcohols shower gels), or >10 ppm in ‘leave-on’ products (such as perfumes). Compliance and an all-trans helical structure (PPII) in aqueous solutions. One can induce a fold- with this directive therefore requires that these compounds are identified and quan- ing process by equilibrating polyproline in one environment and then diluting heavily tified accurately. Two-dimensional gas chromatography with time-of-flight mass into the other. These folding processes take place over a much longer timescale spectrometry (GC×GC–TOF MS) is well-suited for the analysis of allergens within than most peptide folding reactions due to high activation barriers. Intermediates complex cosmetics extracts. The enhanced separation provided by the coupling of along the folding pathway from PPI to PPII and PPII to PPI were recently observed two columns of different stationary phase minimizes tedious sample preparation with ion mobility spectrometry-mass spectrometry (IMS-MS).[1] IMS-MS is a gas steps, which can also introduce error into the analytical process. Despite the su- 54 2015 EAS Abstracts November 2015

perior separation of GC×GC, the identification of individual compounds in complex mer coatings and paints is important in the development of applications targeting samples remains challenging when multiple compounds in a chemical class have energy transfer and heat management. Infrared thermography provides emissivity similar spectra, or weak molecular ions. This problem can be addressed by the use measurements of coating and paint systems over extended temperature ranges of soft ionization to reduce the degree of ion fragmentation, but this approach has targeting the specific applications, but is limited by the spectral range of the camera. been cumbersome to implement until now. This poster presents the use of novel Comparison to infrared (IR) specular and diffuse reflectance spectroscopy can be ion-source technology to obtain complementary, simplified soft EI spectra for full used to confirm thermographic results and provides additional information on the characterization of complex fragrances in a single analytical sequence. emissivity properties outside the spectral range of the camera. This presentation provides concept feasibility for a number of commercial paint and coating samples. 376 Hydrogen Production from Solar Water Splitting Over Titanium Dioxide (TiO2) Based Photocatalysts 380 Analysis of Polyatomic Molecules by Use of High-Resolution Nuree Ha, New Jersey Institute of Technology, University Heights, Coherent Three-Dimensional Spectroscopy (HRC3DS) Newark, NJ 07102, Xianqin Wang Angelar Muthike, Spellman College, 350 Spelman Ln. SW, Atlanta, GA Hydrogen from photochemical splitting of water into hydrogen (H2) and oxygen 30314, Peter Chen, Jessica Robinson, Theresa Wells (O2) has gained attention as an ideal future fuel source. Conventionally, hydrogen Gaseous polyatomic molecules can have conical intersections between different is primarily produced from fossil fuels. In this process, fossil fuels are consumed electronic states and therefore produce very dense spectra that are difficult to ana- and carbon dioxide (CO2) is released to air, which increases environmental issues. lyze. HRC3-D spectroscopy is a new technique that uses the 3rd dimension to im- Solar water splitting is an important method of hydrogen production for subsequent prove resolution, reduce peak density, and provide selectivity. Use of the technique use in a renewable energy source such as hydrogen fuel cells. The objective of and spectral interpretation requires identification of the responsible four wave mixing this study is to determine hydrogen production activity from photocatalytic water (FWM) process. To produce a FWM signal, three input beam frequencies are used splitting over a series of titanium dioxide (TiO2) based catalysts. In this study, the (w1, w2 and w3). The detection system is set in such a way that only one frequency activity of Platinum (Pt) supported on different kinds of titanium dioxide (TiO2) were combination w4=w1-w2+w3 is detected. Ten different FWM processes, each with a compared. The catalysts were synthesized with incipient impregnation method and different energy level diagram, can produce this frequency combination. All ten use characterized with carbon monoxide (CO) chemisorption, temperature programmed the same phase matching geometry (collinear). Four of the FWM processes are reduction and desorption, surface area measurement at liquid nitrogen temperature, parametric and six are non-parametric. None of the nonparametric processes are and activity measurement. likely to be fully resonant, which is required to create peaks for HRC3-DS. 377 Water Contact Angles in Pores and Pore Size Distribution of 381 Transmission Raman Spectroscopy as an Alternate Tool to Ordered Mesoporous Silicas Using Combined Water and Nitrogen Traditional HPLC to Determine the Content Uniformity of Solid Adsorption Isotherms Dosage Forms Karthik Jayaraman, Seton Hall University, 400 South Orange Ave., Michelle Raikes, Boehringer Ingelheim, 139 Briar Ridge Rd., Danbury, South Orange, NJ 07079, Alexander Y. Fadeev CT 06810, Reggie Saraceno, Julia Griffen The extent of hydroxylation of silica surface is of utmost importance for its prac- Transmission Raman spectroscopy is an alternate tool to traditional high-perfor- tical applications as the hydroxyls are major adsorption sites on a silica surface. mance liquid chromatography (HPLC) to determine the content uniformity of solid This work is focused on the systematic experimental characterization adsorption dosage forms. This application is in high demand in the pharmaceutical industry of water on a series of well-defined surfaces with known concentration of different and is in step with quality-by-design (QbD) and process analytical technology (PAT) silanol groups, including fully hydroxylated surfaces, surfaces with only isolated and initiatives. A comprehensive study was conducted with a transmission Raman spec- no vicinal silanols, as well as surfaces with no silanols. Adsorption of water on the trometer analyzer (TRS100) from Cobalt Light Systems, Inc., to quantify the ac- adsorbents varied more than one order of magnitude depending on the hydroxyl tive pharmaceutical ingredient (API) content in pharmaceutical formulations such content. Notably, the adsorption stayed below the apparent monolayer capacity for as powder blends, tablets and capsules. Calibration reference samples of powder all the silicas indicating weak adsorption interactions of the water molecules. For de- blend and tablets were selected to build the calibration model utilizing an orthogonal hydroxylated silicas which contain predominantly only isolated silanols, there is very design of experiment (DoE) approach. A correlation was established between the little water adsorption. To our surprise, the amount of water adsorbed on dehydrox- Raman signal and the corresponding reference HPLC assay values to build ro- ylated silicas (at 600-800-1000 oC) was lower than on the hydrophobic surfaces. bust powder and tablet chemometric calibration models using partial least squares Water t-plots (statistical thickness of adsorbed film as a function of relative pressure) (PLS). This study successfully demonstrated the ability of TRS100 system to accu- have been obtained for a wide range of silicas of varying degree of hydration. Using rately predict API concentration in powder and tablet formulations. The study also the t-plots, pore size distributions were determined from water isotherms for a series demonstrated that powder or tablet calibration models can be used interchangeably of SBA-15 and MCM-41 silicas. The contact angles for water in silica pores have for accurate assay prediction of tablet test samples. The specificity, accuracy, re- been calculated using Kelvin equation and the pore size distributions of the silicas peatability and linearity of the TRS method were demonstrated to be comparable obtained from nitrogen adsorption. For mesoporous silicas calcinated at 550 oC, the with traditional HPLC analysis. The high chemical specificity of TRS and the non-in- water contact angles were ~35-40 deg indicating moderately hydrophobic surface vasive and non-destructive nature of the measurement allow this technique can of partially dehydroxylated silica. After 24 hour treatment of silicas in liquid water at be used as an alternate tool to the traditional HPLC for routine rapid quantitative 100 oC, the hydroxyl content increased producing pore surfaces with water contact analysis. angles ~0-10 deg, indicating fully hydroxylated surface wettable by water. 382 New Methodology for Finding Optimal Spectral Matches in 378 KnowItAll® ATR-IR ID Expert™ Polymer Analysis Applications Reference Databases Dana Garcia, Arkema, 900 First Ave., King of Prussia, PA 19406, Farrel Gregory M. Banik, Bio-Rad Laboratories, 2000 Market St., Ste. 1406, Borden, Marie Scandone, Bio-Rad Laboratories Philadelphia, PA 19103, Ty Abshear, Karl Nedwed Vibrational spectroscopy is the method of choice for unknown material identification Optimized curve matching and display is a novel and valuable curve matching and due to its high chemical specificity, relative low cost, ease of operation and avail- visualization methodology. It allows users to identify optimal spectral matches within ability of portable field instruments. Routine reliable identification is made possible reference databases and visualize the comparative results in a way that is more by the availability of search software programs coupled with spectral databases. discernible to the human eye. We discuss multiple corrections that can be applied KnowItAll® attenuated total reflection infrared (ATR-IR) ID Expert is a search data automatically to compensate for differences between spectral instruments, environ- management protocol simultaneously performing single, multi-components search- mental conditions, sample concentration, attenuated total reflectance correction, es and functional group analysis. Within the KnowItAll® software package, ATR-IR and others to optimize the match between spectral curves. ID Expert provides increased efficiencies in saved time and cost by building bridges between user and software, serving as user guide enhancing decision making and 383 Real-Time Monitoring of Moisture Content in Dryers Using NIR advanced tool selection. In this presentation we provide examples of case studies Spectroscopy for polymer analysis. Results are compared with traditional KnowItAll® search ap- Denise Root, Metrohm USA, 6555 Pelican Creek Circle, Riverview, FL proaches. 33578 The Process analytical technology (PAT) and quality-by-design (QbD) initiatives 379 Infrared Thermography and Spectroscopy Emissivity have been of interest for pharmaceutical manufacturing in the last years. Implemen- Measurements tation of PAT-QbD approach involves monitoring and controlling critical process pa- Evan Crocker, Arkema, 900 First Ave., King of Prussia, PA 19406, Dana rameters that influence the critical quality attributes of the product. One of the prime Garcia processes in pharmaceutical solid dosage form is granulation and drying process. Emissivity is the efficiency with which an object emits radiation and is a key prop- With the PAT and QbD initiatives, the United States Food & Drug Administration erty determining energy transfer. Characterizing the emissivity of formulated poly- aims to increase efficiency of the pharmaceutical production by real-time process 55 2015 EAS Abstracts November 2015

