2018 EASTERN ANALYTICAL SYMPOSIUM & EXPOSITION ANALYTICAL SOLUTIONS TO THE WORLD’S PROBLEMS

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EAS invites YOU to be a part of Bioanalysis Capillary Electrophoresis the Technical Program next Chemometrics year. Conservation Science Environmental Analysis Forensic Analysis Contribute a paper for oral or Gas Chromatography poster consideration via our Liquid Chromatography Immunochemistry website. Industrial Hygiene IR/NIR/Raman Spectroscopy Laboratory Automation Laboratory Management Mass Spectrometry Microchemistry Microscopy NMR Spectroscopy Pharmaceutical Analysis Process Analytical Science Quality by Design Quality/Regulatory/Compliance Sample Preparation Science Education Separation Sciences SFC & Size Exclusion Chromatography Surface Science Vibrational Spectroscopy CALL for PAPERS 2019 Opens March 1st 2018 EAS Abstracts November 2018

2018 EAS Abstracts This volume contains the final abstracts for the oral and poster presentations which take place Monday, November 12, through Wednesday, November 14, 2018. 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.

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1 2018 EAS Abstracts November 2018

1 Hyperbranched Anion-Exchange Phases in Ion Chromatography rent efficiency of electrolytic suppressor devices. Additionally, we provide highlights Christopher Pohl, Thermo Fisher Scientific, 1228 Titan Way, Sunnyvale, of our investigations with various design configurations of the electrolytic suppressor CA 94085, Charanjit Saini to understand the root cause of the higher noise phenomena. We discuss two new Since initial work in 2003, hyperbranched anion-exchanged materials have been designs of the suppressor device, one design tailored for improved overall perfor- widely used in ion chromatography. This versatile synthetic platform has been em- mance under constant voltage mode of operation with the typical eluents used in ployed in more than ten different ion-exchange phases. Here we review the design suppressor IC, and the other design tailored for improved noise performance in the and synthesis of hyperbranched anion-exchange phases with a focus demonstrat- constant current mode with carbonate based eluents. ing the relationship between synthetic parameters and the resultant ion-exchange selectivity of hyperbranched phases. Application of hyperbranched phases for a 5 Raman Spectroscopy for Forensic Purposes and Medical variety of applications is demonstrated. Diagnostics Igor K. Lednev, University at Albany-SUNY, 1400 Washington Ave., 2 High-Performance Anion Exchange Chromatography and Pulsed Albany, NY 12222 Electrochemical Detection: An Ideal Couple for Carbohydrate Raman spectroscopy combined with advanced statistics is uniquely suitable for Analysis characterizing microheterogeneous samples. Understanding the structure and (bio) William R. LaCourse, University of Maryland Baltimore County, MS: chemical composition of samples at the microscopic level is important for many CNMS, 1000 Hilltop Circle, Baltimore, MD 21250 practical applications including material science, pharmaceutical industry, etc. We Pulsed electrochemical detection (PED) following chromatographic separation is a have recently demonstrated a great potential of Raman spectroscopy for disease powerful technique that revolutionized the analysis of underivatized polar aliphatic diagnostics and forensic purposes. In this presentation, we will discuss the devel- compounds. PED offers near universal detection of compounds that contain one opment of a new, noninvasive method for Alzheimer’s disease (AD) diagnostics or more alcohol, amine or sulfur moieties. The analytical utility of PED focuses on based on Raman spectroscopy of blood. Near infrared (NIR) Raman spectroscopy the use of potential-time (E-t) waveforms, which combine amperometric/coulometric coupled with advanced multivariate statistics was utilized for differentiating patients detection with alternated anodic and cathodic polarizations that maintain reproduc- diagnosed with Alzheimer’s disease, other types of dementia and healthy control ible electrode activity. From an historical perspective, waveform development has subjects with more than 95% sensitivity and specificity. When fully developed, this been premised on understanding the tenets of anodic oxygen transfer reactions at fast, inexpensive noninvasive method could be used for screening at risk patient noble metal electrode surfaces. Hence, a series of different waveforms (e.g., PAD, populations for AD development and progression. Raman spectroscopy has already IPAD, etc.) has been reported in the literature. The majority of effort in past years found numerous applications in forensic science providing confirmatory identifica- has almost exclusively focused on quantitative (i.e., sensitivity and lower limits of tion of analytes. The technique is non-destructive, rapid and requires little or no detection) measurements rather than qualitative information. The full benefit of PED sample preparation. Furthermore, portable Raman instruments are readily available for carbohydrate analysis is only realized when it is coupled with high-performance allowing for crime scene accessibility. We have recently demonstrated that Raman anion exchange chromatography (HPAEC). In this talk, the ideal coupling of HPAEC microspectroscopy can be used for the identification of biological stains at a crime and PED will be juxtaposed with the advantageous coupling of academia and indus- scene indicating the type of body fluid. In addition, peripheral and menstrual blood try that made it all possible. HPAEC-PED literally transformed carbohydrate anal- as well as human and animal blood can be differentiated. Phenotype profiling based ysis, and its collective impact on research across numerous industries has been on Raman spectroscopy of dry traces of body fluids is the next exciting challenge. significant. An historical overview of PED development will be presented with em- We will also discuss the application of Raman spectroscopy for detection and char- phasis on the mechanistic discoveries and applied utility at each stage of evolution. acterization of gunshot residue (GSR). Along the way, applications involving pharmaceutical and biological samples will be used to highlight the quantitative and qualitative aspects of this powerful technique. 6 T-Jump Resonance and Normal Raman Determination of Reaction Coordinate of Thermoresponsive Hydrogel Volume Phase 3 Open Tubular Capillary Ion/Liquid Chromatography: The Transition Challenges and the Rewards Sanford A. Asher, University of Pittsburgh, Rm. 701, 219 Parkman Purnendu K. Dasgupta, University of Texas - Arlington, 700 Planetarium Ave., Pittsburgh, PA 15260, Tsung-Yu Wu, Alyssa B. Zrimsek, Sergei V. Pl, Dept. Chem UTA, Arlington, TX 76019 Bykov, Ryan S. Jakubek Some 35 years ago, in addressing the future of open tubular liquid chromatography, The best-known examples of smart, responsive hydrogels derive from poly(N-iso- Jorgenson and Guthrie were prescient in identifying sensitive detection as the domi- propylacrylamide) (PNIPAM) cross-linked polymer networks. These hydrogels un- nant barrier in the path of its success. Techniques such as photoionization detection dergo volume phase transitions (VPTs) triggered by temperature, chemical, and/or and mass spectrometry, it was hoped, would be attractive solutions. PID has not environmental changes. PNIPAM hydrogels can undergo more than 50-fold volume quite lived up to that promise. Some types of nanospray mass spectrometry does changes within ~1 μs intervals. Studies have tried to elucidate the molecular mecha- today have the requisite sensitivity to be applicable, but at current costs, this would nism of these extraordinarily large responses. Nevertheless, the molecular reaction represent a high barrier of entry. Based on some comments by Golay, multicapillary coordinates that drive the VPT remain unclear. Using visible nonresonance Raman chromatography (MCC) was proposed as a solution also early on to remove the temperature-jump (T-Jump) spectroscopy and UV resonance Raman T-jump we de- extreme volume limitations during detection, with the hope that improved precision termined the molecular ordering of this VPT. The PNIPAM hydrophobic isopropyl in manufacturing technology will make this possible. In this talk I would like to ex- and methylene groups first dehydrate with time constants of 109 +/- 64 and 104 plore if MCC can do any better, if it is possible somehow to use optical absorbance +/- 44 ns, initiating the volume collapse of PNIPAM. The subsequent dehydration of detection with short paths that are still sensitive enough or to create reliable shorter the PNIPAM amide groups is significantly slower (360 +/- 85 ns). This determination path data from longer path absorbance measurements and whether amperometry of the ordering of the molecular reaction coordinate of the PNIPAM VPT enables the / admittance / conductance detection provides niche interim solutions for specific development of the next generation of super-responsive materials. analytes. Is ion chromatography at a unique place because of ease of detection? 7 Applications of Vibrational Optical Activity for the Elucidation of 4 Recent Advances in Suppressor Technology for Ion Molecular Stereochemistry Chromatography Laurence A. Nafie, Syracuse University, MS: 1-014 CST, Department of Kannan Srinivasan, Thermo Fisher Scientific, 1228 Titan Way, Sunny- Chemistry, Syracuse, NY 13244-4100 vale, CA 94085 Vibrational optical activity (VOA) is comprised of two principal spectroscopies, one The technique of suppression as presently implemented in ion chromatography in the infrared (IR) region of the spectrum known as vibrational circular dichroism (IC) with an electrolytic membrane suppressor offers continuous operation with the (VCD), and the other in Raman scattering, usually with visible laser excitation, highest dynamic suppression capacity thus enabling the use of high capacity col- known as Raman optical activity (ROA). Both VCD and ROA are difference spec- umns and gradient mode of operation. For anion analysis applications with hydrox- tra with respect to left and right circularly polarized radiation and probe molecular ide eluents, the observed noise performance is typically in the less than 1 nS/cm stereochemistry in a unique way by distinguishing mirror-image molecules with mir- regime. The noise performance, however, is dependent on the applied current and ror-image (oppositely signed) VOA intensity about zero difference. Achiral mole- the current efficiency of the suppressor device. For carbonate based eluents the cules do not exhibit VOA which provides specific sensitivity to chiral molecules. VCD noise performance with electrolytic suppressors is higher and can be in the 5 nS/cm and ROA are complementary with relative strengths and weaknesses in a similar regime. However, with chemical suppressors, the noise performance with carbonate way to the relative strengths and differences of IR and Raman spectroscopy. In based eluents is significantly lower than electrolytic suppressors and in the less than this presentation three applications of VOA will be highlighted. The most important 1 nS/cm regime. In the chemical mode of operation, the noise is not impacted by the application of VCD is the determination of absolute configuration (handedness) of suppressed conductivity background; however, leakage of the chemical reagent can chiral molecules in solution by comparing measured to calculated VCD. This ap- compromise the operational dynamic capacity of the suppressor and the detection plication is now pervasive throughout the pharmaceutical industry and is slowing sensitivity. In this presentation, we review various approaches to improving the cur- making its progress among chemists in academia. For ROA, applications focus on 2 2018 EAS Abstracts November 2018

the conformations of biological molecules, and most recently to higher-order struc- in-source fragmentation spectra for this experiment. A normal and three succes- ture in biopharmaceuticals as a sensitive diagnostic of protein stability under stress. sively higher cone voltage spectra are used to generate entries in the drug-of-abuse Finally, we return to VCD to describe its unique sensitivity to supramolecular chirality library. A reverse search program has been developed to permit rapid data analysis amyloid fibrils, a level of chirality that is intermediate between the chirality of the producing a red-light warning / green light warning after each sample analysis. Upon constituent amino acids and the chirality observable with atomic force microscopy detection of the presence or absence respectively of drugs present in the sample. (AFM) or scanning electron microscopy (SEM). Recently we have added over 100 novel fentanyl analogs to our mass spectra li- brary of 700+ drugs-of-abuse. These analogs are being produced as Certified Ref- 8 Forensic Science R&D Programs at the National Institute of erence Materials and production level drugs-of-abuse by our collaborators at Cay- Justice: Opportunities for Novel Spectroscopic and Analytical man Chemical. This library is modeled after the DART Forensic library offered by the Techniques Applied to Forensic Problems National Institute of Standards and Technology (NIST). Previously, we have shown Gregory Dutton, National Institute of Justice, 810 7th St. NW, utilizing source inlet voltages of 15 V, 30 V, 50 V and 70 V with our WATERS QDa Washington, DC 20531 mass detector provided sufficient precursor and fragment ion information required The National Institute of Justice (NIJ) is the research, development and evaluation for identifying and differentiating a wide range of drug compounds. Results of an agency of the United States Department of Justice. NIJ maintains a leading federal internal cross-validation study using the library search for determination of multiple program of external funding for research & development (R&D) in the forensic sci- drugs in samples, samples containing structural isomers demonstrate the capability ences. The portfolio spans a broad range, from applied research across the scienc- of the program to readily distinguish these compounds with high match probability. es, to the development of prototype devices, and the evaluation of novel instruments and methods. Since 2009, NIJ has funded over $203M of external R&D to support 12 The Growing Phenomenon of the Epidemic of Synthetic Opioids the advancement of accuracy, validity and efficiency in the forensic sciences. Foren- and Forensic Science: Impact and Response sic science is a collection of applied disciplines that draws from all branches of sci- Erin Worrell, Cuyahoga County Medical Examiner’s Office, 11001 Cedar ence. Nevertheless, practicing forensic scientists most often tend to be concerned Ave., Cleveland, OH 44106 with the detection, collection, separation, and analysis of biological and chemical The Medicolegal Death Investigation (MLDI) Department at the Cuyahoga County samples. Due to the unique circumstances of forensic evidence, there is an ongoing Medical Examiner’s Office, is responsible for taking reported death calls and inves- need for these analyses to be done on ever smaller, degraded, compromised or tigating scenes as necessary under Ohio statutes within the county jurisdiction for mixed samples. At the same time, increased backlogs in operational forensic labs a population of 1,3 million people. In 2013 Cuyahoga County reported 194 drug create a need to increase the speed and decrease the cost of analyses. These related overdose deaths. Over the next four years the number of drug overdose needs drive NIJ’s continuing R&D investments in analytical chemistry and bioanal- deaths has risen at a shocking rate. In 2016 Cuyahoga County reported 666 drug ysis. Advances in applied spectroscopy, mass spectrometry, microscopy and micro- related overdose deaths and in 2017 reaching a total of 727 drug overdose deaths. fluidics, among other tools, have already yielded or show promise for successful The nature of this increase has been tied to the increased use and abuse of cocaine, application to forensics. An overview of NIJ’s R&D portfolio is presented, highlight- opiates, and designer drugs including fentanyl related analogs (i.e., Fentalogs). This ing relevant examples in trace evidence (fibers, glass, paint, dust, microbes, etc.); has caused a shift in the paradigm of a MLDI approach to on scene investigation, controlled substances and toxicology; and forensic biology and DNA. safety considerations, and the nature of interactions with law enforcement, family, witnesses, EMTs and medical personnel. The evolution in how the scene investi- 9 NJ’s Drug Monitoring Initiative (DMI): Understanding NJ’s Drug gation is carried out considering the presence of the evolving nature of designer Environment, Public Health, and Laboratory Collaboration drug agents, including highly potent designer fentalogs, has required a changing Adam Polhemus, New Jersey State Police, PO Box 7068, West Trenton, approach to the process of investigating death scenes. These new agents are not NJ 08628 only available on the street, but also through mail and internet sources. Parapher- No abstract submitted by the author. nalia is also changing. Careful questioning on scene witnesses and better scene awareness facilitates a careful and productive scene search, providing a greater 10 Challenges and Insights in LC-MS-MS and LC-TOF-MS Analysis of potential in finding evidence which improves efforts in MLDI determination of a drug Isobaric Compounds in the Opioid Class related cause of death (COD). Safety, involving the use of appropriate personal Michael E. Lamb, NMS Labs, 3701 Welsh Rd., Willow Grove, PA 19090, protective gear, proper technique in handling evidence, having all personnel orga- Donna M. Papsun, Barry K. Logan nized working in sync on scene, and having antidotes available to personnel, is also The current opioid epidemic and the involvement of fentanyl derivatives and other essential in this new paradigm. novel illicit opioids presents unique challenges during toxicological analysis. Con- stantly changing trends and the discovery of completely novel substances necessi- 13 The Foundational Role of Forced Degradation in Stability-Indicating tates continuous updating and evaluation of the analytical method and scope used Method Development: Potential Pitfalls in their identification. Many of these substances, however, are either isomers or Steven W. Baertschi, Baertschi Consulting LLC, 3967 Chadwick Dr., are structurally similar making their separation and quantitation more challenging. Carmel, IN 46033 In developing public health responses, drug user education, harm reduction, and As outlined by ICH Q1A(R2) and other regulatory guidances, stress testing (aka, scheduling, enforcement, and interdiction it is crucial to be able to definitively iden- forced degradation) studies are required to establish the stability-indicating “power.” tify these substances and to analytically differentiate them. High-performance liquid While much has been written about this topic in the last 20 years, there are many dif- chromatography/time of flight-mass spectrometry (LC-TOF-MS) is used in many ficulties and “pitfalls” awaiting the analytical chemist embarking on the development laboratories for comprehensive drug screening applications, including novel drug of a stability-indicating method. This talk focuses on some of these difficulties to identification. A limitation of LC-TOF-MS, which relies on identification of the com- illustrate the importance of well-designed studies, experimental interpretation, and a pound by the accurate mass of the parent drug, includes the risk of not resolving chemistry-guided approach to method development. Examples are provided to illus- isobaric compounds; further, structurally similar compounds may have very similar trate the concepts in the context of both drug substances and formulated products. retention times. To address this, samples screening positive for these closely related drugs are confirmed and quantitated using High-performance liquid chromatogra- 14 Accelerated Degradation of Pharmaceuticals in Leidenfrost phy/tandem mass spectrometry (LC-MS-MS). During confirmation testing of novel Droplets and its Potential illicit opioids, such as U-47700, carfentanil, and furanylfentanyl, investigations of Yangjie Li, Purdue University, Department of Chemistry, 560 Oval Dr., the identity of potential interferents have led to the discovery of additional emergent West Lafayette, IN 47907, Yong Liu, Hong Gao, Roy Helmy, W. Peter substances and/or unknown metabolites of fentanyl related substances, prompting Wuelfing, Christopher J. Welch, R. Graham Cooks updating and expansion of the scope of testing in the confirmation panel. Analysis Forced degradation, a method of studying the intrinsic stability of pharmaceuticals, and differentiation of closely related and isobaric drugs in the opioid class requires usually takes multiple days for the reaction between an active pharmaceutical in- careful scrutiny of all case data, awareness of the long list of possible isomers and gredient and the degradation reagent to occur to a desirable extent, and for liquid isobars, careful validation, and regular updating of the scope of analysis. chromatography ultraviolet (LC-UV) and LC-mass spectrometry (MS) product anal- ysis. Tests using three chemical degradations showed that the reaction/analysis se- 11 Analysis of Novel Opioids using a Direct Analysis in Real Time quence was accelerated and so completed within minutes using Leidenfrost droplet (DART) Equipped Mass Spectrometer reactors and nanoelectrospray ionization MS analysis. The same reactions were Brian D. Musselman, IonSense, 999 Broadway, Ste. 404, Saugus, MA undergone in the levitated droplets as seen in traditional bulk solution experiments. 01906, Frederick Li, Paul Kennedy, Stephen Shrader, Chris Snyder, By manipulating droplet size, levitation time and whether or not make-up solvent is Mishka Repaska added, control of reaction rate and yield is achieved so that forced degradation be- Opioid abuse is a growing epidemic and the constant stream of new fentanyl-an- tween 10% and 20% could be obtained at a desirable speed. Evidence is provided alogs presents a challenge to investigators. A direct analysis in real time (DART) that interfacial effects contribute to rate acceleration, considering that temperature ionization source equipped mass spectrometer (MS) is used to collect intact and and concentration effects were not involved. Requiring much smaller amounts of 3 2018 EAS Abstracts November 2018

pharmaceuticals compared to the traditional method, this combined method of ac- (STs). These powder patterns typically span breadths of one to several MHz, mak- celerated reaction and analysis is fast and reliable. It is currently being extended to ing their acquisition challenging; as a result, there have been very few reports of 10B conduct accelerated forced degradation of sterile formulated peptides. SSNMR in the literature.[1] In this lecture, I discuss the development and application of 10B solid state (SS)NMR for the purpose of characterizing short-range order in 15 Method Specificity: Forced Degradation Study Justifications from glasses and related amorphous solids. I demonstrate that high-quality 10B SSN- an Established Products Perspective MR spectra can be acquired using the WURST-CPMG pulse sequence, and with Neal Adams, Pfizer Inc., MS: PORT-259-175, 7000 Portage Rd., strategic transmitter positioning and spectral processing, such experiments can be Kalamazoo, MI 49001 completed in less than five minutes! It is also demonstrated that 10B SSNMR spec- An integral part of active pharmaceutical ingredient (API) and drug product ana- tra are very useful due to 1) easier extraction of quadrupolar parameters from the lytical method development is understanding what can or could interfere with your broad spin-3 patterns, 2) absence of spectral features arising from dipolar couplings component of interest (Specificity) and being able to measure all potential degra- to other nuclides, and 3) potential use for probing molecular-level dynamics in a dation products that could occur over the shelf life of the product (mass balance). A manner akin to 2H and 14N SSNMR. thorough evaluation of Specificity for API and drug product assay methods involves Reference: drug substance process impurities, excipients (if used), and potential degradation [1] Holland D. et al. J. Glass Sci. Technol. 2007, 48, 1. products. Evaluating the first two, process impurities and excipients, are easily accomplished; however, an evaluation of potential degradation products involves 19 Characterization of Formulated Pharmaceuticals Using Fast MAS some thought. Additionally, more focus has been placed on Specificity, via forced 1H Solid-State NMR Spectroscopy degradation studies, by regulatory authorities. This talk focuses on addressing David A. Hirsh, Iowa State University, 1605 Gilman Hall, 2415 Osborn the details outlined in ANVISA RDC 53/2017. This Resolution was approved for Dr., Ames, IA 50011, Anuradha V. Wijesekara, Scott L. Carnahan, Jo- regulation of the reporting, identification and qualification of degradation products seph W. Lubach, Karthik Nagapudi, Aaron J. Rossini in medicines According to RDC 53/2017, the evaluation of Specificity via forced The identification of solid active pharmaceutical ingredients (APIs) with suitable degradation studies consists of two parts, a theoretical part and an experimental properties is critical to the successful development of dosage formulations. Sol- part. Integral to each of these parts, is the pharmaceutical relevance of a particular id-state nuclear magnetic resonance (SSNMR) spectroscopy is widely employed to degradation pathway to the final drug product dosage form, packaging, and storage detect different API solid forms and probe local structure and intermolecular interac- conditions. Suggestions for utilizing scientific rationale for justification of forced deg- tions within APIs. However, SSNMR experiments on dosage formulations with low radation information for established products are presented. API loading are challenging due to low sensitivity and the presence of interfering signals from excipient molecules. In this presentation, we show that fast MAS (i.e., Perspectives on ANVISA’s RDC 53 Forced Degradation 16 νrot = 50 kHz) 1H SSNMR experiments can be applied to rapidly characterize pure Requirements APIs and APIs within dilute (< 15 wt-%) commercial formulations. We exploit combi- Leonardo Allain, Merck & Co, 770 Sumneytown Pike, West Point, PA nations of selective saturation and excitation pulses, two-dimensional NMR spectra 19486 and spin diffusion periods to eliminate interfering NMR signals from the excipients The Brazilian Health Authority, ANVISA, has introduced new requirements in the and obtain diagnostic SSNMR spectra of the API within dosage forms. Such spectra regulation RDC53 for how the DS and DP should be evaluated in forced degrada- can be used to positively identify the form(s) of the API in a dosage formulation even tion studies to demonstrate that the DP and related substances assay methodology in dilute formulations. Finally, we demonstrate that these methods can distinguish offers appropriate selectivity and mass balance. Unlike previous regulations by oth- between polymorphs of an API and quantify the fraction of a particular phase within er health authorities and the ICH, the RDC53 prescribes more directly how these a formulation. Fast MAS 1H SSNMR spectroscopy is generally applicable for the studies should be conducted, offering an augmented set of conditions to be applied identification and quantification of solid APIs in dosage forms and will help acceler- for both the DS and DP, such as: oxidation by Fe(III), a set goal of 10% degradation, ate the development of pharmaceuticals. chemical stresses performed directly in the DP and placebo matrix in addition of the DS, encompassing all potencies of the DP in the stress study, mass balance 20 High-Resolution Proton-Detected and Multidimensional Solid- assessment, and evaluation of thermal stresses at high and low humidity. This talk State NMR of Pharmaceuticals Utilizing Ultrafast Magic Angle describes these augmented set of stress conditions and targets, and discuss their Spinning of 60-111 kHz value in uncovering the relevance of key degradation routes, using recent case Xingyu Lu, Merck & Co., 2000 Galloping Hill Rd., Kenilworth, NJ 07033, studies. Yu Tsutsumi, Karen C. Thompson, Haichen Nie, Jean-Paul Amoureux, Gary E. Martin, Robert T. Williamson, Wei Xu, Yongchao Su Correlating Structure and Mobility Information to Functional 17 Solid dosage formulation represents a major category of modern medications. As a Properties of Pharmaceutical Formulations noninvasive and high resolution analytical technique, solid-state nuclear magnetic Eric J. Munson, Purdue University, Department of Industrial and resonance (ssNMR) plays an indispensable role in investigating molecular struc- Physical Pharmacy, Heine Pharmacy Bldg., 575 W Stadium Ave, West tures, dynamics and interactions of solid-state pharmaceutical materials in pharma- Lafayette, IN 47907 ceutical sciences. The molecular investigation in these multicomponent and natural Advanced analytical techniques can provide unique insight into the composition and abundance pharmaceutical materials offers both technical challenges and oppor- properties of pharmaceutical formulations. In particular, both structural and mobility tunities for this advanced technique. Recent advances in proton detection under information can be obtained about the formulations, but the challenge is to relate ultrafast magic angle spinning (MAS) of 60-111 kHz open a new avenue of char- that information to functional properties such as physical and chemical stability, dis- acterizing natural abundant pharmaceutical materials. We aim to advance these solution rate, and processing parameters. We are currently using solid-state nu- sensitivity and resolution enhanced techniques cooperated with multinuclear and clear magnetic resonance (NMR) relaxation times to measure particle size, crystal multidimensional ssNMR spectroscopy in pharmaceutical sciences. For example, defects, and chemical impurities in crystalline materials. In addition, we have been we have developed three-dimensional (3D) proton-detected multinuclear correlation using relaxation times to predict protein stability in lyophilized formulations, as well techniques for analyzing pharmaceutical structures. The Merck product, aprepitant, as measuring protonation states of probe molecules, which may be representative is characterized at molecular level in both crystalline active pharmaceutical ingredi- of the pH of the frozen solutions. Finally, we are using relaxation times to determine ent and nanoparticulate formulation (EMEND®) by the proposed 3D ssNMR. These phase separation and predict stability in amorphous formulations. efforts provide new opportunities for ssNMR to investigate quantification, polymor- phism, phase conversions, interaction in low-drug loading formulations at a high Boron-10 Solid-State NMR: Developments of Techniques for Rapid 18 resolution and relatively high-throughput Spectral Acquisitions and Applications to Disordered Solids Robert W. Schurko, University of Windsor, 401 Sunset Ave., Windsor, 13C NMR-Based Methodologies for Solving Challenging ON N9B3P4, Canada, Lucas D.D. Foster, Adam R. Altenhof, Stanislav 21 Stereochemical Problems L. Veinberg Ikenna E. Ndukwe, Merck & Co., 126 E. Lincoln Ave., Rahway, NJ Boron has two nuclear magnetic resonance (NMR)-active nuclides, 10B (I = 3) and 07065, Andrew Brunskill, Donald R. Gauthier, Yong-Li Zhong, Mikhail 11B (I = 3/2). 11B is normally chosen for boron NMR studies owing to its higher nat- Reibarkh, Yizhou Liu, Gary E. Martin ural abundance (n.a.), larger gyromagnetic ratio (γ), and moderate nuclear quadru- Determining the stereochemistry of proton-deficient molecules is challenging us- pole moment (eQ). This latter property generally results in relatively narrow central ing conventional nuclear magnetic resonance (NMR) methods – including Nuclear transition (CT, +1/2 ↔ -1/2) powder patterns, which are typically acquired using 11B Overhauser Effect (NOE) and proton-dependent J-based configuration analysis magic-angle spinning (MAS) experiments. By contrast, 10B is considered to be an (JBCA). The problem is exacerbated when only one stereoisomer is available. unreceptive NMR nucleus, due to its lower n.a., lower γ, and larger eQ. Since 10B Alternative methods based on the utilization of 13C-13C homonuclear couplings is an integer-spin nucleus, 10B SSNMR spectra do not have narrow CT patterns; measured at natural abundance[1] and residual chemical shift anisotropy measure- rather, they consist of six overlapping patterns arising from the satellite transitions 4 2018 EAS Abstracts November 2018

ments[2] in conjunction with density functional theory calculations are illustrated with ologist’s recorded number of colony forming units (CFU) can be considered to be a proton-deficient model compound.[1] someone’s (skilled technician) interpretation of the number of colonies on the plate. References: Experience has shown that different technicians (each skilled) can and frequently do observe different counts on the same sample Tablet Disintegration testing is [1] Saurí, J. Parella, T. Williamson, R. T. and Martin, G. E., Magn. Reson. Chem., done using equipment like DisiTest 50. The disintegration of the tablet is often timed 2017, 55, 191–197 manually by using visual verification. A visual verification of an endpoint of tablet [2] Liu, Y. Cohen, R. D. Gustafson, K. R. Martin G. E. and Williamson, R. T., Chem. disintegration isn’t very traceable and accurate and also takes away valuable time Commun., 2018, 54, 4254-4257 of the scientist, there is also a second person verification involved.

22 Exploring Complex Conformational Dynamics in hDM2 Inhibitor by 26 Automated Design of Experiments: A Multivariate Approach to NMR and DFT TPW Method Optimization Alexei V. Buevich, Merck & Co., 2015 Galloping Hill Rd., Kenilworth, C.J. Moynihan, SOTAX Corporation, 2400 Computer Dr., Westborough, NJ 07033 MA 01581 A novel active pharmaceutical ingredient (API), a potent inhibitor against hDM2-p53 “Doing more with less” is an ongoing theme in both research and development (R&D) protein-protein interaction, has been discovered at Merck. It binds to the hDM2 pro- and quality control (QC) analytical laboratories. High quality, consistent results are tein with high affinity and is active against a wide spectrum of human cancer cells required not optional, especially in regulated environments. Robust analytical meth- containing wt-p53, resulting in growth arrest and cell death. Preliminary liquid chro- ods are essential to meeting these goals. Developing robust methods requires sig- matograph mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) nificant effort from the ever diminishing R&D resources. Further challenges arise in studies of the hDM2 API in unbound form showed the presence of multiple forms. situations where they are sample limited. Systems for automating routine sample Comprehensive NMR studies of hDM2 inhibitor revealed complex conformational analysis have evolved over the years. The SOTAX Tablet Processing Workstation behavior of the API, which included two dynamic processes: fast rotamer exchange (TPW) design facilitates a fully automated, multivariate approach to method opti- and slow atropisomerization of two highly stable conformations. Unanticipated at- mization in addition to providing solutions for routine sample analysis. Users can ropisimerizm in hDM2 API has been rationalized by the density functional theory readily input factor combinations from various design of experiments software pack- calculation. ages directly into the TPW software at run time. Running these experiments via the TPW on-line high-performance liquid chromatography (HPLC) Interface to Waters 23 From Traditional Small Molecules to Novel Liquid Crystal EmpowerTM chromatography data software (CDS) further adds efficiency. All sam- Formulations in Drug Development: A Solid-State NMR Journey ple preparation data is securely transferred directly to the EmpowerTM CDS as the Anuji Abraham, Bristol-Myers Squibb, 1 Squibb Drive, New Brunswick, sample preparation process progresses, enabling the TPW to execute the various NJ 08901 factor combinations and the HPLC analysis completely unattended. The journey and exploration of solid-state nuclear magnetic resonance spectrosco- py (ssNMR) in understanding complex materials are still ongoing and the good news 27 Automated Sample Preparation Directly from Closed Sterile is that pharmaceutical companies are more routinely using under-utilized ssNMR Finished Pharmaceutical Product Vials methodologies to understand novel/complex molecules and formulations in small, Orane White, Merck & Co., MS: RY80T-A145, 126 E. Lincoln Ave., Rah- milla and macromolecules. The talk takes you through a journey of exploring ssNMR way, NJ 07065, William Stevens, Edward Mularz to understand small, complex molecules and novel formulations like liquid crystal MK-7655A drug product consists of a sterile powder for injection presented in a formulations. pharmaceutical vial. Sample preparation for drug product testing has posed multiple challenges due to high complexity of the sample mixture for the triple-combination 24 Quantitative NMR Spin-Diffusion and Relaxation Analysis of therapy (requires serial dilution), unstable nature of the actives, limited solubility, Inhomogeneous Mixing in Pharmaceutical Amorphous Solid and the potential human exposure to allergens. To overcome these challenges and Dispersions on the sub-100-nm Scale ensure an efficient, safe and precise sample preparation procedure for commer- Pu Duan, Brandeis University, Department of Chemistry, 415 South cial drug product testing at the manufacturing site, an automated procedure was St., Waltham, MA, 02453, Yongchao Su, Matthew S. Lamm, Wei Xu, developed on a LEAP sterile automation platform to perform end-to-end sample Fengyuan Yang, Klaus Schmidt-Rohr preparation directly from the manufactured pharmaceutical vial to a chilled sample Phase separation is a known concern in amorphous pharmaceutical dispersions, vial, ready for high-performance liquid chromatography (HPLC) analysis. The LEAP which often has direct impacts on the physical stability and bioavailability of the PAL3 was selected as the development platform because of its smaller footprint, active pharmaceutical ingredients (APIs) within the drug formulation. While mixing versatile liquid handling capabilities and customizable robotic tools designed for homogeneity has been extensively studied, quantifying inhomogeneity on the < flexible configuration. For this application, the PAL3 uses an XYZ robotic armto 100-nm scale is technically challenging, due to limitations of commonly adopted quantitatively transfer the drug product powder from the pharmaceutical vials for analytical methods such as differential scanning calorimetry (DSC) and high-res- precise dilution by volume with a syringe tool and a vortex station. The diluted final olution imaging. Here, we employed nifedipine (Nif) – polyvinylpyrrolidone (PVP) solution is then transported to an HPLC vial in a chilled chamber ready for testing. amorphous solid dispersions (APDs) as a model system. Using double 1H-filtering Proof of concept of the automated approach has been achieved by demonstration of and 1H spin-diffusion solid-state nuclear magnetic resonance (ssNMR) experiments equivalency to the manual method for content uniformity testing. Further optimiza- with 13C detection, we confirmed homogeneous mixing in a spray-dried 5:95 Nif:PVP tion efforts are underway to reduce occasional carry-over from concentrated sample sample, while we showed for the first time the partial but not homogeneous mixing in solutions and shorten the overall cycle time. The automated sample preparation samples made by hot-melt-extrusion (HME). We utilized the 1H inversion-recovery method will be implemented at the Merck commercial manufacturing site in 4Q- 1 experiment to document the H spin-lattice relaxation (T1,H) behavior of Nif and 2018, which will in turn bring unique advantages in efficiency, safety and precision

PVP. We observed a single T1,H in the 5:95 Nif:PVP spray-dried sample, indicating to commercial testing of an important Merck product. homogeneous mixing, while differential T1,H relaxation in HME samples document- ed partial mixing, both as predicted from the spin-diffusion experiments. The sub- 100-nm domain structure deduced from ssNMR was confirmed by surface imaging 28 Automated Capabilities for Complex Material Handling within with an atomic force microscope (AFM). We have analyzed the spin-diffusion and Analytical Testing relaxation behaviors using a general two-mixed-phases model, showing consistent Paul DiGregorio, Chemspeed Technologies Inc., 113 N Center Dr., compositions from both NMR methods. North Brunswick, NJ 08902, Diana Curren The pharmaceutical industry faces increased demand for innovative medicines 25 Application of Computer Vision and Robotics for Lab Automation against various diseases. Historically, most automation has focused on liquids-based Janakiraman Gopinath, Merck & Co., 21 Puddingstone Rd., Morris workflows. However, there is an increasing need in finished drug products to auto- Plains, NJ 07950, Prashant Shah mate the handling of complex materials and workflow steps that goes beyond sim- This presentation focuses on how Computer Vision and AI along with Robotics were ple liquid handling. Currently, most of these materials and workflow steps are still used to enable automation and improve accuracy in Merck Research Labs with the handled manually. This binds highly qualified resources to very time-consuming, following challenges: The Tablet Processing Workstation (TPW) is a fully automat- tedious and error-prone tasks. To overcome these challenges, Chemspeed devel- ed sample preparation system for pharmaceutical dosage forms (tablets, capsules, oped several innovative approaches to handling these materials and automating powders and liquids). For solid dosage forms, incomplete disintegration may re- the associated workflow steps. This presentation highlights several of these capa- sult in incomplete extraction of the active substance form the tablet. Microbiology bilities including: 1) automated complex material handling (solids, slurries, viscous Research labs have tasks which are often performed using the naked eye (i.e., materials), 2) formulating and mixing materials; 3) analytical sample preparation counting microbiological colonies on a plate), since it requires some sort of decision (filtration, blow-down, solid products, precipitation); and 4) integrated third party in- making based on human intelligence and the circumstances, However, a microbi- struments (rheology, dual asymmetric centrifuge). The purpose of this presentation 5 2018 EAS Abstracts November 2018

is to present an overview with innovative examples of how complex workflows in the testing of salmon otoliths for aquatic species bioanalysis and human fingernail ele- pharmaceutical R&D industry can be optimized through automation. mental analysis for nutritional zinc deficiency diagnosis will be described.