analysis and control. Near-infrared spectroscopy (NIRS) is well accepted as a po- was performed with a sample mass of 100 pg. The TXRF set up is now available to tential PAT analyzer due to its rapid and nondestructive technique that additionally all users and is currently the only available synchrotron based total reflection x-ray requires no sample preparation. In this poster, implementing on-line NIR for the dry- spectrometer of its kind. ing process to yield better process control and endpoint determination is examined. 387 Diagnostic of Li-Ion Electrode Ageing Using None Destructive 384 Spectral Response of Osmium Carbonyl Imidazole Complexes to Elemental and Species Imaging Polyanions Ursula E. Fittschen, Washington State University, PO Box 644630, Mehrun Uddin, St. John’s University, 8000 Utopia Pkwy, Jamaica, NY Pullman, WA 99164, Ulrike Boesenberg, Zac Gotlib, Owen Neill, 11439, Enju Wang, Elise Megehee, Cody Piotrowski Magnus Menzel, Juergen Janek, Mareike Falk, Rolf Simon Polyanions are long chain polymers that carry multiple negative charges. Many nat- Micro X-ray fluorescence (MXRF) allows for spatial resolved elemental analysis ural polymers such as DNA, heparin, carrageenan and chondroitin sulfate carry and imaging of condensed matter. X-ray probes in general provide high penetration negative charges due to their phosphate, sulfate or carboxylate groups that dissoci- depth and therefore allow for three-dimensional (3-D) observation of representa- ate around neutral pH. These high negative charges on the polymer make them ac- tive parts of a system, e.g., battery electrodes. Modern synchrotron sources easily tive components in many biological processes, thus controlling their concentration is offer spatial resolutions of ca. 100-500 nm. Whereas laboratory based instruments very important. However, since these biopolymers are mostly non-active in electro- typically have a spatial resolution in the mesoscopic range, i.e., 10-30-µm focal chemistry and spectroscopy, their detection poses great challenge to bioanalytical diameter size. Using micro-X-ray fluorescence and micro- X-ray absorption near chemists. Our approach is utilizing a luminescent Os(II) complex as a marker for de- edge structure spectroscopy (XANES) at a synchrotron source, changes in elemen- tection of these spectroscopy inactive polyanions. Osmium(II) carbonyl complexes tal distribution and redox species correlated to the aging process of LiNi0.5Mn1.5O4 with two phenanthroline and an imidazole group exhibit moderate emission intensity Li-ion battery electrodes were studied with a spatial resolution of 0.5-µm at PetraIII in the visible region. The intensity is up to 20 times enhanced by polyanions includ- P06 (DESY, Hamburg, Germany) [1]. For comparison the same electrodes were ing heparin, carrageenan, chondroitin sulfate, and DNA samples. The enhancement also imaged using scanning electron microscopy (SEM) X-ray emission analysis is due to the fact that the osmium complexes electrostatically bind to the anion sites wavelength dispersive spectroscopy (WDS). The method confirmed the presence and may consequently intercalate into the cavity of the polyanion fold. This presen- of Ni “hot spots” in the aged electrode. However, the SEM/WDS measurements tation also discusses the influence of the substitutions at the imidazole ring in Os(II) were hampered by the roughness of the composite electrode and the measuring CO complex on its binding to the polyanions. These studies could potentially lead to time had to be extended to account for the low count rates of Ni and Mn Kα lines. the development of sensitive detection methods for these polyanions. Lab based 3-D confocal micro-XRF (CMXRF) laboratory based instrumentation will be established at Washington State University in the near future and will allow for 385 Application of Low and Mid Frequency Raman for Characterization spatial resolution of ca. 20x20x20µm3. of Amorphous-Crystalline Indomethacin Reference: Michaella E. Raglione, University of Delaware, 11 Matthew Rd., [1] U. Boesenberg, M. Falk, C. G. Ryan, R. Kirkham, M. Menzel, J. Janek, M. Fröba, Hillsborough, NJ 08844 G. Falkenberg, U. Fittschen, Correlation between chemical and morphological Vibrational (infrared and Raman) spectroscopy has been utilized for decades in heterogeneities in LiNi0.5Mn1.5O4 spinel composite electrodes (LNMO) for lith- identifying compounds using the mid-frequency range, which is characteristic of ium ion batteries determined by micro X-ray fluorescence analysis, Chem. Ma- the atom vibrations within the molecule. These spectroscopic techniques probe ter., Just Accepted Manuscript • DOI: 10.1021/acs.chemmater.5b00119 the molecular structure and local environments providing valuable insight into both the amorphous and crystalline states of molecular substances. Improvements in 388 Imaging ATR Analysis of Active Component Distribution in “Soft technology over the last several years have improved the quality and availability Chew” Formulations of low-frequency Raman (LFR) instrumentation. The LFR spectral region provides Ronald L. Rubinovitz, Thermo Fisher Scientific, 4410 Lottsford Vista insight into the lattice vibrations of molecular crystals providing the scientist a tool for Rd., Lanham, MD 20706 directly monitoring the intermolecular interactions in the solid-state. The LFR spec- The combination of Fourier transform infrared (FT-IR) microspectroscopy and map- tral region can also provide some information on the short range order that exist in ping has enabled better understanding of both the composition and distribution of solution and the solid amorphous state. The more intense LFR bands provide great- individual components in systems as diverse as biological, mineral, polymer, and er sensitivity for detecting the onset of crystalline formation in an amorphous matrix. pharmaceutical. Although the speed and utility of this type of analysis has benefited This study utilized the model system indomethacin (IND), whose different crystalline from the developments in imaging through the use of detector arrays, the fundamen- forms are well characterized. The LFR spectra of the forms showed very distinctly tal sampling considerations of FT-IR are still relevant. In this study, the advantages and intense bands for the crystalline and amorphous forms. The observations from of attenuated total reflection (ATR) microscopy are applied to representative dosage this study, demonstrated that LFR is better suited than the mid-frequency range for forms where the active ingredient is dispersed in a “soft chew” matrix. This alter- monitoring changes in form and amorphous content. native formulation is becoming increasingly popular; however it presents sampling challenges when micro FT-IR measurements are considered. Specifically, these 386 Setup and Characterization of Synchrotron Radiation Induced samples are difficult to cut thin enough for transmission analysis and simply press- Total Reflection X-Ray Fluorescence X- Ray Absorption Near Edge ing them flat enough for transmission destroys the distribution information within Structures at BESSY II BAMLine the sample. Reflectance spectra tend to be of poor quality. ATR measurement of Ursula E. Fittschen, Washington State University, PO Box 644630, a sample cross section would seem to offer straight-forward solution since sample Pullman, WA 99164, Zac Gotlib, Ana Guilherme, Martin Radtke, thickness is not an issue and spectral quality is expected to be quite satisfactory. Heinrich Riesemeier, Sebastian Böttger, Dominique Rosenberg, Peter Unfortunately, as these sample formulations tend to be sticky, the occurrence of Wobrauscheck “sample carry over” make single point ATR mapping difficult. However, the use of an Synchrotron radiation based total reflection X-ray fluorescence spectroscopy imaging ATR accessory which makes contact with the sample just once while still (SR-TXRF) is an analytical technique allowing for non-destructive simultaneous permitting measurements across the sample surface removes this difficulty. Results multi-element analysis of minute amount of a sample (<1 mg). Coupled with X-ray demonstrating the capability to map or image active and major dosage form com- absorption near edge structure spectroscopy (XANES) this technique now provides ponents across the sample surface are presented using univariate and multivariate information about the electronic structure of the elements of interest. SR-TXRF and analysis methods. SR-TXRF/XANES advantages lie in the fact that limits of detection are in the low femtogram and pictogram (pg) region respectively. Whereas using laboratory TXRF 389 A New Calibration Free Powder Blend Monitoring Approach Using setups, typical lower limits of detection fall in the picogram range. However, XANES Entropy requires tunable radiation only available at synchrotrons, but relatively simple sam- Shikhar Mohan, Duquesne University, 600 Forbes Ave., Pittsburgh, PA ple preparation and the ability for trace elemental analysis are two examples that 15282, Anik Alam, James K. Drennen III, Carl A. Anderson highlight the usefulness of this technique. Here we present the installation of an Powder blending is a critical unit operation in the manufacturing of solid-oral dos- automated TXRF system the BAMLine of the Berlin electron storage ring (BESSY age. Monitoring a powder blend process is important as the process needs to be II). The set up allows users to record SR-TXRF and SR-TXRF-XANES spectra with stopped at an appropriate end-point for efficiency and quality control purposes. A high reproducibility on 30 mm diameter quartz sample holders. The sample holder widely applied technique for this purpose is near-infrared spectroscopy (NIRS). Both has been designed to allow synchrotron light to interact with the sample at a grazing qualitative and quantitative models for the active pharmaceutical ingredient (API) angle of below 0.1°, with the detector set up in the polarization plane of the incoming can be built from NIRS to monitor a powder blending process by using chemo- radiation. First results of testing of pre-prepared nickel samples of known concen- metric techniques such as partial least squares (PLS). However, in order to build tration (10 pg and 100 pg) and NIST reference samples showed detection limits of these models, calibration blend runs need to be performed. A new calibration free Ni in the femtogram range. TXRF-XANES studies on various rhenium compounds approach using entropy calculations of NIR spectra was studied. In order to test allowed the speciation of 1 ng Re in an unknown rhenium complex, also Ni XANES this approach, a system including phenytoin (API) and additional excipients (mi- 56 2015 EAS Abstracts November 2015

crocrystalline cellulose, lactose, and magnesium stearate) was blended. The end ues of 100 samples collected throughout the batches. The models were validated point times determined from entropy monitoring was compared to end point time by testing NIR results with additional granulation batches to primary laboratory data. determined by the PLS model. Moving standard deviation was used on the same The moisture models, spectral regions from 1870-1970 nm corresponding to the – data for comparison. The moving standard deviation approach gave an end-point OH combination band were analyzed using principal component analysis. The spec- of less than two minutes; while the entropy approach and PLS model gave similar tral baseline changes were correlated to particle size. R2 of calibration, root mean end points of approximately twenty minutes. The PLS model gives a more accurate square error of calibration, and root mean square error of cross validation values are assessment of the blend homogeneity as this model is based on previous experi- reported for each model. The results showed that NIRS with partial least squares ments. Therefore, the calibration free entropy approach more accurately determined regression can be used to determine, in real-time, the moisture content and particle the end-point of blending compared to the moving standard deviation approach. size of granules after using appropriate preprocessing methods. 390 A New Variable Selection Method for Vibrational Spectroscopy 393 Automated Microwave Sample Preparation of Difficult Petroleum Based on Principal Variables Based Matrices Yuxiang Zhao, Duquesne University, 600 Forbes Ave., Pittsburgh, PA Bob Lockerman, CEM Corp., 3100 Smith Farm Rd., Matthews, NC 15282, Shikhar Mohan, James K. Drennen III, Carl A. Anderson 28104 Vibrational spectroscopy (near-infrared, NIR/Raman) has been adopted as a rapid Sample preparation for metals analysis of petroleum products provide many chal- and non-destructive analytical tool in many fields. In the pharmaceutical industry, lenges. Samples are difficult to digest completely which can lead to background multivariate calibration models, built with NIR/Raman spectra, are commonly used interferences and sample sizes are typically small which prevents analysis at lower for quantification of drug amount. In many cases, model performance is improved if detection limits. We will show a novel automated microwave digestion system and variables/wavelengths specific to the response are selected and the non-informative prepare samples such as bunker oil, Kevlar, catalysts and other difficult materials variables/wavelengths are removed. An innovative variable selection method based encountered in the energy sector. We discuss methods used to provide clear di- on principal variables was developed and investigated. Raman and NIR spectra of gestions as well as analyze these samples by inductively coupled plasma optical multicomponent tablets were the data for this study. Interferences included fluores- emission spectrometry and discuss the results obtained. cence in the Raman spectra and baseline shifts due to density changes in the NIR data. The new method, along with two other common variable selection methods 394 A Modular Low Frequency EPR Spectrometer for Studying Objects (forward interval partial least square (FiPLS) and genetic algorithms (GA)), was ap- with Cultural Heritage Significance plied to the Raman and NIR datasets. Drug weight percentage models were created Lauren E. Switala, Rochester Institute of Technology, Magnetic for both the NIR and Raman datasets. Model performance of the three selected Resonance Laboratory, 54 Lomb Memorial Dr., Rochester, NY 14623, variable models and a full wavelength model were compared. Models built with se- William J. Ryan, Merlin Hoffman, Wyatt E. Brown, Nick Zumbulyadis, lected variables from each technique showed reduced prediction error compared Joseph P. Hornak, to full wavelength model. The model performance of the innovative method, FiPLS, We present a modular low frequency electron paramagnetic resonance (LFEPR) and GA were comparable. The innovative method was demonstrated to be a com- spectrometer made of a 30cm diameter solenoidal electromagnet and interchange- putationally efficient (compared to FiPLS and GA), user friendly (minimal user se- able sample probes, operating at a range of frequencies from 100-500 MHz. The lected parameters) and robust (against non-linearity) automated variable selection two kinds of sample probes are single-turn-solenoids (STS) and surface coils (SC). technique. The STSs hold 1.5cm diameter, 3cm long tubes containing the sample. The 2.9mm and 3.2cm diameter SCs can be placed on the surface of a larger object, allowing it 391 Structural Analysis of Conjugated Aromatic Compounds Using to be studied non-destructively and non-invasively. The spectrometer is controlled THz-Raman Spectroscopy by a LabView program that can be used for several acquisition techniques: conven- Anjan Roy, Ondax Inc., 850 E. Duarte Rd., Monrovia, CA 91016, James tional field swept from 0-18.7 mT, time-resolved spectroscopy at a fixed field value, Carriere, Christopher Meyers and field-cycled spectroscopy. The versatility of this instrument allows us to study Low frequency Raman spectroscopy (Thz-Raman) is a particularly useful technique different objects in useful ways. The SCs have been used to study pottery artifacts, for exploring the structural properties of materials because the low energy vibration- as well as to scan a barcode (printed with ferro/ferrimagnetic toner). The STSs have al transitions in this spectral region provide contributions from both selected mo- been used in a study of clays, and the effect of firing temperature and composition lecular vibrations and crystal lattice vibrations. The crystal lattice vibrations involve on their LFEPR signal, as well as a study of marble regarding its signal’s dependen- movements of the entire molecule with respect to other molecules in the crystalline cy on frequency and orientation with respect to the applied magnetic field. Examples solid, thus providing a more direct probe of intermolecular interactions and structure. of each are presented. In particular, this region of the spectrum is quite sensitive to conformational changes of the molecule such as with different polymorphic forms of the same material or the 395 Determination of Bisphenol A in Water Using Cloud Point Extraction effect of co-solvents/co-crystals. To better understand the relationship between a Coupled with Surface Enhanced Raman Scattering material’s physical structure and its low vibrational energy modes, we analyzed the Uttam Sharma Phuyal, Tennessee Technological University, 1 William L. polycyclic aromatic hydrocarbon family of materials containing compounds such as Jones Dr., Cookeville, TN 38505, Andrew Callender Naphthalene, Biphenyl and Anthracene. With increasing molecular complexity, the With advent of modern, compact, low-cost and field portable Raman spectrometers, corresponding measured peak positions for each material are compared with the there has been growing interest in Raman analysis of environmental pollutants. Still crystalline structure parameters of the organic molecular crystals and theoretical the method is limited by low sensitivity particularly of very dilute samples in aqueous calculations from the literature. We then differentiate the bands which derive from environment. Application of metal nanoparticles to enhance Raman scattering (i.e., molecular vibrations and crystalline lattice vibrations and identify trends. surface-enhanced Raman spectroscopy (SERS)) may address the problem, but col- loidal SERS substrates are difficult to produce reproducibly and nanofabricated sol- 392 Application of In-Line and At-Line NIR Spectroscopy for Moisture id substrates are costly to prepare. Here, we present cloud point extraction coupled Content and Particle Size Determination in Fluid Bed Granulation with SERS substrate as a simple, rapid and inexpensive analysis of Bisphenol A in Ahmed Shawky, University of Maryland, 20 N Pine St., Baltimore, MD water. The extraction effects of parameters such as surfactant concentration, ionic 21201, Stephen W. Hoag, Maissa Y. Salem, Eman S. Elzanfaly, Ahmed strength, incubation temperature and pH are discussed. Also, the SERS effects of E. El Gindy, Keith Freel, Ahmed Ibrahim the gold nanoparticles in analysis of Bisphenol A in water are presented. Fluid bed granulation is a process widely used in the pharmaceutical industry to pro- duce solid dosage forms. In-line and at-line near-infrared (NIR) spectroscopy were used to determine the granule moisture content and particle size; this information 389 The Versatility of Portable Raman in Reaction Monitoring can be used to gain a better process understanding and improve process control. Thomas Padlo, B&W Tek, 19 Shea Wy., Newark, DE 19713, Katherine Traditional particle size and moisture analysis methods are off-line, time consum- Bakeev, James K. Murray ing and have a relatively high cost per analysis so are not well suited for transient Raman spectroscopy is a well suited spectroscopic technique for process develop- measurements to monitor trends or be used for process control and optimization. ment and control within development labs as well as chemical, pharmaceutical and However, rapid at-line or in-line NIR spectroscopy, a process analytical technology, other industries. We show the monitoring of the synthesis of a privileged structure can be used to simultaneously measure both physical and chemical properties. The for medicinal chemistry. This work demonstrates the utility of a high resolution por- purpose of this work was to evaluate both in-line and at-line NIR methodologies table Raman spectrometer as a simple and versatile tool for monitoring the progres- for real-time predictions of moisture and particle size during the entire granulation sion of a reaction using univariate analysis such as peak trending, as well as mul- process – both the spraying phase and drying phase. The secondary nature of NIR tivariate analysis approaches to predict the end point of chemical reactions. Using measurements, requires the development and validation of NIR methods based on portable Raman systems allows the end user to be able to make measurements primary methods. Regression models were developed using NIR spectra acquired in the lab, but also provides the ability to make measurements at-line or on-line during 15 different granulation batches with corresponding primary laboratory val- in small scale pilot plants or be implemented in large scale production sites. For 57 2015 EAS Abstracts November 2015