29 Laser-Ablation Laser-Induced Fluorescence Spectroscopy in a 32 Leveraging Laser Ablation-ICP-MS for Pharmaceutical Analysis Difficult Matrix Lydia Breckenridge, Bristol-Myers Squibb, 1 Squibb Dr., New Bruns- Jonathan A. Merten, Arkansas State University-Jonesboro, PO Box 419, wick, NJ 08903 State University, AR 72467, Chris P. Jones, Patrick D. Tribbett, Anna G. Laser ablation (LA) is a method of sampling solids that incorporates a pulsed laser Anders to produce nanoparticles and larger aggregates that are subsequently swept to an Laser-induced breakdown spectroscopy (LIBS) is known for its sample-prepless inductively coupled plasma (ICP) for atomization and ionization, followed by detec- rapidity, multielement capability and middling limits of detection. Laser-ablation tion by mass spectrometry (MS). Since its inception in 1985 it has become a highly laser-induced fluorescence spectroscopy (LA-LIF), also known as laser-ablation versatile technique for both elemental and isotope specific analyses in a variety of laser-excited atomic fluorescence (LA-LEAF) maintains LIBS’ rapidity, while im- different industries. Desirable features of the technique include significant sensi- proving limit of detections (LODs) through improved selectivity and reduction of the tivity, a linear dynamic range of up to 12 orders of magnitude, little-to-no sample continuum background. The athermal LIF excitation mechanism, particularly with preparation and micro-destructive sampling. Despite the relative ease with which Stokes-shifted measurements, can reduce LODs by up to two orders of magnitude. certain laser ablation systems can be hyphenated with existing ICP-MS instruments Traditionally, pulsed, tunable dye lasers have been used for selective reexcitation in that are traditionally present in most pharmaceutical atomic spectroscopy laborato- LA-LIF, though a number of recent publications have employed spectrally broader ries, LA-ICP-MS has not been leveraged to its fullest potential in the industry. The optical parametric oscillators. Since resonance transitions tend to be in the ultra- ease of solid sample analysis, with practically no preparation or sample expendi- violet (UV), LIF adds significant complexity in either case. However, the uniquely ture, makes it an especially desirable technique for atomic pharmaceutical analy- broad spectra of excimer lasers can provide conveniently direct reexcitation of a sis. This presentation demonstrates how Bristol-Myers Squibb (BMS) has adopted limited number of elements. Argon fluoride excimers lase from ~193.1-193.7 nm and this technique for a number of critical applications, including qualitative elemental provide simple, robust access to a slice of the vacuum ultraviolet otherwise inacces- comparisons between foreign matter and suspected contaminants, full elemental sible to dye lasers and optical parametric oscillators (OPOs). Though argon fluoride fingerprinting of solid samples that may not be amenable to dissolution or digestion (ArF) lasers have been used for nonselective reexcitation in LIBS plasmas in the and characterization of various different types of packaging materials such as glass past, my laboratory is working towards selective measurement of arsenic, carbon and plastics. Typical LA-ICP-MS challenges as they pertain to pharmaceutical anal- and platinum in difficult matrices using ArF LA-LIF from a modified excimer. Iron is ysis such as deleterious matrix-effects during quantitative analysis are addressed. a notoriously difficult matrix; its “rich” spectral background tends to limit LODs. We report here on LA-LIF measurement of arsenic, carbon and platinum in this and 33 Capture of Electrochemically-Generated Fleeting Carbazolium other complicated matrices. Radical Cations and Elucidation of Carbazole Dimerization Mechanism by Mass Spectrometry 30 Overcoming Matrix Challenges in XRF Hao Chen, Ohio University, 136 Clippinger Laboratories, Athens, OH Sharla Wood, Bristol-Myers Squibb, 1 Squibb Dr., New Brunswick, NJ 45701 08901 The capture of reactive intermediates is important for the elucidation of reaction Traditionally, metals analysis in pharmaceuticals is conducted using plasma-based mechanisms. We report the first observation of electrochemically generated, short- techniques, including inductively coupled plasma mass spectrometry (ICP-MS) lived radical cations of carbazole and two N-substituted carbazole derivatives by and inductively coupled plasma optical emission spectroscopy (ICP-OES). These mass spectrometry (MS). In addition, the carbazole dimerization mechanism was techniques offer sensitivity, selectivity, and precision; however, they often require investigated and found to involve the reaction of one radical cation with one neutral lengthy sample preparation and trained analysts. As sample matrices in the pharma- molecule rather than the previously proposed coupling of two radical cations. ceutical industry evolve in complexity, so does the growing need for solid analysis techniques that do not require liquid sample preparation, including dissolution or 34 Identification of Ortho-Substituted Benzoic Acid/Ester Derivatives digestion. Analytical techniques that can be performed on solids are highly desirable via the Gas-Phase Neighboring Group Participation Effect in (+)- owing to the reduced introduction of error or contamination from sample prepara- ESI High Resolution Mass Spectrometry tion, reduced consumption of solvents and reagents, and a rapid analysis times. Huaming Sheng, Merck & Co., MS: RY818-C214, 126 E. Lincoln Ave., Time is an important consideration during the pharmaceutical development process, Rahway, NJ 07065 and often a quick estimate of metal concentration is all that is necessary when de- Benzoic acid/ester/amide derivatives are common moieties in pharmaceutical com- ciding the next step in the optimization of a synthetic process. X-ray fluorescence pounds and present a challenge in positional isomer identification by traditional (XRF) provides an alternative approach to determine elemental content. While an tandem mass spectrometric analysis. A method is presented for exploiting the gas- XRF may not afford the same level of sensitivity as ICP-MS or ICP-OES, it is rela- phase neighboring group participation (NGP) effect to differentiate ortho-substituted tively inexpensive, easy for an untrained individual to use, and requires little to no benzoic acid/ester derivatives with high-resolution mass spectrometry (HRMS1). sample preparation. One disadvantage of using XRF in the area of pharmaceutical Significant water/alcohol loss (>30% abundance in MS1 spectra) was observed for development is the requirement for matrix matching standards to samples in order ortho¬-substituted nucleophilic groups; these fragment peaks are not observable for to obtain an accurate result. Because the sample matrix is constantly changing, ma- the corresponding para and meta-substituted analogs. Experiments were also ex- trix matching is a goal difficult to obtain. Progress towards determining appropriate tended to the analysis of two intermediates in the synthesis of suvorexant (Belsom- general matrices are presented. ra®) with additional analysis conducted with nuclear magnetic resonance (NMR), density functional theory (DFT) and ion mobility spectrometry – mass spectrometry 31 Laser-Induced Breakdown Spectroscopy as a Tool for Rapid (IMS-MS) studies. Significant water/alcohol loss was also observed for 1-substituted Elemental Bioanalysis 1, 2, 3-triazoles but not for the isomeric 2-substituted 1, 2, 3-triazole analogs. IMS- Steven J. Rehse, University of Windsor, Department of Physics, 401 MS, NMR and DFT studies were conducted to show that the preferred orientation Sunset Ave., Windsor, ON N9B3P4, Canada, Alexandra Paulick, of the 2-substituted triazole rotamer was away from the electrophilic center of the Christopher Heath, Robert Valente, Paul Dubovan, Kevin Beaugrand, reaction whereas the 1-subtituted triazole was oriented in close proximity to the Mark Armstrong, Doris Rusu center. Abundance of NGP product was determined to be a product of three factors: The use of laser-induced breakdown spectroscopy (LIBS) in biomedical and biolog- 1) proton affinity of the nucleophilic group; 2) steric impact of the nucleophile; and 3) ical applications has increased dramatically in the past decade. It is the potential proximity of the nucleophile to carboxylic acid/ester functional groups. to make rapid in-vivo or in-situ diagnostic analyses that is driving the interest in medical and biomedical applications. The ability to make localized point elemental 35 High-Throughput Analysis: Where Mass Spectrometry Fits composition measurements (requiring only micrograms of sample) on small fragile Jessica Lin, Genentech, 465 East Grand Ave., South San Francisco, CA biological specimens with minimal or no sample preparation is a primary factor in 94080, Colin Masui, Kelly Zhang the technique’s application to bioanalysis. In this talk I will introduce the technique High-throughput experimentation (HTE) has gained increased popularity in locat- of LIBS, specifically as it may be applied for bioanalysis. I will attempt to provide a ing successful reaction conditions for synthesis of highly functionalized drug can- summary of current LIBS applications related to the analysis of clinically relevant didates. A significant challenge is the high-throughput analysis, where quantitative systems and I will present our group’s efforts to develop a near real-time bacterial information on starting material consumption and product yield, and qualitative classification system that can test arbitrary fluid biospecimens. I will discuss our characterization of reaction side product profile are both needed in a short analy- strategies for easily obtaining adequate signal to noise in bacterial LIBS spectra sis timeframe to accommodate the large amount of samples. Liquid chromatogra- by isolating and concentrating bacteria from fluid suspensions that mimic clinical phy-ultraviolet (LC-UV) has been conventionally used, but the throughput is limited specimens. Other bio-inspired applications we have investigated including the rapid where it can take a whole day or a few days to complete LC analysis of a 96-

6 2018 EAS Abstracts November 2018

well plate or 384-well plate. Furthermore the methodology may not be suitable for 39 Native N-Linked Glycan Analysis by High-Performance Anion- compounds without a UV chromophore. The recent years have seen application of Exchange Chromatography with Pulsed Amperometric Detection high-throughput mass spectrometry such as ESI-MISER-MS (multiple injection in a (HPAE-PAD) and Mass Spectrometry (MS) single experimental run), Rapid Fire MS, and MALDI-TOF-MS. The high-throughput Parul Angrish, Thermo Fisher Scientific, 1214 Oakmead Parkway, MS has drastically reduced the analysis time from days to hours or even minutes, Sunnyvale, CA 94087, Yongjing Chen, Zoltan Szabo, Jim Thayer, and offered unique selectivity with accurate mass or multiple reaction monitoring Yan Liu (MRM). However, ion suppression is a significant issue, making both impurity profil- There are now a number of glycoprotein therapeutics with over 30 approved glyco- ing and absolute quantitation difficult. A third strategy of ultrafast-ultra performance protein-based biodrugs on the market and this number is rapidly increasing. There liquid chromatography mass spectrometry (UPLC-MS) leverages ultrafast UPLC are various techniques, either alone or in combination, which are used in the char- method to resolve components and mitigate ion suppression, and operates MS at acterization of these biomolecules with each offering their own benefits. Among all, a scan/MRM mode, where scan enables impurity profiling, and MRM allows tar- there’s one technique based on the principles of ion chromatography, HPAE-PAD. geted quantitation. Medium throughput may be achieved with characterization of a HPAE-PAD is a widely used technique for determining an extensive set of carbo- reaction plate in hours. This study compares the three methodologies in selectivity, hydrates, supporting simultaneous separation and detection of these analytes with- throughput, repeatability, impurity identification capability, quantitation accuracy and out the need for derivatization. Herein, we are demonstrating three workflows for range, and discusses the situations where each methodology fits. rapid native glycan analysis using – 1) HPAE-PAD using dual eluent generation cartridges (Dual EGC) mode; 2) HPAE-PAD coupled to a Q-Exactive Orbitrap mass 36 Opportunities for Method Development by Using Various Mass spectrometer; and 3) HPAE coupled to a Q-Exactive Orbitrap mass spectrometer. Spectrometric Ionization Techniques Dionex ICS-6000 system in Dual EGC mode enables the analyst to run gradient Norman H. Chiu, University of North Carolina-Greensboro, 301 McIver methods using an isocratic pump, offering improved reproducibility, eliminating St., Greensboro, NC 27412 manual preparation of eluents, and minimizing pump maintenance, thus maximizing The development of soft ionization techniques, namely electrospray ionization (ESI) instrument uptime. Dual EGC mode shows excellent performance for HPAE-PAD and matrix-assisted laser desorption/ionization (MALDI), were the game changers of complex carbohydrates. The chromatographic resolution of glycans is based on in the recent history of mass spectrometry. Due to its simplicity, compatibility to the number of sialic acid units, branch and positional isomerism and the presence/ liquid chromatography and availability in the commercial market, ESI and its related absence of core or outer arm fucose. The resulting PAD glycan profiles are repro- techniques have become the dominant ionization techniques in mass spectrometry. ducible, and the Q-Exactive MS and MS/MS acquisition supports identification of In order to address specific analytical challenges in our research for method devel- the native glycans, including several isobaric forms, as these are separated by the opment, which exclude the yield of target ions, the unique characteristics of various CarboPac PA200 column. The method described here supports highly informative ionization techniques have been explored. Two of our ongoing projects focus on 1) glycan analysis without introducing labeling bias. addressing the shortfall in ion mobility calibration, and 2) developing a high-through- put method for screening cellular toxicity that result from exposing specific cell cul- 40 Analysis of Vanilla Extract by the Molecular Ionization Desorption tures to known or unknown chemical materials. Analysis Source for Mass Spectrometry Ciara N. Pitman, University of Maryland-Baltimore County, 1000 Hilltop 37 High-Throughput Ion Mobility Mass Spectrometry Sequencing of Circle, Baltimore, MD 21250, Joshua A. Wilhide, William R. LaCourse Cyclic Peptides Mediated Through Oxazolidinone Ring Opening The Molecular Ionization Desorption Analysis Source (MIDAS), which is based on Ryan D. Cohen, Merck & Co., MS: RY800-D133, 126 East Lincoln Ave., desorption atmospheric pressure chemical ionization (DAPCI), is a flexible mass Rahway, NJ 07065, Hader E. Elashal, Heidi E. Elashal, Monika Raj spectrometry (MS) source that can be used to analyze liquids, solids, and gaseous Cyclic peptides are attractive targets for drug discovery due to enhanced proteolyt- samples with little to no sample preparation. The MIDAS is designed to control the ic stability, permeability, and binding affinity versus their linear analogs. However, positioning of numerous sample platforms (i.e., 96 well plate, 384 well plate, TLC one hurdle for their drug discovery is sequencing difficulties of both naturally-occur- plate, glass slides) for rapid sample analysis. For example, the MIDAS is able to ring cyclic peptides and those synthesized via a one-bead one-compound (OBOC) scan a developed TLC plate rapidly, produce a chromatogram, and determine the approach. Here we developed a dual ring opening/cleavage methodology for se- identity of peaks through their mass ions. In this presentation, the analytical utility quencing of cyclic peptides by selective modification of serine, threonine, glutamic of MIDAS is highlighted with the forensic analysis of vanilla extract, which may be acid or cysteine residues to an oxazolidinone-type moiety. Formation of this moiety subject to adulteration due to its high cost. Imitation and pure vanilla extracts were increases the susceptibility of the amide bond at the N-terminus towards hydrolysis purchased from local stores. The samples extracts were spotted on a 96-well plate and leads to the opening of a cyclic peptide to its linear counterpart. The resulting and assayed using MIDAS in the positive mode. In both samples, vanillin ([M+H]+ linear peptide can then be sequenced in one minute by a liquid chromatography, ion ~153) was an identifiable peak, but the imitation spectrum also contained ethyl van- mobility, tandem mass spectrometry (LC-IM-MS-MS) method. The addition of ion illin ([M+H]+ ~ 167). The presence of ethyl vanillin allows for the rapid determination mobility spectrometry allowed for greater throughput by reducing chromatographic of adulteration with the much less expensive imitation vanilla. Additionally, differ- run time via markedly increased peak capacity. Libraries of up to 100 cyclic peptides ences in mass spectra from a variety of pure vanilla extracts may provide insight containing different combinations of amino acids were synthesized and sequenced to country-of-origin for each extract. Other application of interest to the food and to determine the robustness of this methodology as well as compatibility with free flavoring industry are also reviewed. amino acid side chains. The process was then automated through the use of de novo sequencing software exhibiting an accuracy greater than 98%. Finally, we ap- Abstract withdrawn by the author. plied to the method towards sequencing several isolated macrocyclic peptides and 41 lasso peptides. 42 Synthesis, Photocatalytic Properties and Langmuir-Blodgett Film 38 Glass or Plastic? The Challenges and Solutions of Analyzing Photoelectrochemical Behavior of CdS Nanoparticles with Mercury by ICP Hydrophilic or Hydrophobic Organic Shell James A. King Jr., Inorganic Ventures, 300 Technology Dr., Momoka Nagamine, Adelphi University, 255 Nassau Blvd., Garden Christiansburg, VA 24073 City, NY 11530, Justyna Widera-Kalinowska, Magdalena Osial, Pawel The quantitative analysis of Mercury is critical to the environmental community due Krysinski to the element’s toxicity in humans and animals and prevalence in certain foods It is necessary to reduce greenhouse gas emissions to limit global man-made cli- and drinking water. For inductively coupled plasma (ICP) optical emission and ICP- mate change. To achieve this goal, alternative energy sources to fossil fuels must be mass spectrometry users the quantification of Mercury is often accompanied by provided. Solar energy, especially quantum-dot-sensitized solar cells, has gained unique challenges due to its chemical nature, and identifying these obstacles plays growing attention as one of the most effective renewable energy sources. In this a key role in working towards analytical solutions. Mercury issues related to chem- research, cadmium sulfide (CdS) nanoparticles with varying stabilizing shell were ical form in solution, sample preparation, storage, and matrices are discussed with successfully synthesized, providing them with hydrophilic or hydrophobic properties. troubleshooting suggestions provided. Additionally, sources of error involved with The as-synthesized nanoparticles with those properties were studied by scanning analysis as a result of the working solutions moving through the instrument are electron microscope, dynamic light scattering, and Fourier transform infrared spec- also discussed; factors such as adsorption to plastic introductory components and troscopy (FT-IR) analysis. Aqueous suspension of the hydrophilic nanoparticles was volatilization play significant roles in an analyst’s ability to perform a proper analysis used for band gap evaluations. The photocatalytic activity of the aqueous suspen- of Mercury. sion was studied in the presence of methylene blue by means of ultraviolet (UV)-Vis spectroscopy. It is concluded that the synthesized nanoparticles catalyzed the deg- radation process of methylene blue under the UV light irradiation. In addition, hydro- phobic nanoparticles were spread onto the free aqueous interface of the Langmuir trough with subsequent Langmuir-Blodgett transfer on the indium tin oxide surface. 7 2018 EAS Abstracts November 2018

Photoelectrochemistry of such layers was then studied in relation to the number of partment, a unidirectional valve, and a simple press fit connector for use with a transferred layers and compared to the drop-casted nanoparticle samples. The re- digital syringe drive or an automated sample preparation workstation. The use of sults showed the increase of the current density with the increase of the transferred positive displacement digital syringes ensures precise and reproducible flow kinet- layers of nanoparticles. ics, providing the opportunity to perform time dependent assays, such as speeding up protein digests. 43 Automated Recycling Chromatography Fabrice G. Gritti, Waters Corporation, 34 Maple St., Milford, MA 01757 47 Rapid Automated Sample Preparation for the Extraction of Semi- Pharmaceutical regulations require structure elucidation by nuclear magnetic reso- Volatile Organic Compounds from Soil nance of all impurities present in the active pharmaceutical ingredient (API) solution. Alicia D. Stell, CEM Corporation, 3100 Smith Farm Rd., Matthews, NC Over 1 mg of impurity with purity > 90% are needed. Serious separation/preparation 28106, Candice A. Olsson, Brittany A. Leffler problems occur when the impurity nearly co-elutes with the API and when it is pres- There is an ever-growing presence of organic compounds in the environment pro- ent in trace amounts. For the lack of resolution, current techniques such as simulat- duced from both human activity and natural processes. Many of these organic com- ed moved bed or steady-state recycling chromatography are failing to fully achieve pounds can be harmful to our health and are listed by the Environmental Protection this task. In this poster presentation, we report on the design of a research prototype Agency as priority pollutants. A number of these pollutants exist in a subset know as recycling (for ultra-high resolution) liquid chromatography system coupled to a col- semi-volatile organic compounds. There is an increasing need to regulate and mon- umn manager (for temperature control and automation) and to a fraction collector itor these semi-volatile organic compounds in soil. Traditional methods to extract (for sample preparation). The integrated system is tested for the isolation and prepa- these compounds from soil, such as Soxhlet, can be time consuming and tedious. ration of an unresolved impurity (abundance < 1/1000 relative to the API estradiol). Presented here is a new rapid automated extraction method in which 30 g of soil An operating pressure of 6000 psi, two 4.6 x 150 mm columns packed with 3.5 um is extracted in 5 min; this includes cooling, filtering, and washing. This is method is Sunfire-C18 particles, a mixture of acetonitrile and water (65/35, v/v) as the eluent simple, fast and effective for many environmental extractions, including the difficult (0.7 mL/min), 100 uL injection volume of a 10 g/L API stock solution, 6 cycles, and a extraction of semi-volatile organic compounds. run time of 23 min generate an optimum production rate of 0.5 microgram per hour. 48 Diving Deep – Continued Studies of the Longitudinal Diffusion 44 Modernization of USP Methods Using Ion Chromatography (IC) for Coefficient in Liquid Chromatography Active Pharmaceutical Ingredient (API) Determination Dwight Stoll, Gustavus Adolphus College, 800 West College Ave., Saint Hua Yang, Thermo Fisher Scientific, 1214 Oakmead Parkway, Peter, MN 56082, Devin Makey, Huiying Song, Monika Dittmann, Gert Sunnyvale, CA 94085, Jingli Hu, Jeff Rohrer Desmet, Deirdre Cabooter IC methods have been proposed to replace existing titration-based methods in The longitudinal diffusion coefficient (i.e., the b-term in h vs. ν relationships) is the monographs as part of the United States Pharmacopeia (USP) modernization effort. single remaining source of axial band broadening in the absence of a flow. A de- Here we show evaluations of two IC methods from USP monographs. The 1st is tailed understanding of the b-term is essential when studying mass transfer mecha- assay of Sodium Thiosulfate and ionic impurities in Sodium Thiosulfate using IC with nisms in high-performance liquid chromatography (HPLC). In spite of more than five a Dionex IonPac AS12A column and suppressed conductivity detection. The assay decades of study of the b-term by hundreds of investigators, there are still important method is linear (r2=0.999) over the established analytical range of 0.2-200 mg/L. It unanswered questions about this peak broadening mechanism. In this presentation is sensitive (LOQ at 0.2mg/L), accurate (intraday and inter-day accuracy 99–106%), we describe recent efforts in our laboratories to refine and develop approaches precise (precision <0.3%), and specific for sodium thiosulfate determination. The that will yield deeper understanding on this topic. First, we summarize the results Sodium Thiosulfate ionic impurities method is linear over the established analyt- of a study designed to compare commonly used approaches to experimental de- ical range for impurities (chloride: 0.04-2.00 mg/L r2=1.00, sulfite: 0.1-5.0 mg/L, termination of b-term values, namely: 1) peak parking; 2) fitting of experimentally r2=0.9998 and sulfate: 0.5-10.0 mg/L r2=1.00). It was sensitive (LOQ of chloride obtained plate height curves; and 3) the dynamic method. These methods were =0.01, sulfite =0.2 and sulfate = 0.05 mg/L), and accurate (recovery 85–108%). assessed based on their mutual agreement, intra- and inter-day variability and mea- The 2nd method is the assay of zinc oxide (USP General Chapter <591>). Using a surement/analysis time. Using these criteria we find that the peak parking approach Dionex IonPac CS5A column with pyridine-2,6-dicarboxylate (PDCA) as the eluent is preferred because it is highly reproducible (relative standard deviation < 1%) and and 4-(2-pyridylazo) resorcinol) (PAR) as a post column reagent, zinc is separat- the instrument time required to make the measurements is reasonable. Second, ed from transition metals and quantified using absorbance detection at 530 nm. recently we have been using the peak parking approach to study the effects of chro- This work demonstrated that adding acetonitrile to the sunscreen before adding 6 matographically relevant variables on the b-term, including the test analyte, mobile N hydrochloric acid is important for the preparation of water-resistant sunscreen phase composition, stationary phase chemistry, and particle pore size. In this way, a samples. The method is linear ranging from 0.1 to 30 µg/mL (r2=0.9995), accurate first attempt is made to rationalize the parameters influencing the b-term in packed (96-101% of label), precise (RSD=0.3-1.4%). Overall both methods had good per- particle columns. formance showing the value of IC to USP modernization. 49 A Diphenyl Bonded-Phase on Wide Pore Superficially Porous 44 Combining Liquid Chromatography and Pyrolysis-Gas Particles for Efficient Separations of Proteins Chromatography-Mass Spectrometry for the Characterization of Edward A. Faden, MAC-MOD Analytical, 103 Commons Ct., Chadds PPE Based Copolymers Ford, PA 19317, Stephanie Schuster, William Miles, Brian Wagner, Ben Merle Corazon R. Ahearn, SABIC, 1 Noryl Ave., Selkirk, NY 12158, Libert, Barry Boyes Allison Caron and Carnegie Mellon University Superficially porous particles (SPPs) of silica, with shells of wide pore size (HALO Liquid chromatography (LC) such as gel permeation chromatography (GPC) is a 1000 Å particles) demonstrate excellent reversed phase separations of large bio- widely used technique to determine the molecular weight distribution of polymers. molecules (>50,000 MW). Until recently, bonded phase choices for wide pore SPP Pyrolysis gas chromatography-mass spectrometry (GC-MS) has been valuable in have been limited to various alkyl chain types. In the current work, an aromatic providing chemical composition of polymers via its thermal decomposition profile. bonded phase is compared to C4 and C18 alkyl chain bonded phases. The features By combining the two techniques, one can achieve further characterization to aid in of these different bonded phases were characterized using small molecule organic understanding the polymerization process, the effect in formulation and the product probes and a variety of proteins. Measurements of column efficiency, retention, se- performance of copolymers. Previous work has shown GPC in combination with lectivity, and protein recovery reveal that small molecule separations do not predict Pyrolysis-GC-MS technique as a quantitative tool by leveraging quantitation of the the results for protein separations. Detailed comparisons of large pore SPP bonded main peak (i.e., monomer) formed from the pyrolysis. In this work, we were able to phases for separations of monoclonal antibodies (mAbs), in particular IgG1s and verify the combined techniques as a quantitative tool in determining the weight per- IgG2s, suggest distinct benefits of the Diphenyl phase for certain specific mixtures. cent of homopolymers contained in a copolymer. The applicability of this technique In some cases advantages of the Diphenyl phase include improvements in pro- to copolymer systems was also demonstrated, yielding several main components tein recovery and changes in selectivity. Examples of high-resolution separations of other than the expected monomer units. IgG1s are shown for preferred different mobile phase organic and acidic modifiers. The HALO 1000 Å Diphenyl bonded phase column exhibited excellent stability in 46 Express Protein Digestion by Automated, Micro Reaction aggressive use conditions, while maintaining the high efficiency for separations of Cartridges several protein mixtures. Anne Jurek, EST Analytical, 503 Commercial Dr., Fairfield, OH 45014, Peter Dawes 50 Applications of Augmented Reality (AR) Technology in Scientific The rapid identification and qualification of biomarkers are of increasing significance Education and Technical Learning in clinical settings, and this is achievable on small sample sizes by integrating the Helen Zhang, DISTAT Co., 222 State St., Kennett Square, PA 19348 ePrep customizable micro solid phase extraction devices (µSPEed) with mass Augmented Reality (AR) is an immersive technology that overlays digital world onto spectrometry. The µSPEed cartridges are comprised of a packed sorbent bed com- the physical world. Using either mobile device or head mounted display device, 8 2018 EAS Abstracts November 2018

information and data are superimposed in user’s field of view of the physical world. 54 Electrochemical-MS (EC-MS) Approach for Forced Degradation Leveraging the visualization advantage and close connection to real world environ- Studies ment, AR technology inherently has great potential in delivering better training and Nicholas Santiago, Antec Scientific, One Boston Place, 26th Fl., Boston, education experience. The revolutionary advantage of AR is that information and MA 02108 knowledge can be delivered in-context and in-situ. The learning environment could Stability studies at varied temperature, humidity and purposeful degradation exper- be virtually anywhere anytime, from individual level to group setting, from labora- iments using chemical and thermal methods are widely applied in Pharmaceutical tory to field study. Various user case studies include superimposing data/images industry to study the stability and degradation of active pharmaceutical ingredients and providing instructions/guidance onto the real world environment. Based on the (API) and formulated drug products. Many degradation reactions occur by REDOX learning experience from these cases, training and education are proved to be more mechanisms and with the availability of electrochemical (EC) flow-cells, a fast and immersive, intuitive, and interactive. Some studies have shown that, efficiency of convenient method of studying these reactions is now available. By online coupling training can be reduced significantly using AR tool. of EC cells with liquid chromatography-mass spectrometry (LC-MS), immediate identification and quantification of the degradation product profiles under varied ex- 51 Integrating Clinical Analytical Research Projects into Chemistry perimental conditions becomes possible. In this study, examples are shown were Curriculum electrochemistry was used to generate oxidative degradation products of pharma- Yuegang Zuo, University of Massachusetts-Dartmouth, Dept. of ceutical compounds. The compounds were oxidized at different potentials and pH. Chemistry & Biochemistry, 285 Old Westport Rd., North Dartmouth, The oxidative products formed were identified and structurally characterized by MA 02747 LC-MS-MS. Results from electrochemical oxidation were compared to those from Chemistry, especially analytical chemistry, plays a critical role in almost every sci- chemical oxidation and from accelerated stability studies. The electrochemical ap- ence and engineering disciplines, and it becomes an important part in addressing proach (in-electro) proved to be very useful as an oxidative stress test. All oxidation and resolving broader societal concerns. Thus, today’s chemistry education must products observed under accelerated stability studies could be generated. In com- be more relevant to modern analytical laboratory practices. Although traditional parison to chemical degradation tests electrochemical degradation has the advan- undergraduate chemistry curricula provide a solid foundation in the fundamental tage of being much faster and does not require strong oxidizing agents. Moreover, principles of analytical chemistry, they do not formally value practical skills that it enables the study of different operating parameters in short periods of time and enable students to adapt and be successful in today’s rapidly changing and com- optimization of the reaction conditions to achieve different oxidative products mix- petitive analytical workplace. To bridge the current gap between “real work” expe- tures. Furthermore, the use of electrochemical degradation allows for immediate up riences and university training in analytical chemistry, the author has incorporated scaling and production of mg quantities of degradants (electrochemical synthesis), real world clinical analytical research projects [R. Zuo et al., 2015, Food Chemistry to generate sufficient reference material for full characterization by other spectro- 182, 242-245; C. Wang et al., 2012, Food Chem. 132, 1420-1428] into analytical scopic techniques, e.g., nuclear magnetic resonance. chemistry curriculum to prepare our students academically for what the real world wants from them. The incorporated clinical analytical research projects challenged 55 Detection of Neutral CO Lost during Ionic Dissociation Using students to think creatively and improved their skills in communication, teamwork, Atmospheric Pressure Thermal Dissociation Mass Spectrometry and problem-solving. The details of this innovative curriculum project are given at (APTD-MS) the presentation. Pengyi Zhao, Ohio University, 393 Clippinger Laboratory, Athens, OH 45701, Travis A. White, R. Graham Cooks, Qinghao Chen, Yong Liu, 52 Mass Spectral Analysis of Fragrances Hao Chen Anna Swyers, Widener University, One University Pl., Chester, PA Elucidation of ion dissociation patterns is particularly important to structural analysis 19013, Shirley Fischer-Drowos by mass spectrometry (MS). However, typically, only the charged fragments from Fragrances are often comprised of complex notes of various chemicals. The com- an ion dissociation event are detected in tandem MS experiments; neutrals are not position of these materials is designed to yield favorable scents, leading to desir- identified. In recent years, we have developed an atmospheric pressure thermal able consumer products. However, the analysis of these items can be challenging. dissociation (APTD) technique that can be applied to dissociate ions at atmosphere Mass spectrometry can be used to validate compositional analysis. To develop a pressure and thus provide one way to characterize neutral fragments. In this paper, viable methodology, components used to mimic these notes are initially evaluated. we focus on the detection of neutral CO resulting from amino acid and peptide ion Pure samples of fragrances, such as vetiver acetate, java were run on the mass dissociation. In the first set of experiments, several protonated amino acids (e.g., spectrometer. Then, a blend of these chemicals is made, in order to assess the +1 ion of phenylalanine) were found to undergo loss of a neutral(s) of total mass 46 efficacy of this methodology in determining compositional analysis. Spectral data Da, a process leading to iminium ion formation. We successfully detected the neu- was successfully collected with subsequent identification of peaks. This project is tral species CO by using a CO sensor, ultraviolet-visible (UV-Vis) and MS analysis made possible by a collaboration with the Extrel CMS Corporation, which provided following selective CO trapping with a rhodium complex. The capture of CO from the instrument to Widener University for development of real-world applications that dissociation of protonated amino acids supports the assignment of the loss of 46 Da can be used in academic settings to develop workplace and graduate school skills to neutral losses of CO and H2O, rather than loss of formaldehyde or dihydroxycar- utilizing high impact practices. bene, other possible fragmentation pathways that have been subject of debate for a long time. In a second experiment, we used the APTD method in combination with 53 Strategies to Evaluate and Monitor Forced Degradation Studies the CO detection technique, to demonstrate the formation of CO in the conversion of Using a Dual Detection (UV-MS) System b ions to a ions during peptide ion dissociations. These results showed the potential Paula Hong, Waters Corp., 34 Maple St., Milford, MA 01721, Zhimin Li, of APTD in the elucidation of ion dissociation mechanisms, using simple home-built Patricia R. McConville apparatus. Forced degradation studies are typically performed to understand the degradation pathway of pharmaceuticals. Given the range of impurities and their chemical and 56 Analysis of Food Samples Using Thin Film Solid Phase physical properties, a single detection technique may not be adequate to accurately Microextraction (TF-SPME) and Thermal Desorption GC-MS measure all of the degradants. Specifically, when ultra-violet (UV) detection is used Laurel A. Vernarelli, GERSTEL, Inc., 701 Digital Dr., Ste. J, Linthicum, alone, non-chromophoric species and/or co-elutions may be missed. To address MD 21090, Jackie Whitecavage, John Stuff the challenges of measuring and quantifying degradants, a dual detection system Food products are routinely monitored for quality, authenticity, and safety. Analysis consisting of a photodiode array (PDA) and a mass detector (MS) will be used to an- of food products may also be necessary due to off flavor or odor complaints. The alyze a stressed drug substance. While UV is typically used to assess and measure aroma and flavor profiles of each product are unique and made up from a variety of degradants, mass spectrometry allows for detection by an orthogonal technique and semi-volatile and volatile compounds including aldehydes, ketones, acids, alcohols, provides information to aid in characterization. For example, mass detection will be terpenes, esters and other trace level components. The wide range of concentra- used to measure any non-chromophoric degradants that may be produced. The tion, polarity and functional groups composing a flavor/aroma profile can make the impact of missing degradants will be assessed. In addition, orthogonal detection will analysis of the sample difficult. Techniques which are simple, use little or no sol- also be used to illustrate the impact of co-elutions on mass balance determinations. vent and encompass a wide range of analytes are desirable. Thin film solid phase By assessing peak purity using both MS and UV, the final separation for the active microextraction (TF-SPME) is an extension of regular SPME. TF-SPME is more pharmaceutical ingredient and its degradants can be optimized to ensure no co-elut- sensitive than regular SPME due to the increased surface area and phase volume. ing peaks. The addition of MS information, whether for non-chromophoric species The TF-SPME device is a 20 mm x 4.8 mm carbon mesh sheet which is impreg- or co-elutions, will allow for a more complete evaluation and more comprehensive nated with a sorptive phase. The TF-SPME devices can be used in headspace or understanding of the degradation pathway. immersive modes. In headspace mode, the TF-SPME device is suspended above a solid or liquid sample in an enclosed vial. In immersive mode, it is placed directly in a liquid sample. In both cases, the sample is agitated by stirring. After extraction,

9 2018 EAS Abstracts November 2018

the devices are blotted dry and placed in an empty thermal desorption tube. They peptide variants were synthesized and assessed in various process related condi- are analyzed by thermal desorption gas chromatography mass spectrometry (GC- tions: bicarbonate buffer (pH 8.3), PBS buffer (pH 7.4), and other pH conditions (2.0, MS). This technique is simple to use and requires no solvent. This work shows the 5.5, and 7.5). High resolution liquid chromatography-mass spectrometry (LC-MS) ap7lication of TF-SPME to the analysis of aroma and flavor components in a variety was used to assess deamidation and other chemical degradation products, and size of food matrices. exclusion chromatography (SEC) was used to assess aggregation. SEC coupled with MS and multi-angle light scattering (MALS) detectors were used to identify 57 Characterization of LC Packing Materials by NMR Solvent different aggregates. The results of this study, in combination with other critical at- Relaxation at High Field tributes like binding affinity and peptide activity, has led to the selection of the most Xiangjin Song, Waters Corporation, 5 Technology Dr., Milford, MA 01757 stable peptide variant. Surface chemistry (functional groups, polarity, hydrophobicity, etc.), and porosim- etry (surface area, pore volume, pore diameter, etc.) are important properties for 61 Determination of Aminoglycoside Antibiotics by LC-PAD liquid chromatography packing materials. Although various mature analytical instru- Jingli Hu, Thermo Fisher Scientific, 1214 Oakmead Parkway, B10, ments are available, nuclear magnetic resonance (NMR) solvent relaxation method Sunnyvale, CA 94085, Jeff Rohrer has its unique strength in evaluation of particle surface and porous structure more Aminoglycoside antibiotics are valuable in the treatment of serious infections. Differ- close to application conditions. This technology gained great success at low mag- ences in antimicrobial potency and toxicity necessitate the limitation and control of netic field. Bench-top even portable devices have already been commercialized. impurities in commercial samples. Ion-pairing reversed-phase liquid chromatogra- However, at high magnetic field (>3T, typical for superconducting magnets used phy is widely used to separate aminoglycosides and the separation has been paired for NMR spectrometers) this approach encounters technical difficulties, including with pulsed amperometric detection (PAD), a type of electrochemical detection. The increased spin-lattice relaxation time (T1), less toleration of field heterogeneity thus Dionex IonPac AmG-3μm C18 columns are designed for ion-pairing reversed-phase field locking and shimming can no longer be skipped as in low field applications, as separation of aminoglycoside antibiotics. The column is packed in a PEEK column well as severe radiation damping. The presented work explores ways to overcome body rather than stainless steel. A stainless steel column can release significant these obstacles at high field for solvent relaxation analyses, including using mixed levels of metals, particularly when corrosive eluents are used, and this can cause deuterated and protonated NMR solvents, etc. The feasibility and complexities of problems for electrochemical detection. Three aminoglycosides were analyzed to the new approaches were tested by Waters chromatographic packing particles. demonstrate the performance of this column with PAD. The analysis of gentamicin sulfate is described in the U.S. and European Pharmacopoeias (USP and EP). In 58 Carryover Improvement Achieved Through Instrument Design addition to using the USP/EP method, we modified the USP/EP monograph mobile Changes and Needle Wash Optimization for HPLC Systems phase in two ways. Method A uses a simple mobile phase, 100 mM trifluoroacetic Amanda Dlugasch, Waters Corp., 34 Maple St., Milford, MA 01757, acid (TFA), while Method B is a fast method that has 2% acetonitrile in the mobile Jennifer Simeone phase. We also evaluated the analysis of etimicin sulfate based on the ion-pair- Sample carryover is a common problem for analytical labs and may adversely af- ing high-performance liquid chromatography (HPLC)-PAD method described in the fect chromatographic methods. Sample carryover occurs when material from an China Pharmacopoeia. The EP monograph for spectinomycin sulfate contains an injection is present in subsequent injections. There are several factors that can assay method based on ion-pairing HPLC-PAD. We modified the mobile phase in influence carryover including the chemistry of the sample analyte as well asthe the EP monograph to a simple mobile phase (0.1M TFA). All three aminoglycoside injector design of the high-performance liquid chromatographic (HPLC) system. The evaluations showed good performance of the column for ion-pairing HPLC-PAD with injector design is important not only to minimize the potential for carryover, but also the possibility of simpler mobile phases than currently used. to properly clean the components of the needle that are vulnerable to contamination. A HPLC system based upon a flow through-needle design is capable of providing 62 Triboluminescence (TL): Fast Detection of Crystallinity within improved carryover performance due to the interior of the needle being washed by Amorphous Solid Dispersions the mobile phase during the analysis and the exterior of the needle being washed Julie M. Novak, Merck & Co., Inc., MS: WP78-21, 770 Sumneytown with an appropriate wash solvent. Modifying the injector design provides a signif- Pike, West Point, PA 19486, Casey J. Smith, Scott R. Griffin, Julia K. icant reduction of the sample carryover with optimal washing procedures of the White, Garth J. Simpson, Siwei Zhang, Zhen Liu, Timothy Rhodes needle. The needle wash, therefore, is an important factor that is often overlooked Triboluminescence (TL) is shown to enable selective detection of trace crystallinity during method development. With an optimal needle wash solvent and the new de- within nominally amorphous solid dispersions (ASDs). ASDs are increasingly used signed injector, carryover can be significantly reduced. In this study, we examine the in pharmaceutical formulations for solubility enhancement; the efficacy of which improved carryover obtained after design optimization of the HPLC system injector. can be negatively impacted by the existence of trace crystallinity introduced during Along with the injector design, optimization of the needle wash solvent will be evalu- manufacture or storage. Instrumentation for TL analysis of powders and slurries ated in order to show the importance of the needle wash composition. A wide range has been developed along with methodology to deconvolve photon events to en- of sample types are analyzed to demonstrate the injector design improvements, as sure TL signal optimization. TL monitoring of the model compound griseofulvin in an well as how the needle wash solvent is sample dependent. ASD has been demonstrated with limits of detection of 140 ppm. TL measurements of a series of APIs, excipients, and ASDs were conducted to reveal the effect of 59 Fully Automated Determination of pH in Cell Culture Media Using crystalline structure on TL responses and the limits of detection in ASDs. Separate Flow Cell Technology studies of the particle size dependence of sucrose crystals and the dependence on Kerri-Ann Blake, Metrohm USA, 9250 Camden Field Parkway, polymorphism in clopidogrel bisulphate particles are both consistent with a mech- Riverview, FL 33578 anism for TL closely linked to the piezoelectric response of the crystalline fraction. Accurate pH measurement is paramount for pharmaceutical product development. Whereas disordered polymeric materials cannot support piezoelectric activity, mo- This measurement can influence fermentation, cleaning, purification, and stability. It lecular crystals produced from homochiral molecules adopt crystal structures that also provides the framework for liquid separation techniques. For cell culture media, are overwhelmingly symmetry-allowed for piezoelectricity. The mechanism of TL on the pH value should remain stable over time to encourage cell growth. This poster the polarity generation of crystals during fracture is discussed. Consequently, TL describes the automated determination of pH in cell culture media using a custom may provide a broadly applicable and simple experimental route for sensitive detec- autosampler with pH flow-through cell. The system was designed to extract samples tion of trace crystallinity within ASD formulations. from a septum sealed vial, determine the pH, and self-clean between samples. Elec- trode stability and product precision data was collected across three instruments 63 Automated Rapid Drug Extraction at Trace Levels from Serum and over two days with <0.05 pH variation. Blood Using a Novel Small Particle Micro-SPE Cartridge Anne Jurek, EST Analytical, 503 Commercial Dr., Fairfield, OH 45014, 60 Assessment of Deamidation and Aggregation of Peptide Variants Peter Dawes Eileen Zhao, Genentech Inc., 1 DNA Way, MS: 432A, South San Sample preparation, from extraction, concentration to dilution and isolation is vital for Francisco, CA 94080, Mohammad Al-Sayah reliable and accurate analyses. Sample preparation is often the most time consuming Peptide therapeutics have become very popular as they can provide novel treat- physical job a chemist performs. Automation of sample preparation minimizes errors ment options. Depending on the peptide amino acid sequence, peptides can under- and frees analysts from liquid handling tasks. The ePrep Sample Preparation Work- go different chemical degradation pathways such as: oxidation, deamidation, race- station provides a simple and effective way to introduce automation into a laboratory. mization, dimerization, isomerization…etc. Additionally, peptides have the tendency Micro solid phase extraction (µSPEed) is an effective sample preparation technique to self-associate, hence forming, dimers, oligomers and higher molecular weight using small bed volumes and small (<3µm) particle size enabling greater concentra- aggregates. Peptide aggregation can be reversible or irreversible which could be tion factors and therefore smaller sample volume analyses than other forms of SPE detrimental for drug development. This work discusses the deamidation and ag- with further advantages in speed, efficiency and solvent use. Blood is well understood gregation of a lead peptide candidate which were observed during the chemical to be a complex and troublesome matrix that requires significant sample preparation, synthesis as well as the release of the peptide from the formulated product. Different the use of µSPEed has the potential to simplify this, streamlining lab procedures. 10 2018 EAS Abstracts November 2018

64 Analysis of Dibenzo[a,l]pyrene in Marine Sediment Samples via conditions, although at higher buffer concentrations and lower percentages of the High-Performance Liquid Chromatography – Laser Excited Time aqueous mobile phase component, longer equilibration times may be required. Resolved Shpol’skii Spectroscopy Ahmed Comas, University of Central Florida, 4000 Central Florida 67 Fast HPLC Separation of Triptans in Plasma on a ZirChrom®-PBD Blvd., Orlando, FL 32816, Jennifer Ferrante, Anthony Santana, Andres Column D. Campiglia Julie A. Jenkins, ZirChrom Separations, Inc., 617 Pierce St., Anoka, MN Eco-toxicological studies attribute a significant portion of the biological activity of 55303, Richard A. Henry polycyclic aromatic hydrocarbons (PAHs) contaminated samples to the presence Triptans, a class of serotonin antagonists, are often prescribed for the acute treat- of high molecular weight PAHs (HMW-PAHs), i.e., PAHs with MW greater than 300 ment of migraine headaches. Traditional high-performance liquid chromatography g.mol-1. Since the carcinogenic properties of HMW-PAHs differ significantly from (HPLC) analysis of triptans has been complicated by the fact that they are very isomer to isomer, it is of paramount importance to determine the most toxic isomers basic drugs. The amine moieties have a strong affinity for silanol groups present even if they are present at much lower concentrations than their less toxic isomers. on silica-based HPLC columns, causing poor peak shape which can lead to poor One of the problems that confronts chromatographic methodology for the analysis speed, quantitation and reproducibility. Changing operating conditions to improve of HMW-PAHs arise from the large number of MW isomers with co-eluting times peak shape can decrease silica column lifetime. This poster demonstrates benefits, and similar, often nearly identical, fragmentation patterns. Previous work in our lab including good selectivity and increased speed of analysis, using a ZirChrom®-PBD showed the feasibility to determine Dibenzo[a,l]pyrene, Dibenzo[a,e]pyrene, Diben- column over a typical C18 column for the challenging analysis of triptans in rat plas- zo[a,i]pyrene, Dibenzo[a,h]pyrene, and Naphto[2,3-a]pyrene isomers in high-per- ma.[1] Another advantage of using ZirChrom® columns is their unparalleled chemical formance liquid chromatography (HPLC) fractions via laser-excited time-resolved and thermal stability, which enables a larger range of operating conditions. Method Shpol’skii spectroscopy (LETRSS).[1] Herein, we expand the HPLC-LETRSS ap- development with a typical C18 column can be challenging for structurally similar proach to the analysis of the 23 commercially available isomer with MW 302 g.mol-1. compounds and complex mixtures of neutral and ionic compounds. ZirChrom®’s A thorough investigation of HPLC retention times, ultraviolet–visible absorption and zirconia-based HPLC columns can help solve these difficult separation problems. room-temperature excitation and fluorescence spectra provide the qualitative pa- The unique surface chemistry of zirconia must be considered and well understood rameters to screen HMW-PAHs in HPLC fractions. LETRSS is then used to analyze to achieve optimal results in method development. In some cases, reversed phase Dibenzo[a,l]pyrene in marine sediment samples of the Gulf of Mexico. interactions can be isolated, while in other situations, the polar and ionic sites of zirconia can be advantageous. An overview of how to best use ZirChrom® columns Reference: are included, and Chrom U will be introduced as an online learning tool to develop [1] W.B. Wilson, B. Alfarhani, A.F.T. Moore, C. Bisson, S.A. Wise, A.D. Campiglia, and troubleshoot HPLC methods on zirconia. Talanta, 148, 444-453, 2016 Reference: 65 The Hydrophobic Subtraction Model of Reversed-Phase Selectivity [1] Ahmed, S; Atia, N.N., Journal of Pharmaceutical and Biomedical Analysis, 143, – Principles and the Public Column Database 241-251 (2017). Dwight Stoll, Gustavus Adolphus College, 800 West College Ave., Saint Peter, MN 56082, Tina Dahlseid 68 New Wide Pore Epoxy Activated Monolithic Silica: Attach any There are currently over 1,000 commercially available columns for liquid chroma- Ligand to Prepare Your Own Column tography that can be classified as reversed-phase (RP). On one hand this is great Egidijus Machtejevas, MilliporeSigma, Frankfurter 250, Darmstadt in the sense that this number of columns provides users with many options when 64293, Germany, Benjamin Peters it comes to choosing a column for an application involving a RP separation. How- Bio-therapeutics such as bio-engineered drugs, peptide therapeutics, and complete ever, the number of options can also be overwhelming, which motivates the use of field of biotechnology represents the promise of new medical treatments for the new tools to support rational choices when picking columns. In the early 2000’s Snyder, millennium. Especially the HPLC is the mostly used analysis method. Most import- Dolan, and colleagues developed the Hydrophobic Subtraction (HS) model of re- ant for the HPLC analysis are the properties of the column. As the rule of thumb it versed-phase selectivity with the goal of providing a means to assess the similarities is widely accepted, that in order for the separated molecules not to be influenced and differences in selectivity between RP columns. In this presentation we will re- by size exclusion process the pore should be at least 10 times bigger than the mol- view the physicochemical principles of the HS model, and discuss some of its known ecule. Therefore about 100 kDa molecule would require around 300 A pores. Chro- limitations. We will also describe applications of the model for choosing columns of molith columns already shown great potential and superiority compared to standard similar selectivity (e.g., for identifying substitute columns during method validation), silica particles. In contrast to conventional packed-particle columns, wide pore (300 or columns of different selectivity (e.g., for choosing columns to be used for evalu- A) monolithic silica columns are made of a single continuous-bed rod of high purity ating method specificity). An important outcome of this work has been the develop- porous silica that is then bonded with C18, C8, C4, epoxy and Protein A. Monolithic ment and maintenance of a freely available public database of characterization data columns remove backpressure as the primary consideration in method development for the columns that have been characterized to date with the HS model. Currently and give back the flexibility of choices in selectivities. Because they have no indi- the database is populated with data for over 700 columns, and it continues to grow vidual particles to shift or break, column performance is very consistent over much steadily as manufacturers introduce new stationary phases to the market. Drawing longer lifetimes, making them ideal for relatively ”dirty/ matrix rich” sample analysis. on these data, we present trends in the characteristics of RP columns introduced Their high permeability also makes them very forgiving of shortcuts and timesaving over the past several years. in sample preparation as well as easier to aggressively flush out to reequilibrate. This presentation guides you through the posibilities of monolithic silica materials. 66 Investigating the Effects of Chromatographic Parameters on New possibilities of modifying epoxy activated columns are highlighted, for example Column Equilibration in Isocratic and Gradient HILIC Separations creating online column reactors, chiral or protein columns. Alex Nasseh, MAC-MOD Analytical, 103 Commons Ct., Chadds Ford, PA 19317, Geoffrey M. Faden, Edward A. Faden, Alan Mckeown 69 High-Performance Separations Using 100% Aqueous Mobile Phase Hydrophilic interaction liquid chromatography (HILIC) is a complex separation tech- Compatible Superficially Porous Particle Columns Coupled with nique, with analyte retention based on potential combinations of polar interactions, Mass Spectrometry electrostatic interactions and analyte partitioning into the adsorbed water layer at Thomas M. Waeghe, MAC-MOD Analytical, 103 Commons Ct., Chadds the stationary phase surface. The complex retention mechanisms combined with Ford, PA 19317, Chuping Luo, Justin Godinho, Ben Libert, Stephanie HILIC being a relatively young technique, means that knowledge and experience Schuster, Barry Boyes amongst analysts can be relatively low. This often leads to challenges with obtaining A challenge for analysis of highly polar compounds by high-performance liquid chro- stable retention times and developing robust HILIC methods. HILIC separations are matography (HPLC) is low retention on reversed-phase columns. To increase reten- often reported as requiring long equilibration times to achieve reproducible retention tion, low concentration of organic solvents are used, potentially at the expense of times. Insufficient column equilibration is often a contributing cause of problems en- phase stability, referred to as dewetting. The HALO AQ-C18 is a superficially porous countered with many HILIC separations. The aim of the work presented in this post- particle packing material specifically designed to address stability under highly aque- er is to investigate critical HILIC separation parameters and how they affect the time ous conditions, while maintaining high retention and high separation efficiency of required for column equilibration. Three stationary phases with differing stationary polar compounds. One such class of polar compounds that have been investigated phase chemistries (acidic, basic and neutral), which are broadly representative of in detail are the purines and their metabolites. An example studied in detail includes many HILIC phases on the market, were tested under a variety of chromatographic the enzyme guanine deaminase (GD). GD catalyzes purine catabolism of guanine conditions and the equilibration times required to achieve stable retention times into xanthine and ammonia. GD levels are high in telencephalic regions and low in assessed. For isocratic separations, the mobile phase pH, buffer concentration and white matter and cerebellum tissues in mammalian brain. The significance of this the percentage of the aqueous component of the mobile phase were investigated. expression pattern on synaptic physiology is uncertain. Here, we present a sensitive In general, it was found that rapid column equilibration was achievable under most and selective liquid chromatogrpahy-mass spectrometry (LC-MS) assay for GD to 11 2018 EAS Abstracts November 2018

improve understanding of the pharmacology of this enzyme. Using 100% aqueous analysis was done using Mascot server and Scaffold 4.1 software. In addition, two conditions and no ion pairing reagents, the column exhibited no evidence of phase other JTB isoforms will be investigated and their possible cellular function(s) will collapse typical of traditional C18 sorbents. Highly reproducible analyses in addition be compared with the functions of the wild type JTB. These studies could help us to robust wash methods were conducted in less than four minutes. This allowed for elucidate the mechanism through which JTB induces cell proliferation and test the rapid screening and replicate enzymatic assays run in parallel. The high-throughput JTB protein as a potential drug target for malignancies with overexpression of the and reproducible separations observed in 100% aqueous mobile phases afforded protein. by this stationary phase combined with highly selective and sensitive mass spec- trometry is demonstrated by a range of additional highly polar analytes. 73 Investigation of the Molecular Changes in Rat Atria during Obstructive Sleep Apnea Using Mass Spectrometry based 70 Differentiating Isomeric Deprotonated Glucuronide Drug Proteomics Metabolites via Ion/Molecule Reactions in Tandem Mass Devika Channaveerappa, Clarkson University, CU BOX 5810, 8 Spectrometry Clarkson Ave., Potsdam, NY 13699, Jacob Lux, Meredith McLerie, Brian John Y. Kong, Merck & Co., MS: RY818-B112, 126 E. Lincoln Ave., K. Panama, Masonic Medical Research Laboratory, Costel C. Darie Rahway, NJ 07065, Zaikuan Yu, Mckay W. Easton, Edouard Niyonsaba, Obstructive sleep apnea (OSA) affects up to 24% of the adult population and is Xin Ma, Ravikiran Yerabolu, Huaming Sheng, Tiffany M. Jarrell, associated with several atrial diseases. It is characterized by transient cessations Zhoupeng Zhang, Arun K. Ghosh, Hilkka I. Kenttämaa in respiration lasting >10 seconds due to narrowing or occlusion of the upper air- Glucuronidation, the most common conjugation reaction in phase II of drug metabo- way during sleep. Although clinical evidence linking OSA to proarrhythmaic atrial lism, produces water soluble substrates known as glucuronides. In some instances changes is well known, the molecular mechanisms by which OSA causes atrial dis- where a parent drug, or its intermediate, contains both O- and N-heteroatoms, glu- ease remain elusive. We have initiated a recently developed rat model which closely curonidation can occur at either site, producing glucuronides with the same molecu- recapitulates the characteristics of OSA, to study OSA-induced cardiac changes. lar weight. Analytical techniques commonly used for the identification of isomers like Male Sprague Dawley rats, aged 50-70 days, received surgically implanted tracheal these include X-ray crystallography and NMR spectroscopy. However, due to the balloons which were inflated to cause transient airway obstructions. Apnea groups need for high quantities of relatively pure compound, utilizing these techniques may experienced 60 apneas per hour of either 13 seconds (moderate apnea) or 23 sec- not be feasible for elucidating structures of trace compounds in complex metabolite onds (severe apnea) for 2 weeks and 8 hours per day. Control rats received surger- mixtures. A tandem mass spectrometric method based on a novel gas-phase ion/ ies but no inflations. Proteomics analysis was done on the rat atria homogenates molecule reaction of deprotonated O- and N-glucuronide drug metabolites with tri- to identify dysregulated proteins in moderate and severe apnea when compared to chlorosilane (HSiCl3) was explored in a linear quadrupole ion trap mass spectrom- control. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) eter that readily enables the differentiation of O- and N-isomers. Upon reactions of was performed and the gel bands were trypsin digested to obtain the peptide mix- HSiCl3 with deprotonated O- and N-glucuronides, a product ion which corresponds tures. The peptides were analyzed by a Nano Acquity UPLC coupled with Xevo G2 to a HSiCl3 adduct that has lost one HCl molecule ([M-H+HSiCl3-HCl]) was ob- Mass Spectrometer. The proteomics analysis revealed that 3 of the 9 enzymes in served. However, the major product ion observed upon reactions of HSiCl3 with glycolysis and 2 proteins related to oxidative phosphorylation were down-regulat- deprotonated N-glucuronides was a diagnostic HSiCl3 adduct that has lost two HCl ed in the severe apnea group. In contrast, several structural and pro-hypertrophic molecules ([M-H+HSiCl3-2HCl]). This product ion was not observed for deproton- proteins were up-regulated with chronic OSA. The data suggests the chronic OSA ated O-glucuronides. Proposed reaction mechanisms were explored with quantum causes protein changes which lead to cessation of glycolysis, a diminished capac- chemical calculations at the M06-2X/6-311++G(d,p) level of theory to validate each ity to generate reducing equivalents (i.e., NADH) as well as promotion of cardiac reaction pathway. hypertrophy.

71 Characterization of Anionic and Cationic Metabolites in a Single 74 GC-MS-Based Untargeted Metabolomics Workflow for Biomarker Embryonic Cell (Xenopus laevis) Using CE-ESI-MS Discovery in Crohn’s Disease Erika P. Portero, University of Maryland, Chemistry Department, 8051 Xin Zheng, Thermo Fisher Scientific, 2215 Grand Ave Pkwy, Austin, TX Regents Dr., College Park, MD 20742, Peter Nemes 78728, Suresh Seethapathy, Jason Cole Metabolic profiling of individual cells during embryogenesis provides molecular Crohn’s disease (CD) is a subtype of inflammatory bowel diseases (IBDs) often information to enhance our understanding of developmental processes. However, thought to be a result of genetic predisposition and environmental factors. Despite detection of anionic compounds using capillary electrophoresis mass spectrometry extensive research, the etiology and pathogenesis remain largely unknown. Gas (CE-MS) presents a challenge due to electrical discharges during electrospray ion- chromatography-mass spectrometry (GC-MS) is one of the most commonly used ization, lowering technical reproducibility and hindering results interpretation. Here, analytical platforms for metabolomics studies due to its high sensitivity and low we enabled the characterization of anionic and cationic metabolites from single cells detection limits. In this study, 18 individual serum samples including six healthy with high technical and biological reproducibility. First, we used fabricated borosil- donors, six CD donors not taking any medications, and six CD donors treated with icate microcapillaries to withdraw ~10 nL cellular content of the ventral V1L cell in infliximab were derivatized, and analyzed on a GC coupled to a quadrupole-Orbitrap the 8-cell Xenopus laevis (frog) embryo. We extracted small molecules using 40% mass spectrometer with electron ionization interface. Compound identification was acetonitrile and 40% methanol and measured them using a custom-built capillary achieved using spectral deconvolution software equipped with a GC-Orbitrap™ electrophoresis- electrospray ionization (ESI) platform coupled to a time-of-flight high-resolution accurate mass (HRAM) metabolomics library. Chemical ionization mass spectrometer. To maintain electrospray stability in the negative ionization was also performed to generate pseudo-molecular ion information for unknown mode, we used a controlled nitrogen gas flow at the inlet of the mass spectrometer. identification. Excellent mass accuracy (< 1ppm) and ultra-high resolution (>60,000) We detected ~200 different cationic molecular features and ~150 anionic features, offered on the Orbitrap™ analyzer allowed confident molecular formula elucidation of which 70 cationic molecular features and 25 anionic features were identified with for novel biomarkers. Significantly changed metabolites such as amino acids and high confidence as small molecules (metabolites) based on accurate mass mea- TCA cycle metabolites were detected in CD donor serums. Differential analysis was surements, tandem MS, migration time comparison to standards and MS–MS/MS employed on detected metabolite perturbations by using Thermo Scientific™ Com- databases. Furthermore, pathway analysis conducted on the resulting metadata re- pound Discoverer™ software. These metabolomic changes are often associated vealed the arginine and proline metabolism pathway (p-value 2.24 x10-12) as well with inflammation. These inflammation-driven changes were minimized when CD as the glycine, serine, and threonine metabolism pathway (p-value 5.13 x10-10) donors were treated with infliximab, an immunosuppressive drug prescribed for CD. of significant coverage, suggesting the importance of these pathways during early For Research Use Only. Not for use in diagnostic procedures. development of the embryo. 75 Transfer and Scaling of a USP Assay for Quetiapine Fumarate 72 Mass Spectrometry Based Proteomics to Investigate and across Liquid Chromatographic Systems Characterize the Jumping Translocation Breakpoint (JTB) Protein Amanda Dlugasch, Waters Corp., 34 Maple St., Milford, MA 01757, Using Cancer Cell Lines Jennifer Simeone Madhuri Jayathirtha, Clarkson University, Box 5810, 8 Clarkson Ave., Pharmaceutical companies often follow current United States Pharmacopeia (USP) Potsdam, NY 13699, Devika Channaveerappa, Kangning Li, Costel methods for the analysis of raw materials and finished products. Many USP meth- C. Darie ods employ high-performance liquid chromatography (HPLC) columns and condi- MCF7 breast cancer cell lines were transfected with the sense and antisense ori- tions which use high flow rates and result in long runtimes. Although the conditions entation of the JTB cDNA in HA, His and FLAG tagged CMV expression vector. are selected for HPLC columns, the methods are often capable of being run on The expression of JTB was confirmed by western blotting. Proteins extracted from lower dispersion, higher pressure systems such as UHPLC and UPLC systems, as transiently transfected cells were separated using sodium dodecyl sulfate–poly- long as the system suitability requirements are met. In addition, the USP has out- acrylamide gel electrophoresis (SDS-PAGE) and the in-gel digested peptides were lined in the USP General Chapter <621> acceptable method adjustments to scale analyzed by a Nano Acquity UPLC coupled with Xevo G2 Mass Spectrometer. Data isocratic methods to provide the same if not improved performance. These allow- 12 2018 EAS Abstracts November 2018

able adjustments include scaling particle size and column dimensions to maintain L/ 80 Introduction to and Overview of Green Analytical Chemistry dp ratio, where L is the length of the column and dp is the particle size of the pack- Joe Foley, Drexel University, Department of Chemistry, 305 Disque Hall, ing material, and adjusting the flow rate and injection volume accordingly. In this 32 South 32nd St., Philadelphia, PA 19104 study, the USP assay method for quetiapine fumarate, an anti-psychotic drug, will The origins of green chemistry and its application to analytical chemistry, i.e., green be evaluated on a wide range of systems including an HPLC, a UHPLC and a UPLC analytical chemistry (GAC), are introduced. Metrics that have been proposed to liquid chromatographic system. This isocratic method will be evaluated under the objectively quantify the “greenness” of a variety of analytical techniques are then conditions as stated per the USP Monograph: Quetiapine Fumarate (USP40-NF35 surveyed. While two key analytical techniques, sample preparation and liquid chro- Page 5939). After evaluation of the original method on all systems, the method matography, have received the most attention with respect to GAC and are em- will be scaled following the USP allowable changes. The results will demonstrate phasized in the remaining talks in this session, it is nonetheless possible to apply the ability to decrease run times and improve throughput by scaling a method to a a green approach to other analytical techniques, and this is the emphasis in the column which uses a smaller particle size. current presentation. Finally, two alternatives to multidimensional liquid chromatog- raphy, one of which was developed in our laboratory, are briefly introduced. 76 New Insights into Marine Aerosols by Mass Spectrometry Kimberly Prather, University of California-San Diego, 9500 Gilman Dr., 81 Recent Developments on Solid Phase Microextraction, a Green #0314, Dept. of Chemistry, La Jolla, CA 92093 Sample Preparation Tool for On-Site, In-Vivo, and Complex No abstract submitted by the author. Matrices Analysis Nathaly Reyes-Garcés, University of Waterloo, 200 University Ave. 77 Nanoparticles in the Air We Breathe: Pristine or Polluted? West, Waterloo, ON N2L 3G1, Canada, Janusz Pawliszyn Murray V. Johnston, University of Delaware, Department of Chemistry The development of analytical workflows capable of providing high quality data with and Biochemistry, Newark, DE 19716 lower environmental impact is garnering a wealth of interest in the scientific com- Airborne nanoparticles strongly influence climate and human health. Understanding munity. Undoubtedly, significant achievements in this regard are attainable through these impacts requires knowledge of how particles form and grow. This presentation the implementation of greener sample preparation approaches that enable a sig- draws from recent ambient measurements and laboratory studies by our group that nificant reduction in organic solvent usage. Solid phase microextraction (SPME), a help elucidate chemical mechanisms of nanoparticle growth from small molecular sample preparation tool that combines sampling and sample preparation in a single clusters to a particle that is large enough to serve as a seed for cloud droplet forma- step, has demonstrated its usefulness in the quantitative determination of a broad tion. These studies rely on the development and use of advanced mass spectrom- range of analytes, while significantly reducing the use of organic solvents. Due to etry techniques, and they give insight into what’s in the air we breathe – whether in its simplicity, SPME facilitates on-site analysis which allows for bypassing the step a pristine or polluted location. of taking the sample to the lab in several cases. Recent advances done in SPME involve the introduction of biocompatible coating materials that not only permit the 78 Widening the Window for Environmental Analysis: On-Line HPLC direct immersion of SPME devices in complex matrices but also offer high selectivity Monitored by 21 Tesla Fourier Transform Ion Cyclotron Resonance for small compounds. Such coatings, in combination with chromatography and/or Mass Spectrometry mass spectrometry instrumentation, have facilitated the development of fast and Alan G. Marshall, Florida State University, NHMFL, 1800 E. Paul Dirac robust methodologies for the analysis of multiple analytes in environmental sam- Dr., Tallahassee, FL 32310, Martha L. Chacon Patino, Yuri E. Corilo, ples, food commodities, biological matrices, and living systems, among others. This Christopher L. Hendrickson, Logan C. Krajewski, Sydney F. Niles, presentation discusses the latest progress on the development of SPME devices Jonathan C. Putman, Steven M. Rowland, Donald F. Smith, Rebecca L. (geometries and coatings) and their application in different challenging analytical Ware, Chad R. Weisbrod, Ryan P. Rodgers scenarios. Special emphasis is given to the features that make of SPME a green Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR MS) offers at sample preparation technique. least 10X times higher mass resolving power than any other mass analyzer, and is thus the mass analyzer of choice for complex mixture analysis. Accurate (sub-ppm) 82 Greenness through Modernized Separation Methods: Introduction mass measurement yields elemental composition, numbers of N, O, and S (hetero) of the Analytical Method Greenness Score (AMGS) Calculator for atoms (i.e., compound “class”), number of rings plus double bonds (DBE, or “type”), Greener Methods and carbon number distribution (yielding the degree of alkylation) for molecules up Michael B. Hicks, Merck & Co., MS: RY801-C101, 126 East Lincoln to ~1,500 Da. We have assigned more than 125,000 peaks in a single petroleum Ave. Rahway, NJ 07738, William Farrell, Christine Aurigemma, Laurent 9.4 T FT-ICR mass spectrum. Data may be visualized from various graphical im- Lehman, Kelly Nadeau, Lauren Weisel, Heewon Lee, Carol Moraff- ages scaled according to ion relative abundance: e.g., class distribution, Kendrick Gingsburg, Mengling Wong, Paul Ferguson plot, van Krevelen plot, DBE vs. carbon number, etc. Environmental applications Green and sustainable efforts have been emphasized for over 25 years to develop include characterization of oil:water emulsions, alternative fuels, crude oil plumes and manufacture pharmaceuticals using earth-friendly and environmentally respon- and chemical evolution of oil spills. This talk reports recent FT-ICR MS instrumen- sible means. These include the major analytical chemistry techniques used for phar- tation developments, including imprinted polymers to extract naphthenic acids, and maceutical characterization. Over the last decade, high-pressure liquid (HPLC) and on-line liquid chromatography (LC) FT-ICR MS at 21 tesla (highest field for ICR) supercritical fluid (SFC) chromatography techniques have undergone substantial applied to characterization of complex organic mixtures. Acknowledgments: Work improvements to instrumentation (UHPLC; ultra-performance convergence chro- supported by NSF Division of Materials Research through NSF DMR-11-57490 and matography or UPC2) and smaller column particle technologies, all of which lead DMR-1644779, The Gulf of Mexico Research Initiative to the Deep-C Consortium, to faster and more efficient separations while reducing development and validation the Florida State University Future Fuels Institute, and the State of Florida. time for both chiral and achiral analysis methods. The pharmaceutical industry as a whole is gradually transitioning away from traditional chromatographic methodol- 79 Advances in Atmospheric Halogen Chemistry by Chemical ogies that include the use of longer columns, extended run times, toxic non-polar Ionization Mass Spectrometry solvents (e.g., heptanes and hexanes for normal-phase liquid chromatography) Kerri A. Pratt, University of Michigan, 930 N. University Ave., Ann Arbor, and their accompanying large solvent volume consumption while adopting more MI 48104, Angela Raso, Siyuan Wang, Stephen McNamara, Kyle efficient, greener solutions. However, many of these advancements have yet to be Custard, Paul Shepson more broadly applied in later stage development and will require a more global Due to rapid transformation and loss of sea ice in the Arctic, there is an urgent need adaptation to benefit from these improvements. Here, we introduce a new green to characterize the changing atmospheric composition impacted by multiphase re- and sustainable calculator to definitively establish the impact process chemists and actions occurring at the frozen snow-covered sea ice surface. We conduct field- analytical separation scientists will have based on method design and instrument based measurements of trace atmospheric gases, at low pmol mol-1 (ppt) levels, selection choices. Our aim is to provide awareness and guidance to the global in- to gain insights into unique atmospheric halogen photochemistry that dramatically dustry at large to select more modernized column packing chemistries, materials impacts the fates of greenhouse gases (ozone and methane) and atmospheric pol- and instruments to ultimately allow for shorter methods, more modernized safer lutants (mercury and hydrocarbons). During three recent field deployments of our and more environmentally-friendly solvent alternatives with comparable separation chemical ionization mass spectrometer near Utqiaġvik, Alaska in the polar spring, efficiencies. we measured several bromine, chlorine, and iodine-containing trace gases for the first time in the Arctic troposphere. Notably, we discovered the photochemical snow- 83 Greener Process Characterization of Biotherapeutics Using Multi pack production of BrCl, Cl2, and I2, similar to our previous finding for Br2. The Attribute Method importance of these species in springtime Arctic atmospheric composition and their Bhumit Patel, Merck & Co., MS: K15, B-404C, 2015 Galloping Hill Rd., levels in the near-surface troposphere is discussed. Kenilworth, NJ 07033, Yi Wang, Mark Brower, Yan-Hui Liu, Douglas Richardson Therapeutic monoclonal antibodies are complex heterogeneous biologics. In-depth 13 2018 EAS Abstracts November 2018

characterization and monitoring of the critical quality attributes (CQAs) of these 87 Mechanistic Aspects of Chiral Discrimination with Sulfated beta biotherapeutics is essential to ensure that the protein drug products meet desired Cyclodextrin quality during manufacturing process and for use in clinical trials. Therefore, numer- Nelu Grinberg, Grinberg Consulting, Mail-stop: 274 Aspetuck Ridge Rd., ous analytical assays are needed for characterization and release. High-resolution New Milford, CT 06776, Ling Wu, Nizar Haddad, Boehringer Ingelheim liquid chromatography- mass spectrometry based multi-attribute method (MAM) has Pharmaceuticals been utilized for the analysis of multiple attributes simultaneously. These attributes A fast enantiomeric separation of a chiral aromatic amine was achieved, using ul- include post translation modifications (oxidation, deamidation, etc), glycans, frag- tra-high-pressure liquid chromatography and highly sulfated beta-cyclodextrin (S- ments, and process impurities. More importantly, MAM can be used in all stages -CD) as a chiral additive in the mobile phase. The stationary phase consisted of a of biologics development and can potentially replace several less green methods core–shell support with a particle size of 2.7 m. Under these conditions the base- currently used in routine in-process and quality control labs. Data generated from line separation was obtained within 2.5 min. The influence of the concentration of this “greener” MAM method is orthogonal and comparable to conventional assays. the additive, along with the thermodynamics of the separation, was studied. Vibra- Quality attributes were also monitored from process intermediates and stability sam- tional circular dichroism (VCD) spectroscopy was applied to assess the absolute ples by MAM. Lastly, case studies of MAM in continuous processing of biologics, configuration of the two enantiomeric analytes, as well as the interaction of these which combines green analytics with green process, are presented. enantiomers with the Sulfated beta Cyclodextrin (S-beta-CD). The VCD results re- vealed that S-beta -CD undergoes a temperature-induced conformational change. 84 Ultrafast Chiral Separations for High-Throughput Enantiopurity Further, VCD experiments indicate that the interactions of the two enantiomers with Analysis the S-beta -CD occur through an inclusion of the aromatic part of the analyte, as well Erik L. Regalado, Merck & Co., 126 E. Lincoln Ave., Rahway, NJ 07065, as through electrostatic interaction between the protonated amine and the sulfate Gregory F. Pirrone, Alexey A. Makarov, Leo A. Joyce, Christopher J. groups located at the narrow part of the S-beta -CD. Molecular mechanics calcula- Welch, Daniel W. Armstrong, Chandan L. Barhate tions performed according to the VCD results are consistent with experimental data, Recent developments in fast chromatographic enantioseparations now make providing further evidence of these interactions. high-throughput analysis of enantiopurity on the order of a few seconds achievable. Nevertheless, routine chromatographic determinations of enantiopurity to support 88 Emerging Methods in 19F-NMR to Characterize Proteins and Small stereochemical investigations in pharmaceutical research and development, syn- Molecules thetic chemistry and bioanalysis are still typically performed on the 5–20 min times- Haribabu Arthanari, Harvard Medical School/DFCI, MS: LC-3311, 450 cale, with many practitioners believing that sub-minute enantioseparations are not Brookline Ave., Boston, MA 02215 representative of the molecules encountered in day to day research. In this study Fluorine-19 (19F) nuclear magnetic resonance (NMR) is emerging as a novel we develop ultrafast chromatographic enantioseparations for a variety of pharma- method to detect weak interactions in biomolecules and to study the structure and ceutically-related drugs and intermediates, showing that sub-minute resolutions are dynamics of proteins and nucleic acids. The power of 19F labeling in protein appli- now possible in the vast majority of cases by both supercritical fluid chromatography cations has been demonstrated by the study of G-protein-coupled receptor (GPCR) (SFC) and reversed phase liquid chromatography (RP-LC). Examples are provided dynamics using site-specific-CF3 labeling to monitor conformational changes and illustrating how such methods can be routinely developed and used for ultrafast by measuring kinetic and thermodynamic parameters that dictate function. 19F is high-throughput analysis to support enantioselective synthesis investigations. not naturally present in biomolecules, rendering 19F NMR background-free and thus especially useful for in-cell NMR experiments, as signals from 1H, 13C and 85 Circular Dichroism Spectroscopy as a Tool to Solve Complex 15N typically suffer from background signals generated by other endogenous bi- Stereochemical Problems in the Pharmaceutical Industry ological molecules. 19F is also a key nucleus in NMR based fragment screening Leo A. Joyce, Merck & Co, MS: RY800-D370, 126 E. Lincoln Ave., using 19F bearing fragments and in the investigation of small-molecule interactions Rahway, NJ 07065 with proteins. Though the chemical shift of the 19F nuclei is extremely sensitive to The last few decades has seen numerous asymmetric synthetic advances, with a the surrounding electronic environment, its large CSA and the broad chemical shift litany of novel and innovative synthetic methodologies reported. While these de- range limit the use of 19F nuclei. Here we present novel NMR methods to combat velopments have given researchers the ability to access many different structural these two challenges of detecting 19F. motifs, the determination of absolute configuration still remains an important and challenging task. Traditionally, optical rotation (OR) has been used as a way to track 89 Nonuniform Sampling for Sensitivity Enhancement in absolute configuration in synthesis. This approach can prove problematic when the Multidimensional NMR purity of the compound is not taken into consideration, with small impurities giving Jeffrey C. Hoch, University of Connecticut Health, 263 Farmington Ave., rise to erroneous results. The use of circular dichroism spectroscopy, both vibra- Farmington, CT 06030 tional (VCD) and electronic (ECD) varieties, has emerged as a robust technique The use of nonuniform sampling (NUS) is becoming increasingly common to en- well-suited to address these challenges. This approach combines computational hance resolution or save measuring time in multidimensional nuclear magnetic and experimental routines to avoid errors and ensure that assignments are made resonance (NMR) experiments. NUS can also be used to enhance sensitivity, by correctly. This presentation covers the application of these techniques within the concentrating sampling on parts of the signal where it is strongest, and using time context of organic synthesis. We highlight cases where the assignment of abso- saved by not sampling some tuples of indirect evolution times to perform additional lute configuration led to a better understanding of the stereochemical course of signal averaging. Linear theory predicts the sensitivity gains over time-equivalent reactions. Then we turn our attention toward challenging substrates where initial uniform sampling are related to the amount of signal power captured by the sam- efforts to make an assignment were unsuccessful. Finally, we cover recent efforts pling scheme, relative to the noise power sampled, and imply that gains can only to obviate preparative separation and provide direct confirmation and quantitation be achieved when the signal envelope is not constant. Additional sensitivity gains of compounds in a mixture. may accrue from nonlinearity of non-Fourier methods used to analyze NUS data. Relevant aspects of NUS and non-Fourier spectrum analysis are discussed, and 86 Insights on Chiral Recognition for Enantiomeric Separation on strategies for improving sensitivity described. An example of nonlinear improvement Teicoplanin Columns in sensitivity will be presented. Ling Wu, Boehringer Ingelheim Pharmaceuticals, 900 Ridgebury Rd., Ridgefield, CT 06877, Nelu Grinberg, Shengli Ma, Sherry Shen, Nina 90 Fast NMR Techniques for Structure Elucidation of Small Molecules Gonella, Heewon Lee, David Bell Eriks Kupce, Bruker UK Ltd., Banner Lane, Coventry, CV4 9GH, United The chiral separation of propranolol was studied on two macrocyclic antibiotic Kingdom phases, Teicioplanin (T2) and Teicoplanin Aglycone (Tag) columns. It was found Getting the most information out of experiments in the shortest possible time has that the retention order of the two enantiomers was reversed on T2 compared to always been of interest in nuclear magnetic resonance (NMR). One successful ap- TAG when using methanol/water as a mobile phase. A conformational change of proach to achieve this has been merging two or more experiments into a single T2 was observed and it was studied using vibrational circular dichroism (VCD). To experiment or recording them in parallel and thus extracting several spectra from understand the chiral recognition mechanism, the thermodynamic parameters of the the same data set. The recent introduction of NMR spectrometers with multiple separation were also studied for the two stationary phases. VCD was further applied receivers opens new possibilities of simultaneous recording spectra from different to assess the interaction of these enantiomers with the T2 and TAG columns. nuclear species. Here we propose several new experiments for structure elucida- tion of small molecules along with new techniques for reducing the measurement time needed to obtain their structure. Two new and related methods, 2BOB and H2OBC, for tracking out the backbone of protonated 13C nuclei are presented. We also propose a 13C detected experiment, the (H)CNMQC pulse sequence for measuring one-bond and long range 15N-13C scalar coupling constants in small organic molecules at the natural isotopic abundance. The core 2D NMR techniques 14 2018 EAS Abstracts November 2018

routinely employed for small molecule analysis and structure elucidation, such as 95 Universal Detection of Body Fluid Traces In-Situ with Raman HSQC, HMQC, HMBC, COSY, NOESY, TOCSY, and similar, can be recorded in Hyperspectroscopy for Forensic Purposes a single measurement by combining up to five pulse sequences into a single su- Gregory Mclaughlin, University at Albany-SUNY, 1400 Washington Ave., per-sequence dubbed NOAH (NMR by Ordered Acquisition using H-detection). In Albany, NY 12222, Marisia Fikiet, Masahiro Ando, Hiro-o Hamaguchi, this way the data collection time may be dramatically reduced allowing fast structure Igor Lednev elucidation of organic molecules from a single measurement. The application of the Raman spectroscopy has been a boon to the field of forensic science. One area that NOAH-3 and NOAH-4 experiments is exemplified here through their use in comput- this technique shows exceptional promise is in body fluid identification and charac- er-assisted structure elucidation. terization, but substrate interference remains a major impediment to its practical implementation. Here, we present an approach for the universal detection of body 91 Advanced NMR Techniques for Challenging Structural Assignment fluids regardless of the substrate. This approach, which is based on Ramanhy- Problems perspectroscopy and multivariate curve resolution (MCR), was applied to datasets Gary Martin, Seton Hall University, 400 South Orange Ave, South representing simulated semen evidence. In every instance, the signal of the fluid Orange, NJ 07079 was extracted and matched to a reference of semen. This approach has immediate No abstract submitted by the author. application for body fluid detection and allows for a universal, automatic, nonde- structive, on-field method for confirmatory identification of body fluid traces ata 92 Forensic Application of Attenuated Total Reflection Fourier crime scene. This approach is applicable where an analyte is either a minor contrib- Transform-Infrared (ATR FT-IR) Spectroscopy for Bloodstain utor or spatially distributed on a strongly interfering substrate. Analysis Ewelina M. Mistek, University at Albany-SUNY, 1400 Washington Ave., 96 Blood Alcohol and Inhalant Analysis by Gas Chromatography - Albany, NY 12222, Igor K. Lednev Vacuum Ultraviolet Spectroscopy Bloodstain identification and characterization is one of the most important steps James A. Diekmann III, VUV Analytics, 715 Discovery Blvd., Cedar during forensic investigation involving violent acts. Current standard methods em- Park, TX 78613, Jack Cochran ployed for the analysis of blood samples are destructive and time-consuming. Here, The analysis of ethanol and abused inhalants in blood is a common routine per- attenuated total reflection Fourier transform-infrared (ATR FT-IR) spectroscopy was formed by laboratories across the nation. Analysis of these volatile organic com- used for characterization of bloodstains in a confirmatory, nondestructive, and rapid pounds (VOCs) is often done with static headspace (SHS) – gas chromatography manner. This technique, in combination with advanced statistical analysis, shows (GC) – flame ionization detection (FID). Given that FID is non-specific, two different potential for differentiating between human and animal (non-human) blood as well GC runs may be required to mitigate coelutions, which can inhibit analytical through- as distinguishing between menstrual and peripheral blood. Partial least squares put and could lead to false positives without a confirmatory technique, such as mass discriminant analysis (PLSDA) models demonstrated complete separation between spectrometry. Vacuum ultraviolet (VUV) spectroscopy is a new, fast-acquiring, sen- human, cat, and dog. The model showed 100% accuracy for classification predic- sitive GC detector that collects absorbance spectra from 125nm to 240nm. Absor- tions of external unknown blood donors. Another PLSDA model demonstrated entire bance in this region of the electromagnetic spectrum is suitable for GC amenable distinction between menstrual and peripheral blood with accurate classification of all compounds, producing unique spectral fingerprints. VUV spectroscopy is not limited external samples. This study demonstrates ATR FT-IR spectroscopy’s great poten- by carrier gas flow rates, allowing for chromatographic compression which decreas- tial for nondestructive and rapid bloodstain analysis. The method shows potential es run times and increases throughput. Deconvolution can also be accomplished for the implementation of bloodstain analyses both at the crime scene and in the lab for coeluting VOCs because of their unique spectral fingerprints, even for isomeric due to availability of portable ATR FT-IR instruments. species. This study focuses on coupling SHS-GC with VUV as an alternative solu- tion to current blood alcohol and inhalant analysis techniques, using a single-column 93 The Detection of Organic Gunshot Residue Using Raman solution. The unique and robust spectral fingerprints are used both to identify VOCs Spectroscopy and Fluorescence present in samples containing inhalants and ethanol, and to quantify each com- Shelby R. Khandasammy, University at Albany-SUNY, 1400 Washington pound of interest using the Beer-Lambert law. Ave., Albany, NY 12222, Alex Rzhevskii, Igor K. Lednev Gunshot residue (GSR) is an important type of trace evidence that is produced 97 Solid Phase Microextraction-DART-MS Screening for Controlled upon the firing of a gun and is comprised of two main components: organic gunshot Dangerous Substances in Complex Matrices residue (OGSR) and inorganic gunshot residue (IGSR). OGSR originates from the Eileen Eubank, MRIGlobal, 65 W. Watkins Mill Rd., Gaithersburg, MD propellant of an ammunition cartridge, meanwhile IGSR originates from the primer. 20878, Janelle Newman, Jeremy Zehr, Joseph Trimboli Traditionally, scanning electron microscope/energy-dispersive X-ray (SEM-EDX) Testing physiological samples for drugs of abuse is responsible for a large number has been used to examine IGSR and thus to overall confirm the presence of GSR. of forensic analyses each year. A major challenge in these cases is that no single However, with recent changes to the elemental composition of IGSR particles in the rapid, broad-spectrum technique is available for screening of biological samples for form of lead-free ammunition types new methods for GSR analysis are necessary. drugs of abuse and associated metabolites. MRIGlobal has addressed this need OGSR detection and identification have become areas of interest as OGSR parti- through development of a screening method that combines solid phase micro-ex- cles may provide an important source of complementary evidence to IGSR anal- traction (SPME) with direct analysis in real time mass spectrometry (DART-MS). yses. In this study, OGSR detection and characterization methods were explored Samples are simultaneously extracted and pre-concentrated using SPME. This using Raman spectroscopic and fluorescence characterizations. increases the concentration of analyte introduced to the mass spectrometer and reduces the background associated with common biological matrix interferences, 94 Raman Spectroscopy for Forensic Bloodstain Identification: such as urea in urine, while minimizing hands-on sample preparation time. Di- Method Validation vs. Environmental Interferences rect-immersion SPME was employed to screen controlled substances and their Robert B. Rosenblatt, University at Albany-SUNY, 23-84 28th St., metabolites. DART-MS analysis of the SPME fiber allows sample introduction in Astoria, NY 11105, Kyle C. Doty, Lenka Halámková, Igor K. Lednev ambient pressure and provides target identification based on exact mass measure- The identification of bodily fluid (BF) stains at a crime scene is a necessary part of ments from time-of-flight mass spectrometry. This experimental approach resulted evidence evaluation, but currently has many inherent complexities. This research in an extremely rapid and highly discriminatory analytical method that minimizes proves that Raman spectroscopy is a more viable method for testing BF stains than the need for multiple immunoassay and chromatography-based screening panels. other methods currently in use, specifically for it not being susceptible to false posi- tive assignments. Currently, different confirmatory tests need to be used for each of 98 Evaluating Practical Uses of Molecular Isotopic Engineering (MIE): the five most common BFs (blood, saliva, semen, sweat, and vaginal fluid). These Authenticity, Security, and Intellectual Property Considerations tests also do not serve the purpose of identifying unknown BFs. Raman spectros- John P. Jasper, Molecular Isotope Technologies LLC, 8 Old Oak Lane, copy is advantageous due to its chemical selectivity, providing unique spectra for all Niantic, CT 06357, Peter Farina, Ann Pearson, Peter S. Mezes, Anthony substances analyzed and the ability to correctly identify all types of BFs. By building D. Sabatelli statistical models, and comparing spectra of BFs to potential false positive sub- Molecular isotopic engineering (MIE) is the directed stable-isotopic synthesis of stances, all BF stains can be correctly identified with one test. This study specifically chemical products for reasons of product authentication and of product security, analyzed twenty-four substances that may be misclassified as blood due to their and also for intellectual property considerations. We report here the successful appearance or if known to provide a FP result with currently used tests. directed synthesis of naproxen from its immediate precursors. We find excellent correspondence between the observed and predicted stable-isotopic results (d13C, d18O, and dD). The observed carbon-isotopic results are readily explained by the laws of mass balance and isotope mass balance. By contrast, the oxygen- and hydrogen isotopic results require an additional assessment of the effects of O and H exchange, presumably due interaction with reaction-solution water. A previous, 15 2018 EAS Abstracts November 2018