known reactions which are repetitively performed, or for continuous on-line process 402 Accurately Characterizing Materials Showing Edge Fracture monitoring of reactions, the present approach provides a convenient alternative to James P. Eickhoff Jr., Anton Paar USA, 10215 Timber Ridge Dr., bench top systems. Ashland, VA 23005 A number of instabilities within a material make it difficult to accurately characterize 397 Benefits and Risks to the Laboratory in Buying Pre-Owned the mechanical properties using standard rheological geometries. One such insta- Analytical Instrumentation bility is called edge fracturing that commonly occurs in polymer melts and solutions. Jon Welsh, Agilent Technologies, 2850 Centerville Rd., Wilmington, DE This behavior also occurs at high deformations or shear rates, which are typically 19808 measured to mimic processing responses. Edge fracturing is characterized as the Today’s laboratories are constantly being asked to increase productivity and capa- deformation of the free surface between the upper and lower geometry. Likewise, bility with ever decreasing budgets. One area where a lab can save costs is buying when edge fracturing occurs second flows develop within the sample. These sec- used analytical equipment. While this can save money there are major risks in- ondary flows propagate radially into the sample, resulting in measuring errors of the volved with this type of purchase. This paper discusses the different options avail- mechanical properties of the material. To minimize the influence of edge fracturing able to laboratories from resellers to original equipment manufacturers (OEMs) and a specific measuring system, consisting of a cone and a partitioned plate is used. A the benefit and risks associated with each. series of samples were measured rheological with both a standard cone-plate con- figuration and the cone-partitioned plate (CPP) configuration. It is illustrated that the 398 The Impact of Environmental Conditions on Pipetting Performance results obtained with the CPP setup not only match the standard measurements in of an Automated Liquid Handling System the linear viscoelastic region, but also provide a better characterization of a material George Rodrigues, Artel, 25 Bradley Dr., Westbrook, ME 04092, John at higher deformations where edge fracturing is observed. Thomas Bradshaw Liquid handler performance can be optimized by adjusting various well-known sys- 403 Creation of an Electronic Logbook for GMP-GLP Laboratories tem operating parameters. But the effect of environmental conditions on liquid han- Robert Falana, Bristol-Myers Squibb, 1 Squibb Dr., New Brunswick, NJ dling quality is often underappreciated or ignored. This presentation illustrates the 08903, Steven Hoffman, John Rumney, Joel Young, Leticia Quinones effect of temperature and humidity on the volumetric performance of a common Multiple, mature metrology applications exist for the management of instrument pipetting robot. Seven volume configurations under nine environmental conditions and equipment inventory and their calibration activities in a regulated environment. were tested. Test volumes ranged from 1 µL to 1,000 µL. Temperatures of 15 °C, Here we describe how the Biovia inventory tool (CIMS) is configured to manage 22.5 °C and 30 °C were selected as test points, while relative humidity was studied the laboratory instrument inventory and calibrations with the unique advantage of at 30%, 55% and 80%. The impact of these environmental conditions on pipetted integration with the electronic laboratory notebooks (ELNs). This electronic logbook volumes was measured, and the resulting accuracy and precision are presented. becomes the cache for instrument related data during the instrument life cycle in- The delivered volume results are explained in terms of a parameter called “evapo- cluding qualification, daily checks, status control, planned and unplanned mainte- ration potential” which may be helpful in predicting an ideal parameter set. These nance, re-qualification and decommissioning under full audit trail. The integration of results provide quantitative justification for testing and optimizing liquid handling the electronic logbook with the ELNs provides visibility to prevent use of not-ready systems under the environmental conditions prevailing at the user laboratory. status instruments and facilitates integrated review of records supporting an analyt- ical result, thus achieving increased preservation of data integrity. The flexibility to 399 Withdrawn by the author. accommodate differences in practices due to regulatory requirements (good manu- facturing practices, good laboratory practice, the United States Food and Drug Ad- 400 An Inexpensive, Programmable System for Prototyping Instruments ministration, European Medicines Agency) or approved procedures are illustrated. and Computerizing Outdated Hardware Scot D. Abbott, Phoenix First Response, 25 Allegheney Pl., Glassport, 404 Simultaneous Determination of Reduced and Oxidized Chemical PA 14540, Ryan L. Taylor Species in a Mixture Using Cyclic Voltammetry A major challenge for developing instrumentation and measurement systems is the Jinmo Huang, The College of New Jersey, PO Box 7718, Ewing. NJ cost (time and money) of high quality computerization for projects. The common 08628 routes commercially available until now have been either 1) expensive software Cyclic voltammetry has been widely used to characterize the electrochemical reac- linked to expensive proprietary input/output devices, 2) low quality input/output de- tivity of a chemical species. It can also be used to quantify a chemical species by vices, and/or 3) programming in a specialty language. These factors have hindered measuring either reducing or oxidizing diffusion current. Because both the reduced or stopped many development efforts because the timescale, programming costs, and the oxidized forms of a chemical species are coexisting in many chemical sys- and relative inflexibility of these routes are prohibitive. In our laboratories, we have tems, it is essential that the both forms are determined simultaneously. In this re- needed to computerize several different kinds of devices and develop flexible user search, cyclic voltammetry is used to investigate the possibility of the simultaneous interfaces at modest cost. We make specialty instruments and also have old instru- analysis. The chemical system used in this study is a ferrous/ferric or cysteine/ ments that needed to be computerized. To do this, we needed to have the ability cystine mixture. The experimental parameters and results are demonstrated and to take in several forms of data, provide real time graphics for the user, and use discussed. PCs running several different versions of Windows® (32 and 64 bit) and Office®. We have developed a high-performance instrumentation-oriented system which is 405 New Platform for Development of Chiral and Achiral HPLC Methods very easy to program, inexpensive, takes many forms of input (analog, digital and Margaret Z. Figus, Merck, 126 E. Lincoln Ave., Rahway, NJ 07065, frequency), and has several forms of output. We have found this approach very Fred Mattrey, Frank Bernardoni, Timothy Nowak, Cindy Novicky, Jinjian workable in our labs. It could be well-applied in several areas, such as developing Zheng, Lin Wang, Weidong Tong, Robert Hartman, Vincent Antonucci and prototyping new instrument personalities, and also reviving old instrumentation A systematic workflow for selecting chromatographic conditions to cover a broad that has been abandoned for controller problems or software issues such as oper- selectivity range and provide a foundation for identifying high-quality high-perfor- ating system. Examples and data are provided. mance liquid chromatography (HPLC) methods is presented. Additionally, a strategy to methodically characterize and evaluate new commercial columns for potential 401 Unattended, Representative Sampling for a Wide Range of inclusion in screening and for wider application in small molecule drug substance Chemical Reactions development is discussed. The end product is an integrated workflow complete with Jane Riley, Mettler Toledo AutoChem, 7075 Samuel Morse Dr., recommended stationary/ mobile phase screening, tools for in-silico optimization, Columbia, MD 21046, Vaso Vlachos and an overall quality-by-design approach to method development. Orthogonal Sampling of chemical reactions for off-line analysis to determine reaction progress retention mechanisms and innovative method development with software assist- or impurity profiles is standard practice. However, the sampling process is not al- ed optimization using ACD LC simulator and Drylab are described. Case studies ways a precise operation and can be challenging with reactions at elevated tem- demonstrate efficient approaches toward faster, more robust and methodical meth- peratures, or reactions of a heterogeneous nature. Delays in quenching can lead to od development. variable results and inaccuracies in the analytical information gained. EasySampler was designed to eliminate these challenges by providing an automated and robust 406 Are you Compliant to the New USP Guidelines for UV-VIS? inline method of taking representative samples from reactions, even at extreme con- Birgit Pils, Mettler Toledo, Sonnenbergstrasse 74, Schwerzenbach ditions. Its unique patented probe enables the capture and immediate quenching of 8603, Switzerland, Gustavo Sierra reaction samples. Immediate quenching of the sample provides a truly representa- In ultraviolet–visible (UV-VIS) spectroscopy, regular performance verification is es- tive sample which is particularly advantageous for monitoring low temperature or air sential to ensure accurate and reliable instrument performance. Widely accepted sensitive chemistry. Multiple customer examples are presented where automated guidelines for performance verification of spectrophotometers are described in the sampling provides greater accuracy and precision over samples taken off-line. United States Pharmacopeia (USP). The recommended tests include the check of photometric accuracy and repeatability, wavelength accuracy and repeatability, 58 2015 EAS Abstracts November 2015