cooperative study with the United States Food and Drug Administration- Division of study of materials, mainly by NMR spectroscopy sometimes augmented with other Pharmaceutical Analysis showed that individual manufacturers of naproxen could techniques. On moving to Delaware, he became a member of SAS that brought as- readily be differentiated by their stable-isotopic provenance (d13C, d18O, and dD; sociations with many that broadened his view of science and continued to keep his Wokovich et al., 2005). Results from two out of three of the naproxen samples pro- research vital. The talk provides glimpses of the travels during that time, including duced for this study correspond well to the naproxen results observed in the coop- a few dead ends erative study. A third does not readily correspond to any of the samples observed in the cooperative study because no associated naproxen appears to have produced 102 Applying Vibrational Spectroscopy in Usual and Unusual Ways in from such a starting material. The general correspondence of the present and pre- Pharmaceutical Sciences vious naproxen data illustrates the plausibility of MIE. We suggest that MIE can be John Wasylyk, Bristol-Myers Squibb, One Squibb Dr., New Brunswick, readily employed in the bio/pharmaceutical industry without alteration of present NJ 08901 manufacturing processes other than isotopically selecting and/or monitoring reac- Many early scientists who studied vibrational spectroscopy had to develop and build tants and products. their own instruments to be able to record their measurements which created a host of additional challenges, thus hindering accurate data that could be compared from 99 Confirmatory Method Optimization for the Analysis of Thirty lab to lab. It was not until the mid-twentieth century that companies rose to the chal- Fentanyl Analogues via Gas Chromatography-Mass Spectrometry lenge of developing optical instruments and eventually created the some of the first Delilah DeWilde, Cedar Crest College, 6228 Simms St., Arvada, CO spectrometers that could expand their scope and versatility. With the development 80004, Matthew Wood, Thomas Brettell, Thomas Pritchett of these commercial spectrometers it became popular to determine the “fingerprint” Forensic drug chemists are responsible for reporting the composition of commonly for any molecule. Over the last quarter of a century, the application of spectrometers seized drugs. The recent opioid epidemic has resulted in many commonly seized has exponentially grown, from identifying pure molecules to complex mixtures to drugs such as heroin, cocaine, and marijuana being observed laced with fentanyl following ‘live’ chemical reactions to identifying trace molecules on material on a and fentanyl analogues. The United States Drug Enforcement Agency (DEA) in a conveyer belt. The talk focuses on some of the more recent advances of vibrational 2017 report about new psychoactive substances (NPS) drugs reported a 117% in- spectroscopy and how it has positively impacted our knowledge in several fields crease from 2016 in opioid drugs, of which fentanyl accounted for 66% of those drug including fundamental chemistry and pharmaceutical sciences. We have come a identifications. The fentanyl epidemic includes fentanyl analogues that are being long way since Isaac Newton first applied the word ‘spectrum’ to describe the rain- synthesized in clandestine laboratories, some of which may not be considered ille- bow of colors when observing light through a prism and based recent advances, gal due to lag time for legislation. The close structural similarity of these analogues we anticipate the expansion of applications of spectroscopy to not slow down in makes the identification challenging. Drug chemists typically use gas chromatog- the near future. raphy-mass spectrometry (GC-MS) to identify these compounds in drug seizures. This presentation discusses a confirmatory method using GC-MS that has been 103 NYSAS Celebrates 60+ Years of Scientific Collaboration and developed for the analysis of 30 fentanyl analogues. The fentanyl standards includ- Discusses the Impact of Chemometrics on the Evolution of Raman ed in optimizing this method were fentanyl, crotonyl fentanyl, acetyl fentanyl, butyryl Instrumentation fentanyl, para-fluorofentanyl, meta-fluorofentanyl, ortho-fluorofentanyl, cis-3-meth- Howard Mark, Mark Electronics, 69 Jamie Court, Suffern, NY 10901, yl fentanyl, trans-3-methyl fentanyl, para-fluorobutyryl fentanyl, meta-fluorobutyryl Fran Adar, Deborah Peru fentanyl, ortho-fluorobutyryl fentanyl, acryl fentanyl, valeryl fentanyl, isobutyryl fen- First, the New York Section of the Society for Applied Spectroscopy (NYSAS) will tanyl, carfentanil, ocfentanil, cyclopropyl fentanyl, alfentanil, sufentanil, remifentanil, take a trip down memory lane and share some of the history behind the inception W-15, 4-ANPP, para-methoxybutyryl fentanyl, thiofentanyl, beta-hydroxythiofen- of SAS and how this Regional Section strives to meet the objectives of the organi- tanyl, alpha-methyl fentanyl, beta-methyl fentanyl, and furanyl fentanyl. A Thermo zation. We will next introduce two members who will discuss topics they have spent Scientific Trace 1310 gas chromatograph outfitted with two flame ionization detec- a lifetime mastering and using to help others. Contrary to some popular beliefs, the tion (FID) detectors and coupled with a Thermo Scientific ISQ LT single quadrupole sciences of Statistics and Chemometrics are not antagonistic to each other. Perhaps mass spectrometer was utilized to improve resolution between fentanyl standards to some chemometricians’ chagrin, Chemometrics is actually a branch of statistics. via split injection. Optimizations also focused on instrumentation variables such as In this talk, we will present an overview of the history of these two sciences and the split ratio, injection temperature, injection volume, and the oven program. explore their similarities and differences. One of the fields that has benefited greatly from the application of chemometrics is Raman spectroscopy and will be covered 100 Another Diamond Anniversary – Diamond Optics for Infrared next. A large number of technologies starting in the 1980’s enabled the practicality Spectroscopy Applications of Raman instrumentation in the hands of the analytical scientist. These included the David W. Schiering, Czitek, 6 Finance Dr., Danbury, CT 06810, John development of sharp edge and notch filters, air-cooled diode lasers with outputs A. Reffner between 850 and 350nm, multichannel detectors (IPDA and then CCD), and pow- The physical, material, and optical properties of diamond provide the most perfect erful desk-top computers. Along with these hardware developments came software optical material for infrared (IR) spectroscopy. The history of the use of diamonds for acquisition and treatment of spectra with increasing sophistication. In particular, for IR spectroscopy coincides with the history of the Society for Applied Spectros- chemometric analysis provides the ability to take advantage of large data files of copy. The first demonstration of the utility of diamond optics was reported in 1959 in complex spectra that are difficult to manage spectroscopically. Several fields where the pioneering work of Lippincott, Van Valkenburg, and Weir. These investigators, this has had an impact will be discussed. and most others until the mid-1980’s, were concerned with high pressure studies of chemical compounds. These fascinating works have illuminated the geochemistry 104 The Evolving Global Regulatory Environment - Strategic and astrochemistry fields. Although the potential of diamond cells for analytical stud- Considerations ies was recognized early, use was limited until the development of Fourier transform Kimberly Belsky, Mallinckrodt Pharmaceuticals, 1425 U.S. Route 206, IR (FT-IR) micro-analysis methods. Less expensive diamond cells were developed Bedminster, NJ 07921 for use in microscopes and beam condensers and first popularized diamond optics The evolving global regulatory environment is providing new opportunities to ad- for analytical studies. Diamond internal reflection elements (IRE) for attenuated total vance drug development and approval. At the same time, there are risks that must reflection (ATR) microscope objectives soon followed. The 1990’s saw the first use be considered as well. Evaluating these changes and integrating into the regulatory of synthetic diamonds in optical accessories for FT-IR spectrometers. Diamond ATR strategy is critical to reducing regulatory burden and mitigating unexpected regu- probes were developed to study chemical reactions under harsh conditions. The first latory hurdles. This presentation provides highlights of new and emerging health diamond ATR accessories for FT-IR spectrometers appeared in the mid-1990’s and authority initiatives to transform drug development and approval while maintaining diamond ATR spectroscopy is now the most popular method for qualitative analysis. the balance of safety and efficacy. The durability of diamond optics also provided the impetus for developing portable and hand-held FT-IR instruments in the 2000’s. This presentation reviews the his- 105 Stability Compliance for Combination Products - A Medical Device tory of the instrumental methods development and application of diamond optics in Perspective IR spectroscopy, covering pioneering work in high pressure and analytical studies. Laure L. Larkin, Ethicon Inc., MS: Bldg 60, Q-108C, PO Box 151, US Rt. 22 West, Somerville, NJ 08876 101 Fifty Years of NMR Spectroscopy and 40 Years of SAS It is not uncommon for medical device expert project teams to find that combination Cecil Dybowski, University of Delaware, Department of Chemistry and product projects land them in a regulatory cross-hair. This happens in-spite of the Biochemistry, Newark, DE 19716 fact that; by design, the medical device current good manufacturing practices (cG- In 1968, a young senior at the University of Texas started doing nuclear magnetic MP’s) allow a significant amount of strategic implementation and risk based decision resonance (NMR) experiments with Professor Charles Wade. Through a graduate making. The drug and biologic industry has very prescriptive compliance approach- career at Texas and then a postdoctoral stint at the California Institute of Technology es and Medical Device professionals are well served if they can attend conferences and a professorial career at the University of Delaware, he has been involved in the and partner intrinsically with their drug/biologics peers. In the medical device world, 16 2018 EAS Abstracts November 2018

our project teams are frequently rewarded for a Time 0 world view. Taking a United function to upper management? You are not alone. This presentation will share States (US)-centric perspective, a “combi-product newbie” thoughtfully reviewing experiences in the journey to measure and communicate value. As a coaching and 21CFR210 and 21CFR211 will find no mention of the globally harmonized regula- consulting company, Sustainable Transformations will share what works and what tory requirements from International Council for Harmonisation (ICH) such as Q1A. does not and will provide a communication template to consider for use in your This simple ICH requirement miss represents a huge gap for the combi-product organization to communicate your function’s value. newbie and could be a catastrophic product killer. Additionally, there is the some- what ambiguous regulatory path. Both the US Food & Drug Administration and the 110 Maximizing Effectiveness when Working with an External Contract International Regulating Bodies, recognize that there is a need to use scientific and Laboratory technically valid approaches to build in flexibility, so long as patient safety and effica- Jonathan Chun, Alliance Technologies, 9 Deer Park Dr., Ste. B, cy remain paramount. This discussion covers the combination product compliance Monmouth Junction, NJ 08852 aspects that can be missed even though “good faith” diligence is exercised during Companies often work with contract laboratories (CRO’s) or consultants to provide a medical device company’s first effort. It also covers examples of some strategic services that are not available internally, require special instrumentation, are not approaches and scientifically/technically justified variations to the prescriptive drug/ routine, or are more timely. Scientists and engineers often face difficult and chal- biologic regulations. lenging problems whether they are developing or producing a consumer product, pharmaceutical or specialty chemical, or troubleshooting a process or a customer 106 Regulatory Expectations in the GMP Pharmaceutical Laboratory complaint. Over the past 15 years, Alliance Technologies has developed an ap- Gayle S. Lawson, United States Food & Drug Administration, U.S. proach to problem solving that must take into account technical challenges, busi- Customhouse Room 900, 200 Chestnut St., Philadelphia, PA 19106 ness drivers, time, and budget. The most successful projects typically involve a The good manufacturing practice (GMP) analytical pharmaceutical laboratory has partnership between client and contract lab based on a few key principles. In this one sample to assure that production got it right. Understanding the laboratory con- presentation we share some practical ways to better work with a contract laboratory trols required to operate in a GMP-compliant analytical pharmaceutical laboratory or consultant to tackle difficult technical challenges. is essential to achieving regulatory and consumer confidence in a drug product. This session reviews regulatory expectations for the GMP-compliant pharmaceuti- 111 The Simplest, Most Effective, and Least Expensive Lab Safety cal laboratory and provide an overview of the laboratory controls needed to support Program GMP-compliant analytical work. The session includes a discussion on what a US James A. Kaufman, Laboratory Safety Institute, 192 Worcester St., Food & Drug Administration inspection team looks for in the laboratory including de- Natick, MA 02482 viation and out-of-specification (OOS) investigations, laboratory records, and other This interesting and entertaining presentation confronts one of the more common controls to ensure the accuracy of results and the integrity of data. We review the excuses for not having or improving the analytical laboratory safety program ... “it significance of maintaining a GMP-compliant laboratory and endeavor to bridge the costs too much.” This is simply not true. Excellent lab safety programs do not need gap between the laboratory and the patient. to cost large amounts of money. Ten simple concepts are presented to demonstrate this important theme. These are the critical components for an effective lab safe- 107 Drug Development Strategies to Meet New Regulatory Expectations ty program. Participants learn how to create a more effective lab safety program –Recent Case Studies on API Form, Elemental Impurities and without a purchase order or requisition. They learn how to evaluate their own lab Mutagenic Impurities Controls safety program using LSI’s 33 component check list. Whether you prefer to do it Yan Wu, Merck & Co., MS: WP78-210, PO Box 4, West Point, PA 19486, qualitatively or quantitatively, this presentation is guaranteed to make your program Lisa Wright better. Some programs are excellent. They will learn how to make them even better. The challenges pharmaceutical industry is facing have grown dramatically in recent Some are just at the beginning. They will find out how easily improvements can be years not only due to increased complexity in drug development but also public ex- achieved. You don’t want to miss this opportunity for a highly informative, worthwhile pectations for safer and more cost-effective drugs. As a result, regulatory agencies and enjoyable learning experience. continuously update their expectations in order to ensure product quality and safety throughout drug development and manufacturing process to put the patient first. In 112 Kinetic-Equilibria Modeling Strategies for Lab-on-a-Molecule this presentation, several recent case studies will be discussed to share drug devel- Probes opment strategies related to active pharmaceutical ingredients (API) form control, Fereshteh Emami, Southeastern Louisiana University, Department of elemental impurities control and mutagenic impurities control during drug develop- Chemistry & Physics, 10878 127W Pursley Hall, Hammond, LA 70402 ment and regulatory filings. In order to solve the challenge of low oral bioavailability This study focuses on the requirements for multianalyte detection and quantification of drug candidates with poor aqueous solubility, different enabled formulation tech- in competitive assays of lab-on-a-molecule probes through kinetic-equilibria model- nologies including using spray-dried dispersion (SDD) and hot-melt extrusion (HME) ing. According to literature, lab-on-a-molecule probes respond to all analytes in one are becoming more popular in drug product development. Demonstration of satis- solution. Hence, orthogonal and quantitative detection of two or more analytes by a factory control strategy on API form control throughout product lifecycle for these single probe is quite challenging. Unrestricted detection assay examples include 1) types of drug products is regulatory expectation. A couple of case studies are given true lab-on-a-molecule probes, which are able to quantitatively sense two or more to discuss how control strategies are developed to meet regulatory expectations on analytes in a multianalyte mixture, 2) pseudocompetitive multianalyte probes that these compounds. Other case studies are also given to discuss drug development exhibit different responses to two or more analytes but fail in a fully competitive and filing strategies to meet regulatory expectations regarding elemental impurities quantitative assay, and 3) non-competitive multianalyte probes that display different (ICH Q3D) and mutagenic impurities in pharmaceuticals (ICH M7). responses to two or more analytes, but no competitive assay is provided. Using model based global analyses for complete sets of measurements taken under dif- 108 Perspectives in Managing Analytical Activities in an all Outsourced ferent initial concentrations, pH, etc., these probes are critically evaluated to opti- and Global Pharma Model mize their operation on multianalyte mixtures in a fully quantitative and orthogonal Shirley A. Rodriguez, Shire, 730 Stockton Dr., Exton, PA 19341 manner. This lecture presents topics related to interactions and experiences when managing analytical activities in an outsourced model for the development and manufactur- 113 Immunoaffinity Capillary Electrophoresis for the Determination of ing of pharmaceuticals. The main topic focuses on method transfers from contract Protein Biomarkers of Disease in Biological Fluids. Maximizing manufacturing organization (CMO) to CMO: establishing transfer criteria, facilitat- Benefits and Minimizing Harm ing and handling of information sharing between external laboratories and lessons Norberto A. Guzman, Princeton Biochemicals, Inc., PO Box 7102, learned from one external manufacturing site to another for a new product recently Princeton, NJ 08543 launched. Other topics related to data integrity, handling deviations and laboratory The formation of some diseases may start as early as childhood and be manifested investigations, managing continuous process improvement initiatives and monitor- 30 to 50 years later. Therefore, one of the main goals of system medicine is to be ing performance of external partners and contract laboratories located worldwide able to diagnose an illness ideally before symptoms are manifested using a panel are also presented. The lecture is from the perspective of the speaker after tran- of crucial biomarkers. This strategy is central to attaining early information on the sitioning from in-house analytical research and development laboratories to an all diagnosis and prognosis of an unhealthy condition, and thus preventing the progres- outsourced model for late stage and commercial analytical. sion of a disease. Furthermore, with the results provided by selective biomarkers it is possible to maximize the therapeutic effect and minimize the occurrence of 109 Musings on the Measurement and Communication of Value for adverse drug reactions by identifying the correct pharmaceutical agent and dose Central Analytical Functions for each patient. An ideal biomarker analyzer should have the advantage of yielding Mark S. Kennedy, Sustainable Transformations, 4 Sunrise Court, West diagnosis with accuracy, rapid analysis time, high sensitivity, and be cost-effective Grove, PA 19390 in order to be the cornerstone of efficient clinical management of diseases. Addition- Have you struggled to communicate the value of your non-revenue-generating ally, it should be an affordable and easy to implement technique in resource-limited 17 2018 EAS Abstracts November 2018

settings. In this presentation, I discuss the determination of peptide biomarkers in tibody. Plasma samples containing the ADC are mixed with the capture antibody, biofluids using a miniaturized immuno-analytical portable instrument. The principle and residual plasma proteins are removed. Purified ADC is eluted from capture by which this instrument operates is based on a two-dimensional technology known antibody under acidic conditions, and then disulfide bonds are reduced under acidic as immunoaffinity capillary electrophoresis (IACE). IACE consists of the use of im- conditions to give intact heavy and light chains with conjugates. The mAb subunits mune-capture techniques found in some immunoassays, such as enzyme-linked are analyzed by LC-MS. Mass analysis occurs at the precursor level over a range immunosorbent assay (ELISA), and pairs it with a high-resolution analytical separa- of approximately 1100-2200 m/z. Subunit masses of ~25 kDa (light chain) and ~50 tion technique, such as capillary electrophoresis.[1] kDa (heavy chain) are readily determined along with small molecule conjugate lev- Reference: els. Based on the conjugates observed on each subunit, the whole-molecule DAR can be calculated. For pharmacokinetic quantitation, the LC area under the curve [1] Guzman NA, Guzman DE. Archives of Biomedical Research, Volume 1, Issue of a single m/z charge state of the un-conjugated subunit mass can be used to fit 1, 2018. unknown samples to a standard curve of ADC spiked in plasma. Mass monitoring of conjugates allows for determination of biotransformation of the small molecule. 114 Optimizing HPLC Separation Performance for Peptides and Other Mid-Size Molecules 117 Electro-Flow Asymmetric Field Flow Fractionation Characterization Richard A. Henry, Independent Consultant, 983 Greenbriar Dr., State of the NIST Monoclonal Antibody Standard RM 8671 College, PA 16801, Justin M. Godinho, Joseph J. DeStefano Robert Reed, Postnova Analytics Inc., 230 South 500 East, Ste. 110, Peptides and small proteins are important to pharmaceutical and biotechnology re- Salt Lake City, UT 84102, Soheyl Tadjiki, Thorsten Klein search. While they are inherently just linear chains of amino acids, these molecules The National Institute of Standards and Technology (NIST) monoclonal antibody configure into many complex structures that vary from low MW (<1 kDa) to very high (mAb) standard reference material provides a platform for evaluating methods used MW (>100 kDa). High-performance liquid chromatography (HPLC) is a preferred to characterize physicochemical and biophysical attributes of other mAbs and large tool for rapid peptide and protein analysis. In HPLC methods, native structures are biomolecules. In this study, the NIST mAb was used to evaluate separation pa- denatured under mobile phase conditions that often include strong acids and organ- rameters for asymmetric flow field flow fractionation – multi angle light scattering ic solvents. Whether columns having totally porous particles (TPP) or superficially (AF4-MALS) analysis of mAbs, especially in comparison to size exclusion chroma- porous particles (SPP) are chosen, the pore-size should be significantly larger in tography (SEC-MALS). The amount of aggregates present in the NIST mAb was diameter than the largest target analyte for highest column performance. Optimum measured to be 10% by FFF with refractive index detection, whereas SEC-refractive efficiency cannot be achieved if peptides and proteins are restricted from full access index (RI) did not detect any aggregates. This non-detect by SEC-RI is possibly to stationary phase within pores. This paper clearly demonstrates the impact of due to loss of aggregate material on the SEC column during separation. A new size-exclusion on reversed-phase separations of molecules in the molecular weight module for AF4 using an auxiliary electric field for separation by macromolecule range of 500 Da to 20 kDa. Poor performance is often caused by restricted acces- surface charge, electro-flow AF4 (EAF4) was employed to measure the electropho- sibility to relatively smaller pores. This can be alleviated by changing to larger pore retic mobility of the NIST mAb during size separation. The EAF4-measured value for columns having an equivalent stationary phase. SPP columns with various pore electrophoretic mobility of the NIST mAb in 1x phosphate buffered saline was -1.68 sizes and stationary phases were used in the study. +/- 0.05 µm cm s^-1 V^-1.

Effects of Photosensitized Lipid Oxidation on Supported Lipid 115 118 Bead-Extraction and Heat-Dissociation (BEHD): A Novel Way to Bilayer Formation and Structure Overcome Drug and Matrix Interference for Small Biotherapeutic Nathan Wittenberg, Lehigh University, 6 East Packer Ave., Bethlehem, Modality such as Domain Antibody PA 18015, Ashley Baxter, Michael Farley, Joseph Saba Weifeng Xu, Bristol-Myers Squibb, MS: 14-08, RT 206 and Province Lipid oxidation by reactive oxygen species has a number of negative consequenc- Line Rd., Princeton, NJ 08543, Michael Sank, Renuka Pillutla es in biology. Additionally, photosensitized lipid oxidation has been explored as a Biological therapeutics are foreign antigens to the patient immune system and can way to trigger drug release from liposomal containers. In this work we explore the induce an undesirable immune response resulting in the formation of anti-drug effects of photosensitized lipid oxidation on the interactions of phospholipid vesicles antibodies (ADA), which in turn leads to a wide range of side effects. Cell-based with SiO2 surfaces. Using quartz crystal microbalance with dissipation monitoring functional neutralizing antibody (NAb) assays are preferred to characterize neutral- (QCM-D), we determined that light exposure radically alters the pathway of sup- ization activity of ADA against the biotherapeutics, but are often vulnerable to drug ported lipid bilayer (SLB) formation for vesicles that contain boron-dipyrromethene interference, as well as interferences from numerous serum factors, such as growth (BODIPY)-conjugated fluorophores. Zwitterionic vesicles typical form supported factors and disease-related cytokines. Biotin-drug extraction with acid dissociation bilayers on SiO2 through a two-step adsorption-rupture pathway. However, after (BEAD) has been successfully applied to extract ADA, thereby removing drug and illumination with broadband visible light, vesicles containing unsaturated lipids and other interfering factors from human serum samples. However, the harsh acid used BODIPY conjugated to lipid tailgroups (BODIPY-PC) rupture immediately upon en- in the extraction procedure can cause irreversible denaturing of NAb and lead to countering the SiO2 surface. Alteration of vesicle rupture pathways is dependent underestimated NAb measurement. Here we describe a new scenario where ac- on the identity and position of the fluorescent moiety, along with the unsaturation id-dissociation is not optimal for a PEGylated domain Ab. We further demonstrate of background phospholipids. Vesicles containing unsaturated phospholipids and that heating at 62 ºC can not only dissociate drug/ADA immune complex but also phospholipids with BODIPY or NBD conjugated to their fatty acid tails show the most selectively and irreversibly denature domain Ab drug due to much lower thermal pronounced effects. On the other hand, vesicles containing lipids with Texas Red stability of a domain Ab, compared to that of full antibodies. The irreversible dena- and NBD conjugated to their head groups show negligible differences compared turing of the drug is favorable since reduced amount of functional drug in the sample to controls. Vesicles of saturated phospholipids and BODIPY-PC are insensitive to facilitates the equilibrium towards the formation of immune complex between ADA illumination. Inclusion of an antioxidant (alpha-tocopherol) in illuminated vesicles and added biotinylated drug thus increases the recovery of ADA from samples. We with BODIPY-PC causes a reversion to the two-step adsorption-rupture pathway, call this new procedure biotin-drug extraction with heat dissociation (BEHD), which indicating this effect is mediated by lipid oxidation. Finally, illumination of SLBs con- could be applied to any new modality of biotherapeutics with much lower thermal taining unsaturated lipids and BODIPY-PC causes rapid tubulation, which can be stability than that of the full antibody. eliminated by antioxidants. Thus SLB tubulation is likely another effect of photosen- sitized lipid oxidation. 119 Multiplexed Residual Process Impurity Monitoring in Antibody- Drug Conjugates by Charged Aerosol Detection 116 A Generic mAb Subunit LC-MS Assay for In-Vivo Drug-to-Antibody Steven Chin, Genentech, MS: 89, 1 DNA Way, South San Francisco, Ratio and ADC Concentration Determination in Pre-Clinical Studies CA 94080, Tao Chen John F. Kellie, GlaxoSmithKline, 709 Swedeland Rd., King of Prussia, Antibody-drug conjugates (ADCs) are hybrid therapeutic modalities designed to PA 19406 improve cancer treatment through precision killing with fewer side effects. They Antibody-drug conjugates (ADCs) have emerged as promising drug candidates are typically constructed with a potent cytotoxic drug covalently conjugated to a and are routinely tested pre-clinically. The mAb and small molecule conjugates can specific monoclonal antibody through a carefully designed linker. These multiple often bring analytical challenges in pre-clinical testing, such as linker stability or components make ADC manufacturing a complex multi-step process, through which small molecule metabolism. Furthermore, in the discovery space, benchmarks of a variety of potential impurities can be introduced. Effectively monitoring and con- biomolecule quality must be determined. For ADCs, such determinations may not trolling these impurities are required to confirm product quality, to assure patient be straightforward with data from traditional ligand binding assays or monitoring safety, and to demonstrate regulatory compliance. The wide range of potential im- free small molecule. We have developed a generic, anti-human mAb liquid chroma- purities with distinct physicochemical properties (polarity, charge states, lack of UV tography-mass spectrometry (LC-MS) assay to determine in-vivo drug-to-antibody absorption, etc.) make monitoring of residual impurities in ADCs quite challenging. ratios (DARs), pharmacokinetic concentration, and biotransformation information. In this work, a hydrophilic interaction chromatography (HILIC) coupled with charged Briefly, streptavidin magnetic beads are coupled to biotinylated anti-human IgG an- aerosol detector (CAD) method was developed for multiplexed detection of residual 18 2018 EAS Abstracts November 2018

process impurities in ADCs. Nine reagents commonly used in ADC manufacturing, application of these methods for uncovering novel targets of clinically used kinase including antimicrobial agents added in cell culture, reducing/oxidizing/capping inhibitors and for revealing novel drivers and suppressors for melanoma metastasis agents needed in the conjugation process, and stabilizing agents introduced in for- will also be presented. Through this presentation, I hope to illustrate that quantitative mulation development were separated within fifteen minutes. This method utilizes proteomics constitutes a power tool for discovering novel nucleic acid- and nucleo- a silica-based mixed-mode column with both cation-exchange and anion-exchange tide-binding proteins and for revealing their functions in cells. properties operated in reversed-phase mode under HILIC conditions. Method pa- rameters were optimized with regard to mobile phase (buffer ions, ionic strength, 123 Quantification of Protein Post-Translational Modifications Using pH), mobile phase gradient, and column temperature. This method was prequali- Stable Isotope and Mass Spectrometry fied and has been successfully applied to detect residual impurities in ADCs with Grace Xinzhao Jiang, Amgen Inc., One Amgen Center Dr., Thousand excellent selectivity, sensitivity, and reproducibility. It presents a simple, fast, and Oaks, CA 91320 generic approach for simultaneously monitoring multiple residual process impurities With the increased attention to quality-by-design (QbD) for biopharmaceutical prod- to speed up ADC development. ucts, there is a demand for accurate and precise quantification methods to monitor critical quality attributes (CQAs). To address this need we have developed a mass 120 Green Chemistry: The Missing Elements spectrometry (MS) based method to quantify a wide range of post-translational John Warner, Warner Babcock Institute for Green Chemistry, 100 modifications (PTMs) in recombinant proteins using stable isotope-labeled internal Research Dr., Wilmington, MA 01887 standard (SILIS). The SILIS was produced through metabolic labeling where 15N Imagine a world where all segments of society demanded environmentally benign was uniformly introduced at every nitrogen atom in the studied proteins. To enhance products! Imagine if all consumers, all retailers and all manufacturers insisted on the accuracy of the method, the levels of PTMs in SILIS were quantified using or- buying and selling only non-toxic materials! The unfortunate reality is that, even thogonal analytical techniques. Digestion of an unknown sample mixed with SILIS if this situation were to occur, our knowledge of materials science and chemistry generates a labeled and a non-labeled version of each peptide. The non-labeled would allow us to provide only a small fraction of the products and materials that and labeled counterparts co-elute during reverse phase high performance liquid our economy is based upon. The way we learn and teach chemistry and materials chromatography (RP-HPLC) separation but exhibit a sufficient mass difference to science is for the most part void of any information regarding mechanisms of toxicity be distinguished by MS detection. With the application of SILIS, numerous PTMs and environmental harm. Green Chemistry is a philosophy that seeks to reduce or can be quantified in a single analysis based on the measured MS signal ratios of eliminate the use of hazardous materials at the design stage of a materials process. 15N-labeled versus the non-labeled pairs. Several examples using microbial and It has been demonstrated that materials and products CAN be designed with neg- mammalian-expressed recombinant proteins demonstrated the principle and utility ligible impact on human health and the environment while still being economically of this method. The results indicate that SILIS is a valuable methodology in address- competitive and successful in the marketplace. This presentation will describe the ing CQAs for the QbD paradigm. history and background of Green Chemistry and discuss the opportunities for the next generation of materials designers to create a safer and more sustainable fu- 124 Applications of Mass Spectrometry in Biologics Drug Discovery ture. and Development Yongsheng Xiao, Shire, 200 Shire Way, Lexington, MA 02421 121 Making the Case for Multidimensional Liquid Chromatography in Development of protein therapeutics, especially monoclonal antibodies (mAbs), has the Search for Biomarkers gained significant momentum in recently years. The developments in instrumen- Mark R. Schure, Kroungold Analytical, Inc., Theoretical Separation tation and bioinformatic tools have made mass spectrometry (MS) the method of Science Laboratory, 1299 W. Butler Pike, Blue Bell, PA 19422 choice for comprehensive protein characterization. However, complete and detailed Determining the presence and concentration of specific molecules as indicators of structural characterization of protein therapeutics, such as intact mass, peptide health and specific diseases has revolutionized medicine. Although many definitions mapping, disulfide mapping and glycan analysis by mass spectrometry (MS) analy- of the term “biomarker“ exist [1], the general idea is that some material or compound sis is still a significant challenge due to the lack of appropriate experimental protocol is present in a body fluid that reveals the presence of disease. Finding “smoking and software. Here, we give two examples to demonstrate the applications of Mass gun” biomarkers, those which signal an impending disease state, may be problem- Spectrometry in Biologics Drug Discovery and Development. Frist, we report an atic in that some of these may be present at low concentrations that reside within integrated de novo antibody sequencing strategy by MS analysis to reliably deter- the noise threshold of a detector. It is this theme that draws us to ask the question, mine antibody sequence with 100% accuracy. Although the amino acid sequence of “How many biomarkers may be present below the limit of detection and buried in an antibody is mainly obtained by DNA sequencing, sometimes de novo antibody the noise?” Some of these issues have been raised by Enke and Nagels[2] in their sequencing is required when the original cell line or cDNA is not available. We have analysis of levels of natural substances. I will extend this discussion with recent demonstrated, for the first time, 100% amino acid sequencing of a monoclonal an- work from my laboratory and my many collaborators. Although this is a simple ques- tibody, including identification of Leu/Ile residues, exclusively by means of mass tion, chromatography has a well-known problem: even with long columns and slow spectrometry. In second example, we have successfully determined the disulfide velocity, the component saturation is so high in a body fluid that even with high reso- structure for murine Meteorin by liquid chromatography (LC)-MS analysis of pep- lution mass spectrometry, large amplitude signals swamp out neighboring low-level tides generated by trypsin plus endoprotease-Asp-N. For proteolytic peptides linked signals causing loss of potential biomarker detection. How bad is the loss? This will by more than one disulfide bond, we used electron transfer dissociation (ETD) to be shown by generating chromatograms with statistical distributions of peak heights partially dissociate disulfide bonds followed by high-energy collisional dissociation and retention times. One potential aid to this is the use of two-dimensional liquid (HCD) to determine disulfide linkages. Furthermore, In-Source Reduction (ISR) was chromatography to reduce the saturation of components coming into the detector. also used for complete disulfide mapping analysis. Our approach could be applied In so many different ways, slowing down the chromatography allows one to get to study the disulfide structure for any protein therapeutics. more effective information per unit time. In this presentation, which relies heavily on modeling, the pathways to component density reduction is explored and practical 125 Novel Approaches toward Analysis of Glycolipids estimates reveal the difficulty of finding low-level biomarkers. Qibin Zhang, University of North Carolina-Greensboro, 500 Laureate Way, Ste. 4226, Kannapolis, NC 28081 References: Glycosphingolipids play essential roles in biological processes and are involved in [1] K. Strimbu, J. A. Tavel, What are Biomarkers? Curr. Opin. HIV AIDS 2010 5(6) various pathologies such as cancer, neurodegeneration and autoimmune diseas- 463-466. es. The function of glycosphingolipid is determined by both the glycan and lipid [2] C, G. Enke, L. J. Nagels Undetected Components in Natural Mixtures: How structure, which necessitates characterizing glycosphingolipid as intact molecular Many? What Concentrations? Do They Account for Chemical Noise? What Is species. In this presentation, I will introduce the recent developments in our group Needed to Detect Them? Anal. Chem. 2011, 83, 2539-2546. on structural characterization and multiplexed quantification of glycosphingolipid while maintaining the molecule’s structural integrity – an approach analogous to 122 Quantitative Proteomic Approaches for Interrogating Nucleic Acid- the more known top-down approach in proteomics. For multiplexed quantification, and Nucleotide-Binding Proteins a rapid, chemoselective oxidation of sialic acid side chain in gangliosides was de- Yinsheng Wang, University of California-Riverside, Department of veloped, which was followed by ligation with a carbonyl-reactive isobaric tandem Chemistry, Riverside, CA 92521 mass tag (TMT) reagent and subsequent reversed-phase liquid chromatography The functions of nucleotides and nucleic acids involve their interactions with cellular tandem mass spectrometry analysis. Attachment of the isobaric label was observed proteins. In this presentation, I discuss our recent efforts toward the development to improve the ionization efficiency of complex gangliosides using electrospray ion- and applications of quantitative proteomic methods for unbiased, proteome-wide ization. Higher-energy collisional dissociation (HCD) fragmentation of the resulting discovery of proteins that can recognize unique secondary structures of DNA. I TMT-labeled ganglioside ions provided information-rich spectra containing frag- also discuss our recent development of targeted quantitative proteomic methods ments from the glycan, lipid, and TMT reporter ions. This facile approach enabled for interrogating ATP- and GTP-binding proteins at the entire proteome scale. The simultaneous quantification of up to six samples as well as identification of glycan 19 2018 EAS Abstracts November 2018

and lipid compositions in a single injection. Using ozone induced dissociation mass References: spectrometry, we demonstrated that C=C located in the fatty acyl chain is signifi- [1] Hudson, BS Vibrational Spectroscopy Using Inelastic Neutron Scattering: Over- cantly more reactive than that in the long chain base, and C=C at different locations view and Outlook. Vibrational Spectrosc. 2006, 42, 25-32. have characteristic position-specific ions. This capability is very promising toward comprehensive analysis of biologically relevant glycolipids and could enable dis- [2] Hudson, BS Vibrational Spectroscopy via Inelastic Neutron Scattering. In Fron- covery of novel biological functions of previous non-distinguishable C=C positional tiers of molecular spectroscopy. Laane,J Ed; Elsevier: Amsterdam; 2009; pp. isomers of glycolipids. 597-622. [3] Hudson, BS, et al. Infrared, Raman, and Inelastic Neutron Scattering Spectra of 126 Biomedical Applications of SERS: Diagnostics, Metabolomics, Dodecahedrane: an Ih Molecule in Th Site Symmetry, J. Phys. Chem. A (2005), Forensics 109, 3418-3424. INS is non-destructive permitting return of this 160 mg sample, Lawrence Ziegler, Boston University, Department of Chemistry, Boston, the world’s total supply. MA 02215 [4] Dinca, SA, et al, Insulated polyacetylene chains in an inclusion complex by pho- Surface enhanced Raman spectroscopy (SERS) is an attractive methodology for topolymerization, MRS Online Proceedings Library (2015), 1799, 1-6. analytical biomedical applications owing to its rapid, sensitive, specific, easy-to-use, label-free and multiplexing capabilities for molecular detection and identification. [5] Hudson, BS, Polyacetylene: Myth and Reality, Materials. 2018; 11(2):242. We demonstrate that a SERS based platform can be used for rapid, label-free, bacterial diagnostics in human body fluids, tumorigenic cell identification and the 129 Vibrational Spectroscopy of Hydrogen-Processing Enzymes Using identification of trace amounts of human body fluids for forensic applications. In Mössbauer Photons - Why the Fuss About Little Bumps and particular, 785 nm excited SERS spectra provide unique signatures for detecting Squiggles? and identifying bacteria relevant to urinary tract infections, bacteremia and sexual- Stephen P. Cramer, University of California-Davis, 291 Margarita Ct., ly transmitted diseases. A phenotypic SERS based platform can achieve antibiotic Los Altos, CA 94022 susceptibility testing in less than one hour as compared to the 24-48 hour times of [FeFe] hydrogenases (H2ases) are enzymes that catalyze the reversible oxidation traditional cell culturing methods. Understanding the molecular origins of the cel- of H2 and reduction of protons with high turnover frequencies (>10,000 s-1) under lular SERS signatures is crucial for their use in bioanalytical applications. 785 nm physiological conditions, in part due to the unique structure of their active site cofac- excited SERS spectra of bacterial cells are dominated by the stress induced purine tor. This ‘H-cluster’ consists of covalently linked [2Fe]H and [4Fe-4S]H sub-clusters. metabolites of nucleotide degradation in the pericellular region. Similarly, the SERS The catalytic mechanism is not completely understood. Nuclear resonance vibra- spectra of normal and tumorigenic epithelial cells are predominantly determined by tional spectroscopy (NRVS) is a powerful and relatively new way to study Fe in bio- purinergic signally processes in the outer cell wall region, and are the basis for dis- logical systems. In this synchrotron radiation technique, a sample is excited with a tinguishing pathogenic and normal cells, and offers a new opportunity for monitoring ~1 meV bandwidth beam near the Mössbauer resonance, and the delayed fluores- treatment efficacy. The sensitivity and selectivity of SERS provides a new tool for cence is recorded as a function of excitation energy. When applied to Fe-containing on-site forensic identification of trace amounts of human body fluids. Hemoglobin, samples, NRVS is only sensitive to vibrations involving motion of 57Fe. Results are in particular, exhibits a strong SERS signature that has great value for forensic sci- presented on model compounds, small Fe-S proteins, and [FeFe] H2ase, and the ence applications. Highly reproducible redox interactions with the SERS substrate implications for the catalytic mechanism are discussed. The needs and prospects influence the observed hemoglobin spectrum. for future improvements in NRVS experiments are discussed.

Microscale Tools for Precision Medicine 127 Real-Time and Nanoscale Infrared Imaging in the Biosciences 130 Lisa M. Miller, Brookhaven National Laboratory, Bldg. 743, NSLS-II, Ryan C. Bailey, University of Michigan, 930 S. University Ave., Ann Upton, NY 11973 Arbor, MI 48109 Fourier-transform infrared (FTIR) spectroscopic imaging is widely used for studying The concept of personalized medicine is predicated on an ability to comprehend a the chemistry of proteins, lipids, and DNA in biological systems without the need for patient’s disease state in a highly informed manner that ideally illuminates an effec- additional tagging or labeling. While conventional IR microspectroscopy has prov- tive treatment strategy. However, in many cases new technologies are still needed en valuable for resolving the chemical components in biological samples, the long to fully characterize dynamic molecular signatures of disease onset and progres- wavelengths of infrared radiation limit the spatial resolution that can be achieved. sion. To this end, our group is developing multiple technologies that aim to increase In addition, FTIR imaging experiments are often limited to dried samples due to the the depth of biomarker analysis that can be performed in a clinical laboratory setting. significant spectral overlap between water and the protein Amide I band. However, One such technology leverages well-established semiconductor fabrication meth- recent advances in full-field FTIR imaging and near-field nano IR imaging utilizing ods to create highly multiplexed and robust silicon photonic biosensor arrays that a synchrotron source have been used to address these limitations. In this talk, we are extremely sensitive and readily scalable to emerging challenges in point-of-care demonstrate a method to obtain high quality FTIR images at submicron pixel res- clinical diagnostics. In this talk, I describe applications of this technology for cancer olution in vivo over a duration of 18 hours through the use of a custom microfluid- and inflammatory-based diagnostics. ic-incubator and a FTIR microscope coupled to a focal plane array detector and a Miniaturized Devices for the Analysis of Biomolecules Linked to synchrotron light source. With such a system, spatially-resolved, temporal studies 131 Diseases of in vivo protein folding in a cell culture model of amyotrophic lateral sclerosis was Adam T. Woolley, Brigham Young University, Chemistry & Biochemistry performed. In another example, we describe the use of synchrotron near-field na- Dept., Box 25700, Provo, UT 84602 no-IR imaging with a 50 nm beam to the study of amyloid formation in neurites in a Microfluidic systems have been under investigation for over a quarter of a century, cell culture model of Alzheimer’s disease. Taken together, the coupling of advanced with a key focus on disease detection. We have been developing microfluidic de- infrared imaging techniques with a synchrotron light source can provide new ways vices with applications in improving the diagnosis of sepsis and preterm birth risk. of examining the chemistry of biological systems in real time and/or at the nanoscale As part of a collaborative grant we are working to create microchips for diagnosis of for the first time. bacterial blood infections in as little as one hour. We are developing sequence-se- lective extraction of disease-linked nucleic acids from lysed bacteria samples. We 128 Vibrational Inelastic Neutron Scattering Bruce Hudson, Syracuse University, Center for Science & Technology, have extracted and fluorescently labeled DNA on porous polymer monoliths in mi- Syracuse, NY 13244 crodevices with recovery values >80%. We further developed a single-step process [1] Vibrational inelastic neutron scattering (INS) depends for its intensity on atomic mo- for making DNA-linked monoliths for sequence-specific extraction. Our current ef- tions, especially those of hydrogen atoms if present. The interaction of a neutron forts entail creating an integrated system that combines these monoliths with bacte- with an atom depends on the isotope. Deuterium atoms scatter much more weakly ria capture and lysis, as well as with single-molecule optofluidic detection. Moreover, than hydrogen permitting selective enhancement of one component over another. we are developing integrated and automated microdevices that analyze a panel of [2,3] INS intensities are easily and reliability computed from normal mode computations. maternal serum biomarkers linked to preterm birth (PTB) risk. We are currently [4] [1,2] There are no symmetry selection rules for INS spectra permitting observations 3D printing truly microfluidic features in integrated devices for PTB biomarker anal- for modes that are neither infrared (IR) nor Raman active.[3] The INS spectra dis- ysis. These microchips should help to advance disease diagnosis. We acknowledge cussed were obtained for crystalline samples at low temperature. The VISION spec- support of this work from the NIH (R01 EB006124 and AI116989). trometer at the ORNL SNS facility is superior to any other in the world for this appli- References: cation by at least 100 fold. Comparison of an INS spectrum with a simulation using [1] Knob, R. et al. J. Chromatogr. A. 1562, 12-18 (2018). periodic methods permits thermodynamic analysis of polymorphic forms. Solid state elimination polymerization leading to polyacetylene where IR is not possible and [2] Nielsen, A.V. et al. Electrophoresis in press (2018). Raman disappears will be discussed.[4,5] [3] Sahore, V. et al. Anal. Bioanal. Chem. 410, 933-941 (2018).

20 2018 EAS Abstracts November 2018

[4] Beauchamp, M.J. et al. Anal. Bioanal. Chem. 409, 4311-4319 (2017). orthogonality and high peak capacity offered by multidimensional LC. Both speed and quality have been significantly increased. The new strategy and multiplexed 132 Plasmonic Nanobiosensors: From Therapeutic Drug and platform have been successfully applied for routine pipeline projects support and Environmental Monitoring to Optophysiology of Living Cells for quality control purpose. Multiple case studies and future direction are discussed. Jean-Francois Masson, University de Montreal, CP 6128 Succ Centre- Ville, Montreal, PQ H3C 3J7 135 Applications and Method Development of 2D LC for Small Molecule This presentation focuses on applying these concepts for several classes of sensors Pharmaceutical Analysis for monitoring biomolecules, therapeutic drugs, pheromones and for environmen- Pankaj Aggarwal, Pfizer Inc., MS: 8220-4402, Eastern Point Rd., tal contaminants. We have developed a surface plasmon resonance (SPR) and Groton, CT 06510, David T. Fortin, Angel R. Diaz localized (L)SPR) sensing platform based on a small and portable instrument that Liquid chromatography (LC) is the most commonly used technique for purity analy- can be field-deployed. In the first example, this SPR chip was integrated witha sis of pharmaceuticals, which is necessary for ensuring product quality and patient RDX-selective molecularly imprinted polymer to detect RDX at ppb levels directly in safety. The LC method development for pharmaceuticals mostly comprises of ex- natural waters. The system was deployed to a Canadian army base for monitoring tensive column screening with different mobile phase combinations to optimize the the level of RDX in proximity of training grounds. This system was tested on several specificity and sensitivity of the high-performance liquid chromatography (HPLC) trips in different environmental conditions and results were in good agreement with method used for the analysis. However, the pharmaceutical samples can be re- high-performance liquid chromatography performed in a laboratory. Clinical sensing ally complex and it’s a good practice to confirm the absence of co-elution using in crude biofluids is a common challenge to different biosensing platforms. To pre- a complimentary technique or approach such as peak-purity assessments. Peak vent nonspecific adsorption of serum, a series of peptide monolayers were synthe- purity is commonly assessed via spectroscopic and mass homogeneity; however sized and tested in crude serum. Based on this, competition assays were validated these require that the co-elulting analytes have different responses such that they for therapeutic drug quantitation, such as methotrexate with the SPR sensors. The can be flagged by the software. Two-dimensional liquid chromatography (2D LC) methotrexate assay was tested at a local hospital and was cross-validated with the has emerged as a solution to many of these issues encountered in analyzing phar- current state-of-the-art fluorescence polarization immunoassay analyzer commer- maceutical samples. This presentation covers method development strategies for cially available. Lastly, we are currently exploring the concept of optophysiology small molecules using automated 2D LC with maximum orthogonality. The potential using plasmonic nanopipettes for monitoring living cell secretion events. Plasmonic use of in-silico prediction tools for orthogonal method selection is presented. Case nanopipettes were developed based on the decoration of patch clamp nanocapillar- studies showing the implementation of 2D LC for analyzing peak purity, regio-isomer ies with Au nanoparticles. The plasmonic nanopipette is thus competent for dynamic co-elution, and on-column degradation are discussed. surface-enhanced Raman spectroscopy measurements in the liquid environment near cells. This nanobiosensor was tested with the detection of small metabolites 136 Recent Advances in Resolving Power and Detection Sensitivity of near living Madin-Darby canine kidney strain II (MDCKII) cells and of neurotransmit- Two-Dimensional Liquid Chromatography for Bottom-Up Analysis ters released by neurons. of Therapeutic Proteins Dwight Stoll, Gustavus Adolphus College, 800 West College Ave., Saint 133 Rapid Dialysis-based Binding Measurements with 3D-Printed Peter, MN 56082, Hayley Lhotka, David C. Harmes, Ben Madigan, Integrated Membranes Gabriel Leme, Gregory Staples Dana Spence, Michigan State University, 775 Woodlot Dr., East Lansing, The biopharmaceutical industry relies on separation, identification, and quan- MI 48824, Cody Pinger, Andre Castiaux titation of peptides produced from enzymatic digestion of protein therapeutics to Protein-ligand binding assays facilitate the understanding of biomolecular interac- demonstrate product knowledge to regulatory agencies. Peptide-level characteri- tions and classical equilibrium dialysis methods are often used for accurate deter- zation is implemented throughout the drug development process, including during mination of binding properties. While accurate, the long equilibration times associ- optimization of cell culture conditions, and monitoring the stability of formulations. ated with the technique (>6 hours) hinder throughput. Here, in an attempt to gather Mass spectrometry is an indispensable tool for these applications. Nevertheless, high-accuracy results while reducing total analysis time, our group has been produc- improvements in separation methods to simplify samples presented to the mass ing various filtration methods that rely on simple membrane-containing tools that are spectrometer generally improve the quality of identification and quantitation of re- fabricated by polyjet 3D-printing. A minimal portion (1-2%) of the solution containing sults. Demand for increased separation power on shorter timescales is building for the binding analytes of interest is driven through the membrane pores and collect- multiple reasons. Among these factors are the interest in using liquid chromatogra- ed for analysis. Specifically, the device was used to investigate the binding affinity phy-mass spectrometry (LC-MS) for host-cell protein (HCP) characterization, the between Zn2+ and either human serum albumin or a commercially purchased gly- emerging multi-attribute method (MAM), and development of peptide-level process cated human serum albumin. Both of these ligand/protein binding systems have analytical technologies. In this presentation we describe the results of recent efforts implications in type 1 diabetes. The device was then used to investigate the binding to optimize a two-dimensional liquid chromatography (2D-LC) approach involving between the various albumin types and C-peptide, the 31 amino acid peptide that is reversed-phase separations in both dimensions – the first at high pH and the second co-secreted with insulin from pancreatic beta cells. Results for albumin/Zn2+ bind- at low pH. We have explored a number of variables in search of conditions that pro- ing obtained using these methods (Kd = 5.77 ± 0.19 × 10-7 M) were statistically vide a good compromise of method reliability, chromatographic resolving power, and equivalent with results reported using other methods. The binding affinity of C-pep- detection sensitivity. These include second dimension cycle time, column length, tide to normal albumin (Kd = 2.4 ± 0.3 x 10-6 M) agreed with values reported in the particle size, and second dimension flow rate. Under these optimal conditions we literature using standard techniques. Unlike Zn2+ binding, the binding of C-peptide find that post-second column dispersion is especially important in ways that are not to normal human serum albumin was statistically equal to its binding to glycated typically encountered in conventional LC experiments. Finally, we demonstrate the human serum albumin (77.7 ± 6.2 % and 78.8 ± 7.4 %, respectively), suggesting utility of this high performing method for the discovery of low-concentration peptides that C-peptide replacement therapy in people with T1D may be strongly dependent in complex protein digests. upon the characteristics of Zn2+ binding to human serum albumin. 137 Adding Mass Detection to a USP Method Using Heart-Cutting Multi- 134 Recent Advances of Multidimensional HPLC: Beyond Peak Dimensional Liquid Chromatography Capacity and Orthogonality Margaret Maziarz, Waters Corporation, 34 Maple St., Milford, MA 01757, Kelly Zhang, Genentech, 1 DNA Way, South San Francisco, CA 94080, Claude Mallet, Paul Rainville, Mark Wrona Jessica Lin, Sam Yang, Midco Tsang United States Pharmacopeia (USP) compendial methods are routinely adopted Recent years have seen a leap of applications of multidimensional high-performance by pharmaceutical companies during the development and quality control of drug liquid chromatography (HPLC) to resolve real world problems. The applications of substances and finished drug products. Often, these methods use mobile phases multidimensional HPLC in pharmaceutical industry have advanced from research to with non-volatile buffers, which are not suitable for mass detection. Any modifica- routine use. This is largely due to the commercialization of multidimensional HPLC tions to these methods will require revalidation. In this study, we demonstrate that instruments, better user friendly software, a large diversity of columns to choose a heart-cutting multi-dimensional liquid chromatography with At-column dilution and the appealing results from academia research. Many applications have been enables use of mass detection directly with the unmodified methods. We use this reported using multidimensional separation to increase peak capacity or enhance approach to add mass detection to a USP monograph for lidocaine and prilocaine selectivity by using orthogonal separation in each dimension. These applications cream that utilizes a non-mass spectrometry (MS) compatible buffer to confirm include but not limited to resolving peak co-elution, removing matrix interference identity of an impurity peak. An impurity peak observed in the assay preparation including using non-volatile mobile phases for mass spectrometry, chiral separation, solution is heart-cut from the USP method run in the 1st dimension, transferred to profiling complex sample components, etc. To accelerate drug discovery and devel- the holding loop, and loaded onto a mixer. The heart-cut volume is diluted with an opment speed, while maintain the high quality of data, we developed a new strat- aqueous solvent, refocused onto the trap column and transferred to the 2nd dimen- egy of using platform multidimensional HPLC for sample impurity profiling without sion under MS compatible conditions for analysis by mass detection. The workflow compound specific method development. This strategy is based on but beyond the and fraction collection is controlled by switching the positions of two valves of the 21 2018 EAS Abstracts November 2018

column manager. The development of conditions for maximum peak trapping and the crystallization of FFA in a confined environment and assign the ordered and separation conditions is performed following a method optimization protocol. The disordered domains. protocol evaluates the effect of dilution solvent (loading) pH, trap column chemistry and mobile phase for 2nd dimension separation on the efficiency of peak trapping. 141 Structural Details from Quadrupolar Solid-State NMR of Solution- The mass spectra data obtained from the 2nd dimension analysis are used to con- Processed Thin Films from Group 13 Oxide Molecular Precursors firm identity of the impurity peak. Sophia E. Hayes, Washington University, Dept. of Chemistry, 1 Brookings Dr., St. Louis, MO 63130, Jinlei Cui, Yvonne Afriyie, Cory K. 138 Dynamic Disorder in Regulation of HIV-1 Maturation by Integrating Perkins, Douglas Keszler Solid State NMR, cryo-EM, and MD We have conducted a series of studies by gallium (71Ga and 69Ga) and aluminum Caitlin M. Quinn, University of Delaware, Dept. of Chemistry and (27Al) nuclear magnetic resonance (NMR) on a combination of thin metal-oxide Biochemistry, Newark, DE 19716, Mingzhang Wang, Huilan Zhang, films and the molecular (cluster) precursors used in their preparation. The clusters Rupal Gupta, In-Ja Byeon, Eric O. Freed, Peijun Zhang, Juan R. Perilla, themselves are structurally intriguing, being kinetically-trapped species consisting Angela M. Gronenborn, Tatyana Polenova of 13 metal sites, bridged by hydroxide groups, and coordinated with water, with Acquired immunodeficiency syndrome (AIDS), caused by the human immunodefi- the chemical formula: [M13(OH)24(H2O)24](NO3)15, where M is a group 13 metal ciency virus (HIV) is a global pandemic with ~35 million people infected worldwide. (nitrates are charge-balancing species in the solid state). Solutions of these clus- During HIV-1 viral maturation, Gag, the polyprotein which comprises the shell of ters can be spin coated into smooth, uniform metal oxide films (<100 nm thick) the immature virion, undergoes a complex morphological transformation through a onto substrates. This synthesis route involves condensation and formation of the series of proteolytic cleavage steps. A longstanding question in the mechanism of three-dimensional film structure—-local chemical environments can be tracked as maturation is the structure of the C-terminal region of the CA-SP1 maturation inter- the films evolve over the annealing temperature range of 200 °C to 1100 °C. As mediate. Utilizing an integrated approach, combining solid state nuclear magnetic these are amorphous films at temperatures below 900 °C, solid-state NMR is partic- resonance (NMR) and molecular dynamics with information from cryo-EM and solu- ularly informative about the local structure of the sites. We will discuss two studies: tion NMR, we show that in tubular assemblies of CA-SP1 the SP1 peptide exists in characterization of the gallium NMR of the molecular precursors, to show the po- a dynamic helix-coil equilibrium. These conclusions are also supported by dynamic tential for employing CASTEP-NMR, a program to compute quadrupolar tensors to nuclear polarization (DNP) results at cryogenic temperatures, through which we can understand the lineshapes for multiple sites in the clusters; and characterization by obtain information about dynamically disordered regions not accessible with other aluminum NMR of the aluminum oxide films at a range of temperatures to monitor methods. We further demonstrate that small molecule maturation inhibitors such the structural evolution as the films dehydrate, dehydroxylate, and undergo nitrate as Bevirimat (BVM) inhibit the final step of HIV-1 maturation by stabilizing a helical loss. In this latter case, other structural characterization methods fail, and solid-state conformation of SP1. The maturation-arresting SP1 mutation T8I also induces he- NMR provides one of the only “windows” into those processes. lical conformation in SP1 as well as other conformational and dynamical changes throughout the protein. Overall, we demonstrate that the dynamics of CA and SP1 142 Understanding the Depth Response Profile of Transmission Raman are critical for HIV-1 maturation, and small molecules inhibit maturation by disrupting Spectroscopy of Diffusely Scattering Media these motions. Jun Zhao, B&W Tek, 19 Shea Way, Newark, DE 19713 The see-through Raman technology (STRaman) we developed previously for Characterization of Amorphous Pharmaceutical Solids Using NMR through-package-material-identification is applied to quantitative transmission Ra- 139 Spectroscopy man measurements of low concentration active pharmaceutical ingredients (APIs) Joe Lubach, Genentech, MS: 432A, 1 DNA Way, South San Francisco, in pharmaceutical tablets. Due to the uneven distribution of typical APIs in such CA 94080 products, a sufficiently large sampling volume is a prerequisite for an accurate Amorphous solids are now widely used throughout the pharmaceutical industry, pri- quantification. The large and easily variable sampling area and high throughput of marily in cases where absorption of the active ingredient is limited by poor aqueous the STRaman lends itself naturally to this application. Additionally, molecules at dif- solubility and dissolution. Rendering the drug into a high energy disordered state, ferent depths contribute differently toward the total intensity of transmitted Raman molecularly dispersed in a polymeric excipient, can afford faster dissolution and signal, and it is desirable to have a theoretical understanding of how optical prop- higher drug concentrations in solution relative to its crystalline counterparts. The erties of the sample and the sampling optics affect the response profile. Previously, disordered nature of these materials makes them inherently more difficult to study, such studies have relied on numerical simulation. Here, I develop a set of analytical especially when diluted into complex mixtures such as solid dosage formulations. equations based on the Kubelka-Munk model of diffuse reflectance to describe the Solid-state NMR spectroscopy has proven to be a critical tool in the characterization transmitted Raman response of a uniform scattering media as a function of depth. and understanding of these systems. Using a variety of experiments to probe nuclei With this, it is easy to predict how reflectance of photon recycling mirrors affect the such as 13C, 15N, 19F, and 1H, detailed information on the structure and dynam- response curve. Results predicted by the equations are compared with experiments ics of amorphous systems can be obtained. A number of different applications are measured with 0.1 mm depth resolution. highlighted in this talk, including quantitation of crystalline material in an amorphous solid dispersion dosage form, evaluation of drug-polymer miscibility and phase sep- 143 Raman Hyperspectroscopy and Advanced Statistical Analysis: A aration, and investigation of the nature of stabilizing drug-polymer interactions. Novel Universal Method for Disease Diagnostics Nicole M. Ralbovsky, University at Albany-SUNY, 1400 Washington 140 High-Field Solid-State NMR of Disordered Solids Ave., Albany, NY 12222, Lenka Halamkova, Igor K. Lednev Dinu Iuga, University of Warwick, Gibbet Hill Rd., Coventry CV4 7AL, Raman hyperspectroscopy combined with advanced statistics is uniquely suitable United Kingdom for characterizing micro-heterogeneous systems. Understanding the structure and The UK 850 MHz Solid-State Nuclear Magnetic Resonance (NMR) Facility pro- biochemical composition of samples at the microscopic level is important in many vides unique possibilities to investigate, develop and apply advanced solid-state practical applications, including disease diagnostics. The great potential of Raman NMR methods for the structural characterization of disordered solids in applications hyperspectroscopy for medical diagnostics is based on its ability to integrate the across chemistry, materials science, life sciences and environmental sciences. Sev- contribution of multiple biomarkers in a spectroscopic signature. Hyperspectral im- eral projects run at the facility have exploited the increase in sensitivity provided aging collects multiple spectra at different positions on the sample for the purpose by high field and the enhanced resolution of 1H obtained under very fast MAS to of determining the spatial distribution of its components. The spectral differences acquire 1H-1H homonuclear correlation (obtained via DQ or NOESY mechanisms) between two different classes of samples (such as healthy and diseased classes) spectra with increased resolution or to detect challenging quadrupolar nuclei like are related to changes in concentration of specific biomolecules; these changes 14N to reveal one-bond NH connectivities or additionally longer-range NH prox- can lead to the discovery of disease biomarkers. Because these spectral variances imities. After a short overview of such projects (like: structural characterization of are often minute, advanced statistical analysis is used to better identify the differ- lipophilic guanosine supramolecular assemblies, identification of intermolecular hy- ences between classes. The result is a robust statistical model that can predict the drogen bonding in the solid-state structure of an indomethacin-nicotinamide cocrys- classification of an unknown sample, and thus signify whether or not a person has tal and characterization of nitrogen functional groups in pharmaceutical compounds a particular disease. We discuss preliminary results which used this approach for like cimetidine, nicotinamide palmitic acid cocrystal and acetaminophen–polyvin- the development of a minimally invasive method for diagnosing Alzheimer’s disease ylpyrrolidone solid-dispersion) the presentation will focus on further developments based on analysis of saliva. Saliva obtained from patients with Alzheimer’s disease of the D-HMQC experiment with R3 and SR4 heteronuclear recoupling with or with- was successfully differentiated from those with mild cognitive impairment and from out WURST saturation of the satellite transitions and its applications to 35Cl-1H healthy donors with over 95% accuracy. This technique has also been effectively heteronuclear correlation for probing salt formation mechanism in pharmaceuticals. used for differentiating classes of human glomeruli tissue taken from healthy and Furthermore, the benefit of high magnetic field and fast spinning will be exemplified amyloidosis-affected patients. This novel method has a wide applicability for diag- for two other nuclei: 1) 27Al DQMAS spectra are used to study proximities of various nosing any disease that exhibits pathophysiological changes as it progresses. [AlOn] species in tellurite glasses and 2) 19F MAS spectra are used to characterize 22 2018 EAS Abstracts November 2018

144 Machine Learning Algorithms Applied to Raman Spectroscopic was fed to a Thermo Fisher Scientific 11mm twin screw co-rotating extruder for Data the extrusion process experiment. Several process parameters, such as number of Lenka Halamkova, University at Albany-SUNY, 1400 Washington Ave., mixing zones, extrusion temperature, and screw speed were investigated in order to Albany, NY 12222, Igor K. Lednev achieve uniform amorphous APAP distribution in HPMC polymer matrix. A near in- Raman spectroscopy has become increasingly valued in recent years for different frared (NIR) fiber optics probe was installed in the extruder die for in-process moni- application areas where non-destructive, microscopic, chemical analysis and imag- toring of APAP crystalline residual level. Both crystalline and amorphous APAP have ing is needed. Forensic and biomedical applications of Raman spectroscopy utilized strong spectral response in NIR wavelength range. The peak area near 6100 cm-1 for routine analysis require reliable methods for data evaluation. Over the last years, can be used to track the residual crystalline APAP concentration in real time. Ex- advances in the instrumental design of Raman spectroscopy overcame the problem trudate samples were also taken from multiple locations along the extruder screws with high fluorescence, low sensitivity and reproducibility. The advance in the field and at various extrusion conditions for off-line Raman microscopy image analysis of chemometrics provides many effective ways to solve calibration and classification and polarized microscope analysis to exam APAP/polymer mixing uniformity and problems and opens a new perspective for the spectra differentiation and identifi- residual crystalline APAP particle distribution. cation of heterogeneous samples. Raman spectroscopy provides a unique “finger- print” of molecules present in samples. From the Raman spectrum of biological 148 NIR Spectroscopy for Endpoint Analysis in Blending Operations samples, the major compounds like proteins, lipids, carbohydrates, DNA/RNA, etc., Edward Gooding, Viavi Solutions, 1402 Mariner Way, Santa Rosa, CA can be assigned based on their wavelength-specific position. The information from 95407, Brad Swarbrick each peak and their ratios provides indeed useful information, however multivariate Blending is a key process in the production of pharmaceuticals, animal feed, petro- statistical analyses on whole spectra are usually required to discriminate differences chemicals and many other industrial materials. Traditionally, sample homogeneity among individual samples or groups. The high complexity of biological systems and has been monitored using sample thieves combined with atline or offline analysis. large number of variables usually paired with the quite low number of observations However, the introduction of a sample thief into a blending vessel comes with signif- are some challenging characteristics of Raman data. What are multivariate statisti- icant sampling error that is difficult to quantify. Furthermore, in powder blending, a cal techniques that help deal with Raman data effectively? The application of some sample thief can itself disrupt the blend structure, thus requiring extra blend cycles algorithms behind machine learning, as well as the strengths and weaknesses of to reach the endpoint. Spectroscopic methods are also subject to spatial heteroge- different methods are discussed. neity, but by permitting real-time process monitoring, they can be made more robust than thief sampling. In addition, windowed probes do not disturb the uniformity of the 145 Iterative Target Detection for Detection and Classification with an blending process. Near-infrared (NIR) diffuse reflectance spectroscopy is sensitive Application in Hyperspectral Imaging to both the chemical and physical makeup of powder samples via the absorption Neal B. Gallagher, Eigenvector Research, Inc., PO Box B, Manson, WA and scattering coefficients, respectively, of the material studied. Hence, this tech- 98831 nique is especially well suited to monitoring blending of granular solid samples. Classical least squares (CLS) is the tool of choice for detection and classification in We present data acquired with the VIAVI Solutions MicroNIR PAT-W spectrometer hyperspectral images because often target spectra are known but reference values from blending operations on pharmaceutical ingredients and animal feeds. Principal for each pixel are rarely available. Generalized least squares (GLS) is a weighted component analysis demonstrates that blending endpoints can be determined with CLS model used to suppress clutter signal (interferences and noise) while enhanc- substantially less blending and analysis time than in traditional sampling protocols. ing minor target signal. (GLS is also known as the matched-filter and the Aitken es- Using modern process analytical technology software, analytical methods can be timator.) An iterative target detection approach exhibits synergy between GLS and quickly built, calibrated and validated using this approach. the extended mixture model (extended least squares, ELS) to further improve dis- crimination. To enhance visualization of the methodology an example is shown for a 149 Autonomous Spectroscopy for Process Monitoring Landsat 8 image of Lake Chelan, WA, USA. However, the concepts demonstrated Brian G. Rohrback, Infometrix, Inc., 11807 North Creek Parkway S, Ste. are applicable to a wide range of applications including fault detection and classi- B-111, Bothell, WA 98011 fication in the process environment, chemical and medical imaging and forensics. A consortium of companies has undertaken a project to reduce the effort devoted to The distinct advantages over approaches like principal components analysis and producing, maintaining, and stabilizing optical spectroscopy performance in routine partial least squares include interpretability and ease of model updating (adaptabil- quality assessment. Over a five-year period, the group has examined an unprec- ity) relevant for time-series application. The example shown utilizes GLS iteratively edented historical collection of spectra from multiple spectrometers spanning 1-5 in a hierarchical approach to classification, followed by a combined GLS-ELS model years from sixteen oil refineries, with the goal of developing and maintaining stable was used to further split a single class that was otherwise difficult to classify. Both models for long-term deployment. Even though the data are tied to petroleum work, objectives were complicated by the presence of significant interference signal but the lessons learned are true for all applications. The technologies utilized follow a showed good results verified using ground truth. pattern of best practices, including the use of Robust outlier diagnostics, local and hierarchical modeling, and model augmentation. The effort has resulted in signifi- 146 Near Infrared Solutions for Biopharmaceutical Development and cant progress towards automation of model creation, stability, and maintenance in Manufacturing an industrial process. Additionally, we share a comparison of prediction capabilities Adam Hopkins, Metrohm USA, 9250 Camden Field Parkway, Riverview, of different spectroscopy technologies and form-factors that can lead to a significant FL 33578, Mackenzie Speer reduction in deployment and maintenance cost. Near infrared (NIR) spectroscopy has long been used in the traditional pharma- ceutical industry for applications such as wet-granulation and tablet analysis. How- 150 Analytical Solutions to Challenges in Headspace-GC-MS Analysis ever, the emergence of biopharmaceuticals and the continued emphasis on qual- of Volatile Extractable and Leachable Compounds ity-by-design provide many opportunities for NIR spectroscopy. The complexity of Xiaoteng Gong, SGS North America Inc., 75 Passaic Ave., Fairfield, NJ the products and production matrices are difficult to analyze by traditional methods 07004, Dujuan Lu, Danny Hower and nearly impossible to measure in real time except with spectroscopy. This talk Organic volatile impurities (OVIs) are found as residual solvents or degradation demonstrates the capabilities and benefits of NIR spectroscopy for biological and products from packaging materials of pharmaceutical products. These compounds biosimilar production. From materials inspection, to process development, and im- have no therapeutic value and might leach into drug products which can pose safety plementation as process analytical technology we will explore applications at each risks or change the efficacy of the product. It is important to identify andquan- phase of the biopharmaceutical production cycle. tify the volatile extractables and leachables since the presence of these OVIs in pharmaceutical products may be harmful to human health if they exceed the safety 147 Drug Polymorphous Analysis Using Raman and NIR Spectroscopic threshold. Commonly, gas chromatography equipped with a headspace sampler Techniques: an Application in HME Process Optimization and Real- and mass spectrometry (Headspace-GC-MS) is used to analyze OVIs. In extract- Time Monitoring able and leachable studies, it can be challenging to achieve sufficient sensitivity and Herman He, Thermo Fisher Scientific, 4410 Lottsford Vista Rd., selectivity, because of the diversity of unknown analytes potentially present and the Lanham, MD 20706, Mohammed Ibrahim, Rui Chen, Jiaxiang Zhang, complex matrix of the drug products. To address the challenges in the Headspace- Michael Repka GC-MS analysis of OVIs, a method development study was performed to optimize Polymorphous conversion of pharmaceutical active ingredient (API) can dramatical- the headspace sampling parameters with the strategies for developing the method ly affect its solubility and bioavailability. In some cases, the desired polymorphous based on the theory of gas phase chemistry. In this oral presentation, we focus on form of drug substance is even not stable at its pure solid format. With a proper partition coefficient of common OVIs in a variety of matrices, phase ratio, and equi- selection of bonding polymers, a hot melt extrusion (HMT) process can transform librium time, which provide theoretical foundations for incubation temperature, salt the drug polymorphous forms and capsule the drug molecules or particles within addition, and sample/vial volume ratio. In addition, different strategies of preparing its polymer matrix to maintain the structure stability. Pre-blended acetaminophen samples for Headspace-GC-MS analysis of OVIs in packaging components were (APAP) and a cellulose polymer hydroxypropyl methylcellulose (HPMC) powder compared and optimized. 23 2018 EAS Abstracts November 2018

151 An In-Depth Look at Osmium Characterization and Impurity 154 Using Wavelength-Dispersive X-Ray Fluorescence (WD-XRF) as a Determination by ICP Walkup, High-throughput Alternative to Inductively Coupled Thomas J. Kozikowski, Inorganic Ventures, 300 Technology Dr., Plasma Optical Emission Spectrometry (ICP-OES) for R&D Christiansburg, VA 24073 Pharmaceutical Elemental Impurity Applications Osmium has become an element of great interest over the past several years since Tiffany M. Brucker, Vertex Pharmaceuticals, 50 Northern Ave., Boston, its inclusion as a Class 2B element in United States Pharmacopeia (USP) <232> MA 02210, Eric J. Borsje, Henrik T. Rasmussen due to its use as a catalyst in the manufacture of certain drug products and its Heavy metal impurities are critically assessed in pharmaceuticals due to possible known toxicity to humans. Many analysts are struggling with this element due to its toxicological effects. The International Conference of Harmonization (ICH) lists unique chemistry. Nitric acid digestion techniques pose several challenges mainly permitted daily exposures (PDE) for each element, and materials must be within due to oxidation of the metal to osmium tetroxide, which is volatile and toxic to the these specifications.[1] Currently, inductively coupled plasma spectrometry (ICP) is analyst. Proper analytical testing of osmium by inductively coupled plasma (ICP) is the most commonly used technique to measure metals within the pharmaceutical not well understood due to its tendency to stick to plastic surfaces of containers and industry. However, ICP has several drawbacks including, but not limited to, difficult introduction system tubing. It can also give high biased results if any of the osmi- sample preparation (using strong acids, for digestion of samples), sample destruc- um exists as osmium tetroxide when it reaches the plasma due to increased neb- tion, daily calibration, and potential carry-over between samples. Wavelength-dis- ulization efficiency compared to commercially available spectroscopic standards. persive X-ray fluorescence (WD-XRF) is an alternative technique for elemental im- USP <232> emphasizes a risk-based approach and strongly suggests omitting purity analysis used across several industries for a variety of applications. However, the testing of osmium if it is not necessary, but many contract analytical labs are there is limited information evaluating the technique for pharmaceutical applications. being tasked with testing all 24 elements anyway. The focus of this presentation Advantages of WD-XRF include minimal sample preparation, non-destructive anal- is on characterizing trace metals grade ammonium hexachloroosmate(IV) by both ysis, and absence of daily calibration. We evaluate the benefits and capabilities of ICP- optical emission spectrometry (OES) and ICP-mass spectrometry (MS). The WD-XRF, comparing quantitative performance with ICP-OES methods while addi- various challenges of osmium in aqueous media are discussed including long-term tionally investigating the feasibility of creating a walk-up workflow with high-through- stability, storage container material, acid matrix selection, and waste handling. In- put capability. Additionally, we explore the instrument’s internal semi-quantitative strument-specific concerns are addressed as well including interferences by both application. ICP-OES and ICP-MS, introduction system selection, calibration approaches, impu- Reference: rity determination, and washout strategies. [1] ICH Q3D Elemental Impurities Guidance for Industry, September 2015 152 Characterization of Non-Compendial Reference Standards for Impurities: How Good is Good Enough? 155 Challenges in Root-Cause Investigation and Elimination of Artifact Bernard A. Olsen, Olsen Pharmaceutical Consulting, LLC, 520 Kings Peaks in the UPLC Impurity Profile of Compounds with Nosyl Glen Way, Wake Forest, NC 27587, Christian Zeine, Jens Boertz Group For the characterization of primary reference standards for active pharmaceutical Van Truong, Merck & Co., 126 E. Lincoln Ave., Rahway, NJ 07065, Hao ingredients (API) numerous suggestions are available already, for example in the Luo, Robert Hartman general text 5.12. of the European Pharmacopoeia. Also International Conference Compounds containing nosyl groups were found to be reactive toward liquid chro- on Harmonization (ICH) Guideline Q7 refers to reference standards for API. Howev- matography stationary phases with charged surfaces, such as ultra-performance er, for reference standards for impurities, information and guidance is less easily at liquid chromatography (UPLC) column L1 C18+. During the purity testing of an ac- hand, and less detailed. The European Pharmacopoeia states in text 5.12. that “a tive pharmaceutical ingredient (API) intermediate containing bis-nosyl groups using CRS (Chemical Reference Substance, author’s note) corresponding to an impurity an UPLC L1 C18+, 1.6 µm column, four artifact peaks were observed in the chro- is characterized for identity and purity”. The ICH just requires in the impurity guide- matogram. These peaks showed up randomly at different levels, which posed a lines Q3A/Q3B that “reference standards used in the analytical procedures for con- great challenge to quantitate the impurity profiles from batch release, stability, and trol of impurities should be evaluated and characterized according to their intended DoE studies. The following observations led to the determination that the random uses.” National regulatory authorities also do not provide further guidance on the peaks were artifacts of on-column reactivity: 1) they appeared inconsistently and topic. Not surprisingly therefore, approaches to impurity standards vary widely, from varied significantly from analysis to analysis for the same sample; 2) they never both manufacturers of such standards and users as well. This contribution explains appeared in blank injections either before or after sample injection; 3) they did not the different approaches to impurity reference standards. The focus is on analytical show up in the first sample injection on a new column. However, they would ap- characterization, and on the question which level of characterization is adequate for pear quickly and become larger after continuous injections; 4) once present, column the corresponding purpose. It will also point out the risks connected to the quantita- flushing with the high organic component could not remove them. A root-cause in- tive use of an impurity reference standard which from its characterization might be vestigation discovered that the positively charged column surface could induce the more suitable for a qualitative use only. ‘on-column interaction’ between the compound and the stationary phase at low-pH mobile phase conditions to form the artifacts. As part of the column chemistry, the 153 External Reference Standards or Relative Response Factors: surface of the L1 C18+ column is positively charged at low pH while being neutral Considerations for Quantitation of Impurities in Pharmaceuticals from mid to high pH. This column characteristic was evaluated during the root-cause Bernard A. Olsen, Olsen Pharmaceutical Consulting, LLC, 520 Kings investigation to aid finding the solution for this challenging artifact issue. Details of Glen Way, Wake Forest, NC 27587, Christian Zeine, Jens Boertz the root-cause investigation and mitigation strategy to eliminate the artifacts from Accurate and reproducible quantitation of impurities in pharmaceuticals is an import- the impurity profile without requiring significant changes to the validated method ant part of drug development. Impurities must be controlled to ensure the quality of are discussed. the product is as good as or better than the quality of materials used in preclinical safety studies and clinical trials. International Conference on Harmonization (ICH) 156 Application of LC-MS-MS to Study the Photodegradation of Q3A and Q3B guidelines for drug substances and drug products, respectively, pro- Phthalates: Kinetics and Intermediate Degradation Products vide regulatory expectations for investigation and control of impurities. Thresholds Xiaofei Lu, University of Massachusetts Dartmouth, Chemistry and for identification and safety qualification of impurities can be based on relative per- Biochemistry Dept., 285 Old Westport Rd., Dartmouth, MA 02747, centages or directly in milligrams of exposure, depending on the nature of drug sub- Yuegang Zuo stances and products. Therefore, accurate assessment of impurity levels is needed Phthalic Acid Esters (PAEs) have been intensively used as plasticizer and additives to guide development efforts. Accurate impurity quantitation is also needed during in industrial and daily consume products. They can be readily released into the en- the lifecycle of the drug. Determination of impurities for most pharmaceuticals is vironment during manufacture, use, and disposal and have been detected frequent- accomplished by high-performance liquid chromatography (HPLC) with ultraviolet ly in various environmental matrices, including surface waters. Many studies have (UV) detection. Quantitation can be performed versus an external standard of the showed that PAEs possess long-term adverse effects such as reproductive toxicity, impurity itself or by comparison to the response of the active pharmaceutical ingre- carcinogenicity, and chronic organ toxicity. Knowledges on transformation and fate dient (API). If the response (peak area/concentration) of the impurity is different than of PAEs in the environment are urgently needed to assess their risk to human health the response of the API, a relative response factor (RRF) may be applied to report and ecosystem. In this study, an accurate and rapid liquid chromatography mass a more accurate weight percentage result for the impurity. Relative response factors spectrometry (LC-MS-MS) method has been developed to study the photodegra- can be applied whether an area normalization procedure (% of total peak area) or dation of dimethyl, diethyl, and dipropyl phthalate. All phthalate photodegradation a dilute solution of an API standard is used. Several factors influence the choice reactions obeyed pseudo zero-order kinetics. The effects of nitrite, nitrate, humic of impurity quantitation using impurity reference standards or the API with RRFs. acid (HA) and pH on the photodegradation of phthalates were also investigated. This contribution reviews these factors and discusses considerations for making an Both nitrite and nitrate promoted the photodegradation of all the three PAEs. HA at informed choice. low concentration accelerated the phthalate photodegradation, but at high concen- tration HA inhibited the photodegradation. Hydrolysis and hydroxylation products 24 2018 EAS Abstracts November 2018