instrument resolution as well as stray light measurement. Recently, the USP intro- ratios of PMMA to PVP. We form these Blended Polymers by dissolving PMMA and duced a new chapter on ultra-violet visible spectroscopy and adapted the test for PVP individually in ethylene chloride and mixing the appropriate amount of their stray light. Here, we compare the methods for measuring stray light according to ethylene chloride solution to obtain the desired ratio of PMMA/PVP. Then dipcoating the current and previous version of the USP and highlight the advantages of the of the QCM quartz crystal in these solutions produces a film Blended Polymer film new test. with a known ratio of PMMA to PVP. Leaching these Blended Polymer films with water removes the PVP leaving behind a PMMA film whose structure is determined 407 Withdrawn by the author. by ratio of PMMA/PVP in the unleached film. The influence of the PVP’s molecular weight the range (10,000 to 36,000) holding the molecular weight of PMMA constant 408 Design and Development of an Analytical Method for the Detection at 996,000 was also investigated. of Nanoceria Particles Ali Othman, Clarkson University, Dept. of Chemistry and Biomolecular 412 3-D-Printed Supercapacitor-Powered Electrochemiluminescent Science, 8 Clarkson Ave., Potsdam, NY 13699, Gonca Bulbul, Silvana Protein Immunosensors for Cancer Diagnostics Andreescu Karteek Kadimisetty, University of Connecticut, 55 N. Eagleville Rd., Nanoceria is currently used in various catalytic processes due to their unique chem- Unit 3060, Storrs, CT 06269, Islam Mosa, Spundana Malla, Jennifer E. ical and electronic configuration. These particles are characterized by high reac- Satterwhite-Warden, James F. Rusling tivity, and catalytic properties which make them useful for implementation in many A low cost, sensitive, supercapacitor-powered electrochemiluminescent (ECL) pro- practical applications. It has been used as fuel additive, as a fuel-borne catalyst tein immunoarray fabricated by an inexpensive three-dimensional (3-D) printer. The and as abrasives in printed circuit manufacture to decrease the emission of par- immunosensor detects a panel of three prostate cancer biomarker proteins in serum ticulate matter from diesel engines and to lower the generation of diesel exhaust in 35 min. The immunosensor employs screen printed carbon sensors and the 3-D particles (DEPs), but are emitted as cerium oxide nanoparticles (CeO2) along with printed device operated by gravity flow. Sample and reagent delivery and washing DEP in the diesel exhaust. Studies show that these nanoparticles may induce lung are performed using simple operations. Prostate cancer biomarker proteins prostate injury and co-localized in the lung tissues after combined exposure. Moreover, it in- specific antigen (PSA), prostate specific membrane antigen (PSMA) and platelet duced sustained inflammation and surfactant accumulation, and altered the balance factor-4 (PF-4 ) in serum were captured on the antibody-coated carbon sensors of mediators involved in tissue repair process leading to excess collagen deposit followed by delivery of detection-antibody-coated Ru(bpy)32+ (RuBPY)-doped sili- and pulmonary fibrosis. Thus, the release of these nanoparticles into the environ- ca nanoparticles in a sandwich immunoassay. ECL light was initiated from RuBPY ment may cause health concerns. Methods to determine the concentration of these in the silica nanoparticles by electrochemical oxidation with tripropylamine (TPrA) nanoparticles under conditions relevant to environmental and biological systems are co-reactant using supercapacitor power and ECL was captured with a charge-cou- needed to determine the level of exposure and provide concentration limits for toxi- pled device camera. Detection limits of 300-500 fg mL-1 was achieved for the three cological testing. In this presentation, we demonstrate design and development of a proteins in undiluted calf serum. Assays of six prostate cancer patient serum sam- new method for the detection of CeO2. The method is based on the use of different ples gave good correlation with single protein enzyme-linked immunosorbent as- organic ligands such as ascorbic acid that are used to recognize and catalytically says. This type of immunosensing platforms could provide sensitive onsite cancer amplify signals, aiding in the detection of the nanoceria particles in the environ- diagnostic tests in resource-limited settings with the need for only moderate-level ment. The analytical capability of our approach and a potential implementation of training. this method for real world applications are discussed. 413 Microwave Sample Preparation of Infant Formula and Nutritional 409 Simultaneous Microfluidic Assays of CD-62L and IL-6 as Protein Supplements Biomarkers for Metastatic Bladder Cancer Bob Lockerman, CEM Corp, 3100 Smith Farm Rd., Matthews, NC Gayatri S. Phadke, University of Connecticut, 55 N. Eagleville Rd., 28106, Daniel Iversen, Tina Restivo, Ariel Smith Storrs, CT 06268, Jennifer E. Satterwhite-Warden, Dharamainder Dietary supplements are a rapidly growing segment of the food and beverage in- Choudhary, John A. Taylor, James F. Rusling dustry around the world but particularly in the United States. The ingredients which The stage at which bladder cancer is diagnosed is a critical determinant in patient make up the composition of these supplements are sourced from around the globe therapy outcome. Bladder cancer cases currently show a 5-year survival rate of 36% making testing of these products of critical importance. A new United States Phar- for regional and 6% for distant spread of the disease. A previous study identified macopeia method 2232 is in the process of final approval which will standardize L-selectin (CD62L) as a potential protein biomarker indicative of aggressive bladder the testing of these materials. Closed vessel digestion is listed as a way to prepare cancer. This work focuses on quantifying CD62L expression in patients with varied samples using Method 2232 and microwave digestion provides a rapid preparation progression of the disease, as high-grade muscle invasive tumor cases exhibit over- step as compared to other techniques. Limited sample size has prevented its use expression of this protein. We describe here a sensitive amperometric microfluidic for some applications especially for infant formula. We show the possibility of pre- immunoarray for biomarker protein detection, which employs modified sandwich paring a wide variety of supplements and infant formula samples in a microwave immunoassays. Analyte proteins with detection antibodies on enzyme-label dec- system using sample sizes of up to two grams. We illustrate the step wise approach orated magnetic beads are captured online, followed by incubation with surface required to prepare these samples as well as show recovery data for reference bound capture antibody on an 8-electrode screen-printed carbon array. The signal materials as well as spike recoveries for both volatile and non-volatile elements. is obtained by amperometric detection after injection of a hydroquinone/hydrogen peroxide activator/mediator mixture. Current efforts are directed towards simultane- 414 New Applications Using PDMS Over-Coated Adsorbent Based ous detection of ng/ml levels CD-62L along with fg/ml levels of Interleukin-6 (IL-6) Fiber Coatings as a benchmark. The combination of electrochemical detection with microfluidics Len M. Sidisky, Supelco, Division of Sigma-Aldrich, 595 North Harrison provides a cost-efficient, flexible and effective tool for bladder cancer diagnostics. Rd., Bellefonte, PA 16823, Robert E. Shirey, Katherine K. Stenerson, Olga I. Shimelis, Yong Chen, Tyler Young 410 QCM Gas Sensor Based Blended Polymer Films Extraction of analytes out of complex matrices using adsorbent based solid-phase Ho Yeon Yoo, Stanley Bruckenstein Chemical Consulting & Services, microextraction (SPME) fibers can be extremely difficult primarily because of the 115 Foxpoint W., Williamsville, NY 14221, Stanley Bruckenstein binding of the matrix components to the adsorbent particles in the fiber coating. To Porous Poly(methyl methacrylate) (PMMA) films were produced on a quartz crystal minimize this binding a proprietary polydimethylsiloxane (PDMS) layer has been microbalance (QCM) crystal by dipcoating the quartz crystal using solutions contain- applied over the adsorbent-based fiber. The PDMS coating serves as a barrier be- ing PMMA, polyethylene oxide (PEO) or both. After drying, the films were leached tween the adsorbent and the matrix, but allows smaller analytes to migrate through with water to remove the PEO to produce porous PMMA films. The influence of the the coating and be retained on the adsorbent. The overcoat increases the life of molecular weight of PMMA and PEO on the analytical sensitivity these films to water the fiber by minimizing adhesion of the matrix to the fiber coating and making the vapor was studied. The molecular weight of PMMA had a negligible influence on the fiber more durable. In addition, the ends of the fiber become sealed to prevent the water vapor sensitivity of films made from pure PMMA solutions, Films made from matrix solution form wicking into the fiber coating. This helps to improve the re- pure high molecular weight PEO solutions had a much higher sensitivity. producibility of the fiber coating. This presentation compares PDMS over-coated adsorbent based fibers to adsorbent based fibers without the overcoat. A variety of 411 QCM Studies of The Water Vapor Sensitivity of PMMA:PVP Blended analytes and applications are shown. In some cases, the extraction of analytes with Polymer Films the PDMS-OC fibers are compared to the extraction of these analytes using other Minseon Ju, Stanley Bruckenstein Chemical Consulting and Services, techniques. The presentation shows how to optimize methods to enhance recovery 115 Foxpoint W., Williamsville, NY 14221, Ho Yeon Yoo of analytes using the PDMS-OC adsorbent based coatings. Both Poly(methyl methacrylate) (PMMA) and Polyvinylpyrrolidone (PVP) form water vapor sensitive films on a quartz crystal microbalance (QCM) quartz crystal. They also can be mixed together to form Blended Polymers that have a wide range of 59 2015 EAS Abstracts November 2015

415 Extraction and Quantification of Polycyclic Aromatic Hydrocarbons 419 Optimized Supercritical Fluid Extraction of Capsaicinoids from the in Dried Bloodspots on FTA Cards by UPLC-UV Capsicum Annuum Cultivar (Red Pepper) Anthony A. Provatas, University of Connecticut, CESE, 3107 Horsebarn Kenneth J. James, Supercritical Fluid Technologies, 1 Innovation Wy., Hill Rd., Unit 4210, Storrs, CT 06269, Cory A. King, Alexander V. Newark, DE 19711, Kenneth R. Krewson, Andy Cloud Yevdokimov, James D. Stuart, Christopher R. Perkins The relationship between supercritical CO2 extraction solubility parameters and the Persistent organic pollutants are routinely quantified in tissue samples due to their specific surface area of pretreated capsicum annuum cultivar will be examined in tendency for bioaccumulation and the negative effects they can impart on an or- order to obtain the optimum extraction efficiency for the major capsaicinoids includ- ganism’s health. Polycyclic aromatic hydrocarbons (PAHs) are one such class of ing capsaicin, dihydrocapsaicin, and nordihydrocapsaicin. Linearly increasing the compounds, being structurally composed of multiple fused un-substituted aromatic solubility parameter pressure of supercritical CO2 isothermally, increases solvent rings. Owing to their extensively delocalized π systems from their high degrees density and subsequently solvent power leading to increased solubility of non-po- of conjugation, PAHs are extremely lipophilic and are thus readily up-taken into lar capsaicinoid molecules and greater extraction efficiency from the raw cultivar. tissues when present in the environment. Metabolism of these compounds leads to However, in the target extraction zone, linearly increasing the solubility parameter the generation of various carcinogenic, mutagenic, teratogenic, and generally toxic temperature, to take advantage of capsaicinoid phase change, has a net positive derivatives. Screening of blood for persistent organic pollutants, including PAHs, improvement in extraction efficiency at the expense of supercritical fluid density. has traditionally been performed using samples in the form of whole blood or plas- Diffusion and transfer to the bulk solvent phase is increased but not nearly to the de- ma. While using samples in this form is certainly effective, whole blood tends to be gree that specific surface area through cultivar pretreatment facilitates extraction ef- difficult to work with due to its high viscosity and tendency to coagulate: transferring ficiency. Increasing cultivar specific surface area through milling pretreatment yields quantifiable masses or accurate volumes of whole blood can be a tiresome effort. an increase in capsaicinoid extraction efficiency via supercritical CO2 as long as In comparison, blood spotted onto free-to-air (FTA) cards provides the advantage mass transfer is not impeded by high levels of tortuosity. Product and process cost of being comparatively easier to handle and work-up, while using only microliter efficiency of commercial extraction is a key economic element in the value chain of volumes of blood and retaining high analyte recovery. Using an ultra-pressure liquid natural substance production from not only capsicum annuum cultivars but a wide chromatography ultraviolet (UPLC-UV) approach, sample run times are short, and range of agricultural products found globally. detection limits are in the low ppb range. As a result, this novel methodology may be an attractive alternative for environmental sampling and analysis. 420 EMR-Lipid: Highly Selective Matrix Removal for Multi-Residue Analysis in Complex Samples 416 CIC – Combustion Ion Chromatography – Old Wine in a New Bottle Derick Lucas, Agilent Technologies, 2850 Centerville Rd., Wilmington, Stuart J. Procter, Metrohm USA, 6555 Pelican Creek Circle, Riverview, DE 19808, Bruce Richter, David Long, Limian Zhao FL 33578, Kendra Cox, Jay Gandhi, Jay Sheffer Current methodologies for multi-residue analysis often implement a general ex- Since the 1950s, off-line combustion techniques like the Wickbold apparatus and traction followed by analysis with a selective instrument such as liquid chromatog- Schoeninger flask have been popular sample preparative methods for petrochemi- raphy tandem mass spectrometry (LC-MS-MS), gas chromatography (GC)-MS-MS, cal and petroleum products. However, these techniques are very labor intensive and GC-MS, etc. Although these techniques are simple and ideal for the extraction of are not cost effective in fast paced high-throughput laboratories in modern times. diverse analyte groups, they also extract a large amount of matrix and tradition- The fully automated combustion ion chromatography (CIC) system presented here al cleanup sorbents struggle to effectively and selectively remove these unwant- combines a highly efficient combustion system with the separation power of ion ed interferences. Complex samples high in lipids are particularly problematic as chromatography (IC). CIC allows for the simultaneous speciation of halides (F, Cl, co-extracted matrix can cause poor reproducibility, ion suppression/enhancement, Br and I) and sulfur compounds (as sulfate) from sub-ppm to per cent levels in any changes in analyte response over time, and more instrument maintenance. Agi- combustible sample matrix. Various applications for real world samples like diesel lent Bond Elut enhanced matrix removal – lipid (EMR-Lipid) represents the next fuel, gasoline, petroleum products, and polymers are presented. generation of sample preparation technology; providing selective lipid removal for complex samples without analyte retention. EMR-Lipid is available in a convenient 417 Automated On-Line Extraction and Chromatography with dispersive solid-phase extraction (dSPE) format and is amenable to widely accept- Supercritical Fluids ed workflows such as QuEChERS (quick, easy, cheap, effective, rugged, and safe) William Hedgepeth, Shimadzu Scientific Instruments, 7100 Riverwood and protein precipitation. Data demonstrates the performance benefits achieved by Dr., Columbia, MD 21046, Ken Tanaka cleaner sample extracts using this new material in applications involving multi-class, A new analytical system was released this year which combines online automation multi-residue analysis for pesticides, veterinary drugs, and mycotoxins in complex, of sample preparation, separation, and analysis. The system uses supercritical car- high fat samples. Dramatic improvements are achieved for matrix removal, analyte bon dioxide for the extraction and analysis of target compounds from a variety of recovery, and reproducibility compared to currently available cleanup sorbents. The sample matrices. The system can provide reduced sample preparation times with high performance and selectivity of EMR-Lipid make it an attractive option for lab- less solvent waste, while improving the reproducibility of results. The poster de- oratories seeking to simplify sample preparation for fatty samples, while enhancing scribes the automated process of the system and present a number of applications analytical and instrumental integrity. from sample matrices such as food, polymers, and pharmaceuticals to show the utility of the system. 421 Application of a Unique Microwave Digestion Technology Coupled with ICP-MS for the Determination of Key Elements in Dietary 418 Automated Desorption, SPE Extraction, and LC-MS-MS Analysis of Supplements Dried Blood Spots Reynhardt Klopper, Anton Paar USA, 10215 Timber Ridge Dr., Ashland, Fredrick D. Foster, Gerstel, 701 Digital Dr., Ste. J, Linthicum, MD 21090, VA 23005, Paul Dodson John R. Stuff, Edward A. Pfannkoch Dietary supplements are regulated by the United State Food and Drug Administration The extraction of dried blood spots (DBS) typically involves manual intervention. (FDA) and can be found in various forms, including tablets and powders, consisting First, a small disc is punched out of the center of a dried blood spot card. Following of vitamins, minerals, herbs or botanicals. Under the Dietary Supplement Health and solvent extraction of the sample, it is also common to include further cleanup using Education Act of 1994 (DSHEA), dietary supplement manufacturers are responsible solid phase extraction to improve detection limits or exchange solvents for com- for ensuring the safety of products before market release. Elements such as Zn, Mg patibility with subsequent chromatographic separations. Modern analytical labs are and Se are added for nutritional purposes, and need to be determined for labeling looking to automation to help reduce solvent usage and increase sample throughput confirmation. The presence of certain toxic elements, e.g., Pb, Hg is highly regulated while ensuring the high quality of the resulting data. A single robotic X-Y-Z coordi- and may not exceed set concentration levels. A number of analytical techniques are nate autosampler commonly used for sample introduction in gas chromatography used for dietary supplement testing, the most common being inductively coupled (GC) or high-performance liquid chromatography (HPLC) can be used to perform a plasma mass spectrometry (ICP-MS) coupled with microwave sample digestion. wide variety of sample preparation techniques using a single instrument and con- In this study we illustrate how a new and unique microwave digestion technology trolling software. This sampler can also be configured as part of the liquid chro- allows for the efficient and fast processing of dietary supplements. The compact matography mass spectrometry (LC-MS-MS) system. In this report, the complete and lightweight Multiwave GO microwave system features the patent pending automation of dried blood spot analyses by the robotic autosampler is discussed. directed multimode cavity (DMC) technology, which enables focused application of Examination of a new, automated DBS analyzer that allows samples to be rapidly microwave energy to a compact cavity system, while allowing for the simultaneous and effectively desorbed, extracted, and injected into a LC-MS-MS system, is de- processing of up to 12 samples in 18 minutes, yielding clear solutions of multi-gram scribed. Automated DBS extraction methods for a variety of analytes from different samples. matrices are examined and resulting precision and accuracy data are provided.