were identified as two major categories of intermediate in the photodegradation of 160 Egg as a Medium in Ancient Mycenaean, Greek and Roman phthalates. Painting, Norman Muller, retired Princeton University Art Museum, Elm Dr., 157 Old Museum Collections as a Source for New Chemical Analyses Princeton, NJ 08544 Jennifer A. Loughmiller-Cardinal, University at Albany-SUNY, 1117 Life While encaustic is a well-known paint binder in ancient Roman Fayum portraits Sciences, Albany, NY 12222, Igor Lednev on thin wood panels, little attention has been given to the fact that egg (tempera) Archaeologists have, for the past 150 years, collected and stored an enormous was a more common paint binder among the ancient cultures of the Mediterranean number of artifacts. Meanwhile, most museum collections have remained underuti- region, extending back to 1300 B.C. The scientific identification of egg as an an- lized, under-studied, and neglected. While the methods of analysis have changed cient paint binder begins in the late 1970s, and over the years dozens of studies dramatically over this past century, application to ancient materials, especially or- have appeared in scientific journals identifying the binder. From 2001 to about 2008, ganic residues, remains underdeveloped. With the rapidly expanding analytical research on thirteen ancient Roman paintings on wood panel, and fragments of techniques designed to identify organic and inorganic food residues, the material them, were analyzed for medium, using gas chromatography/mass spectroscopy housed in museums offer ideal study cases. Traditionally, most methods entailed (GC-MS) for the first four samples, and high-performance liquid chromatography sample taking by scraping or drilling, and ultimately the destruction of the residue (HPLC) for the remaining samples. All were found to contain egg, often with an sample, this therefore precludes the analysis of a significant portion of rare, pre- addition of glue. cious, or fragile artifacts. Raman spectroscopy does not require samples to be taken from the ceramic, but can be directly applied to the artifact itself in a nondestructive 161 Mass Spectrometry Study of Organic Gunshot Residue way. This also eliminates the time and cost of sample preparation. Further, Raman Jillian Mizak, West Chester University of Pennsylvania, 750 South spectroscopy is now more commonly available and generally employed in museums Church St., Schmucker Science South 333, West Chester, PA 19383, for pigment and material analyses. A great number of archaeological specimens Monica Joshi would be added to the list of potential candidates for analysis if a non-destructive Gunshot residue (GSR) consists of inorganic residue, originating from the primer, method could be applied. This presentation is a discussion of recent applications of and organic residue that originates from the combustion of propellant. Traditionally, Raman hyperspectroscopy on organic food residues towards these goals forensic detection of GSR has focused on the scanning electron microscopy ener- gy dispersive X-ray spectrometry (SEM-EDX) study of the morphology and chem- 158 Investigation of Painted Decorations and Soluble Nylon Coating on ical composition of characteristic particles formed by the primer residue. Recently, Japanese Sugito at the Philadelphia Museum of Art there has been increasing interest in the organic component of GSR due to several Georgia A. Arbuckle-Keil, Rutgers University, 315 Penn St., Camden, reasons, particularly the changing composition of primers. Concurrent analysis of NJ 08102, Beth A. Price, Kate Duffy, Matthew Dustin, Peggy Olley, Katie inorganic (IGSR) and organic GSR (OGSR) generally involves splitting the sample Shulman, Wei Kao, Felice Fischer or performing a stub and swab collection. However, methods that are able to detect Elaborately painted, cedar wood sliding doors (sugito) graced Japanese castles, both in a single collection are preferable. This presentation discusses the use of during the Edo period. Twenty large sugito, ranging in size from 63 ½ x 62 inches mass spectrometry to detect and identify organic GSR components from cartridges, (pair) to 70 x 68 inches (individual door) are in the Philadelphia Museum of Art GSR collection stubs and swabs. SEM-EDX is used to monitor collection of GSR (PMA). Several analytical methods have been applied to identify the constituents, on the collection devices. The study aims to determine the feasibility of a single pigments and binders, in the painted decorations. Detection of metal(s) in pigments, collection dual analysis method for gunshot residue. Direct probe and solid phase for example, was performed via a portable X-ray fluorescence (XRF) analyzer. Fur- microextraction mass spectrometry (SPME-MS) sample introduction techniques for ther characterizations were made using vibrational spectroscopy (Fourier transform mass spectrometry provide rapid and sensitive confirmation of target compounds. infrared microspectroscopy (MFTIR) and Raman microspectroscopy) and scanning Diphenylamine and its derivatives, ethyl and methyl centralite, akardite-II, di-, tri- electron microscopy (SEM) with energy dispersive spectroscopy (EDS) applied to nitrotoluene are target OGSR compounds in this study.[1-2] paint cross-sections. Interestingly, the sugito at the PMA were coated in the 1960s References: with soluble nylon to protect and consolidate the decorated areas. Although soluble nylon was favored for its flexibility, clarity and adhesive properties, it is no longer [1] Maitre, M., Kirkbride, K. P., Horder, M., Roux, C., & Beavis, A. (2018). Think- used as it ages poorly and becomes insoluble. A soluble nylon product, noted in ing beyond the lab: Organic gunshot residues in an investigative perspective. the art conservation literature, was marketed under the trade name Calaton CB Australian Journal of Forensic Sciences, DOI: 10.1080/00450618.2018.1457718 by Imperial Chemical Industries, Ltd. (ICI). Calaton CB is a N-methoxy methylated [2] Taudte, R.V., Beavis, A., Blanes, L., Cole, N., Doble, P., & Roux, C. (2014). De- derivative of nylon 6,6. Vibrational spectroscopy, thermal analysis and pyrolysis gas tection of gunshot residues using mass spectrometry. BioMed Research Inter- chromatography mass spectrometry (Py-GCMS) indicates that the polymer coating national, 2014, 1-16. on the PMA sugito is a derivative of nylon 6,6. This investigation is of significance in- ternationally as the sugito in Nagoya and Nijo Castles, Japan are national treasures 162 The Prevalence of Male DNA Under a Females Fingernails and cannot be sampled. Alexis Baxter, Cedar Crest College, 100 College Dr., Allentown, PA 18104, Janine Kishbaugh 159 Spectroscopic and Diffraction Analysis of Verdigris Pigment & The frequency of finding a male DNA profile under a female’s fingernails isun- Alteration Products on Organic Substrates (Paper and Gum Arabic) known. When a victim comes into contact with a male assailant during a violent or Marcie B. Wiggins, University of Delaware, 005 Lammot duPont sexual assault, DNA genotyping is used to find male DNA on the victim and identifies Laboratory, Newark, DE 19716, Emma Heath, Karl S. Booksh, Jocelyn the perpetrator. However, finding male DNA may not necessarily reflect the context Alcantara-Garcia on how the transfer occurred. Considering that DNA methodologies have improved Many organic cultural heritage objects exhibit discoloration and extreme degrada- with sensitivity, transfer may occur through casual contact. In addition, secondary tion as a result of the presence of copper-based pigments, such as copper acetate and even tertiary transfer may be detected. Assessing the random nature of male (verdigris). This study focuses on the degradation of neutral and basic verdigris to female transfer is the basis for this study. Female participants in this study either pigments with respect to its organic substrates, cellulose and gum Arabic. Whatman lived with or frequently encountered male individuals in their daily lives. A female filter paper treated with neutral and basic verdigris and 0-10% gum Arabic binder that lives alone was used as a control. All samples were collected via cutting with were artificially degraded. These samples were monitored with X-ray diffraction and fingernail clippers. All nails from one hand were combined and analyzed together. Fourier-transform infrared (FTIR) and X-ray photoelectron (XPS) spectroscopies. DNA from these samples were then extracted using a Qiagen DNA Investigator Both neutral and basic verdigris form an intermediate form of basic verdigris as a kit, followed by quantitation via real-time PCR using SYBR-Green and Alu-primers. first degradation product before continuing to degrade to copper oxide. Although Amplification was performed with the PowerPlex® Y23 system. Genotyping was changes seem independent of their organic substrates, the presence of organic performed using an Applied BioSystems 3130XL and fragment analysis performed material facilitated conversion. The addition of gum Arabic reduced the copper spe- with GeneMapper® IDX v1.5. Of the 42 samples tested, 90% produced partial pro- cies, however degraded samples show copper (I) species convert back to copper files from single and multiple contributors. Over half of the samples had multiple (II) species. These findings are pivotal to understand the degradation pathways of contributors. In some cases, profiles were linked to male co-habitants. Since only verdigris, a pigment that has a documented contribution to the decay of cellulose partial profiles were obtained in this study, it is hypothesized that full male profiles artifacts. These results are the beginning of more in-depth studies that aim to under- obtained during casework is more indicative of forceful contact. stand fundamental copper-organic reactions of other materials at risk. 163 The Stability of Synthetic Cathinones of Toxicological Interest Lexus R. Rutter, Arcadia University, 450 S. Easton Rd., Glenside, PA 19038, Karen S. Scott, Heather L. Ciallella Analytical chemistry faces increasing challenges arising from the degradation of analytes over time. Having knowledge of stability of these analytes in solvents and 25 2018 EAS Abstracts November 2018

toxicological samples is therefore of the utmost importance. Control standards are compounds that can have a significant odor impact. This talk showcases Dow’s typically sold or prepared in solvents like methanol or acetonitrile meaning it is im- odor analysis capabilities and how they are used to solve problems and advance portant to understand the stability of analytes in these solvents as well as toxicologi- product development with examples from our coatings, packaging, and personal cal specimens including blood and urine. Previous research in our lab has evaluated care businesses. the stability of alpha-pyrrolidinopentiophenone (α-PVP) mephedrone, methylene- dioxypyrovalerone (MDPV), and, naphyrone in various conditions to determine if 167 GC Inlet Liner Selection for Split and Splitless Analyses degradation occurs and under what environmental conditions. It was found that the Linx K. Waclaski, Restek Corporation, 110 Benner Circle, Bellefonte, drugs manifested a range of instability mainly at room temperature that warranted PA 16823 additional studies. This research aimed to fill the gaps in previous research by eval- At the heart of the gas chromatography (GC) inlet is the inlet liner, which plays a uating the stability of the four synthetic cathinones with different storage conditions critical role in the vaporization and transfer of the sample to the column. Selecting and matrices. In study one, we evaluated the stability of all four synthetic cathinones an appropriate inlet liner for an analysis can depend on a number of factors, includ- under light and dark storage conditions at room (20 °C), fridge (4 °C) and freezer ing injection type, nature of the sample, and analyte properties. This presentation (-20 °C) temperatures over 30 days in methanol and acetonitrile. In study two, we examines choice of liner geometry based on injection type and sample, for opti- evaluated the stability of MDPV in urine at room (20 °C), fridge (4 °C) and freezer mized analyte recoveries, ensuring an efficient transfer to the analytical column. (-20 °C) temperatures over 30 days. In study three, we evaluated the stability of The exploration of liner geometries includes discussion of special shapes/features, α-PVP in blood and urine at room (20 °C), fridge (4 °C) and freezer (-20 °C) tem- volume, and the use of glass wool packing. The importance of inertness will also be peratures over 30 days. The results of the three studies indicate variable stability explored with respect to the physical interactions that can occur within a GC inlet. among the drugs and the matrices investigated. 168 Boost Productivity and Laboratory Efficiency for VOCs Analysis in 164 Role of Insects in Human Identity Water and Soil by Advanced Purge & Trap Gas Chromatography- Shayna L. Gray, The Pennsylvania State University, Forensic Science Mass Spectrometry (P&T-GC-MS) Technology Program, 329 Whitmore Laboratory, University Park, PA 16802, Scott Carlos F. Garcia, Thermo Fisher Scientific, 2215 Grand Ave. Parkway, Lindner, Reena Roy Austin, TX 78728, Amy S. Nutter, Jacob Rebholz, Tammy J. Rellar, Tim Evidence such as blood or saliva containing deoxyribonucleic acid (DNA) can be Anderson, Lori Dolata, Daniela Cavagnino helpful in linking an individual to a crime scene. Traditionally, DNA is obtained from Environmental testing laboratories are routinely involved in monitoring water and body fluids deposited on substrates commonly encountered at crime scenes. One soils contamination from chemicals commonly found in industrial products or con- of the more unusual sources that can be used for generation of DNA profiles are sumer goods, with the aim to control and minimize people exposure to possible toxic insects such as mosquitoes or flies. These insects are not generally included as compounds. Volatile organic chemicals (VOCs) are commonly found as contami- potential sources of human DNA despite the fact that female mosquitoes feed on nants in the environment and therefore heavily monitored in water and soil under human blood and store the blood in their midgut for a period of time after feeding. strict regulations. State-of-the-art approaches are available to help testing laborato- The goal of this research was to obtain forensic short tandem repeat (STR) DNA ries in delivering high-confidence results more easily and more efficiently, reducing profiles as well as detect single nucleotide polymorphism (SNP) from the blood in time waste and keeping costs down. This paper illustrates how advanced technol- the abdomen of mosquitos. STR analysis using two direct amplification kits and ogy is applied to the analysis of VOC in environmental matrices, offering enhanced SNP analysis using a sequencing panel were performed. Analysis of blood included analytical performance, extended uptime, and a streamlined workflow. both single sources and mixtures of human blood. It was concluded that DNA con- tained in the abdomen of the mosquito can be used to determine the donors’ identity 169 Non-Contact Submicron Spatial Resolution IR Spectroscopy and using both the STR and SNP technologies. The results indicated that profiles ob- Imaging of Pharmaceutical Samples tained from the blood in the mosquitoes were consistent with profiles obtained from Curtis Marcott, Light Light Solutions, 2558 High Hammock Rd., reference blood of the same source. DNA from mosquitoes euthanized at different Seabrook Island, SC 29455, Eoghan Dillon, Debra Cook, Craig Prater, time intervals was assessed for degradation and it was determined that DNA was Neil Lewis completely degraded when blood remained in the abdomens of these mosquitoes Many pharmaceutical ingredients have sub-micron particle sizes. The chemical for 72 hours. nature of the interactions between active pharmaceutical Ingredients (API) and ex- cipient components plays an important role in both the formulation and end use 165 Ghost Peaks and Artifactully Increased Impurity Peaks in Both performance of pharmaceutical products. Ingredient distributions, molecular level Direct Injection and Headspace GC Analyses due to Thermo interactions, as well as tablet coating materials all affect the timing and effectiveness Lability of Analytes and/or a Common Sample Diluent (Resubmitted) of the release of the API into the patient. A new infrared spectroscopic approach Min Li, Huahai US Inc., 2001 Eastpark Blvd., Cranbury, NJ 08512 that uses the photothermal infrared (PTIR) response of the sample is discussed. A In both direct injection and headspace gas chromatography (GC) analyses, ghost visible laser is used to sense the photothermal response when a tunable IR pump peaks and artefactually increased impurity peaks are observed from time to time, laser wavenumber is absorbed by molecular vibrations in the sample. The resulting particularly for analytes with thermo lability. Such events can trigger unreal and un- spatial resolution is thus determined by the diffraction limited spot size of the visible desirable out-of-specification (OOS) investigations in a good manufacturing practice laser (around 500 nm) independent of the IR wavenumber. In addition, this visible (GMP) environment. Investigation of such events are usually not straightforward optical response is measured in a reflection geometry, resulting in IR spectra that and sometimes can be quite challenging. In this presentation, we present sever- match those acquired in transmission mode, even for thick samples. Several ap- al case studies in which the root causes of these events were thoroughly investi- plications of PTIR analysis of pharmaceutically relevant samples are discussed.. gated, leading to the elucidation of the underlying molecular mechanisms of these events. The case studies covered range from thermo decomposition of analytes 170 Non-Contact Sub-Micron Infrared Spectroscopy Using Visible in GC injector port to sample diluent (DMSO) decomposition (usually catalyzed by Probe Detection the presence of the analytes). Various tools were utilized during the investigations, Eoghan Dillon, Photothermal Spectroscopy Corp., 325 Chapala St., including GC-mass spectrometry and the use of stable isotope labels. The results Santa Barbara, CA 93101, Craig Prater, Curtis Marcott and methodology utilized would be helpful for a systematic investigation of such Infrared spectroscopy is among the most common chemical characterization tech- artefactual events in the future. niques, having been used for many decades now. However, two limitations have prevented its adoption in many applications: the spatial resolution is limited diffrac- 166 Development of New Products and Odor Problem Solving Using tion physics to around 5 micrometers; and, thin (or diluted) samples are needed to Gas Chromatography-Mass Spectrometry-Olfactometry Analysis minimize infrared (IR) band saturation. This talk focuses on a new technique that Michelle Gallagher, The Dow Chemical Company, MS: S1117, 400 uses the photothermal response of a sample to eliminate both of these limitations. Arcola Rd., Collegeville, PA 19426, Elizabeth Snow, Jim DeFelippis, Jim A visible laser is used to sense the photothermal response when a tunable IR probe Bohling, Paul Doll, Melissa Leach laser wavenumber is absorbed by molecular vibrations in the sample. The resulting Gas chromatography-mass spectrometry-olfactometry analysis (GC-MS-O) is a spatial resolution is determined by the diffraction limited spot size of the visible laser technique used to determine the retention time of odorants in complex mixtures and (around 500 nm) independent of the IR wavenumber. The visible optical response combines high resolution chromatographic separation with human olfactory abilities. is measured in reflection mode, resulting in IR spectra that match those acquired in GC-O has been widely practiced in the food and flavor industry, but can also be transmission mode, even for thick samples. Since it works in reflection mode, it can very useful in other industries such as the chemical industry as will be discussed in be used for IR spectroscopy of surfaces that don’t lend themselves to an attenuat- this presentation. At the Dow Chemical Company, GC-O has been utilized to help ed total reflection (ATR) type of contact method. The photothermal response has innovate new products and solve critical odor issues in our products, raw materials already been measured by an atomic force microscope (AFM) probe and shown to and customer samples. Our instrumentation combines two-dimensional separa- have an IR spatial resolution of 10 nm. However, the use of an AFM requires some tions, along with GC-MS-olfactory analysis, allowing us to identify critical trace level degree of AFM related sample preparation, while this new method is easy to use 26 2018 EAS Abstracts November 2018

and will work on samples prepared for IR and Raman microscopy. Several exam- sis on a NanoAcquity UPLC coupled to a Q-TOF Ultima API mass spectrometer ples of this new sub-micron non-contact methodology in polymer, pharmaceutical (both from Waters Corp). To complement this, the sera were also digested by tryp- and life science applications are presented. sin in-solution followed by nanoLC-MS-MS analysis. The initial scout runs for the in-solution digestion and nanoLC-MS-MS analysis are complete. EWe identified po- 171 Examining Performance of Confocal Raman Microscope Using tential dysregulations of proteins involved in blood clotting, the complement system, Nanocarbon Materials and the immune system due to legacy chemical exposure. Additional more in-depth Alexander Rzhevskii, Thermo Fisher Scientific, 2 Radcliff Rd., analyses are currently underway. Tewksbury, MA 01876 Recent technological developments in Raman microscopy and imaging for the char- 174 Dual nESI–DIMS-MS for Measurement of Differential Lipid acterization of objects on a submicrometer scale dictate the need for materials and Expression methods that could be used to test confocality and spatial resolutions of Raman James E. Keating, University of North Carolina-Chapel Hill, Dept. of microscopes in order to comprehensively evaluate their capabilities and objectively Chemistry, Chapel Hill, NC 27599, Gary L. Gish compare the microscopes with each other. Historically, the resolution targets such Current lipidomics workflows are centered around acquisition of large data sets fol- as the Si knife edge and the wafer for testing, respectively, the lateral (horizon- lowed by lengthy data processing, which reveals information and trends that require tal) and the axial (vertical) spatial resolutions, were readily available and provided follow-up experiments. A dual nanoelectrospray – differential ion mobility spectrom- satisfactory approaches for checking overall microscope confocality and identifying etry – mass spectrometry (dual nESI-DIMS-MS) platform was developed to perform performance issues (e.g., a malfunctioning sampling stage or a poor optical align- real-time comparisons between samples, providing information that determines the ment). However, the targets are not well suitable for reckoning a true “instrument direction of an experiment as its being performed. Syncing the control of nanospray- function” of the confocal Raman microscope and, thus, cannot be “ideal standards” ers and DIMS compensation field to the accumulation portion of the ion trap scan to evaluate the spatial resolutions achieved in a given instrument and to serve as function enables unified control of the platform for differential mobility separation, benchmarks for the instrument comparison. In this work, the results of Raman hy- acquisition of full scan mass spectra, data comparison and follow-up tandem mass perspectral imaging of a single individual Carbon Nanotube and a single layer of spectrometry experiments. The separation mechanism of DIMS (in-space versus Graphene on a glass substrate and buried between glass layers are reported. The the in-time separation of conventional ion mobility) is conveniently coupled to the materials can be referred to as “ideal standards” for the spatial resolutions tests dual nESI source as transmission of target species is controlled by a compensa- because they represent the structures with the dimensions significantly smaller than tion voltage rather than a retention/drift time after injection (as in chromatography, the diffraction limited volume of a focused laser beam and the high enough Raman low-field ion mobility). This voltage can be scanned to transmit ions of successive scattering cross-sections to ensure reliable measurements. The results show that differential mobilities or specifically stepped to transmit previously identified target the standards can be used for a comprehensive examination of the confocal per- ions. The dual nanospray source is designed to collect data from two samples (con- formance, accuracy and reproducibility of high-end Raman imaging microscopes. trol and experimental) at each compensation voltage, providing measurement of differential expression. The dual nESI-DIMS-MS platform can be operated in an 172 Dynamic Fluorescence Measurements of Rose Bengal untargeted (ramped over range of voltages) or targeted (selected voltages) mode. Photooxidation The dual nESI-DIMS-MS/MS platform was validated through measurement of lipid Yinan Zhang, University of Delaware, 17 Marvin Dr., Apt. B2, Newark, species in biological media extracts with both simulated (lipid standards spiked in) DE 19713, Sharon L. Neal and real (environmentally stimulated) differential expression. Rose Bengal (RB) is a halogenated fluorescein dye, a highly efficient photosen- sitizer that has been used in a variety of environmental and biomedical applica- 175 Digital Breast Cancer Diagnosis and Microenvironment Analysis tions, such as photodynamic therapy. It is well known that RB is readily excited to using Chemical Imaging the triplet state and converts ground state molecular oxygen to singlet oxygen by Shachi Mittal, University of Illinois at Urbana Champaign, Beckman energy transfer. While oxygen photosensitization by RB has been widely studied Institute for Advanced Science and Technology, Champaign, Illinois, under various conditions, the sensitivity of the photooxidation to the solvent and 61820, Kevin Yeh, Andre Kadjacsy-Balla, Rohit Bhargava the potential of counter ions has made mapping the interaction of processes that The current histopathological tools are lacking in quantitatively estimating disease occur during photooxidation challenging. The potentially dramatic effects solvent progression starting from benign intraductal breast proliferations to tumor invasion. amphiphilicity and compartmentalization can have on the physical and chemical This can primarily be attributed to the limited use of multiple tissue compartments in properties of solubilized molecules is well documented, but studies of the impact observing, understanding and treating cancer due to the lack of technology to cap- of solvent microheterogeneity on RB photooxidation are limited. In this paper, we ture its detailed morphological and biochemical signatures. The proposed approach report our progress using frequency-domain dynamic multivariate fluorescence of chemical imaging coupled to pattern recognition tools integrates the structural measurements to investigate the photooxidation of RB in microheterogeneous me- and molecular tissue signatures for extensive profiling of the tumor and its associat- dia. Octanol is a biorelevant, microheterogeneous solvent that is commonly used to ed microenvironment. Early and precise tumor detection can greatly improve patient model the hydrophobicity of biological systems in experimental and computational outcome as survival rates and prognosis is highly dependent on the stage of the studies. Fluorescence emission-decay measurements can be arranged in matrix tumor. In the proposed study, breast tissue microarrays spanning different disease format, making them amenable to numerical analysis, enabling computational isola- states were imaged using chemical imaging. Corresponding H&E stained images tion of the spectra and decay profiles of components present in the photoirradiated were annotated by a pathologist to be used as ground truth to train a machine samples. Varying the photoirradiation conditions (source power, solvent, co-solubili- learning model for identifying different cell types. This method was then used to zation with oxygen traps) reveal mechanistic details via their impact on the contribu- build digital protocols for differentiating between cancerous and intraductal lesions tion of sample components. The spectra and kinetics of RB and its photoproducts in to overcome some of the challenges in the current standards of patient care. Dis- octanol will be compared to those observed in isotropic solvents, including aqueous ease signatures apparent both in epithelium (tumor) and stroma (tumor microenvi- buffer and butanol. ronment) have been modelled to build a comprehensive diagnostic and prognosis tool. Further, the developed approach was extended to discrete measurements to 173 Effects of Legacy Chemical Exposure in the Great Lakes Ecosystem achieve clinically feasible times for patient studies. This technique opens new ave- on the Human Proteome nues for digital and rapid cancer assessment for improved patient care. Emmalyn J. Dupree, Clarkson University, 8 Clarkson Ave., Potsdam, NY 13699, Bernard Crimmins, Thomas Holsen, James Pagano, Brooke 176 Raman spectroscopy and MCR-ALS Applications for Polymorph Thompson, Krista Christensen, Michelle Raymond, Jon Meiman, Costel Identification in Chinese Architectural Paints C. Darie Marcie B. Wiggins, University of Delaware, Dept. of Chemistry, The Great Lakes fish are a bountiful and healthy food source rich in omega 3 fatty Newark, DE 19716, Liu Mengyu, Liu Chang, Catherine Matsen, Karl S. acids utilized by many that reside adjacent to the lakes, but there are concerns Booksh about the risks of contaminant exposure through fish consumption. An anonymized In-depth analysis of copper-based pigments provides information on both the origin study was performed where sera from fish consumers in the Great Lakes basin were of these blue and green materials as well as insights for preserving cultural heritage analyzed for legacy chemicals such as polychlorinated biphenyls (PCBs) and or- objects. These pigments have a tendency to degrade and interact with the surround- ganochlorine pesticides (OCPs). Samples exhibiting the lowest concentrations were ing organic media. Chinese architectural paints are an example of several different adopted as controls and the highest were considered the exposure group. Legacy “copper green” pigments. Initial identification of copper chloride trihyrdoxide poly- chemical analysis was performed using Centers for Disease Control and Prevention morphs in the artwork found in the Forbidden City’s Lin Xi pavilion (Beijing, China) methods employing isotope dilution mass spectrometry. Proteomics analysis was prompted subsequent investigation into decorations from 12th to 19th centuries. performed by albumin depletion and separation by sodium dodecyl sulfate poly- Hyperspectral Raman imaging of these Chinese architectural paint cross-sections acrylamide gel electrophoresis (SDS-PAGE), followed by the in-gel trypsin digestion were analyzed with multivariate curve resolution-alternative least squares (MCR- and nano-liquid chromatography tandem mass spectrometry (LC-MS-MS) analy- ALS) to distinguish component spectra of atacamite and botallackite polymorphs 27 2018 EAS Abstracts November 2018

and their relative ratios. The differences in the polymorphs present and their relative 180 In-Situ Derivatization of Flavor Additives in E-Cigarette Liquids ratios speak to the origin of these pigments, which are currently being investigated. during Nano-Electrospray Ionization Mass Spectrometry This study explores the transition from mined “mineral green” to synthesized “cop- Megan R. Ogorchock, University of North Carolina at Chapel Hill, Dept. per green” pigments, and introduces insight into the pigments’ role in the overall of Chemistry, Chapel Hill, NC 27599, Tavleen K. Kochar, Gary L. Glish stability of the paint. Chemicals used as flavorings in e-cigarettes, such as diacetyl, cinnamaldehyde, benzaldehyde, and vanillin, have recognized associations with respiratory disease 177 Microfluidic Single-Cell Analysis of Oxidative Stress in when inhaled. Diacetyl is associated with bronchiolitis obliterans, more commonly Dictyostelium discoideum known as “popcorn lung.” Exposure to cinnamaldehyde has been shown to com- Jessica T. Duong, Trinity College, 300 Summit St., Hartford, CT 06106, promise the functionality of immune cells. Benzaldehyde has been found to have Kathy Rodogiannis, Michelle L. Kovarik cytotoxic and apoptotic effects. Vanillin has led to altered airway epithelial response Single cell analysis is necessary to investigate the role of cellular heterogeneity in on a cellular level. These small carbonyl molecules can be analyzed using mass stress responses. In this study, microfluidic chemical cytometry was used to analyze spectrometry (MS) by direct infusion, but are often detected with low sensitivity. single cells of the unicellular organism Dictyostelium discoideum. The reactive ox- Alternatively, derivatization of the analyte can be utilized prior to MS analysis to ygen species indicator dicholorodihydrofluorescein was used to report on oxidative enhance sensitivity for detection. One example of analyte derivatization involves stress levels, and carboxyfluorescein was utilized as an internal standard to account using Girard’s reagents. Girard’s Reagent T (GirT) and Girard’s Reagent P (GirP) for differences in loading and retention. The effect of peroxide treatment on single undergo a Schiff-base formation with a carbonyl compound and provide the analyte cells was assessed through microfluidic chemical cytometry. Cells were incubat- with a permanent, positive charge that increases ionization efficiency and sensitive ed with the dyes before being lysed by an electric field on a microfluidic device; detection by MS. Typically, derivatization reactions are run for hours at elevated dyes from the cells were then electrophoretically separated and detected by fluo- temperatures to get efficient conversion to products. However, recently it has been rescence. Based on data from 353 cells, the peak area ratio of dichlorofluorescein demonstrated that reactions can be completed on-line on a microsecond timescale to carboxyfluorescein increased from untreated cells (1.69 ± 0.89) to cells treated in a microdroplet generated through nano-electrospray ionization (nESI). Herein, we with 40 mM hydrogen peroxide (5.19 ± 2.72), as expected with increased oxida- demonstrate simultaneous derivatization and analysis of carbonyl compounds using tive stress. Additionally, the variance of the data increased with oxidative stress. nESI-MS. The derivatization kinetics of GirT and GirP reacting with these harmful These results suggest that Dictyostelium discoideum may possess adaptive, het- carbonyl flavorings were studied in the generated microdroplets. erogeneous responses to peroxide-induced oxidative stress. Future work is directed towards replicating the experiment with alternative sources of oxidative stress to 181 LabVIEW Controlled Instrumentation for the Acquisition and determine the conditions under which increased variance occurs. Automation of Time-Based Fluorescence and Phosphorescence Measurements 178 HOMEChem (House Observations of Microbial and Environmental Anthony M. Santana, University of Central Florida, 4000 Central Florida Chemistry): Preliminary High-Resolution Aerosol Mass Blvd., Orlando, FL 32816, Khang D Trieu, Stacy M. Wise, Andres D. Spectrometry Results from an Indoor Air Field Measurement Campiglia Campaign Research in our group has focused on exploring the full potential of high-resolution Erin F. Katz, Drexel University, 3141 Chestnut St., Philadelphia, PA photoluminescence spectroscopy with a significant selectivity enhancement based 19104, Michael R. Giordano, Benjamin S. Werden, Laura Ampollini, on information-rich multidimensional data formats. Herein, we present an improved Delphine K. Farmer, Marina E. Vance, Peter F. DeCarlo version of a previously developed instrument for the acquisition of high-resolution Despite years of outdoor air quality research geared towards improving human multidimensional photoluminescence data.[1] The tunable dye laser originally used health, it is estimated that 90% of our lives in America are spent indoors, and conse- in the previous system was replaced with a solid-state laser that allows continues quently, most exposure to outdoor pollutants occurs in the built environment. More- excitation between 200 and 1000nm. The use of a half-wave plate, a neutral density over, activity indoors significantly effects the concentrations and chemistry of gas filter and a polarizer provides constant intensity across the entire excitation range and particle phase constituents of indoor air. A recent field measurement campaign with an excitation band-pass equal to 0.32nm. Photoluminescence is collected with that took place in June 2018 in Austin, TX called HOMEChem (House Observations the aid of a spectrograph and an intensified-charge-coupled device (ICCD). The of Microbial and Environmental Chemistry) aimed to explore the effect of every- spectrograph is equipped with two diffraction gratings consisting of 1200 grooves. day activities such as cooking, cleaning, and the use of personal care products on mm-1 and 1800 grooves.mm-1. The 1200 grooves.mm-1 provides a limiting res- the chemistry of the indoor environment. This project brought together a myriad of olution of 0.32nm. This resolution includes cross talking among four CCD pixels. state-of-the-art advanced analytical techniques including high resolution gas and Correction factors were experimentally obtained to minimize gain variations across particle phase mass spectrometers deployed to measure outdoor air, indoor air, and the active area of the CCD. LabVIEW Schemes were developed in-house to allow interactions between the two. We present preliminary high-resolution aerosol mass for the automation of data acquisition and processing. Individual structures were spectrometry and gas phase spectroscopy results from HOMEChem with a focus on programmed to provide spectral correction and ease collect two-dimensional (2D) chemistry and concentrations during cooking events. During a simulated Thanksgiv- excitation and emission spectra, lifetime decays, wavelength time matrices (WTMs), ing meal preparation and consumption, concentrations of indoor particulate matter excitation and emission matrices (EEMs) and time-resolved excitation and emission were observed to be roughly 100 times that of outdoor. These particles were com- matrices (TREEMs) in the fluorescence (ps – us) and phosphorescence (ms – s) prised of black carbon, chloride, organic aerosol, and more. High concentrations of time domains. Examples of these data formats will be presented for the analysis of gas phase ammonia (~100 ppb) and carbon dioxide (~4000 ppm) were observed organic pollutants in Shpol’skii matrixes. as well. We anticipate that the results of this study will influence how we view and Reference: control our indoor spaces to reduce exposure to indoor air pollution. [1] A.D. Campiglia, A.J. Brystol, S. Yu, Anal. Chem. 2006, 78, 484-492 179 Characterization of CZE-MS Detection and Quantification of Beta- N-Methylamino-L-Alanine in Plasma Samples 182 Spectroscopic and Computational Studies of PRODAN Kaylie I. Kirkwood, North Carolina State University, 2620 Yarbrough Matthew J. Phillips, University of Delaware, 105 Lammont Dupont Dr., Raleigh, NC 27607, Joshua Beri, Allyson L. Mellinger, Michael S. Laboratory, Newark, DE 19711, Swapnil Baral, Edward Lyman, Bjoern Bereman, David C. Muddiman Baumeier, Lars Gundlach We describe an optimized method for the detection and quantification of the neu- 2-propionyl-6-dimethylamino naphthalene (PRODAN) is used in membrane re- rotoxin beta-N-methylamino-L-alanine (BMAA) using a chip-based capillary zone search due to the sensitivity of its emission spectrum to changes in the environment. electrophoresis mass spectrometry (CZE-MS) platform. Resolution of BMAA and Typically, the fluorescence intensity at two wavelengths is analyzed to draw con- its structural isomers, AEG, 2,4-DAB and BAMA, was achieved using this method clusions about water content, for example, of the PRODAN’s micro-environment. in less than 5 minutes. Various parameters were optimized using parallel reaction However, both polarity and hydrogen bonding ability of the environment play roles monitoring (PRM) in order to improve the limits of detection and quantification of in PRODAN’s emission behavior; PRODAN was studied in a variety of solvents and BMAA and increase the number of MS2 scans. A positive control standard consisting mixtures, using steady state spectroscopy and time resolved techniques as well as of a mixture of peptides containing BMAA misincorporations was synthesized from computational methods to bring a new perspective to the discussion of the nature of α-casein and characterized. Additionally, CZE-MS and liquid chromatography-MS the excited states of PRODAN and the factors which give rise to them. methods were developed for future application to a large cohort of amyotrophic lat- eral sclerosis (ALS) patient plasma samples for targeted and untargeted analyses. 183 Use of FT-IR Spectroscopy to Classify and Discriminate Food Protein Powders Ronald Rubinovitz, Thermo Fisher Scientific, 4410 Lottsford Vista Rd., Lanham, MD 20706 Variations in food protein powders can arise from factors such as their origin and

28 2018 EAS Abstracts November 2018

the method of extraction and isolation. As a consequence, the protein component of the in-situ measurement of metabolites of interest in cell culture media were devel- these products are in turn expected to vary in structure. Additionally, these products oped. Two of the most commonly monitored parameters are glucose, as the major contain also non-protein components (lipids, carbohydrates, proteins, salts, etc.) carbon and energy source, and lactate, as a metabolic product. Therefore, two am- introducing still more variation. In the manufacturing environment, it is important to perometric enzymatic sensors specific towards these key analytes were developed. quickly characterize materials in terms of identity and quality. Therefore, the prac- Glucose and lactate sensors were based on screen-printed miniaturized technology ticality of Fourier transform infrared (FT-IR) to rapidly classify protein powders of which allowed collecting samples from the media in order of microliters, without dis- particular type (whey protein versus rice protein, for example), detect differences turbing the cells. The proposed glucose and lactate sensors were able to distinguish between suppliers of nominally similar proteins, and estimate lot to lot variation can differences in glucose and lactate levels in cell media 24 hours after the cells have be highly advantageous. FT-IR spectroscopy makes such classification possible been seeded. Due to the quantitative establishment of the uptake of glucose and due to its ability to measure proteins as well as the additional components in these production of lactate it was possible to effectively control the growth process stages. powders. Beyond just detecting the presence of proteins, FT-IR has been used to characterize the secondary structure of the proteins through the analysis of the am- 187 Analysis of Greenhouse Gases Using the Shimadzu GC-2014 with ide I band at about 1650 cm-1 .Thus, in addition to principal component analysis the ARC Jetanizer for CO2 and CO In-Jet Methanization (PCA) of the entire mid-IR region containing information related to overall product Ian W. Shaffer, Shimadzu Scientific Instruments, 7102 Riverwood Dr., composition, this study also focuses on the amide I region by PCA and curve-fitting Columbia, MD 21046, Kyle O. Reddick, Yuan Lin, Martin D. Smith, methods as a means to emphasize protein secondary structure differences as a Allison M. Mason means to effective classification based on product type and vendor. Greenhouse gas monitoring include the measurements of carbon monoxide, carbon dioxide, nitrous oxide, and methane. Low sensitivity of CO2 and CO through using 184 Multimodal Tissue Segmentation of Prostate Cancer Biopsies thermal conductivity detection (TCD) methods and the lack of detection from flame Using Chemical Imaging ionization detection (FID) makes detection sensitivity a challenge. Methanization is Anirudh Mittal, University of Illinois at Urbana-Champaign, 405 N. a method using a nickel catalyst to convert CO and CO2 into methane which can be Mathews Ave., Urbana, IL 61801, Shachi Mittal, Kevin Yeh, Jennifer detected using FID, a detector known to exhibit high levels of sensitivity and a broad Pfister, Rohit Bhargava range of linearity. Traditionally, methanization must be performed using a separate American Cancer Society estimates that there will be about 160,000 new cases and unit added prior to the FID for operation which can be costly, provides limited linear- approximately 30,000 deaths from prostate cancer in 2018, making it the second ity, and is highly susceptible to degradation from sulfur containing compounds and leading cause of cancer deaths in American men. Therefore, early and precise tu- oxygen. Activated Research Company (ARC) has developed an in-jet methanizer mor detection becomes an important step to greatly improve patient outcomes as (Jetanizer) which can be installed with no modification or permanent alteration to survival rates are highly dependent on the stage of the tumor. For instance, the rela- the instrument setup. The Jetanizer can allow for low level detection of CO and CO2 tive 5-year survival rate for local and regional stage prostate cancer is nearly 100% with linearity extending into percent level concentrations with less susceptibility to but drops drastically down to 29% for distant stage prostate cancer. The current catalysis poisoning by high oxygen samples allowing for a simplified flow path with methods of evaluating prostate cancer biopsies using standard histopathology are increased robustness. The goal of this analysis is to demonstrate the capability of limited by long turnaround times, lack of standardized assessment techniques, high the Shimadzu Greenhouse Gas Analyzer with the ARC Jetanizer. cost and inadequate accuracy. To overcome the above problems, we apply a meth- od based on chemical imaging coupled with artificial intelligence (AI) techniques to 188 Removing Polycyclic Aromatic Hydrocarbons (PAHs) by differentiate between epithelial and stromal tissue compartments; further segment- Adsorption onto Silica Gel Treated with LipophIlic Carboxylic ing them into tumor regions and the associated microenvironment respectively. This Acids automated cancer diagnosis platform can be used to help pathologists in faster Jessi Dolores, Manhattan College, 245 East 110 St., Apt. 3A, New York, screening and making more confident decisions. Also, the proposed approach gen- NY 10471, Jianwei Fan erates label free spectroscopic images while keeping the sample unperturbed in any Polycyclic aromatic hydrocarbons (PAHs) are a group of non-polar organic mole- way making it accessible for any further patient study. cules composed of two or more aromatic rings. Generated primarily from incom- plete combustion of coal and vehicular exhaustion, PAHs are both mutagenic and 185 Binary Spectronephelometry (BSN) of Bacterial Cultures: carcinogenic, and the United States Environmental Protection Agency (EPA) listed Noninvasive Quantitative Raman Spectroscopy in Optically Thin or sixteen PAHs as priority contaminates in ecosystem. This project used lipophilic Dilute Two-Phase Samples carboxylic acid treated-silica gel as the adsorbent to remove PAH from aqueous Steven Ortiz, Syracuse University, 0-014 Center for Science and solutions. The purpose of pre-treating silica gel with lipophilic acids is to increase the Technology, Dept. of Chemistry, Syracuse, NY 13244, Richard surface hydrophobicity for better attracting nonpolar PAH molecules. Using ultravio- McDonough, Paul W. Dent, Jerry Goodisman, Joseph Chaiken let spectroscopy to monitor the lipophilic acid concentrations it shows that the acids Chemical analysis of turbid samples usually requires filtering/centrifugation thereby are partially loaded on the silica gel. Using fluorescence spectroscopy to measure altering the sample and rendering further experiments on that sample impossible. the concentration of PAH it shows that the treated silica gels can remove PAH from Monitoring the time development of bacterial cultures for experimentation, biopro- the aqueous solution much better than the untreated does. The PAH adsorption cess control or other applications using conventional approaches requires physical capacities are quantitatively determined for various treated silica gels which reveals sampling thereby risking contamination and consuming the culture. Developments the dependence of the capacity on the structure and functional group of the lipophilic in enabling technologies including lasers, detection and spectroscopic filters has carboxylic acids. stimulated innovation in development of algorithms that render optical density measurements i.e., OD600 obsolete in some useful situations. We demonstrate 189 An Acoustic Levitator for Heterogeneous Chemical Reactions that binary spectronephelometry (BSN) has the same sensitivity and precision for Beni B. Dangi, Florida A & M University, 1530 S. MLK Blvd., Jones Hall population monitoring as OD600 but with simultaneous metabolic monitoring. BSN 219, Tallahassee, FL 32307, Jordan Dixon involves probing the culture with near infrared laser light and collecting Rayleigh/ An acoustic levitator holds small solid/liquid sample in given gas medium allowing Mie scattered light i.e., elastically scattered light simultaneously with collecting fluo- containerless and bulk-free conditions to study surface-gas reactions. The acoustic rescence and Raman scattered light i.e., inelastically scattered light. Implementing levitator, in combination with a high speed CCD camera, was utilized to study evap- this approach using commercial off the shelf components we show that bacteria oration kinetics of pure methanol and hexafloro isopropanol (HFIP) solvents. Rates number density can be monitored simultaneously with glucose consumption and of evaporation were calculated from size measurements of the droplet over time at the production of polysaccharide mucous materials in various media. From the point ambient conditions. Two different regimes of evaporation kinetics were measured of view of Raman spectroscopy with our current level of signal to noise, complex experimentally for both solvents. Polymer solutions of polyethylene oxide (PEO) in growth medium e.g., Lysogeny Broth (LB) is consumed as a single component ac- methanol at various concentrations were prepared. These solutions were also mon- cording to principal component analysis (PCA) of growth curves for Escherichia coli itored over time to extract the evaporation kinetics data. Total evaporation times for and Pseudomanas aeruginosa. various microliter volumes of methanol were measured and compared under normal laboratory conditions, and under acoustic levitation. Significant difference in evap- 186 Easy Monitoring of Cell Culture Growth using Disposable oration time and trend were observed. These studies provide crucial data for future Electrochemical Enzymatic Sensors experiments, such as crystallization of proteins and polymers from solutions under Harihara Subramanian Narayanan, Metrohm USA, 9250 Camden Field microgravity conditions. Furthermore, the acoustic levitation will be utilized in trap- Parkway, Riverview, FL 33578, Pablo Fanjul-Bolado ping solid/liquid samples under controlled gas and radiation conditions to explore Determination of cell growth and viability is crucial for bioprocesses monitoring. Tra- heterogeneous chemistry relevant to planetary and exoplanetary atmospheres. ditionally, the control of the cell culture growth is performed by cell counting, which can be a time-consuming procedure that requires an expert user. Thus, in order to take the cell culture control to the next level, electrochemical enzymatic sensors for 29 2018 EAS Abstracts November 2018