60 2015 EAS Abstracts November 2015

422 Assuring Water Quality with Advanced Analytical Methods dotoxin. Depending on the size of the system, the number of sampling sites and the Sut Ahuja, Ahuja Consulting, 1061 Rutledge Ct., Calabash, NC 28467 sampling schedule, this testing can be very burdensome to the quality control labo- More than one billion people in the world today drink unclean water from various ratory, or if outsourced, can be extremely expensive, often running into thousands of sources. These sources are exposed to multiple contaminants including metals. dollars a week, money which can be well spent otherwise. In addition, the results of Arsenic contamination of groundwater has become a major problem worldwide, in- these tests are not readily available in order to allow a rapid response to excursions cluding the United States. Arsenicosis, resulting from drinking arsenic-contaminated in the system. The time frames may be days to weeks before the data are available. water, affects around 200 million people worldwide; it can lead to a protracted and In addition, total organic carbon analysis and conductivity are so sensitive that just excruciating death. This problem is most pronounced in Bangladesh and India. To sampling the system changes the results. These two particular methods lend them- follow up on the earlier initiatives in Dhaka, Paris, Atlanta, New Dehli, and Somer- selves to on line analysis. They are fully automated and the relevant USP chapters set, a workshop was recently held in India to find solutions to metals contamination were written with the understanding and expectation that they would also be used of water. This was followed by a symposium in the spring 2015 ACS meeting in for on line systems. This talk focuses on the background of these tests, as well as Denver. Discussion focuses on the contamination of groundwater by arsenic, the the implementation of these tests in a pharmaceutical grade water system. ultratrace analytical methods used for monitoring arsenic, and the best options for remediation. Solutions that offer significant improvements in water purification tech- 426 Understanding the Mechanism of Drinking Water Disinfection nologies, at reasonable costs, are highlighted. Organic By-Products: Analytical Technology Partners with Organic Chemistry 423 Novel Cation-exchange Phases for the Analysis of Alkali Metals Daniel Norwood, 24 Weantinock Dr., New Milford, CT 06776 and Alkaline Earth Cations in Drinking Water No abstract submitted by the author. Chris A. Pohl, Thermo Fisher Scientific, 1228 Titan Wy., Sunnyvale, CA 94085 427 In-Vitro In-Vivo Correlation in a QbD Environment Stationary phases from the earliest days of ion chromatography utilized poly- Raimar Loebenberg, University of Alberta – DDIC, 3-143K Katz Bldg., mer-based stationary phases incorporating sulfonic acid type cation-exchange Edmonton, AB T6G2E1, Canada sites. However, this type of stationary phase had a number of disadvantages includ- Drug development in a quality-by-design (QbD) environment requires to set product ing: poor chromatographic performance for aromatic solutes, incompatibility with target profiles and to identify critical product or process parameters. The quality of mobile phases containing organic solvent and very high affinity for divalent cations. a product is planned rather than tested into the finished product. To archive such The latter property made it impractical to elute and resolve both monovalent and goals computer simulations are increasingly used. This talk shows how computer divalent cations with acceptable resolution. In 1987 Professor Gerard Schomberg models can be used to predict drug plasma profiles and how this can be used to (Chromatographia 23 (1987) 465) demonstrated the utility of using carboxylic acid identify clinically relevant product specifications. One example is dextromethorphan, based weak acid cation exchange phases as an alternative to sulfonic acid station- this drug is a weak base with a high pKa above 7. Substances like that are subject ary phases. By utilizing a combination of low pH and chelating mobile phase addi- to lysosomal trapping which means that the drug gets fast absorbed but appears tives, elution with suitable resolution of common monovalent and divalent cations only slowly in the systemic circulation due to its prolonged trapping in lysosomes. of interest in ion chromatography was achieved for the first time. In the following Without accounting for this trapping period wrong dissolution specifications might years, weak acid cation-exchange materials of this sort came to be nearly exclu- be chosen to achieve an In-vitro in-vivo correlation (IVIVC). This talk explains how sively utilized for such applications. Weak acid cation-exchange has a number of the knowledge gained by computer simulations can be used to select appropriate disadvantages as well. First, the chromatographic performance carboxylic acid type performance specifications cation-exchange phases are inferior to what is achievable when using sulfonic acid type cation-exchange media. Second, with carboxylic acid type cation-exchange 428 Biorelevant Dissolution Measurements at the Drug Solubility Limit: phases it is difficult to achieve adequate resolution of sodium and ammonium ion Optimizing Exposure Rankings which is important for the analysis of trace ammonia in groundwater. In the present Paul Harmon, Merck, 770 Sumneytown Pike, West Point, PA 19486 work we describe a hybrid material that combines the best aspects of both types of A simple biorelevant dissolution methodology is described which maximizes the stationary phases. ability to make predictive human/animal area under the curve (AUC) rankings for formulated Biopharmaceutical Classification System (BCS) II/IV dosage forms. The 424 Recent Developments in Analyzing Ionic Components in Drinking critical aspect of the methodology is to have only as much (or less) formulated drug Water in the Fassif stage of the experiment that can actually be dissolved in the Fassif Kannan Srinivasan, Thermo Fisher Scientific, 1228 Titan Wy., medium. This insures the effective dissolution rate for the entire (formulated) drug Sunnyvale, CA 94088, Rong Lin, Herbert Wagner particle distribution is actually measured and compared. For low solubility drugs, Analysis of trace ions has been challenging in ion chromatography (IC), and is usu- this often means only a sub-portion of a single dosage form can be solubilized in ally accomplished by the introduction of a large volume of the sample either directly (for example) the typical 500 or 900 mls of fasted state simulated intestinal fluid onto the separator, or through a preconcentration step using a concentrator column. (FaSSIF). In this context, the SGF (non-absorptive phase) of the simple two-stage While the approach works well for samples containing trace ions, with samples con- experiment is introduced as a means to homogeneously disperse the disintegrated taining matrix ions the approach is challenging and can result in poor detection of tablet/dosage form, which allows for a representative portion to be removed and trace ions. In samples containing high concentration of matrix ions such as drinking diluted into an appropriate volume Fassif dissolution media to obtain the predictive and waste water samples, in the direct injection approach overloading effects can dissolution rate data. Dissolution rate calculations for dispersed drug particle distri- ruin the separation and detection of trace components. Similarly concentrating in butions (DDD+ software) are compared to actual drug particle dissolution data un- the presence of matrix ions can also concentrate the matrix ions and elute the trace der the method conditions and confirm the predicted sensitivity to drug particle size. components. In this presentation we review current methods in pursuing trace anal- The methodology is applied to three different formulations of Compound 1 (BCS IV) ysis in the presence of matrix ions. A matrix diversion approach called matrix elimi- and a suspension vs. tableted form of Compound 2. The relative human AUC data nation ion chromatography (MEIC) that allowed sensitive and selective detection of for these formulations is shown to compare favorably with the relative biorelevent trace components will be discussed. In this method a large loop injection was used dissolution rates obtained by the subject methodology. in the first dimension followed by analysis on a high capacity column. The matrix ions are diverted to waste while focusing the ions of interest onto a concentrator 429 Practical Approach for Developing Dissolution Methods to Support column. The concentrated ions are analyzed using a capillary column of a different Clinical Relevant Specifications selectivity in the second dimension. With a concentration sensitive detector such Jian-Hwa Han, Abbvie, 1 North Waukegan Rd., North Chicago, IL 60064, as conductivity excellent sensitivity was accomplished. Analysis of trace levels of Patrick Marroum, Greg Webster, Nikoletta Fotaki, Limin Zhang, Xujin Lu perchlorate, bromate and haloacetic acids with MEIC is shown here in the presence After many years of research on biorelevant dissolution methods, the industry is still matrix ions in reagent and drinking water samples. unclear on how to develop suitable in-vitro drug release/dissolution testing proce- dures which can provide good discriminatory power for setting up clinically relevant 425 Qualification of On-Line Total Organic Carbon and Conductivity specifications. Some dissolution procedures utilize specialized chemicals/materials Monitoring for a High Purity Pharmaceutical Water System (i.e., biorelevant dissolution media), unique mechanical design for different hydro- Chris Knutsen, Bristol-Myers Squibb, 1 Squibb Dr., New Brunswick, NJ dynamics and/or mass (liquid/solid) transport to better match human physiological 08901 conditions. All of these are valuable tools for predicting the drug product perfor- Pharmaceutical grade water systems must be continually monitored in order to mance during the development stages; however, the most ideal scenario is to have monitor the functioning of the system as well as to assure compliance to the relevant a quality control-friendly dissolution method that is able to reject the bad samples compendial monographs. Many of these systems are closed loops. The systems which may fail clinical end points (e.g., not bioequivalent). The method development are typically monitored for at least conductivity, total organic carbon and microbial efforts need to be started as early as possible with good collaborations among ana- bioburden. In addition, some water systems will require monitoring for bacterial en- lytical/formulation/ pharmacokinetic groups. Fully understanding the product design 61 2015 EAS Abstracts November 2015