190 Abstract withdrawn by the author tablished which ensured a robust SFC method with increased confidence in the final method. In addition, method run time was reduced from 110 minutes to 3 minutes. 191 Selective Liquid Phase Hydrogenation of Cinnamaldehyde Using 195 The Application of Photometric TOC Test Kits and a Metal Salts and Pd/Al2O3 Spectrophotometer for the Analysis of Cleaning Validation Samples Jessica M. Miller, Cedar Crest College, 100 College Dr., Allentown, PA Bruce Herzig, MilliporeSigma, 2909 Highland Ave., Norwood, OH 18104, Lindsey A. Welch 45212, William Sebistian The purpose of this research project is to examine the different hydrogenation Cleaning validation is an important part of the production process and is used in pathways of cinnamaldehyde, an α,β-unsaturated aldehyde, guided by metal salt the production of pharmaceuticals, over-the-counter (OTC) formulations, personal additives. When cinnamaldehyde is hydrogenated the aldehyde can be reduced to care, and food products. This process ensures that all residues of previously made form an unsaturated alcohol, and then subsequently reduced the saturated alcohol. batches and cleaning agents have been removed from the process equipment. Cinnamaldehyde was hydrogenated using commercial Pd/Al2O3 in the presence of Total organic carbon (TOC) is often used as a detection for cleaning validations. varying hydrated metal chlorides (FeCl2, NiCl2, CuCl2, SnCl2, AlCl3, FeCl3, and This poster demonstrates using a photometric test kit and ultraviolet-visible (UV-Vis) CoCl2), and metal acetates (Ni(CH3COO)2, Cu(CH3COO)2, and Co(CH3COO)2), spectrophotometer from MilliporeSigma for this type of testing. in isopropyl alcohol at moderate temperature in 1 atm H2. Products were analyzed using gas chromatography mass spectrometry and gas chromatography infrared 196 A Simple and Rapid Method for Detecting the Pesticide Fipronil on spectroscopy. It was observed that when only Pd/Al2O3 was used it was selective Egg Shells and in Liquid Eggs by Raman Microscopy towards the unsaturated alcohol and when a metal chloride was added the selectiv- Qin Tu, Northwest A&F University, c/o University of Massachusetts- ity towards the unsaturated cinnamyl alcohol was increased in a trend correspond- Amherst, 99 Southpoint Dr., Apt E, Amherst, MA 01002, Lili He ing to hard-soft acid base theory (HSAB). This hypothesis that HSAB guides the Introduction: The European “poisonous eggs” incident was the largest European additives’ effect on selectivity was tested by using the corresponding metal acetate food scandal in 2017 and involved at least 40 countries (Reuters, 2017). Millions salts. The acetate salts led to results similar to the use of Pd/Al2O3 in the absence of eggs have been recalled across Europe with losses expected to run into the mil- of additives. Selectivity significantly shifted from hydrocinnamaldehyde toward the lions of dollars. Experts analyzed egg products imported from the Netherlands and cinnamyl alcohol with the addition of hard acids. Spent catalysts were analyzed discovered that some eggs were contaminated with an insecticide called fipronil. In with X-ray photoelectron spectroscopy to elucidate the effect of metal salts on the this study, we developed an innovative approach to detect trace amounts of fipronil catalyst. recovered from chicken egg shells and liquid eggs using Raman microscopy. To the best of our knowledge, there are no reports in which describe the utilization of 192 The Effects of Solvents on the Hydrogenation of α-Methyl-Trans- Raman spectroscopy to detect or characterize fipronil. This is also the first study in Cinnamaldehyde with the use of Metal Chloride Additives which simple sample preparation methods for the recovery of fipronil from chicken Kirsten R. Replogle, Cedar Crest College, 100 College Dr., Allentown, egg shells and in liquid eggs for Raman microscopic detection have been described. PA 18104 Purpose: The aim of this study was to utilize Raman microscopy for simple, rap- The compound α-methyl-trans-cinnamaldehyde is often used in fragrances and fla- id, and sensitive detection of fipronil on chicken egg shells and within liquid eggs. vorings. It also serves as a model compound for selective hydrogenation. Results Methods: Fipronil rapidly crystallizes when solubilized in water-acetone solution of the investigation of the hydrogenation of this compound using Pd/C in various (1:6, V/V, acetone/water). Here, fipronil crystals were concentrated and recovered solvents and hydrated metal chlorides will be presented. The solvents used were through filtration using acetone. Fipronil crystallization upon a gold-coated glass isopropyl alcohol (IPA), toluene, cyclohexane, and acetonitrile. The hydrated metal slide was achieved by acetone-evaporation for Raman analyses. Results: The limit chlorides used were FeCl3·6H2O, SnCl2·2H2O, CuCl2·2H2O, and NiCl2·2H2O. of detection for fipronil detected by this approach was 0.32 mg/kg in liquid eggs and Reactions were carried out at atmospheric pressure using a hydrogen balloon. 0.065 mg/m2. This method has demonstrated a strong capacity for the rapid (<30 Progress was monitored with a gas chromatography mass spectrometry (GC-MS). min) detection of fipronil on chicken egg shells and in liquid eggs. Both nonpolar aprotic solvents, cyclohexane and toluene, were more selective than the polar protic solvent, IPA. Between the two nonpolar aprotic solvents, toluene 197 Poly(diallyldimethylammonium chloride) Treatment of Cotton produced a higher percent conversation under all conditions tested. In all solvents Surfaces. A Raman Study tested, SnCl2·H2O had the lowest percent conversion. FeCl3·6H2O was the least Dana Garcia, Arkema Inc., 900 First Ave., King of Prussia, PA 19406, selective metal chloride tested in all solvents. Toluene, cyclohexane, and IPA were Pan Pan all non-selective towards the unsaturated alcohol product. Comparison of these re- Confocal Raman spectroscopy was utilized to examine interactions between sults to those of cinnamaldehyde may explain the mechanism of hydrogenation for poly(diallyldimethylammonium chloride) and cotton. Based on the unique structur- these aromatic aldehydes. al probe associated with Raman spectroscopy, we identified transition peak shifts and peak shape changes associated with cotton after treatment with poly(diallyldi- 193 Analysis of Biodiesel-Diesel Blended Fuels Using Gas methylammonium chloride) consistent with complex formation. These changes are Chromatography and Chemometric Methods absent if other treatments are applied to the cotton surface. The poly(diallyldimeth- Helen V. Tsiagras, College of the Holy Cross, 1 College St, PO Box ylammonium chloride) association with cotton results in a fixative characteristic for 2868, Worcester, MA 01610, Karina L. Ramos, Alicia M. Riddell, Amber stained and dyed materials M. Hupp Biodiesel offers a renewable source of energy from various plant and animal-based 198 Depth Profiling Composition and Color of a Surface Treated feedstocks. It is now a viable option over fossil fuels for clean energy that reduces Thermoplastic Material carbon emissions. The chemical composition of biodiesel fuels is identified through Michael L. Hall, SABIC, 1 Noryl Ave., Selkirk, NY 12158, Nancy Jestel the presence of fatty acid methyl esters (FAMEs) that vary in composition based on Plastic hybrid designs for electronic applications need high bonding strength. The feedstock. In this research, samples were prepared from canola, waste grease, and manufacturing of these products often require surface treatments that can influence tallow feedstocks. Pure biodiesel samples and blended samples of biodiesel and the color and could also adversely affect the chemical stability of the resins in the different locally sourced diesels, were separated using gas chromatography-mass formulation. In this investigation, the composition and color of a surface treated spectrometry (GC-MS) on a RTX-50 mid-polar column. Various concentrations of thermoplastic formulation was assessed as a function of depth. The color was de- biodiesel-diesel blends (B100, B20, B10, B5, and B2) were analyzed using princi- termined using a Ci7800 spectrophotometer and composition assessed by photo- pal component analysis (PCA) to determine variables contributing to differences in acoustic FTIR spectroscopy. The spectral datasets from a microlapping study were sample composition. combined and modeled by multivariate curve resolution. The output from the model provided insight into the visual and chemical changes of the treated formulation as 194 Quality-by-Design based Development of a Fast and Robust a function of depth. Method for Impurity Profiling of Carbamazepine Using SFC and the Fusion QbD® Software Platform 199 Imidazole and RNO method for Singlet Oxygen Detection by Time- Mijo Stanic, Chromicent GmbH, Johann-Hittorf-Str. 8, Berlin, Adlershof resolved, Broadband UV-Vis Absorbance Measurements D-12489, Germany, Alexander Schmidt, Richard Verseput Johanna Herman, University of Delaware, 163 The Green, 173 Brown The general potential of supercritical fluid chromatography (SFC) as alternative to Lab, Newark, DE 19711, Sharon Neal 1 high-performance liquid chromatography (HPLC) was evaluated using the HPLC The reactive oxygen species, singlet oxygen ( O2), has been studied in many fields, method for impurity profiling of Carbamazepine in the current Ph.Eur. monograph. such as pollutant weathering, DNA damage, photodynamic therapy, and polymer Incorporating quality-by-design (QbD) principles to method development using the science. One method widely used to monitor singlet oxygen production is the im- Fusion QbD Software Platform enabled optimizing method parameters for mean idazole and RNO (Imd/RNO) method, originated by Kraljic and El Mohsni. In this 1 performance and robustness. An operating space within the design space was es- method O2 is captured by the ring of imidazole (Imd) resulting in the formation of a 30 2018 EAS Abstracts November 2018

trans-annular peroxide intermediate capable of bleaching p-nitrosodimethylaniline photometry, attenuated total reflectance (ATR) infrared spectroscopy, and scanning (RNO). Though the method has been widely used, including in analyses monitor- electron microscopy/energy dispersive x-ray spectroscopy (SEM-EDX). No sample 1 ing O2 production in complex environments, there have not been many reports preparation was required for any technique. For Raman analysis, each sample was investigating the efficiency of the assay. In this research, time-resolved, broadband placed directly onto a glass slide, where three different areas were scanned sixteen ultraviolet-visible (UV-Vis) absorbance measurements and multivariate data analy- times. For the ATR, each sample was analyzed in triplicate using 32 scans at a res- sis techniques are used to compare the Imd/RNO method in the photosensitization olution of 4 cm-1, with a background taken before each run. For SEM-EDX analysis, of Rose Bengal in phosphate buffer, methanol, octanol, and phosphate buffer sat- each sample was placed on a piece of carbon tape for analysis. Three different 1 urated octanol. The quantum yields (φ) of O2 production in each solvent system areas of each sample were analyzed to obtain the chemical composition. The data were determined via a relative actinometric method with methanol as the reference from all three techniques was determined to be reproducible. The Raman and ATR solvent. The dependence of RNO bleaching on the Imd concentration peaked in spectra were compared to every other spectrum in pairs, of which 90.55% of the the millimolar Imd range in aqueous media. On the other hand, the rate increased Raman spectra and 95.23% of the ATR spectra could be discriminated. Principal continuously in organic media with Imd concentration. These results indicate a dif- component analysis (PCA) was used to analyze the SEM-EDX data, and visual ference in the mechanism of Imd/RNO method in these media that requires further examination of 3-dimensional plots showed that replicates of each sample clustered investigation. together and could be distinguished from others. These results show that using a combination of the three spectroscopic techniques shows the potential to differenti- 200 Race Differentiation by Raman Spectroscopy of a Bloodstain for ate between 34 cosmetic foundation samples. Forensic Purposes Ewelina M. Mistek, University at Albany-SUNY, 1400 Washington Ave., 203 Forensic Characterization and Discrimination of Manila Envelopes Albany, NY 12222, Lenka Halámková, Kyle C. Doty, Claire K. Muro, Igor Isabel Sanchez-Melo, University of New Haven, 1 Atwood Pl., Unit K. Lednev 410, West Haven, CT 06516, Virginia M. Maxwell D.Phil, Brooke W. Bearing in mind forensic purposes, a nondestructive and rapid method was de- Kammrath veloped for race differentiation of peripheral blood donors. Blood is an extremely Envelopes can be found at crime scenes when ransom, threat letters or potential- valuable form of evidence in forensic investigations, so proper analysis is critical. ly harmful substances are sent to victims. Therefore, they are important probative Because potentially miniscule amounts of blood traces can be found at a crime items of evidence analyzed by forensic document examiners. Manila envelopes are scene, having a method that is nondestructive, and provides a substantial amount commonly used in the United States to transport or send documents as they are of information about the sample, is ideal. In this study Raman spectroscopy was made of thick and durable paper. Although there are many studies on the foren- applied with advanced statistical analysis to discriminate between Caucasian and sic analysis of office paper and paper-based banknotes, and only a few on white African American donors based on dried peripheral blood traces. Spectra were col- envelopes, there does not exist previously published research on the physical or lected from 20 donors varying in sex and age. Support vector machine discriminant chemical characterization of manila envelopes. The goal of this research was to analysis (SVMDA) was used for differentiation between the two races. An outer loop analyze manila envelopes using analytical methods normally applied for the analy- subject-wise cross-validation (CV) method served to evaluate the performance of sis of paper and adhesives with the purpose of characterizing and comparing those the SVM classifier for each individual donor from the training data set. The perfor- sold by different manufacturers as well as the envelopes included in the same and mance of SVMDA, evaluated by the area under the curve (AUC) metric, showed different batches from the same manufacturer. Samples from five manila envelopes, 83% probability of correct classification for both races, and a specificity and sensi- size 9x12”, from each of the three boxes purchased from ten different brands were tivity of 80%. This preliminary study shows promise for distinguishing between dif- examined in order to evaluate whether there are significant differences that can ferent race donors of human blood. The method provides rapid and reliable results be used for forensic discrimination and/or identification of the manufacturer. The without any preparation, destruction, or consumption of the sample— thus making it analytical methods considered consisted of physical measurements of the enve- an ideal method for use at real life crime scenes. lopes and its folds, color examination, the use of alternate light sources (ALS), and chemical analysis using thin-layer chromatography (TLC), attenuated total reflection 201 Multiple Transfers of Drug Contaminated Prints and their Analysis fourier-transform infrared (ATR FT-IR) spectroscopy, Raman spectroscopy, X-ray with Raman Spectroscopy fluorescence (XRF), and X-ray powder diffraction (XRD). An analytical protocol Victoria R. DePrimo, University of New Haven, 46 Highland Rd., Staten for the forensic analysis of manila envelopes was developed, beginning with the Island, NY 10308, Kenneth Zercie, Pauline Leary, Nicholas Petraco, non-destructive techniques. Lisa Dadio, Brooke Kammrath The aim of this research was to determine if substrate, enhancement technique, and 204 Detecting Opioids and their Metabolites in Wastewater multiple transfers affect the detection and identification of drugs in fingermarks using Madison E. Pursell, Moravian College, Dept. of Chemistry, 1200 Main Raman spectroscopy. This is of great importance in forensic science as fingermarks St., Bethlehem, PA 18018, Alison E. Holliday are one of the most common traces left behind at crime scenes and illicit drugs There were 3,778,000 reported abusers of codeine, morphine, and heroin as of are a significant criminal justice problem. The ability to associate illicit drugs with a 2016 (NSDUH). According to the Centers for Disease Control and Prevention specific fingermark has the potential to put the drugs in the hands of a specific indi- (CDC), there were 29,873 overdose deaths related to these three drugs. This num- vidual. Although trace amounts of illicit materials within fingermark friction ridge de- ber has been climbing for years and is likely to continue doing so. The illicit use of posits have been identified from single, secondary transfers, the amount of transfer these drugs is likely underreported, as this data is obtained using surveys. However, drug contaminated fingermarks remained unknown. Raman spectroscopy has been by testing wastewater for these drugs and their metabolites, we can provide indi- utilized for sample identification by differentiating distinct features between other rect monitoring and estimate the number of people using these drugs in a specific substances and those of a crystalline nature. This research examined the number region. To analyze the wastewater, solid phase extraction, derivatization, and gas of multiple transfers of drug contaminated fingermarks where cocaine was able to chromatography-mass spectrometry are used. Quantification is done using deuter- be detected using Raman spectroscopy. Ten (10) participants imparted drug-con- ated internal standards. We detected codeine, morphine, and heroin’s major metab- taminated fingermarks 20 successive times on a series of different substrates with olite, 6-acetylmorphine, in untreated wastewater from Bethlehem, PA. The limits of specific enhancement techniques. Portable and Benchtop Raman Spectrometers detection are 0.85 ng, 1.2 ng, and 2.4 ng for codeine, morphine, and 6-acetylmor- were employed to assess the number of successive transfers from which drug con- phine, respectively. taminated fingermarks can be detected. By understanding how these illicit materials transfer between individuals and substrates as well as limitations associated, a level 205 Building a Method to Analyze Drugs and Metabolites in Wastewater of certainty can be provided through the substance identification. Therefore, using Effluent Raman spectroscopy to associate illicit drugs within a fingermark has the potential Julie A. Palkendo, Kutztown University, 316 Boehm Science Center, to put the drugs in the hands of a specific individual, aiding forensic scientists two- Kutztown, PA 19530, Megan A. O’Neill fold. Pharmaceuticals and their metabolites in recent years have become labeled as “newly-emerging contaminants” in drinking water by the United States Environmen- 202 Discrimination of Cosmetic Foundations Using Several tal Protection Agency (EPA). The contaminants are actually not new at all, but due Spectroscopic Techniques to the improvements in analytical instrumentation there are now highly sensitive and Jessica McFarland, Cedar Crest College, 100 College Dr., Allentown, selective tools for detection. A likely source of contaminants is believed to be from PA 18104, Thomas Brettell, Megan Zellner, Lawrence Quarino treated wastewater, which flows back into watersheds that downstream communi- Personal care products, such as cosmetic foundations, can be found as trace evi- ties may use as drinking water sources. Current wastewater treatment plants are dence in the form of smears on fabric. If analyzed, this evidence could be used to very capable of removing pathogens, inorganics, and solid materials; however, little corroborate accounts of events. However, little research has been conducted in the is known about if and how a treatment plant’s design impacts the removal of drugs way of distinguishing between foundations for forensic purposes. This study exam- and their metabolites. Most studies in this arena have focused their results on esti- ined 34 foundations from a variety of manufacturers using Raman microspectro- mating the amount of drug use in a given community’s population. In this study, an- 31 2018 EAS Abstracts November 2018

alytes in wastewater effluent were concentrated using solid phase extraction (SPE), pyrene-cis-4,5-dihydrodiol, Benzo[a]pyrene-r-7, t-8, t-9, c-10-tetrahydrotetrol (+/- and a liquid chromatography tandem mass spectrometry (LC-MS-MS) method was ) and Benzo[a]pyrene-r-7, t-8, t-9, t-10-tetrahydrotetrol (+/-). The obtained results developed to quantify over 30 drugs and drug metabolites. In order to assess the provide a solid foundation for future applications of the Shpol’skii phenomenon to method, a sample of wastewater effluent was spiked with a standard pharmaceutical the analysis of B[a]P metabolites in physiological matrixes. mix, amphetamine (AMP), methamphetamine (MAMP), and 11-nor-9-Carboxy-Δ9- THC (nor-THC). All compounds except acetaminophen, ciprofloxacin, and nor-THC 209 A Rewarding Journey into the Disordered World of Ion Conductors were detected. Additionally, unspiked drugs of methadone, codeine, oxycodone, Yan-Yan Hu, Florida State University, Dept. of Chemistry & Biochemistry, oxymorphone, and hydrocodone were easily detected in the wastewater sample. 95 Chieftan Way, Tallahassee, FL 32306 Po-Hsiu Chien, Xuyong Feng, The SPE technique will be modified further to recover all target compounds, and the Jin Zheng multiple reaction monitoring (MRM) method is being refined to better elucidate and Ordered structures are relatively easy to access, to understand, and to appreciate. quantify drug isomers. The beauty of disorder can often be overlooked due to the challenges in revealing its nature. In this contribution, I present the journey of exploring the functional and 206 Evaluation of Luminol Chemiluminescence Enhanced by Silver disordered structures using solid-state nuclear magnetic resonance (NMR). The Nanoparticles with Possible Application to Determination of Cobalt journey started with Prof. Clare P. Grey, for understanding the origin of the addi- in Natural Water Samples tional capacity found in metal oxides when used as anodes in Lithium ion batteries Mary Lynn Grayeski, Marywood University, Science, Math, and Comp. (LIBs). X-ray diffraction or absorption techniques failed to provide insights regarding Sci. Department, 2300 Adams Ave., Scranton, PA 18509, Caitlin Kurey the hypothesized interfacial processes in the metal/Li2O composites. Solid-state Determination of trace metals in natural water samples poses a challenge because NMR was employed to study the model system RuO2. The results revealed that of low concentrations often requiring rather sophisticated methodology. Chemilumi- a major contribution to the extra capacity is due to the generation of LiOH and its nescence detection offers advantages of low detection limits and simple instrumen- subsequent reversible reaction with Li to form Li2O and LiH. Equipped with the tation. This investigation examines the effect of silver nanoparticles on luminol che- knowledge and the acquired tools, I have moved on to adventure on my own. We miluminescence detection catalyzed by metals for possible analytical applications. have devised a tracer-exchange NMR method to probe ion transport mechanisms in Silver nanoparticles strongly enhance the chemiluminescence but varies with added other fast ion conductors, which has promoted our understanding on how structures, metal catalysts. This is primarily due to the differences in the kinetic profiles ob- in many cases defective ones, affect ion transport. This journey has been proven served for different conditions. This enhancement can be explained due to a catalyt- very rewarding. We have identified the functional defects in a group of Na solid elec- ic effect. Enhancement is observed in the presence of cobalt, iron, and copper with trolytes Na3PS4-aXa (X=Cl, Br), maximized the amount of these functional defects, cobalt exhibiting high initial intensities. This chemistry has potential applications to and achieved ionic conductivity enhancement of a few hundred times. We have also the detection of cobalt in natural waters. Advantages and limitations are discussed. ventured beyond defects in ordered structures into the completely “structureless” glass phases. It has led to the discovery of new fast ion conductors that demonstrate 207 Analyzing Nitrosamines in Drinking Water by Solid Phase record conductivity and stability against Li metal. Extraction Followed by Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry 210 Capturing and Quantifying Functional Dynamics in Viral Trevor D. McBrine, University of Connecticut, Center for Environmental Assemblies - Atomistic View from NMR, MD, and QM/MM Sciences and Engineering, Unit 4210, 3107 Horsebarn Hill Rd., Storrs, Tatyana Polenova, University of Delaware, Dept. of Chemistry and CT 06269, Julia M. Lineweber, James D. Stuart, Anthony A. Provatas, Biochemistry, Newark, DE 19716, Caitlin M. Quinn, Mingzhang Wang, Christopher R. Perkins Manman Lu, Juan Perilla, Angela M. Gronenborn Nitrosamines are a group of organic compounds consisting of a nitroso group bond- Recent methodological advances are presented that enable atomic-level charac- ed to an amine. Nitrosamines are commonly found in drinking water as by-prod- terization of dynamics of large biological assemblies of HIV-1 capsid protein. HIV-1 ucts due to the use of chlorine, chloramine, and anion-exchange processes to treat capsids, assembled from ~1,500 copies of the capsid (CA) protein, are an integral surface water sources. The presence of nitrogen precursors from upstream waste part of mature virions. Conical in shape, capsids enclose the viral genetic mate- water disposal allows for the formation of nitrosamines through the reaction of dime- rial (two copies of RNA) together with several proteins that are essential for viral thylamine with dichloramine. N-nitrosodimethylamine (NDMA) is the most frequently replication. In the assembled state, capsids are remarkably dynamic, with the CA detected nitrosamine in water. The presence of these compounds in drinking water residue motions occurring over a range of timescales from nano- to milliseconds. poses environmental and public health concerns because they have been shown These motions are functionally important for capsid’s assembly, viral maturation, to cause high incidences of cancer in both animals and humans. Unfortunately, the and interactions with host factors. An integrated magic-angle spinning (MAS) nucle- low molecular weight of nitrosamines prevents their removal by reverse osmosis, ar magnetic resonance (NMR), MD, and density functional theory (DFT) approach and instead water must be treated with UV exposure. The purpose of this project are presented, to probe the functionally important motions in assemblies of CA and was to determine the concentrations of nitrosamines in drinking water samples us- their complexes with host factors, as well as assemblies of CA-SP1 maturation inter- ing UPLC-MS/MS after a solid phase extraction (SPE) sample preparation step. mediates. The role of dynamic allosteric regulation in capsid’s assembly, maturation, Coconut charcoal SPE cartridges were used to capture the nitrosamines and were and escape from the host factor dependence will be discussed. The integration of eluted with methylene chloride. The eluent volume was reduced on a nitrogen blow- experimental MAS NMR and theory, at classical and quantum mechanical levels, down unit, with the residue taken up in methanol, while ensuring the sample was yields quantitative, atomic-level insights into the dynamic processes that govern the not brought to dryness. The methodology used was validated by method detection capsid’s function. limit, precision and accuracy studies, which displayed excellent analyte sensitivity and recovery. 211 NMR Studies of Paramagnetic Materials: Structure-Activity Relationships in High Energy Li-Ion Cathodes 208 Shpol’skii Spectroscopy of Polar Fluorophores in Polar Solvents Fulya Dogan, Argonne National Laboratory, 9700 S. Cass Ave., Lemont, Mohammadreza Chehelamirani, University of Central Florida, 2550 IL 60439 N Alafaya Trail, Apt. 5103, Orlando, FL 32826, Anthony M. Santana, Researchers worldwide are searching for new electrochemical energy storage ma- Andres Campiglia terials that meet high energy density, life, cost, and safety requirements of batteries The broad nature of excitation and emission spectra often limits the selectivity of for electric vehicle and other high energy applications. Layered lithium transition room-temperature fluorescence spectroscopy. A well-known technique to reduce metal oxide intercalation cathode structures have been widely studied as the next band broadening is Shpol’skii spectroscopy (SS). This technique deals with cryo- generation cathodes for advanced lithium-ion batteries. Structural changes in ma- genic temperatures where samples are frozen to 77K or below. The term Shpol’skii terials require mitigation strategies for electrochemical cycle life, stability and safety matrix refers to a dilute solution of a non-polar fluorophore (guest molecule) in a concerns and generally involve increase of lithium and manganese content and use non-polar solvent (host matrix) where the solvent freezes into an ordered polycrys- of different dopants and surface coatings. However, each change brings additional talline matrix. If the dimensions of the fluorophore and the solvent match up well challenge to understand the structural modifications induces and its relation with enough, guest molecules occupy a small number of crystallographic sites in the materials activity. Therefore, direct observation of local environment changes with- host matrix. Matrix isolation of fluorophore molecules results in vibrationally re- in the bulk and on the surface is crucial. Solid state nuclear magnetic resonance solved excitation and fluorescence spectra with sharp line widths. Unfortunately, the (NMR) is a unique structural probe that can quantitatively characterize local envi- requirement of a specific fluorophore-solvent combination has confined SS to the ronment changes and can be used to monitor the evolution of local order and low analysis of non-polar fluorophores in non-polar solvents. This presentation takes concentration defect formation with the goal of correlating local structural changes the first steps in removing this limitation with the analysis of polar metabolites of with electrochemical activity. For paramagnetic battery cathodes, the presence of polycyclic aromatic hydrocarbons in alcohol matrixes. We investigate the line nar- hyperfine (Fermi contact) interaction helps to directly “see” lattice and non-lattice rowing effect of primary alcohols on the spectral profiles of benzo[a]pyrene (B[a]P) species and separate dopants from coatings, bulk from surface and follow defect metabolites. The studied metabolites include 1-Hydroxybenzo[a]pyrene, Benzo[a] formation. This overview talk on paramagnetic battery systems focuses on applica- 32 2018 EAS Abstracts November 2018

tion of 6Li and 27Al solid state NMR on LiNixMnyCo1-x-yO2 and xLi2MnO3•(1-x) It will be demonstrated that the mechanism of HILIC is increasingly understood, LiMO2 (M=Mn, Ni, Co) types of cathodes to study the evolution of lattice aluminum and indeed that changes in the parameters of the separation (particularly changing and lithium sites upon cycling and correlation of structural reorganization with elec- the stationary phase) can give rise to significant selectivity changes that are not trochemical behavior. possible in RP. 212 Developing and Applying New Tools to Understand How Materials 215 Surface Chemistry Considerations in HILIC: Impact on Solvent for Li and “Beyond-Li” Battery Technologies Function Dynamics and Retention Mechanisms Clare P. Grey, University of Cambridge, Dept. of Chemistry, Cambridge, David S. Bell, Restek, 110 Benner Circle, Bellefonte, PA 16823 CB2 1EW, United Kingdom Hydrophilic interaction liquid chromatography (HILIC) is a complex system involving The development of light, long-lasting rechargeable batteries (and the invention of partition, polar and ion-exchange interactions. Stationary phases employed in the the lithium-ion battery, now 25 years ago) has been an integral part of the por- HILIC mode are highly varied in terms of their dominant retention mechanisms. table electronics revolution. This revolution has transformed the way in which we The interactions exhibited by a given stationary phase are highly dependent on communicate and transfer and access data globally. Rechargeable batteries are preferential solvation of the surface by components of the mobile phase. Solvation now playing an increasingly important role in transport and grid applications, but phenomena and the impact of solvation on dominant retention mechanisms will be the introduction of these devices comes with different sets of challenges. New discussed for several classes of HILIC phases. Method development can be greatly technologies are being investigated, such as those using sodium and magnesium facilitated by understanding the interactions different stationary phases provide and ions instead of lithium, and the flow of materials in and out of the electrochemical by applying that knowledge in an informed manner to the separation task at hand. cell (in redox flow batteries). Importantly, fundamental science is key to producing Matching the target analyte properties to retention mechanisms and maximizing the non-incremental advances and developing new strategies for energy storage and probability of selectivity by choosing stationary phases with the appropriate inherent conversion. This talk focuses on our work on the development of methods that allow mechanisms is demonstrated through a variety of examples to positively impact devices to be probed while they are operating (i.e., in-situ). This allows, for example, method development. the transformations of the various cell components to be followed under realistic conditions without having to disassemble and take apart the cell. To this end, the 216 Evaluation of New HILIC Columns for Pharmaceutical Analysis: application of new in- and ex-situ nuclear magnetic resonance (NMR), magnetic res- Successes and Challenges onance imaging (MRI) and X-ray diffraction approaches to correlate structure and Zachary S. Breitbach, AbbVie, Inc., 1 North Waukegan Rd., North dynamics with function in lithium- and sodium-ion batteries and supercapacitors will Chicago, IL 60064 be described. The in-situ approach allows processes to be captured, which are very The orthogonal nature of hydrophilic interaction liquid chromatography (HILIC) difficult to detect directly by ex-situ methods. Complementary ex-situ investigations separations compared to conventional reversed phase separations has led to an allow more detailed structural studies to be performed, to correlate local and long- increase in the utilization of HILIC. Early HILIC separations and columns have been range structure with performance. replaced with new stationary phases that offer unique selectivity, as well as in- creased column stability. For example, the use of native cyclofructan as a HILIC sta- 213 Hydration of Counterions Plays a Major Role in Retention and tionary phase produced better separations for certain classes of polar compounds Selectivity in Hydrophilic Interaction Chromatography compared to most other existing commercial HILIC phases. Further, the multipoint Andrew J. Alpert, PolyLC Inc., 9151 Rumsey Rd., Ste. 180, Columbia, attachment strategy used to anchor such HILIC selectors has improved stationary MD 21045 phase robustness. Most recently, specialized HILIC columns which are highly effi- The hydration of counterions can have a dramatic impact on retention of charged cient have been developed using core-shell silica. Overall, the superficially porous solutes in hydrophilic interaction liquid chromatography (HILIC) or electrostatic re- particle based columns showed advantages over the fully porous particle columns, pulsion-hydrophilic interaction chromatography (ERLIC). The use of a well-hydrat- in terms of high throughput and efficient separations at high flow rates, allowing for ed cation such as Mg+2 or ethylenediammonium ion (EDA) promotes retention of very fast separations to be performed. With better columns available, many pharma- negatively charged solutes. This was exploited at pH 2.5 to leave peptides with (-) ceutically relevant challenges can be overcome. For example, HILIC allows for the charge only on phosphate, sialic acid, and isoAsp residues, increasing retention of retention of polar targeted impurities and has been applied in the analysis of citric peptides having them. Poorly hydrated trifluoroacetyl- ion (TFA) was used as the acid, acetamide, imidazole, DMSO, and tosic acid. This is particularly advantages anion to decrease retention of all other peptides. With the use of EDA-TFA salt, when the analysis is applied to hydrophobic drug substances which can readily be retention of a singly phosphorylated tryptic peptide was 2.5-4x greater in HILIC than eluted in the void volume of the HILIC column. Also, with the increasing number of the nonphosphorylated sequence and 12-18x greater in ERLIC. With phosphopep- phosphate prodrugs being investigated, HILIC may offer an improved separation tides with a tryptic-type sequence, the range of the increase is proportional to the platform for general related substances, when compared to reversed phase mode. proximity of the phosphate group to the C-terminus, evidence of orientation of the As presented, such applications have come with their share of successes as well peptide during its migration on the column. This is a ~ 4x increase in selectivity for as challenges. phosphopeptides over what had been obtained before, making this approach com- petitive with IMAC and titania-based methods. Unlike those methods, the ERLIC 217 High Resolution Mass Spectrometry Bioanalysis mode permits the separation of phosphopeptides from each other with good se- Qin Ji, Bristol-Myers Squibb, PO Box 4000, Princeton, NJ 08543 lectivity. The use of EDA-TFA prospectively permits detection methods that require Triple quadrupole mass spectrometry has been a gold standard of mass spectro- volatile mobile phases. Preliminary data suggests that the method is also promising metric detection for quantitative analysis since it was invented in late 1970s by for isolation of glycopeptides, especially those containing sialic acid residues. Richard Yost and Christie Enke. Because of its nearly 100% duty cycle of the ion utilization for selective reaction monitoring (SRM) of a “product ion” from a target 214 Hydrophilic Interaction Chromatography: What are Its Advantages analyte, it provides excellent lower limit of quantitation (LLOQ) and specificity for a and Limitations? bioanalytical assay. In recent years, high resolution mass spectrometry has evolved David V. McCalley, University of the West of England- Bristol, Centre for rapidly, and its application for liquid chromatography-mass spectrometry (LC-MS) Research in Biosciences, Coldharbour Ln., Bristol BS16 1QY, United bioanalysis has drawn a significant attention in pharmaceutical/biotech industry Kingdom since this was proposed in early 2010s. The attractive features of the high resolution Hydrophilic interaction chromatography (HILIC) is increasingly accepted as an alter- mass spectrometry for LC-MS bioanalysis include: 1) monitoring a wide mass range native to reversed-phase (RP) LC for the separation of polar and ionized substances of ions without a significant compromising of the sensitivity; 2) achieving excellent that are difficult to retain by RP. For solutes amenable to either technique, HILIC has assay selectivity through high resolution/high mass accuracy detection; 3) acquiring a number of advantages, most of which originate from its use of high concentrations data for qualitative and quantitative (Quan/Qual) analysis simultaneously; 4) im- of organic modifier in the mobile phase. This use results in lower back pressures proving selectivity and sensitivity in the analysis of analytes with no or low abundant enabling faster flow rates, the use of longer columns and better sensitivity in detec- product ions for MRM detection; and 5) monitoring ions with increasing mass range tion systems where evaporation of the mobile phase is employed (as in electrospray and spectral generation rate with newly developed instrument. This presentation ionization mass spectrometry). However, the mechanism of HILIC is perceived to discusses the history and current status of high resolution mass spectrometry instru- be complex and difficult to understand and manipulate. Furthermore, a significant mentation and its applications in LC-MS bioanalysis in supporting drug discovery problem appears to be the amount of time required for stabilization of the system in and development. Additional thoughts on future directions are discussed as well. comparison with RP, which potentially could provide a significant barrier to its use in gradient elution. In this presentation, these potential limitations will be discussed and solutions presented. For example, while full equilibrium in isocratic separations may take up to an hour, a repeatable partial equilibrium can be achieved, suitable for gradient elution, in as little as 5 minutes. Examples of these separations will be given including the analysis of hydrophilic antibiotics and other pharmaceuticals. 33 2018 EAS Abstracts November 2018

218 Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) 221 Wastewater-Based Monitoring of Community Health and Behavior Screening Approaches for Studying Global Conformational Kevin J. Bisceglia, Hofstra University, MS: 151, Dept. of Chemistry, Structures of Peptides/Proteins in Solution Hempstead, NY 11549 Alexey A. Makarov, Merck & Co., MS: RY801-A103, 126 E. Lincoln Municipal wastewater is increasingly recognized as a valuable resource for monitor- Ave., Rahway, NJ 07065, Nicole Schiavone, Gregory Pirrone, Nicholas ing community-level trends in human health and behavior. In the same way that one Pierson, Ian Mangion might conduct a urine test on an individual, one might monitor municipal sewage Methodologies for studying protein higher-order structure in solution can help to for the presence of biomarkers that are indicative of a wide variety of conditions or establish a better understanding of the intrinsic link between proteins’ conforma- activities. In contrast to survey-based methods for obtaining such information, this tional structure and their biological function and activity. In this presentation we will practice - called wastewater-based epidemiology - has the potential to provide data demonstrate the applicability of screening approaches for studying global protein in near real-time and with improved spatial resolution. While still in its infancy, the conformational changes in solution. Several screening approaches were surveyed approach is already being used in several countries to monitor trends in illicit drug, in combination with differential hydrogen-deuterium exchange. We describe an ap- alcohol, and tobacco use. More recently, efforts have been underway to expand its plication of chromatography hyphenated techniques based on coupling as a single focus to consider metrics of diet and nutrition, toxicant exposure, illness, and dis- on-line workflow with differential hydrogen-deuterium exchange (HDX-MS). One ease. This presentation introduces the concept of wastewater-based epidemiology technique joins HDX-MS with size-exclusion chromatography (SEC). A semi-auto- and summarize major advances in the field. It will also address challenges and mated experimental setup based on the use of SEC on-column conditions allowed solutions that have arisen related to sampling, measurement and reporting, and for tracking of protein conformational changes in solution as a function of acetonitrile the extrapolation of biomarker concentration to useful metrics of use and exposure. concentration. In this setup, the SEC protein elution data was complemented by the ΔHDX profile which showed global protein conformational changes as a differ- 222 Analytical Solutions to Regulatory and Other Monitoring Problems ence in the number of deuterons exchanged to protons. Another technique utilized for Food Safety Purposes ultra-high pressure liquid chromatography (UHPLC) to explore compressibility of Steven J. Lehotay, United States Dept. of Agriculture, Agricultural the higher order structure of proteins under increasing pressure detected by HDX. Research Service, 600 East Mermaid Lane, Wyndmoor, PA 19038 It was found that the increase of retention factors upon pressure increase, at con- In keeping with this year’s theme of EAS, “Analytical Solutions to the Worlds Prob- stant flow rate and temperature, was based on reduction of the proteins’ molecular lems,” recent developments in providing analytical solutions to regulatory and other molar volume. The change in the proteins’ molecular molar volume was caused monitoring problems for food safety purposes are presented. Food safety is a funda- by changes in protein folding, as was demonstrated by differential HDX. By mod- mental need for life, and even today, the earliest detection systems to assess food ifying pressure during UHPLC separation, it was possible to achieve changes in safety are universally employed: visual inspection, smell, taste, and bioassay by in- protein folding, which were manifested as changes in the number of labile protons gestion. Today’s analytical solutions are more technologically advanced, entail less exchanged to deuterons, or vice-versa. personal risk, and have many other advantages over traditional bio-based methods. Since the passage of the first in a series of food safety laws in the United States 219 Single-Cell Capillary Electrophoresis Mass Spectrometry Finds in 1906, life expectancy increased from age 47 to 79, and mortality due infectious Cell Heterogeneity in the Developing Embryo and the Central disease fell from 53% to 3% today. Even since the passage of the Food Quality Nervous System Protection Act in 1996 and Food Safety Modernization Act in 2011, foodborne illness Peter Nemes, University of Maryland-College Park, 0107 Chemistry has decreased 40-60% according to estimates by the Centers for Disease Control Bldg., 8051 Regents Dr., College Park, MD 20742, Camille Lombard- (CDC). Improved analytical solutions to food safety analysis has played a role in Banek, Erika P. Portero, Sally A. Moody, Sam B. Choi, Chiara M. Manzini those outcomes. According to the World Trade Organization, global trade of food Characterization of molecular processes in rare cells and cell populations is key to and agricultural products was $3.25 trillion in 2015, and since lives and livelihoods understanding embryonic development and brain function. To assess how differen- can depend on monitoring results, the analytical methods used must be accurate tial gene expression establishes normal cell heterogeneity in the embryo and the and valid for a wide scope of ultra-trace contaminants in diverse commodities. The central nervous system, new analytical instruments are needed, particularly those methods must also be fast, easy, and inexpensive due to the perishability of many capable of analyzing small populations of cells, preferably single cells. High-res- foods. New developments in high-throughput analysis for hundreds of analytes, as olution electrospray ionization mass spectrometry (HRMS) is able to characterize well as needed research gaps, are discussed to better meet food safety analysis metabolites and proteins, but typically requires averaging hundreds of thousands challenges. to millions of cells, thus potentially losing information specific to small populations of cells or individual cells. Here we present a trace-sensitive HRMS approach that 223 Abstract withdrawn by the author. achieves sufficient sensitivity to measure metabolites and proteins in small cell pop- ulations to single cells. The HRMS instrument integrates a custom-built microanalyt- ical capillary electrophoresis platform and HRMS, delivering a ~60 amol sensitivity 224 Analytical Challenges in Studies of Pesticides and Pollinators for metabolites and ~260 zmol lower limit of detection for model peptides. In this Brian Eitzer, The Connecticut Agricultural Experiment Station, 123 talk, we discuss how we utilize this platform to compare the metabolic and pro- Huntington St., New Haven, CT 06511, Kimberly Stoner, Richard Cowles teomic state of single identified cells in the early developing frog (Xenopus laevis) There are many challenges to understanding the risk imposed by pesticides on pol- embryo. Additionally, we present technological developments to extend this micro- linators. One is in the determination of exposure. Bees travel a long distance while analytical platform to small populations of neurons in the mouse brain. Single-cell foraging and make their own choices of which plants to forage from. It can there- HRMS opens new investigative possibilities to help better understand molecular fore be difficult to determine which plant/pesticide combinations can lead to higher mechanisms during normal and impaired development. risk. We have met this challenge by analyzing the pesticide content pollen being brought back into the hive. These bulk pollen samples, which are a notably complex 220 A Brief History of Microflow Chromatography Mass Spectrometry matrix are analyzed using QuEChERS (quick, easy, cheap, effective, rugged, and Michael E. Lassman, Merck & Co., MS: KW K15-MN308, 2000 Galloping safe) and liquid chromatography mass spectrometry for pesticides at parts per bil- Hill Dr., Kenilworth, NJ 07076 lion (PPB) concentration levels. Results have shown that the number of pesticides For significantly more than a decade, practitioners of liquid chromatography mass present, the identity of those pesticides and their concentrations can vary greatly spectrometry have applied their trade to the quantitation of proteins and peptides in both from one location to the next and in time. We then apply a hazard quotient to plasma/serum. The complexity of the sample matrix as well as the extreme dynamic each sample. Next, we have gone back to the highest hazard samples, sorted the range in concentration of analytes poses significant challenges and has required samples by pollen pellet color, conducted palynological analysis, and re-analyzed multiple analytical innovations, of which the advent of low-flow liquid chromatog- the samples. The palynology identifies the amount of various plant species within raphy is potentially one of the most significant. In this presentation, we will review a particular pellet type and therefore we can determine the plant species and pes- seminal publications that highlight the evolution of quantitative protein and peptide ticides generating the highest risk. We are also studying the concentrations of pes- analysis by mass spectrometry and the incorporation of lower and microflow chro- ticides in pollen and nectar of ornamental plants following the normal horticultural matography to achieve greater sensitivity while retaining analytical robustness. use of pesticides. A challenge in this study is the need for very sensitive methods Improvements in instrumentation and a better understanding of how to fully take as hours of time can be required to collect sample sizes of less than a gram. The advantage of these instruments has deepened the depths to which researchers can analytical protocols we use allow us to determine low PPB concentrations in these now probe the human proteome. Here, we also review some of the more recent small sample sizes. literature to highlight how researchers have successfully employed microflow chro- matography to enable ground-breaking research.