and in-vivo performance are critical attributes for dissolution method development. growing area in many pharmaceutical companies. However, releasing and charac- The critical quality attributes (CQAs) including critical material attributes (CMAs) terizing such molecule requires a number of analytical methods. Even for primary and critical process parameters (CPPs) need to be identified to make some atypical structure characterization, a number of different high-performance liquid chromatog- samples for in-vitro release/dissolution method development as well as in-vivo clin- raphy (HPLC) techniques such as ion exchange, size exclusion, reverse phase and ical assessment. Clinically relevant specifications may not be achieved for all drug hydrophobic interaction are involved. Unfortunately, each technique can only pro- products, and Biopharmaceutics Classification System (BCS) classifications may vide limited information based its own separation mechanism. Two-dimensional-LC guide us through the considerations. A high-level strategy for developing dissolution provides the possibility to combine two orthogonal methods and discover the cor- methods from first in human to Phase 3 to establish clinical relevant specifications relation of the data. This correlation provides critical information for characterization. is discussed. 434 Developing Robust Size-Exclusion Chromatography Methods for 430 Biorelevant Dissolution Media and Applications in Pharmaceutical the Analysis of Biotherapeutic Proteins Development - Case Studies Stephan M. Koza, Waters, 5 Technology Dr., Milford, MA 01757 Xujin Lu, Bristol-Myers Squibb, 1 Squibb Dr., New Brunswick, NJ 08903 Size-exclusion chromatography (SEC) is the predominant method of choice for In-vitro dissolution testing is a valuable tool for formulation development and for the analysis of biotherapeutic protein aggregation due to the short analysis times, quality assessment of finished products. Biorelevant in-vitro dissolution can help general simplicity and reliability in the routine performance of the analysis, and the predict the in-vivo performance of the dosage forms, qualitatively forecast pharma- quantitative precision of this method. However, in order to get reliable and repro- cokinetics and bioavailability of drugs, and provide useful information for formulation ducible results appropriate steps must be taken to assure that ionic and hydro- design and optimization. Biological fluids or simulated biological fluids have been phobic interactions between the analyte proteins and the SEC particle surface are considered as the media for biorelevant dissolution because they can mimic the minimized to the greatest extent possible, and that the chromatographic selectivity conditions at the site where most of the drug absorption occurs. This provides a and efficiency of the separation as they relate to pore size and effective column better understanding of the release mechanisms and possible in-vivo behavior of dimensions are appropriate for the intended assay. Finally, it is of critical importance a drug product, thus enhancing the predictive capability of the dissolution testing. that the distribution of proteins and their aggregates as determined by SEC be ver- This presentation describes several case studies using simulated biological flu- ified by complementary methods. In this presentation considerations that should be ids as the media for in-vitro dissolution testing: 1) Use of plasma as a medium for made with respect to developing and characterizing reliable analytical SEC methods the dissolution of a drug in micro suspension intravenous injection formulation to are discussed. Additionally, the transfer of SEC methods between high-performance demonstrate the solubility kinetics and the availability of the drug through fast in vivo liquid chromatography (HPLC) and ultra-HPLC platforms are covered along with drug release; 2) Using fasted state simulated intestinal fluid (FaSSIF) and fed state practical advice on achieving reliable day-to-day SEC method performance in the simulated intestinal fluid (FeSSIF) for prediction of food effects in development of laboratory. a lipid tablet formulation; 3) Application of simulated saliva for in-vitro screening of taste masking efficiency of polymer coated active pharmaceutical ingredient (API) 435 A Fresh Look at the Derivative Quotient Method in Regression in pediatric formulation development. The advantages and benefits of using simu- David W. Hopkins, NIR Consultant, 472 S. Moorland Dr., Battle Creek, lated biological fluids for the biorelevant in-vitro dissolution testing in support of the MI 49015, Karl H. Norris development of a variety of formulations are discussed. The original work of Karl Norris and William Hruschka in applying multi-term linear regression on derivative quotients has been reviewed using new software devel- 431 Determinants of Elution Rates in Preparative Ion-Exchange oped using Matlab. The method starts with full spectra for a set of samples used Separations of Proteins for calibration, and results in one or more terms in a prediction equation where the Abraham M. Lenhoff, University of Delaware, Dept. of Chemical and numerators and denominators of each term are the values of derivatives at select- Biomolecular Engineering, Newark, DE 19716, James M. Angelo ed wavelengths. The derivatives are calculated by the Gap method pioneered by Although the loading properties of chromatographic adsorbents are extensive- Norris, and each derivative is optimized for the gap, center wavelength, and width ly quantified in terms of such measures as the dynamic binding capacity, elution of ‘boxcar’ smoothing. We have extended the original work by utilizing derivatives characteristics, which are essential for resolving different species, have been only up to fourth order. The numerator and denominator derivatives do not have to be of sparsely investigated systematically. We have characterized protein elution from ion the same order. Principles of developing calibration models and validating them are exchangers under the hypothesis that the rate-limiting step is simple pore diffusion presented, using near-infrared (NIR) spectroscopy to measure protein in wheat in of the desorbed protein out of individual resin beads. Although the hypothesis is samples that exhibit a high degree of spectral variation due to particle size variabil- supported by data in some systems, including a monoclonal antibody, other systems ity. Consisting of just one to two terms, the models are very easy to implement. We show much slower elution, for which we have sought mechanistic explanations. We believe that coupling the models with current chemometric methods for qualifying find that the physicochemical properties of individual proteins can have an appre- samples for analysis provides a powerful new technique for routine analysis of sam- ciable effect on elution, including via oligomerization and phase separation. Key ples. This regression method appears to be capable of achieving greater accuracy factors determining the behavior found are the effective porosity and the adsorption than can be obtained utilizing the standard methods of pretreating the spectra and capacity of the material, with low porosities, such as those encountered in poly- regression using polymerase chain reaction (PCR) or partial least squares (PLS), mer-derivatized phases, especially important in causing slow elution. We show that possibly because multiplicative spectral effects are compensated by the quotient judicious manipulation of solvent properties can result in dramatic improvements in terms, while the chemical differences are calibrated by the multiple regression. elution behavior. 436 A Novel Configuration for Near-Infrared Analysis of LPG 432 LC of Therapeutic Monoclonal Antibodies Using Submicrometer Composition and Quality Control in a Refinery Setting Particles Susan Foulk, Guided Wave, 3033 Gold Canal Dr., Rancho Cordova, CA Mary Wirth, Purdue University, 560 Oval Dr., West Lafayette, IN 47907, 95670, Shashi Mistry, Terry Todd, Nate Peters, Dian Wang Xiang Cao, Oyeleye Alabi, Ao Zeng Near-infrared (NIR) spectroscopic process analyzers are commonly used in refiner- Therapeutic monoclonal antibodies is a rapid growth sector in the pharmaceutical ies for a variety of composition and physical property measurements such as octane industry due to their high specificity and low toxicity. Compared to small-molecule number, aromatic content, and distillation properties of feed and finished products. drugs, these present more challenges in characterization due to the many possible Despite wide use, there are few instances of using NIR spectroscopy to measure protein variants and differences in glycosylation, as well as antibody fragmentation properties of LPG (liquefied petroleum gas). The composition analysis of LPG is typ- and aggregation. Chromatography is an indispensable tool in the characterization ically carried out by gas chromatography (GC). In this work, a near-infrared analyzer of drug purity, but proteins have always have always been a challenge in chroma- was installed on the Hydrocracker fractionation unit (HCU) on following columns: 1) tography due to their slow mass transport and strong interactions with surfaces. We Main fractionator (Naphtha), 2) Main fractionator (Light distillate), 3) Main fraction- discuss packing with submicrometer silica particles, chemical modification of the ator (Heavy distillate), 4) Depropanizer bottoms (Butane analysis), 5) Deethanizer silica, and hardware and instrumental contributions to peak broadening to maximize bottoms (Propane analysis) The Suncor Edmonton refinery had installed distillation resolution of representative IgG1, IgG2 and IgG4 samples by reverse-phase liquid analyzers on the first three streams above and GCs on streams 4 and 5 above. Due chromatography, hydrophilic interaction liquid chromatography, and ion chromatog- to the need to improve quality, uptime and to reduce maintenance costs, Suncor raphy. shifted to NIR technology for these measurements. The LPG analyses must work for entire operating range of the HCU as well as assist in liquid and vaporization modes. 433 Bio-Chemical Characterization of Therapeutic Protein by Two- This paper describes the installation and experimental work involved and provide a Dimensional-LC (2-D-LC) System discussion of the LPG NIR data and results. An HCU process unit-wide multi-vari- Shenjiang Yu, Merck, 2000 Galloping Hill Rd., Kenilworth, NJ 07033, able controller is incorporated for quality control using five NIR analyzers. Suncor Chen Zhi, Daisy Richardson, Mohammed Shammen intends to economically optimize the Hydrocracking unit using properties such as Therapeutic protein, including monoclonal antibody (mAb), is one of the most rapidly 90%, viscosity, cloud point, and flash point as needs and seasons demand. The 62 2015 EAS Abstracts November 2015

LPG stream properties are required to control the purity of both Butane and Propane in sensitivity are achieved by Duo LASER excitation, which allows the measurement for the export market as well as to stabilize the process unit. NIR allows Suncor to of Raman spectra within a smaller range of wavelengths. This technique results in achieve speed of quality control at a much reduced cost over the long term. the highest sensitivity across a large spectral range of 300 cm-1 to 3200 cm-1. Thus, weak Raman signals are considered in the verification algorithms and accomplish 437 Ultra-Compact Smart Spectrometers for Food, Agriculture, and maximum unambiguous verification of materials. Pharmaceutical Applications Nada O’Brien, JDS Uniphase, 2789 Northpoint Pkwy., Santa Rosa, CA 441 Drug and Other Finished Product Identification with Handheld 95407 Raman Recent innovations in optical designs have led to the realization of ultra-miniature Claire Dentinger, Rigaku Analytical Devices, 30 Upton Dr., Ste. 2, spectrometers with excellent performance and low-power consumption enabling a Wilmington, MA 01887, Joseph Stoltz, Thomas MacNeil new class of handheld near-infrared (NIR) devices that are controlled by smart mo- Handheld Raman analyzers are very well suited for material identification outside bile devices. The benefits of such devices are numerous: lower cost, much smaller the analytical laboratory. Due to their portability, ease of use and material specific- form factor and ease of model updates across a wired or wireless network, thus ity, handheld Raman instruments are well accepted in the pharmaceutical industry resulting in greater scalability and wider deployment. Another benefit is the ability to for raw material identification (RMID) of excipients and active pharmaceutical in- interconnect and network multiple such devices for a given application which allow gredients (API). The feasibility studies described here investigate applications be- for smart, real-time monitoring, and continual optimization of the analysis. In this yond RMID that can benefit from the speed, portability and reduced fluorescence presentation, we talk about the technology behind a miniature NIR spectrometer interference of a 1064 nm handheld Raman. First a feasibility study using a 1064 that weighs 3 ounces and is less than 2” in diameter. The spectrometer covers nm handheld Raman analyzer to investigate different finished drug products (DP) the wavelength ranges of 950-1650nm in one configuration and 1150-2150nm in formulations is discussed. This looks at distinguishing the DP from corresponding another. We discuss specific fit-for-purpose applications of a handheld version of placebos and the ability of the dose of the API to be determined with the handheld the spectrometer that is tethered to a small tablet computer for non-destructive on- Raman. Secondly, we show some examples of using handheld Raman for detection farm nutrient content analysis of animal feed and show results of detecting fraud of counterfeit pharmaceuticals and other commercial finished products. in adulterated chicken filets. Other application examples in the pharmaceutical in- dustry such as on-line analysis of active ingredient concentration on a tablet-press 442 Continuous Gradient Temperature Raman Spectroscopy (GTRS) of machine and end-point monitoring on a blending process are presented. The spec- Oleic and Linoleic Acids from 100 to +50 °C trometer used on the rotating blender is battery-powered and relies on wireless Walter F. Schmidt, United States Drug Administration, USDA-ARS- communication for instrument control and data transfer. We therefore show how EMFSL, 10300 Baltimore Ave., Beltsville, MD 20705, Leigh Broadhurst, mobility, affordability, and simplicity of these smart spectrometers are democratizing Moon S. Kim, Julie K. Nguyen, Jianwei Qin, Kuanglin Chao, Gary L. the NIR spectroscopy field for a wide range of applications. Bauchan, Daniel R. Shelton The new GTRS technique identifies Raman frequency shifts in solid or liquid sam- 438 Derivatives: What Are We Actually Doing? ples, and correlates them with specific temperature ranges within which flexible James A. de Haseth, Light Light Solutions Instruments, 165 Sunnybrook structures absorb heat. GTRS can easily detect changes that occur within fractions Dr., Athens, GA 30605, Franklin E. Barton II of 1°C/min intervals. GTRS provides rapid and straightforward identification of mo- Derivatization is often used as a form of deconvolution for the enhancement or lecular rearrangements that occur just prior to or at phase transitions. We analyzed elucidation of spectral information. There are many different ways to accomplish the unsaturated fatty acids oleic (OA, 18:1n-9) and linoleic (LA, 18:2n-3) from -100 derivatization and this presentation discusses the differences between the various to 50 °C. 20 Mb three-dimensional data arrays with 0.2 °C increments allowed com- algorithms. These differences are not simply the algorithmic approaches but more plete assignment of solid, liquid and transition state vibrational modes. For OA, large importantly relate to the effects on the spectrum. Some algorithms introduce distor- spectral and line width changes occurred in the solid-state to transition near -4 ºC, tions more than others such as spectral shifts and artifacts. Of course derivatization and the melt (13 ºC) over a range of only 1 °C. Below -7 ºC, the methyl rocking vi- does not simply deconvolve spectral peaks but also deconvolves the noise which brational mode at 895 cm-1 is strong; from -7 ºC to 13 ºC, intensity increases to very invariably leads to a loss in signal-to-noise ratio of the spectrum. In an effort to re- strong; above 13 ºC, intensity falls precipitously and a new, very sharp frequency capture the original signal-to-noise ratio spectral smoothing frequently accompanies arises at 858 cm-1. For LA, major intensity reductions from 200-1750 cm-1, large derivatization. The smoothing algorithms are invariably convolution functions and spectral and line width changes occurred at the melt transition (-7 °C). To -7 ºC, CH2 hence counteract some of the benefits of derivatives. The convolution functions twisting occurs primarily on the carbonyl sided chain: HOOC(CH2)7- and extends also can add spectral errors. Examples of different algorithms and their effects are into CH wagging op at C10. Above -7 ºC, concurrently CH2 rocking at C11 becomes shown with model and experimental spectra. markedly stronger, C=CH rocking ip at C10 and C12 intensifies, and C=CH wagging op at C9 and C13 becomes undifferentiated. LA melting initiates at the diene struc- 439 Noninvasive Glucose Sensing with Miniaturized Integrating Sphere ture, then progresses towards the ends of the molecule. Alexandra Werth, Princeton University, 41 Olden St., Princeton, NJ 08544, Sabbir Laikat, Laura Xu, Kevin A. Bors, Claire Gmachl 443 Through-Container Raw Materials ID Verification Using Spatially Diabetes is a disease which affects over 387 million people worldwide. A portable Offset Raman Spectroscopy (SORS) and accurate noninvasive in-vivo glucose sensor can significantly improve the qual- Matthew Bloomfield, Cobalt Light Systems, 11951 Freedom Dr., Reston, ity of life for many diabetics who draw blood multiple times a day to monitor their VA 20190, Darren Andrews, Pavel Matousek glucose levels. We have implemented a noninvasive, mobile glucose sensor using Growing regulatory pressure and the wider adoption of PIC’s globally is driving the a mid-infrared quantum cascade laser, integrating sphere, and TE-cooled detector. pharmaceutical industry towards 100% container inspection of incoming goods. Recently, using this sensor and partial least squares regression (PLSR) prediction Meeting this demand for increased identity verification has motivated the adoption analysis on human subjects we have achieved accuracies over 80%. The integrat- of vibrational spectroscopy, due to its speed and ease of use. Raman spectrosco- ing sphere has increased the spectral stability from a previous design, allowing us to py has overtaken near-infrared (NIR) due to its increased specificity, simpler mod- use the TE-cooled detector to increase mobility without loss of accuracy. We believe el-building and faster measurement time. these results can be further improved with a custom built, miniaturized integrating sphere which would increase the mobility of the system and sphere throughput. 444 Transmission Raman Spectroscopy: An Alternative Technique for Content Uniformity and Polymorph Quantification of Intact Tablets 440 The Next Generation Handheld Raman Spectrometer: BRAVO for and Capsules Raw Materials Verification Mark Mabry, Cobalt Light Systems, 11951 Freedom Dr., Reston, VA Yan Wang, Bruker, 19 Fortune Dr., Billerica, MA 01821, Tom Tague 20190, Matthew Bloomfield, Julia Griffen, Darren Andrews The recently released Bruker Handheld Raman Spectrometer (BRAVO) with unique Spectroscopic techniques, such as Raman spectroscopy, have a lot to offer in the optical performance (fluorescence mitigation) are designed for efficient and reliable quality control (QC) laboratory for quantitative analysis of pharmaceutical samples. verification of incoming materials, monitor of key manufacturing processes and in- Raman is non-destructive, chemically-specific, faster than high-performance liquid spection of finished products. The common problem of fluorescence with Raman chromatography (HPLC) and near-infrared (NIR) and needs no consumable items. measurement is managed by the patented sequentially shifted excitation (SSE) In conventional back-scattering geometry, quantitative Raman measurements are technique. Raman spectra are acquired at SSE energies using temperature tuned limited by sampling from the front surface, causing sub-sampling errors or problems diode lasers. The applied SSE algorithm takes advantage of the fact that Raman due to coatings. By operating in a transmission configuration, transmission Raman signals exhibit a spectral shift as a function of excitation energy whereas the fluo- spectroscopy (TRS) allows the whole sample to be measured, making it a truly vol- rescence remains constant. The generated Raman spectra are free of fluorescence umetric analysis technique for intact tablets, capsules and other solid dosage forms. and feature a high signal to noise ratio. This allows the identification of a much wider An important application of this relatively new technique is content uniformity (CU), range of raw materials using a handheld Raman system than ever before. Advances for which HPLC is the industry standard. In contrast, TRS requires no expensive 63 2015 EAS Abstracts November 2015

consumables and relatively simple model-building, with limit of quantitation (LOQ) technique and share how this technique was applied to microscopically analyze of <1% in many intact tablets. In routine, batch testing mode, measurement times multiple pharmaceutical dosage forms. RTI can non-destructively generate data and are typically seconds per sample with exceptional reproducibility. Another valuable can be used with existing laboratory equipment (stereomicroscope and microscope use is in quantifying amounts of polymorphs in intact samples. The sensitivity of camera). This technique can potentially be used to identify counterfeit drug prod- Raman scattering to crystal structure, particularly in the low energy phonon mode ucts, check the film coating of a tablet, and provide improved visualization of device region (<200cm-1) makes TRS ideal for measuring residual crystallinity or recrystal- components. lization of active pharmaceutical ingredient in amorphous form. TRS can be used in formulations with limits of detection an order of magnitude better than powder X-ray 448 Ca, Mg, K and Na Determination in Plant Leaves of Arabidopsis diffraction (PXRD), the current standard approach, and comparable with ssNMR. Thaliana Wild Type and a Chloroplast K+ Transport Loss-of- Additionally, on-line CU testing for real time release testing and continuous manu- Function Mutant facturing is realizable with a fully automated TRS tablet testing solution. The prac- Samaneh Tabatabaei, Washington State University, Dept. of Chemistry, tical issues of tablet presentation and positioning are less likely to impact results. 100 Dairy Rd., Pullman, WA 99164, Ricarda Hoehner, Henning Kunz, Ursula Fittschen 445 Quantitative Determination of Cannabinoids in Cannabis Plant Ions play an important role in signal processing and catalysis in biological systems. Material Using High Performance Liquid Chromatography - UV Ion transport across cell membranes is facilitated by a suite of membrane proteins. Diode Array-Mass Spectrometry (Trap) Detector When ions accumulate on one side of the membrane, ion and charge gradients Bhupendra R. Patel, New Jersey Dept. of Health, Public Health & are established that represent potential energy which can be used to drive other Environmental Laboratory, 3 Schawrzkopf Dr., West Trenton, NJ 08628, transport processes or biochemical reactions. Specific membrane proteins are also Daniel J. Wene, Sherman S. Hom, Bahman Parsa used to sequester toxic metals into specialized cellular storage organelles. Metal As of June 2015, twenty three states in the United States have legalized the use ion accumulation occurs at many levels in biological systems from the sub-cellular of Cannabis for medical purposes. The New Jersey Medical Marijuana Program and cellular levels, to the tissue, organ and organism level. We investigated the (NJMMP) was established in 2011. One of the objectives of the NJMMP was to impact of ion accumulation on physiological processes in plant leaves Arabidopsis provide high quality safe Cannabis plant material to qualified patients. An efficient, thaliana. Arabidopsis is an excellent model organism due to its short generation cost effective, and defensible test method for the analysis of cannabinoids with a time (5-6 weeks), a plethora of genetic tools and more importantly, an extensive short sample testing turnaround time was needed to ensure compliance to the NJ loss-of-function or gain-of-function mutant collection. kea1kea2 mutants lacking two Medical Marijuana regulation that any finished medical Cannabis dispensed prod- crucial chloroplast K+ transport proteins were generated. Compared to wild type, uct would have no more than 10% THC. A rapid, high resolution, reversed-phase the younger tissues in these mutants show dramatic reduction in photosynthesis. high-performance liquid chromatography (HPLC) method using a core shell column Micro X-ray fluorescence (µXRF) imaging can reveal the spatial distribution of ele- was developed to determine the potency and profile of cannabinoids in Cannabis ments in the leaves. Using total reflection X-ray fluorescence (TXRF) we quantified plant material. The Cannabis plant material was ground and then extracted with a specific ions contents in plant leaves from different genotypes to test if this phenom- methanol:chloroform mixture. An aliquot of the extract was injected into a HPLC and enon is attributed to a special ion accumulation. Ca, K, Mg and Na were determined the cannabinoids were separated by a reversed-phase core shell C18 column with using TXRF and atomic absorption spectroscopy. Preliminary results showed that mobile phase of methanol and 25 mM aqueous ammonium acetate using a gradient Ca concentration increases with age of the leave while K decreases for wild type program. A diode array UV detector was used for presumptive identification and and mutant. However, this process seems to be significantly more pronounced in quantitation of cannabinoids. Confirmatory identification of the different cannabi- the mutant. noids was performed using a mass spectrometry (MS) detector (Trap). Validation data for both external and internal standard methods and the summary of the results 449 Three-Input-Three-Output (3i3o) Continuous Flow Microfluidic of approximately fifty cultivars of Cannabis plant material submitted by the New Devices as Platforms for Biomineralization Studies Jersey Alternative Treatment Centers are presented. George A. Kumi, Rutgers University-Camden, Dept. of Chemistry, Science Building, 315 Penn St., Camden, NJ 08021 446 Potential Antimicrobial Compounds from Campsis Radicans, There are numerous attributes that make microfluidic devices particularly attractive Bignoniacea for crystal synthesis, such as the small sample size requirement and the efficient Ramya Ayakkad RamKumar, Fairleigh Dickinson University, 1000 River heat transfer traits. In spite of their touted simplicity, single-phase (i.e., ‘non-droplet Rd., Teaneck NJ 07666, Ish Kumar, Alice Benzecry based’) systems are generally avoided because of the potential for crystal adhesion The family Bignoniaceae includes about 120 genera well known for their exten- to the channel walls and the device clogging that can ensue from this adhesion. In sive chemical reservoirs, which includes napthaquinones, iridoids and secondary actuality, these adverse traits can be mitigated or eliminated by the design of appro- metabolites such as alkaloids, glycosides, terpenoids, flavonoids, reducing sugars, priate microfluidic systems. As a demonstration of this strategy, we have developed carbohydrates, quercetin and steroids. Extraction and purification of isolated com- a non-droplet-based device that affords control of where favorable crystallization pounds have been achieved by the usage of column chromatography, thin layer conditions occur within a device, thereby reducing some of the aforementioned chromatography and high-pressure liquid chromatography (HPLC). Up to date, we problematic issues. Using calcium oxalate as a model system in these proof-of-con- have isolated three antimicrobial active compounds. Each compound has been pu- cept studies and employing a range of microscopic methods: optical, infrared, con- rified into several peaks. Compound A contained seven pure peaks, compound B focal Raman, and electron, for crystal characterization, we have also gleaned new contained 4 pure peaks and compound C also contained 4 peaks. Each peak was insights into calcium oxalate crystallization. For example, we have obtained further collected and freeze dried. Compound C peaks (C1, C2, C3, C4) have shown an- supporting experimental evidence detailing how the ratio of calcium ions to oxalate timicrobial activity against Staphylococcus aureus, Bacillus subtilis and Rhizopus ions during calcium oxalate formation influences the shape and size of the crystals nigrican. Compounds A and B peaks are also being tested against gram positive, formed. The rationale for our design and results demonstrating the capabilities of gram negative bacteria as well as fungi. Next each active pure compound will be our device with respect to calcium oxalate crystallization is discussed. In addition, fully identified. design improvements for the next generation of such devices are presented.

445 Applying an Artifact Conservation Imaging Technique to the 450 Morphologically Directed Raman Spectroscopic Analysis of Microscopic Analysis of Pharmaceutical Products Forensic Samples Jennifer A. Sandidge, Merck, 126 E. Lincoln Ave., Rahway, NJ 07065, Brooke W. Kammrath, University of New Haven, 300 Boston Post Rd., Josephine L. Bermudez, Jason D. Ehrick Forensic Science Dept., West Haven, CT 06516, Andrew Koutrakos, Reflectance transformation imaging (RTI), an imaging technique used in museum Josemar Castillo, Joe Wolfgang, Deborah Huck-Jones artifact conservation studies, was utilized for the first time with pharmaceutical prod- Morphologically directed raman spectroscopy (MDRS) is a novel and reliable tool ucts. This proof of concept experiment sought to apply the same principles used that would enable criminalists to obtain more information from forensic samples than to examine large artifacts to examine small pharmaceutical products (500 µm and is currently employed for investigations and adjudications. MDRS combines auto- greater in size) including: powder agglomerate formulations, micro particles and a mated particle imaging and Raman spectroscopy into one instrument. Particle im- tablet at a microscopic level. To generate the RTI, multiple images of each pharma- aging is performed to determine particle size and shape distributions of components ceutical product were captured as the camera and subject remained stationary and in a blended sample. Particle size is an important physical property of particulate the position of the light changed. The images were processed through a mathemat- samples and can be used in conjunction with Raman spectroscopy in the general ical model resulting in the ability to virtually re-light and mathematically enhance the unknown analysis, in addition to the detection of drugs, the mineral content of soils surface of the product. Mathematical models or rendering modes including diffuse and gunshot residue analysis. Although measurement of particle size distributions is gain, specular enhancement, and image un-sharp masking can enhance the per- routinely carried out across a wide range of industries and is often a critical parame- ception of shapes on the surface, adjust the specularity and color data, or increase ter in the manufacture and analysis of many products and substances, it is not wide- the contrast exhibited respectively. This presentation provides an overview of this ly used in the forensic sciences. Raman spectroscopy is used in forensic science to 64 2015 EAS Abstracts November 2015

determine the molecular chemistry of materials because it is rapid, reliable, allows it: precision, accuracy, limit of detection, limit of quantitation and reproducibility is analysis without contacting the sample, is non-destructive, and enables detection at discussed. These techniques can be applied to determine NSAIDs in real samples low concentrations. Combining these two analytical techniques allows the individual such as natural waters, urine or blood. components present within a blend or mixture to be independently characterized and compared. This presentation demonstrates how such a tool can be used to gain 454 Passive Monitoring: A Guide to Sorbent Tube Sampling for EPA a better understanding of mixtures across many areas of forensic science, as it is Method 325 applicable to a range of Raman active samples. Nicola Watson, Markes International, Ste. 130, 2355 Gold Meadow Wy., Gold River, CA 95670, Caroline Widdowson, Charles Haws, Chris Hall 451 Forensic Analyses Using Infrared Spectroscopic Imaging The United States Environmental Protection Agency (US EPA) has proposed new Adam C. Lanzarotta, United States Food and Drug Administration, legislation for US petroleum refineries to control emissions from storage tanks, Forensic Chemistry Center, 6751 Steger Dr., Cincinnati, OH 45237 flares and coking units. When fully implemented, the rule will result in an estimated Macroscopic infrared spectroscopic detection methods are generally fast, require reduction of 5600 tons per year of toxic air pollutants and 52,000 tons per year minimal sample preparation, can be conducted on a wide variety of sample types, of volatile organic compounds (VOCs), improving air quality and protecting public are useful for identifying both active ingredients and excipients, and are able to health for workers and surrounding communities. Integral to the new law will be the differentiate polymorphs as well as salt forms. However, with regard to analyte iden- requirement to monitor air concentrations of benzene at the perimeter fence-line. tification, these advantages are typically only realized for detecting pure compounds Diffusive monitoring has been widely used in a range of air monitoring scenarios, or analytes present above 1% in a non-interfering matrix. Multicomponent samples i.e., occupational hygiene, as well as indoor air and ambient air monitoring. By elim- can be manually separated and individual particles can be identified using Fourier inating the requirement for a sampling pump, diffusive monitoring provides a simple transform infrared microspectroscopy, but this approach is often time consuming, and cost-effective method of collecting the large number of samples required in requires a skilled analyst and can be ineffective when the sample contains small many air monitoring programs. Key applications include personal exposure monitor- particles. In many cases a more efficient and effective approach includes infrared ing, large-scale environmental studies, and indoor air monitoring. Widespread use spectroscopic imaging, which uses a multi-channel detector to collect an infrared over many years has resulted in over 100 published uptake rates (e.g., ISO-16017 spectrum at each spatial location in a two-dimensional region of interest. The advent part 2). This simplifies matters for new users, as often uptake rates are available of infrared spectroscopic imaging turned the most significant disadvantage of macro without the need to determine them experimentally. This paper discusses the ap- infrared spectroscopy, the ability to identify individual ingredients in a multicompo- plication of passive sampling with industry-standard sorbent tubes and factors that nent sample, into an advantage. In addition to the benefits enjoyed by all infrared need to be taken in to account when deploying them. techniques, many analytes can be detected in the presence of each other and low and high concentration analytes can often be detected in a single measurement, 455 Using Direct Mercury Analysis for Mercury Speciation in Marine all without having to modify solvents, concentrations, flow rates, etc., such as with Environmental Samples chromatographic and mass spectrometric detection methods. In fact, a single set of Mike Lindenmuth, Milestone, 25 Controls Dr., Shelton, CT 06484 parameters is often suitable for most samples. This presentation discusses some Inorganic mercury at low concentrations does not cause significant health risks. of the more interesting applications for the analysis of compromised United States However, its conversion to organic methyl mercury by anaerobic bacteria seen com- Food & Drug Administration regulated products. monly in marine environments, makes it extremely toxic. Methyl mercury enters the food chain through the absorption of phytoplankton and its concentration increases 452 Quantification of Morphine from SR Pharmaceuticals in Gastric as we go higher in the aquatic food chain. Consumption of fish having high concen- Fluid via GCMS trations of methyl mercury can cause health issues including chromosomal damage, Ulrich Englich, Syracuse University, Dept. of Chemistry, 1-014 Center reproductive damage, skin reactions etc. Hence, monitoring methyl mercury levels for Science and Technology, Syracuse, NY 13244, Tatiana Del-Solar, in fish has gained significant interest. Many labs interested in analyzing mercury in Michael Hodgman, Michael Holland, Susan M Wojcik, William D. Grant, fish are moving towards the direct mercury analysis technique, as it offers significant Claudia Koraimann, Erich Leitner cost and time savings by eliminating sample preparation, need for digestion equip- Sustained release (SR) morphine pharmaceutical are narcotic pain relievers that ment and waste disposal. However, the direct mercury analyzers available today are are used to manage severe pain in an around the clock manner. Often these medi- designed to analyze total mercury and not methyl mercury. In recent years, methods cations are given orally. Examples include Avinza (King Pharmaceuticals) or Kadian have been developed that allow chemists to extract the organic methyl mercury from (Alpharma, Inc.) Accelerated release of morphine and consequently delayed toxicity an environmental sample and analyze an aliquot of the extracted organic phase, from the formulation can occur if SR pharmaceuticals are taken in connection with which is further analyzed using a direct mercury analyzer. This presentation covers: other medication or drugs of abuse. When patients with overdose symptoms arrive 1) introduction to direct mercury analysis and its advantages over conventional tech- in the emergency room polyethylene glycol electrolyte lavage solution (PEG-ELS) is niques; 2) concept and operating principle of direct mercury analyzers; 3) methods administered to purge the bowel. In a collaborative effort with the Upstate New York to analyze methyl mercury using a direct mercury analyzer; 4) data analysis. Poison Center we have been developing a gas chromatography mass spectrometry (GC-MS) based quantitative analytical protocol to determine the amount of mor- 456 X-Ray Fluorescence Elemental Imaging and Speciation of Li-Ion phine released by 3 different SR pharmaceuticals in simulated gastric fluid. The goal Cell Electrodes and Used for Diagnostics in Total Reflection X-Ray of this study was three-fold: 1) to establish a model for the study of orally adminis- Fluorescence tered SR pharmaceuticals, 2) to study the effect of alcohol as one of the common Ursula E.A. Fittschen, Washington State University, Dept. of Chemistry, co-ingested substances of abuse on the release of morphine, and 3) to determine if 100 Dairy Rd., Pullman, WA 99164, Zachary P. Gotlib, Owen K. Neill, PEG-ELS affects the dissolution rate of SR morphine products. Ulrike Boesenberg, Jürgen Janek, Mareike Falk, Magnus Menzel, Martin Radtke, Uwe Reinholz, Stanisław Nowak, Christina Streli, 453 Analysis of NSAID Residues in Aqueous Samples by SPME Kathryn McKintosh, George Havrilla Coupled to GCxGC-TOF-MS and GC-MS-MS Over the past two decades, X-ray fluorescence (XRF) has experienced a remark- Anumeha P. Muthal, Seton Hall University, Dept. of Chemistry and able evolution in instrumental development including high-performance optics and Biochemistry, 400 South Orange Ave., South Orange, NJ 07079, low-power instrumentation. Research is ongoing in areas such as trace elemental Nicholas H. Snow micro-analysis and two-dimensional (2D) and three-dimensional (3D) X-ray mapping Recently, the residues of non-steroidal anti-inflammatory drugs (NSAIDs) are stud- using micro X-ray fluorescence (MXRF), as well as in synchrotron radiation-based ied as emerging pollutants in water which enter the environment from manufac- MXRF and micro X-ray absorption near edge structure analysis (XANES). Using turing, improper disposal and through human and animal excretion. Multi-dimen- micro-X-ray fluorescence and micro-XANES, changes in elemental distribution and sional techniques such as two-dimensional gas chromatography coupled to time redox species correlated to the aging process of Li-ion battery electrodes were stud- of flight mass spectrometry (GCxGC-TOF-MS) and gas chromatography coupled ied. We found aging of electrodes correlates to inhomogeneities in elemental distri- to triple quadrupole mass spectrometry (GC-MS-MS) have become techniques of bution and state of charge [1]. Total reflection X-ray fluorescence analysis (TXRF) is choice for trace analysis because of their high sensitivity and selectivity. In this work, a micro analytical tool allowing for trace elemental determination of minute amounts solid-phase micro extraction (SPME) was used to extract and inject NSAIDs into of sample. TXRF-XANES allows to determine valence of Fe from amounts as low as GCxGC-TOF-MS and GC-MS-MS without derivatization. A Polydimethylsiloxane/Di- 500 pg in <µg of an aerosol sample. Self-shading is a major limitation in TXRF ap- vinylbenzene (PDMS/DVB)/carboxen SPME fiber showed maximum selectivity for plication. We used full field XRF approach to diagnose shading in model specimens NSAIDs with optimized extraction parameters of 70 oC for 30 minutes, desorption prepared by ink-jet printing [2]. time of 3 minutes and sample pH at 3.2. The two chromatographic techniques are References: discussed in light of their separation and detection selectivity for NSAIDs in complex [1] U. Boesenberg et al. Chem. Mater. 2015. mixtures and analyzed at trace levels. Optimization of analytical figures of mer- [2] U. Fittschen et al. SAB 2014.

65 2015 EAS Abstracts November 2015

457 Homodecoupled 1,1- and 1,n-ADEQUATE: Pivotal NMR Experiments 460 HSQMBC-TOCSY Experiment: Facilitating Chemical Assignment for the Structure Revision of Cryptospirolepine and Structural Elucidation of Small Molecules and Natural Products Josep Sauri, Merck, 126 E. Lincoln Avenue, PO Box 2000, Rahway, Josep Sauri, Merck, 126 E. Lincoln Avenue, PO Box 2000, Rahway, NJ 07065 NJ 07065 Cryptospirolepine is a complex, indoloquinoline alkaloid isolated in late 1991 from A novel nuclear magnetic resonance (NMR) method based on a selective heteronu- extracts of the Ghanaian chewing stick, Cryptolepis sanguinolenta. When the struc- clear single quantum multiple bond correlation (HSQMBC) pulse sequence is pro- ture was reported in 1993, the structure elucidation employed what were then state- posed to obtain both long-range heteronuclear correlations and carbon multiplicity of-the-art nuclear magnetic resonance (NMR) methods and 3 mm micro NMR probe information from a single NMR experiment. Even C/CH2 and odd CH/CH3 carbons technology. Using the NMR methods then available, there were no correlations can be directly distinguished from the relative positive/negative phase of cross- observed to the carbonyl in any heteronuclear multiple bond coherence (HMBC) peaks. The method can be readily extended by a total correlation spectroscopy data that could be acquired. The structure elucidation predated the application of (TOCSY) propagation step via 1H-1H coupling pathways, and it is fully amenable for 1H-15N HMBC methods to natural products by several years. Computer-assisted the simultaneous and precise determination of long-range heteronuclear coupling structure elucidation or CASE methods were in their infancy and incapable of deal- constants. In addition, a broadband HSQMBC-TOCSY experiments is proposed as ing with a molecule of the complexity of cryptospirolepine. Hence, the assembly complementary and highly sensitive approach to obtain weak (nJ<2 Hz) and/or very of the structure hinged on a ROE observed between a vinyl proton and one of the long-range heteronuclear correlations (4J, 5J, 6J) that are usually missing in con- terminal resonances of a four-spin aromatic system and a putative and unusually ventional heteronuclear multiple bond coherence (HMBC)-HSQMBC experiments. strong 4JCH correlation to a quaternary carbon from the same aromatic proton, Several examples are shown to demonstrate how these techniques can become an and the absence of any long-range heteronuclear correlation to the single carbonyl important tool for chemical assignment purposes and structure elucidation of small carbon. Nearly a decade later, several degradants were isolated from the original molecules and natural products. sealed NMR sample, one of which had a structure whose formation could not be ra- tionalized from the reported structure of cryptospirolepine. In light of this history, we 461 Taking NMR Spectroscopy Out of the Lab and Into the “Real now have interrogated a 700 µg voucher sample of cryptospirolepine using 1.7 mm World”: Applications of Cryogen Free NMR in the Manufacturing, MicroCryoProbe™ technology and our recently developed homodecoupled variants Process Monitoring, Quality Control, and, Adulteration Screening of 1,1- and 1,n-ADEQUATE (HD-ADEQUATE) to resolve long-standing structural of Dietary and Nutritional Supplements questions associated with the structure of crytospirolepine. Paul J. Giammatteo, Process NMR Associates, 87A Sand Pit Rd., Danbury, CT 06810, John C. Edwards 458 Trace Level Analysis Using Selective Excitation Proton NMR Compact, cryogen free high-resolution nuclear magnetic resonance (NMR) sys- Spectroscopy: A Tool for Genotoxic Impurity Analysis tems operating between 45 and 200 MHz proton frequencies are becoming more Frank Rinaldi, Bristol-Myers Squibb, 1 Squibb Dr., New Brunswick, NJ readily available. These systems represent break-through technologies in magnet, 08903, Scott A. Miller, Charles Pathirana electronic, and, software designs that enable the ability to utilize these new NMR Nuclear magnetic resonance (NMR) is generally thought of as an insensitive tech- systems in applications such as: 1) traditional analytical NMR Spectrometers, 2) nique; however, due to recent advances in NMR technology, NMR data can be as continuous or stop-flow detectors, 3) as “in-situ” reaction monitoring systems, used for trace level analyses. Although the introduction of cryoprobe technology 4) as fully integrated “at-line” or on-line process control systems. This presenta- has increased the S/N of NMR substantially, this alone is not sufficient to enable tion focuses on the advantageous use of compact NMR systems in manufacturing trace level detection. Coupling selective excitation proton NMR spectroscopy with and process/product monitoring in a variety of nutritional and dietary supplements. high sensitivity cryoprobe technology has enabled us to analyze trace levels, in Examples include: monitoring of batch production of polysaccharide supplements, the low ppm range, of genotoxic impurities (GTIs) in pharmaceutically important process monitoring of fish oil production from raw fish oil through final omega-3 compounds. Regulatory agencies have established stringent guidelines centered on enhanced fish oil supplements, and, rapid screening of herbal dietary supplements controlling the levels of GTIs in active pharmaceutical ingredients (API). Therefore, for adulteration. rapid and robust trace level methods for the determination of GTIs are needed. Using NMR to analyze GTI levels has many advantages, in particular the speed of method development, analysis time and the use of ambient analysis conditions. In this presentation, we demonstrate how selective excitation proton NMR spec- troscopy can be utilized to improve limits of detection. In addition, case studies are presented on how this NMR approach was used to determine GTI levels in the 3 to 10 ppm range relative to the main compound. 459 Beyond Chemical Shifts: Using NMR Relaxometry (a.k.a. Time Domain NMR) as an Effective Analytical Tool in Materials Science, Pharmaceutical, Biochemical, Food, Engineering, and, Medical Applications Paul J. Giammatteo, Process NMR Associates, 87A Sand Pit Rd., Danbury, CT 06810, Mark Manahan New advances in magnet technologies, spectrometer designs and hardware, and, user friendly software, make time domain nuclear magnetic resonance (TD-NMR) a valuable analytical system for the analyses of viscous liquids, emulsions, polymers, complex biological systems, nanoparticles, composites, ceramics, etc. In contrast to high-resolution NMR, TD-NMR is not restricted by the sample’s physical state. While not observing molecular structure information, TD-NMR techniques elucidate the physical characteristics of samples including viscosity, diffusion and porosity, crystallinity, particle size distributions, surface interactions and adsorption/absorp- tion with little sample preparation. In TD-NMR, relaxometry information is obtained using five principle TD-NMR measurements: free induction decay (FID), Hahn Spin Echo, T1, T2, and CPMG (Carr-Purcell-Meiboom-Gill). This presentation describes the TD-NMR measurement protocols for each of the five measurements as well as detail specific examples of their application.

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