34 2018 EAS Abstracts November 2018

225 RTRT as the Final Piece of a Comprehensive Control Strategy in as the new discoveries we have made about the process of metastasis: namely how Continuous Manufacturing tumor cells access the blood stream through microanatomical sites called Tumor Justin G. Pritchard, Vertex Pharmaceuticals, 50 Northern Ave., Boston, Microenvironment of Metastasis (TMEM) (Robinson et al. Clinical Cancer Research MA 02210, Kelly Swinney 2009) (Harney et al. Cancer Discovery 2017). We additionally identify a small mol- Real-time-release testing (RTRT) methods should be developed and implement- ecule inhibitor able to abrogate the function of these sites (Harney et al. Molecular ed in the context of the overall release drug product control strategy. The control Cancer Therapeutics 2017) and then conclude with published and new efforts to strategy includes such elements the design of the manufacturing process, product bring these insights directly into the clinic (Sparano et al. NPJ Breast Cancer 2017). knowledge, in-process testing and the release strategy. The real-time-release test- ing strategy relies not only on the RTRT method but on ensuring the process is with- 230 Chemical Imaging with a Quantum Cascade Laser for Rapid Cancer in control and demonstration of that control through in-process testing. Automated Assessment feedback loops within the manufacturing process, monitoring of the design space, Rohit Bhargava, University of Illinois at Urbana-Champaign, 405 N. and thorough interrogation of the process through in-process testing build a solid Mathews Ave., Urbana, IL 61802, Kevin Yeh, Shachi Mittal foundation for the implementation of real-time-release testing in continuous drug A new possibility in biomedical imaging is emerging in which the intrinsic chemical product manufacturing. In a comprehensive control strategy, only material manu- content of tissue is used to provide contrast in images without dyes or labels. In factured within the design space and conforming to in-process control acceptance contrast to the conventional approach of staining tissue followed by manual assess- criteria may be included in the batch for release consideration. The RTRT methods ment, the new approach utilizes spectroscopic methods. The recorded chemical and strategy are developed in coordination with the overall drug product control information is parsed by computational methods to visualize structure and to provide strategy and manufacturing process. diagnoses. Consequently, the approach is entirely digital and with extensive cancer and its microenvironment contrast in a single imaging measurement. Using artificial 226 Points to Consider in Developing an RTRt Capable NIR Method intelligence for knowledge extraction, a very powerful modality emerges in which Gary McGeorge, Bristol-Myers Squibb, 1 Squibb Dr., New Brunwsick, a single recording of data from unperturbed samples can be related to a variety of NJ 08903 pathophysiologic states. For cancer pathology, both tumor and microenvironment There are very few literature examples of how near infrared (NIR) methods should characteristics (molecular and spatial) can measured at the same time. This opens be developed, validated, deployed and maintained by pharmaceutical producers, new opportunities for insight into disease progression that considers the entire tis- leaving a large gap to be filled by academicians or regulators. Whether there are sue as an integrated system. Designed instrumentation, numerical methods, sam- very few applications or that they are simply not being published is unclear. The ples and statistics all play inter-related roles in the quality of information obtained. In European Medicines Agency guidance for NIR published in 2014 put strict pre- the first part of the talk, we focus on the fundamentals of this emerging technology scriptive expectations on such method development and deployment, resulting in using quantum cascade laser based instruments. In the second, we present case very challenging environment within which to operate. The concerns around this studies of rapid analysis of samples for cancer pathology, in which practical technol- guidance (in-part) appear to have scared industry away from submitting process ogies that can be useful for clinical diagnoses and research are becoming apparent. analytical technology (PAT) applications. In this presentation we initially introduce a real time release testing (RTRt) application within Bristol-Myers Squibb that is 231 Mid-Infrared Spectroscopic Imaging and Its Biomedical used during routine commercial release. Then we break down a variety of topics Applications that needed addressed including how to choose specifications that drive method Rohith Reddy, University of Houston, 4726 Calhoun Rd., Houston, TX performance requirements; model validation and transfer; model maintenance and 77204, Shihao Ran routine process trending. Through this comprehensive discussion one should walk Vibrational spectroscopy using mid-infrared light enables chemical identification of away feeling positive with the view that PAT systems can be deployed in a manner samples. Biomedical samples such as cancerous tissue are chemically heteroge- that is aligned with regulatory expectations. neous and bulk spectroscopy is inadequate to understand such samples. Mid-infra- red spectroscopic imaging (MIRSI) combines the molecular specificity of vibrational 227 PAT Tools Critical for Real-Time Release Testing spectroscopy with the spatial detail afforded by microscopy. This provides chemical Carl Anderson, Duquesne University, 600 Forbes Ave., Pittsburgh, PA maps of objects and has found extensive applications in a variety of fields including 15282 forensics, art analysis, separation sciences, and medical diagnosis. The combina- No abstract submitted by the author. tion of biochemically relevant molecular information along with morphology makes MIRSI especially useful in medical applications such as cancer diagnosis. Fouri- 228 Enabling Real Time Release of Solid Oral Dose Products a case er-transform infrared (FT-IR) spectroscopic Imaging has been the most popular form study of MIRSI. We present a summary of the work on FT-IR imaging over the past two Sarah Nielsen, Janssen Pharmaceuticals, 1000 Route 202 South, decades which has demonstrated that it is possible to use molecular information Raritan, NJ 08530 from MIRSI to diagnose a variety of cancers including prostate and breast can- Janssen Pharmaceuticals has designed and implemented a direct compression cer. We will present and discuss techniques for identifying tissue sub-types as well continuous manufacturing (CM) line for producing commercial product. One of the as cancer grade in a quantitative, label-free manner. New mid-IR sources such as benefits of continuous manufacturing is the ability to make numerous “mini” batches quantum cascade lasers (QCLs) have recently become commercially available and of material which can show the correlation of process conditions and tablet critical have resulted in novel and innovative imaging modalities with important advantages quality attributes. A targeted design of experiments guided by a criticality analysis over FT-IR. Techniques such as discrete frequency (DF)-IR, nano-IR, atomic force can specifically probe how process conditions and tablets physical properties cor- microscopy (AFM)-IR and photo-thermal IR have made it possible to perform MIR- relate to dissolution. This information can then be used for building real time release SI on the nano-scale. However, new techniques introduce new challenges which tests. This talk will give an overview of the development, validation and practical can be understood by modeling and analyzing the underlying optical phenomena implementation of a real time release test for dissolution. and instruments. Models for understanding instrumentation and data are present- ed. Important challenges related to temporal coherence and spatial coherence are 229 Surgical Engineering Enables Intravital Imaging of Mechanisms of discussed. Metastasis in Primary and Secondary Sites David Entenberg, Einstein College of Medicine/Montefiore Medical 232 Noninvasive In-Vivo Peripheral Vascular Spectrometry: And the Center, 1301 Morris Park Ave., Price 202, Bronx, NY 10461 Beat goes on” Metastasis (spread of cancer cells around the body) is responsible for the vast ma- Joseph Chaiken, Syracuse University, Dept. of Chemistry, Syracuse, jority of cancer mortality. Standard methods for studying metastasis either do not NY 13244, Seth Fillioe, Charles M. Peterson, Paul Dent, Sri Narsipur, capture the dynamic process, or are non-physiological (disconnected from rest of Richard Steinmann, James Mostrom, Bin Deng, Jerry Goodisman the living organism). Multiphoton microscopy of living animals (intravital imaging) Improved enabling technologies, understanding of relevant photon-tissue interac- can study cancer live and in real-time. However, technical challenges have limited tions and photochemistry, and experience in laser probing of tissue in vivo continue the duration, the amount, and the locations of tissue that can be measured. To over- to advance progress in noninvasive blood and tissue analysis. We present a new come these challenges, we have employed Surgical Engineering; bringing the skills combination of simultaneous spectroscopic and physical optical measurements im- of the surgeon into the optics laboratory to develop Large-Volume High-Resolution plemented in a commercially and clinically viable form that allows real-time moni- Intravital Imaging (LVHR-IVI) (Entenberg et al. Methods 2017) and a new Window toring of hematocrit (Hct) and vascular volume (VV) in the peripheral vasculature for High-Resolution Imaging of the murine Lung (WHRIL) (Entenberg et al. Nature of finger tips and toes. Combining Hct and VV with conventional vital signs con- Methods 2018). These technologies expand the utility of mouse models by allowing: tinuously in real time provides a more complete and more coherent picture of a 1) imaging of more tissue (and in new locations) than previously possible, and 2) person’s physiological state. Trends within and changes from normal homeostasis direct visualization of the process of metastasis in the breast and lungs, in-vivo, lon- combined with appropriate simultaneous analytics are becoming the most relevant gitudinally, and at single-cell resolution. We present these new technologies, as well observations upon which one can initiate an immediate medical course of action 35 2018 EAS Abstracts November 2018

with continuous quality assessment and control. Establishing the nature of normal trometry (GC-MS). Unfortunately, these measurements are often complicated by homeostasis itself, as accurately and precisely as possible using our measurement the varied and unpredictable responses of different molecules in the GC-MS lead- set, becomes of immediate and predictive necessity. The active clinical context is ing to various method detection limits (MDLs) and tenuous concentration estimates also required to accurately interpret the state of homeostasis and variations and without exhaustive calibrations. Here, we show how companies are using the Pol- changes from indication to indication e.g. nephrology, trauma, cardiovascular dis- yarc/FID combined with GC-MS to improve the accuracy and speed of E&L, and ease, diabetes etc. We will present data defining homeostasis in terms of the normal solvent analyses, and meet the requirements of the United States Food and Drug temporal variation of Hct and VV using measurements on blood in the peripheral Administration (FDA), European Medicines Agency (EMA), International Organiza- small vasculature of sensitivity, precision and temporal bandwidth that has not been tions for Standardization (ISO), and the Product Quality Research Institute (PQRI). previously possible with current technology. We will also discuss how this new ap- Compound independent calibration with a single internal standard improves injec- proach may enable other types of blood and tissue analysis in the normative state tion-to-injection variability (%relative standard deviation) relative to GC-MS alone, as well as the above conditions. yields predictable MDLs for threshold calculations, and significantly reduces the workload when compared with the need for calibration. 233 Bridging the Gaps between Comprehensive Multidimensional Separation Techniques 237 Applied Paint Analysis for Historic Architecture Tadeusz Gorecki, University of Waterloo, Dept. of Chemistry, 200 Tina Reichenbach, 160 Fairview Ave, Ste. 812-136, Hudson, NY 12534 University Ave. W., Waterloo, ON N2L 3G1, Canada, Hei-Yin Chow, The study of historic paints in architectural settings was pioneered in the 1970s, Alshymaa A. Aly as part of a general upswell of preservation-related effort in a post-Penn Station Compared to conventional one-dimensional separations, comprehensive two-di- response to urban renewal threats. The genesis for these early efforts was the goal mensional gas and liquid chromatography offer increased selectivity and peak ca- of thorough documentation of existing fabric for the historic record and as a result, pacity. Both techniques rely on repeated reinjection of fractions eluting from the architectural paint analysis evolved into an exercise in producing reams of detailed first column into a second column of different selectivity. In gas chromatography data to fulfill requirements for Historic Structure Reports or for other regulatory pur- (GC)×GC, this is accomplished with the help of a special interface called a modu- poses. In the decades since, the processes and goals have remained largely the lator; in liquid chromatography (LC)×LC, the modulation process is usually carried same, albeit with the introduction of more sophisticated techniques and imaging. out using multiport valves and sampling loops. The modulation process in GC×GC This presentation challenges the industry to consider practical applications as goals is significantly more complicated than in LC×LC. On the other hand, separation of for studying historic painted surfaces, to better and more efficiently utilize the devel- the individual fractions in the second dimension is straightforward in GC×GC as it oping tools and techniques to answer particular questions, to enlighten mysteries, is carried out under practically isothermal conditions, whereas most LC×LC second to supplement the historic record with site-relevant data, to design and develop. dimension separations are carried out using various types of gradient elution. This puts a limit on practically attainable modulation period lengths, and limits the avail- 238 Miniaturization of Noninvasive Analytical Instruments: A New Day able separation space. In addition, when different modes of LC are used in the two and New Tools for the Paper Conservator dimensions, problems related to incompatibility of the mobile phases used are often Ted Stanley, Collections Conservation, Rare Books & Special encountered. By utilizing the best ideas from each technique, many of these prob- Collections, Princeton University Library, 1 Washington Rd., Princeton, lems can be overcome. In GC×GC, a significant improvement in peak capacity can NJ 08544 be accomplished by using temperature programming in the second dimension. This Various spectrometer manufacturers began making their instruments smaller in size can be done by resistively heating the second dimension column under controlled and more affordable in recent years. On a practical basis, these developments have conditions. In LC×LC, good orthogonality and short second dimension separation made it possible for the instruments to become an integral part of the conservation times can be obtained by using parallel gradients in both dimensions. In the talk, the laboratory. Spectroscopy can be a particularly valuable tool in paper conservation latest results obtained using both approaches are presented. where a wide variety of materials such as cellulose, resins, adhesives, pigments and inks are involved in research and treatment. No longer restricted solely to the 234 Analysis of Stem Cells by Comprehensive Two-Dimensional Gas conservation scientist, the conservator can take advantage of the technology in a Chromatography/Time-of-Flight Mass Spectrometry number of ways. This presentation will highlight three areas where the technology John Dimandja, Georgia Institute of Technology, 5850 Silver Shadow was especially useful. One example will demonstrate spectroscopy’s ability to illu- Ct., Stone Mountain, GA 30087 minate how artifacts are crafted as reflected in the study of a 16th century erasable No abstract submitted by the author. notebook. Another example shows how spectroscopy lends to the body of cultural knowledge by examining the pigments of a rare 16th century Aztec deerskin map. 235 Adsorption Chromatography with new Intuvo GC platform The final example identifies pigments to help form a sensible treatment plan for a Peilin Yang, Dow Chemical Company, 400 Arcola Rd., Collegeville, PA discolored and stained early 19th century hand-colored engraving. The presentation 19426, Jim Luon, Ronda Gras, Yujuan Hua explores the advantages of Fourier transform infrared and Raman spectroscopy in Gas chromatography (GC) is a quintessential enabling analytical technique across practical conservation. multiple spaces for process optimization, process troubleshooting, product quality control, and license to operate. As a result, any improvement in terms of reliability 239 An ESR Mobile Universal Surface Explorer and performance to the technology can have a positive lasting impact. Recently, Joseph P. Hornak, RIT Magnetic Resonance Laboratory, 54 Lomb a new generation of gas chromatograph that is ground breaking, highly reliable Memorial Dr., Rochester, NY 14623 with unsurpassed analytical performance has been commercialized. The new gas Many samples cannot be studied by electron spin resonance (ESR) spectrosco- chromatograph sports many innovative features including uniform planar heating py because they are too large to fit into the cavity resonator of a conventional 9 technology for rapid heating and cooling which can substantially improve sample GHz spectrometer, and too precious to be destructively sampled for study. An ESR throughput, a novel state-of-the-art surface deactivation chemical flow path for trace mobile universal surface explorer (MOUSE), also known as a unilateral ESR spec- chemical analysis, and the leveraging of microfluidic centric technology to eliminate trometer, was constructed for studying this class of sample. The ESR MOUSE can the needs for column positioning. When these performance features were combined nondestructively record a low frequency ESR (LFESR) spectrum of a small, 2.9 with the high degree of selectivity and strong retention characteristic of porous layer mm diameter, 0.3 mm thick, disc on the surface of any size object by placing the open tubular column technology, volatile compounds such as light hydrocarbons of MOUSE against the object. The capabilities of the ESR MOUSE are demonstrated up to C7, primary alcohols, and mercaptans can be well separated and analyzed in a with point spectroscopy of the paramagnetic pigments ultramarine blue, rhodochro- matter of minutes. This analytical approach substantially improves sample through- site, blue vitriol, and terracotta red in linseed oil paints on canvas; one-dimensional put by at least a factor of 10 times when compared to published methodologies. In ESR imaging of electrophotographic toner on paper; two-dimensional ESR imag- addition, the use of porous layer open tubular columns advantageously eliminates ing of magnetic ink on a US one-dollar bill; and point spectroscopy of a Meissen the need for costly and time-consuming cryogenic gas chromatography required for Contemporary Böttger Red Stoneware candlestick. The mobile nature of the the separation of highly volatile compounds by partition chromatography with wall MOUSE will allow the spectrometer to be brought to the sample, thus opening new coated open tubular column technology. In this presentation, system design, key applications of ESR spectroscopy. features, and selected challenging applications are presented. 240 Mobile Analysis in Conservation of Outdoor Surfaces 236 Improving Accuracy and Repeatability with Single Injection MS/ John Scott, New York Conservation Foundation, 17 Battery Place, New Polyarc Split for E&L and VOC Analyses York, NY 10004 Andrew J. Jones, Activated Research Company, 7561 Corporate Way, No abstract submitted by the author. Eden Prairie, MN 55344 The analysis of extractables and leachables (E&L), and residual solvents found in pharmaceuticals is often performed using gas chromatography with mass spec- 36 2018 EAS Abstracts November 2018

241 Public Standards for Radioactive Drugs paint comparison by SEM-EDS, there is little guidance concerning potentially critical Steve Zigler, PETNET Solutions, a Siemens Company, 810 Innovation analytical variables, including: sample preparation, data acquisition, and data eval- Dr., Knoxville, TN 37932, Ravi Ravichandran uation. This presentation discusses the analysis of 300 automotive paint samples, Since 1955, the United States Pharmacopeia (USP) has maintained monographs which were analyzed on a layer-by-layer basis (totaling over 1200 layers) to better and general chapters for radioactive drugs, or radiopharmaceuticals. Over time, understand the impact of these variables and to develop a broader understanding USP standards have evolved to accommodate new technologies, analytical meth- of the range of elemental compositions that each layer of an automotive paint can ods, and products. For example, in the 1980s, the USP developed monographs span. From this dataset, selection criteria have been evaluated for establishing the and general chapters for positron-emitting, PET radiopharmaceuticals used in range of elements that can be detected in automotive paint as a function of layer diagnostic imaging applications. Today, the USP contains monographs for about type (e.g., clearcoat, basecoat, primer). In combination, these experiments will lead 70 radiopharmaceuticals and four general chapter dedicated to radiopharmaceu- to better constraints on the analytical variables of SEM-EDS analysis that impact tical articles. The unique characteristics of radiopharmaceuticals require analytical forensic paint comparisons, a better understanding of compositional variations in methods that meet compendial requirements for specificity, accuracy, linearity, and automotive paints, and, ultimately, more rigorous methods of interpretation for fo- other fundamental aspects of modern analytical methodology while paying special rensic paint comparisons. attention to radiation safety. Chosen methodologies should be suitable so that radio- active waste generation storage and disposal are in accordance with the regulatory 246 The Analysis of Dyed Beaver Furs Using Transmission and expectations. Another key component of this methodology is the use of radiation Fluorescence Micro-Spectrophotometry detection instrumentation and the determination of radiochemical identity/purity and Frani Kammerman, John Jay College of Criminal Justice and CUNY radionuclidic identity/purity. This presentation describes current approaches for an- Graduate Center, 524 W. 59th St., New York, NY 10019, Mircea A. alytical methods used in USP monographs and general chapters. Comanescu, Tiffany J. Millett Animal hair recovered as evidence may play a valuable role in forensic investiga- 242 Impact of a Packaging System on Drug Quality and USP’s Effort to tions. Questioned animal hairs are examined macroscopically and microscopically, Revise its Packaging Standards similarly to the forensic examination of human hair; however, the microscopic com- Desmond Hunt, United States Pharmacopeia, 12601 Twinbrook parison of animal hair bears less significance than that of human hair, and often, Parkway, Rockville, MD 20852 only species determinations can be done. Because of this, atypical characteristics, Plastic and elastomeric packaging materials are used in a variety of ways within the such as dyed furs, becomes utmost important for forensic purposes, because the pharmaceutical sector, from packaging system and medical devices to tubing and dye can be analyzed to provide more information and demonstration of associa- single use components used in the manufacturing process. The question faced by tion/disassociation between questioned and known hairs. Micro-spectrophotometry the pharmaceutical industry is, “What ultimate impact does a plastic or elastomeric (MSP) is an instrumental technique used to gain information on the absorption char- material or component has on the drug product?” Because the makeup of most acteristics, or the true color, of a sample. MSP is a desired technique for examina- packaging materials is proprietary, end-users must rely on their own testing for ex- tion of transfer evidence samples, since it is non-destructive and requires minimal tractables and leachables to determine a material suitable. With a robust Compen- sample preparation. While the human eye can detect differences in color, MSP can dial standard, knowledge of a material can be obtained that will provide the end-user be used to discern nuances of which the human eye is incapable. For this research, with the ability to make more informed decisions about materials during the drug de- dyed beaver pelt from commercial garments were analyzed using transmission and velopment process, which ultimately saves time and effort. Improvements in analyt- fluorescence MSP and the results are presented. While micro-spectrophotometry is ical techniques and industry best practices has made United States Pharmacopeia not a novel method for the analysis of forensic samples, the goal of the research is (USP) take a closer look at our current standards associated with plastic and elasto- to demonstrate its ease of use and continued employment for the potential of asso- meric materials used to package pharmaceutical products and the biocompatibility ciating known and questioned hairs based on their dye characteristics. testing of these materials. This conscious review of these Compendial Standards has led to efforts to update them. Thus, the session gives an overview of the current 247 Microscopy as a Tool in Environmental, Health, and Safety revision efforts, for USP plastic, elastomer and biocompatibility standards. Investigations Andrew Havics pH2, LLC, 5250 E. US Highway 36, Ste. 830, Avon, IN 243 Over-the-Counter Drug Product Standard – USP Initiatives 46123 Sujatha Ramakrishna, United States Pharmacopeia, 12601 Twinbrook In the Environmental, Health, and Safety (EHS) arena, cases vary tremendously but Parkway, Rockville, MD 20852 are driven by client types - individuals, manufacturers, employers, insurance com- No abstract submitted by the author. panies, attorneys, etc. Several cases are presented. including: a woman who ended up in a coma after a powder product application, health questions over downwind 244 Over-the-Counter Drug Product Standard – Industry Initiatives emissions from a manufacturing plant, a co-worker accused of urinating in a cubicle, Kylen W. Whitaker, The Procter and Gamble Company, MS: DV2-3N7, two cases of surface darkening in houses, a couple suspect white powder cases, 8700 Mason Montgomery Rd., Mason, OH 45040 source identification for an indoor air quality case, and an unintentional asbestos Since the inception of Untied States Pharmacopeia (USP) Monograph Modernization exposure in a commercial office building. The examinations conducted included the in 2010, major effort has occurred with respect to updating over-the-counter (OTC) application of polarized light microscopy (PLM), fluorescence microscopy (FlM), mi- drug product monographs. Besides the missing product monographs, the published cro-chemical testing, scanning electron microscopy with energy dispersive X-ray ones lacked organic impurity methods and specifications. Since the request to fill spectroscopy (SEM-EDS) analysis, and transmission electron microscopy (TEM) this gap, some solutions were found while other solutions fell short and uncovered with EDS and selective area electron diffraction (SAED). issues with respect to the complexity of OTC products. Industry has responded with collaborations between companies through the Consumer Products Healthcare 248 Screen Shot Association (CHPA), with the USP and United States Food & Drug Administration Peter Diaczuk, Pedico Research Institute, 329 Whitmore Lab, University (FDA) and by sending in their own product specific monographs. Some of these ap- Park, PA 16802, Xiao Shan Law proaches have resulted in success such as USP General Chapter 227. Some have This research project was generated from a shooting case involving a suspected stalled by creating compliance issues when trying to apply a single method and one bullet hole through a window screen. After passing through the window screen, the set of impurity specifications to dozens of potential products that fit a monograph bullet continued until it encountered an occupant of the premises. When submitted title. The priority for modernization has been focused on analgesics and cough/ for examination, the window screen contained two holes, so it became important to cold actives which combine to account for over $10 billion in US sales alone. This determine whether one or both holes found in the window screen were made by a presentation explains why the normal approach to USP product monographs will bullet(s), and if so, could an angle be approximated (or eliminated) based on the not work and how possible solutions including new monographs forms may work. It shape of the hole. Similar to fabrics, preexisting gaps formed by the warp and weft also focuses on high-performance liquid chromatography (HPLC) methods to look of aluminum wire form the basis of an aluminum window screen. Pieces of screen at these actives and their degradation products in complex matrices. material were fastened to wooden frames to simulate the frame of a typical window and fired upon with different caliber bullets at different velocities and angles, to 245 Examining Elemental Analysis by SEM-EDS in Forensic Paint determine differences in the entry and exit hole deformation. The holes made by Comparisons these test-fired bullets distinctly revealed that one of the two holes in the apartment Christopher S. Palenik, Microtrace LLC, 790 Fletcher Dr., Ste. 106, screen did not result from bullet perforation. Additional test-fired bullets in the exper- Elgin, IL 60123, Ethan Groves iment showed a more elliptical shape with increasing angle away from an orthogonal Forensic paint analysis is among the more commonly encountered types of trace impact, an increased diameter with increasing bullet caliber, and varied results with evidence. Elemental analysis using scanning electron microscopy / energy disper- bullet velocities in different calibers. This last variable, bullet velocity, becomes the sive X-Ray spectroscopy (SEM-EDS) is a well-established technique that is often most challenging aspect to interpret when assessing bullet holes in window screen. used to provide probative information in forensic casework. For the task of forensic 37 2018 EAS Abstracts November 2018

249 Organic Salts: Tunable Materials for Analytical Applications 252 Looks Can Be Deceiving: Spectrochemical Analysis Applied to Isiah M. Warner, Louisiana State University, Dept. of Chemistry, 434 Ocular Surface Phenomena Choppin Hall, Baton Rouge, LA 70803 Frank V. Bright, State University of New York – Buffalo, Dept. of My research group has been exploring the scientific applications of room-tempera- Chemistry, Buffalo, NY 14260 ture ionic liquids (RTILs) for several years. More recently, we have extended the In clinical practice and research, a fluorescent dye called fluorescein is widely used range of these materials to include applications of similar solid phase materials, i.e. by eye care professionals to evaluate the ocular surface and the observed signal organic salts with melting points of solid phase ionic liquids (25 °C to 100 °C) up is often interpreted as damage to ocular surface cells. Over the past several years, to organic salts with melting points of 250 °C. To contrast these new materials with there has been much discussion and debate regarding what has been described as RTILs, we have created the acronym, GUMBOS (group of uniform materials based transient “corneal staining” by fluorescein, which is characterized by an increase in on organic salts). These GUMBOS have the tunable properties frequently associat- asymptomatic, superficial, punctate corneal hyperfluorescence associated with the ed with RTILs, including tunable solubility, melting point, viscosity, thermal stability, use of multipurpose contact lens solutions (MPS). This phenomenon is typically hydrophobicity, and functionality. Thus, when taken in aggregate, GUMBOS allow seen between 30 minutes and 4 hours after soft lens insertion and it resolves after production of solid phase materials which have a wide range of biomedical and 6-8 hours. Although the most intense appearance of this transient corneal staining other scientific applications. Our GUMBOS have led to applications in many areas has been equated to MPS “toxicity” to the cornea, MPS-associated, transient cor- including sensors, cancer therapy, novel approach to antibiotics, and production neal hyperfluorescence with fluorescein is asymptomatic and lasts on average only of tunable nanomaterials (nanoGUMBOS). In regard to nanomaterials (nanoGUM- a few hours, while a true toxicity reaction is typically symptomatic and may require BOS), we believe that our methodology represents an extremely useful approach to several days to resolve. We have set about to determine the origins of transient production of tunable nanomaterials since our materials are designed and assem- corneal staining by using a battery of modern spectrochemical analysis tools. In this bled for specific uses (task specific), rather than adapted for use as is done for many presentation, the speaker will outline the problem in more detail and described the nanomaterials. Selected applications of ionic liquids and GUMBOS, including sen- road we have taken to identify the root causes of staining and to explore the interac- sor applications and cancer therapy, are highlighted in this talk. Particular emphasis tions between the two most common antimicrobial agents found in commercial MPS is placed on a possible new development for selective cancer therapy. products (polyhexamethylene biguanide (PHMB) and/or polyquaternium-1 (PQ-1)) with small unilamellar vesicles and supported lipid bilayers that have been designed 250 Designer Separations with Smart Nanomaterials to mimic the human corneal epithelial cell membrane. Lisa A Holland, West Virginia University, Dept. of Chemistry, Morgantown, WV 26506 253 Aptamers: A Case Study in Chemical vs. Biological Evolution Smart nanomaterials are an innovative alternative to support flexible and repro- Linda B. McGown, Rensselaer Polytechnic Institute, Dept. of Chemistry grammable sample processing and tunable microscale bioseparations. The analyte & Chemical Biology, Cogswell 321, Troy, NY 12180 resolution of biomolecules separated by these nanophases are exceptionally high, Combinatorial chemistry has a decades-long history in drug discovery and thera- yielding separation efficiency up to 2 million theoretical plates. A critical advantage peutics, including its relatively recent application to the selection of oligonucleotide of self-assembled nanophases for efficient separations of biomolecules is the ease affinity reagents, or aptamers, to particular targets for use in biology, medicine and with which they are modified and then introduced, patterned, and replaced in the analysis. The basic tenet of the combinatorial approach is that a sufficiently large capillary. The self-assembled separation is facilitated by different phospholipid library of randomly synthesized molecules will include one or more members with nanogels that form different thermally responsive morphologies, with a liquid-to-gel the desired functionality. Over the years, however, it has become apparent that this transition at ~22 °C. This property makes it easy to load and pattern nanogels in tenet is flawed. This talk discusses reasons for this in the context of aptamer se- narrow microfluidic channels or narrow-bore capillaries at low temperature. The flu- lection to protein targets, contrasting the established, combinatorial selection route ids are then locked in place by raising the temperature to gel the material. Discrete through chemical “evolution” to new approaches that take advantage of naturally zones of nangels are maintained in microscale channels to accomplish traditional occurring oligonucleotides resulting from earth’s long history of biological evolution. gel-based separations but in a microscale format. For example, nanogels of differ- ent viscosity can be patterned to create stacking zones used in sieving and then 254 Learning about the Small Things that have Big Impacts on thermally erased. Nanogels have also displayed a stabilizing effect to enzymes and Individuals’ Lives other proteins, which provides the means to incorporate different affinity reagents Charles V. Morton, Retired, 1018 Leo Way, Oakland, CA 94611 into the separation. This enables cost-effective use of nanoliter volumes of highly Dr. Peter De Forest, has been well known to the New York Microscopical Soci- specific enzymes and antibodies in microscale channels to identify specific proteins ety since becoming a member soon after arriving to teach at John Jay College as well as post translational protein modifications. A wide range of applications are after receiving his Doctor of Criminology degree from University of California (U.C.) discussed to demonstrate this new approach to translate traditional gel-electropho- Berkeley in 1969. To say the 1960s were momentous and turbulent times at U.C. resis separations into a microscale format. The applicability of this technology is Berkeley would be an understatement of sub-microscopic proportions. Peter would demonstrated with glycan and protein separations relevant to next generation phar- enter this period and place in history to pursue a course of study involving microsco- maceuticals. py, microchemistry and forensic science. Although proposed by August Vollmer and Alexander Kidd in 1928, the School of Criminology wasn’t established until 1950, a 251 Spectroscopy through the Microscope: Probing What’s Happening mere 12 years before Peter entered the program. The genesis was even earlier, in Inside Chromatographic Silica Particles 1916 summer session courses formulated by August Vollmer and taught by Vollmer David A. Bryce, University of Utah, Dept. of Chemistry, 315 South 1400 and other U.C. faculty. Peter arrived on campus and was quickly recognized as an East, Salt Lake City, UT 84112, Jay P. Kitt, Joel M. Harris impressive undergraduate student resulting in him being hired as a laboratory aid at Recent breakthroughs in combining spectroscopy with optical microscopy are allow- Paul L. Kirk, Ph.D. and Associates where Peter and I worked until 1969. This added ing chemical analysis to be carried out in fL volumes with unprecedented spatial res- significant legal and practical considerations to our educations at CAL. In 1964 he olution and sensitivity. In this talk, these tools will be applied to investigate interfacial became Dr. Kirk’s teaching Assistant. Our lives became even more intertwined with chemistry that governs retention of molecules within individual chromatographic my marriage to a student and part-time secretary to Paul Kirk and Peter’s marriage silica particles. Confocal-Raman microscopy can be used to interrogate sub-fem- to her sister, Carol, two years later. Carol was also a major part of Peter’s profes- toliter volumes inside of individual reversed-phase chromatographic particles and sional life, being recognized for her contributions to the profession with the creation report interface structure through vibrational spectra of solute molecules retained of the Northeastern Association of Forensic Scientists (NEAFS) Carol De Forest at the alkyl-chain/solution interface. This method is useful for detecting solutes that Student Research Grant. are extracted from a surrounding solution and the kinetics that govern their accu- mulation within a particle. We investigate the structure and properties lipid bilayers 255 The Mystery of the Lost World Trade Center 9/11 Flag: A Trace deposited on pore surfaces of silica; these materials can be used for assessing Evidence Case for the Ages lipid-membrane affinity of analytes on these model lipid-membrane phases. Raman Nicholas Petraco, John Jay College, 240 Abbey St., Massapequa Park, spectroscopy provides insight into conformations of the lipid acyl chains and how NY 11762, Nicholas D. Petraco these change with lipid deposition conditions, temperature, and solute interactions. The flag raised by three New York City firefighters at the WTC “Ground Zero” site Confocal-Raman microscopy probing of these lipid-bilayer supports can also be was discovered missing. The discovery was made when the flag was returned to its used for in-situ, label-free, and quantitative investigation of membrane-localized original owners. They immediately started an investigation which ended after a local protein-ligand interactions. national TV show aired the story of the missing 9/11 flag in October, 2014. A few days later, a flag was turned into a fire house in Everett City, WA, by an unknown man. The local police started an investigation during which they were only able to determine that the questioned flag could be the original 9/11 flag. Nine months after they sent photos and dust samples taken from the questioned flag to John Jay College where research on the ground zero dust was originally conducted and pub- 38 2018 EAS Abstracts November 2018

lished. Forensic scientists at John Jay carried out an examination on the questioned spectrometer to successfully identify falsified medicines in low income country set- specimens, comparing them to their extensive collection of known dust samples tings are also presented. obtained at “Ground Zero,” on 9/11. After comparing the dust from the flag to the known specimens from “Ground Zero” they were able to conclude to a high degree 259 Food Safety Screening with Surface Enhanced Raman of probability that the questioned flag was indeed the original lost 9/11 flag. Spectroscopy: Ensuring Safe Food Reaches Consumers Katherine A. Bakeev, B&W Tek LLC, 19 Shea Way, Ste. 301, Newark, 256 Investigating Potential Mechanisms of Postmortem Hair Root Band DE 19713, Chris Ye, Kevin Hu, Philip Zhou, Qizhen Chen, Jack Zhou (PMRB) Formation Food safety testing is becoming increasingly important as the global food supply JoAnn Buscaglia, Federal Bureau of Investigation Laboratory, MS: is essentially borderless. There are continuing instances of economically-motivat- CFSRU, 2501 Investigation Parkway, Quantico, VA 22135, Jack ed food contamination reported, some that have led to serious incidents including Hietpas, Adam H. Richard death. There is a need for on-site testing methods that can be done rapidly and A postmortem root band (PMRB) is a distinct microscopic feature that occurs in accurately at a cost that can keep food safe and affordable. Perishable foods, such the pre-keratin region of anagen and early catagen hairs derived from deceased as produce must be tested while they are still fresh. The testing must be rapid, and individuals. The finding of a hair containing a PMRB can be quite probative. How- preferably be able to be done throughout the life of the product from farm to table. ever, interpretations of PMRBs have been challenged in criminal court cases (e.g., The testing must also have the sensitivity to detect a range of compounds including Kogut v. County of Nassau, and State of Florida v. Anthony) primarily on the basis pesticide residues, food additives and intentional contaminants, residual veterinary that the formation mechanism is not known. To investigate this issue, detailed ob- medicines as well as other harmful substances. We discuss field screening by a sur- servations were made using high-magnification images of ultramicrotome sections face-enhanced Raman spectroscopy (SERS) method with specialized reagents for of known PMRBs. Microscopical analysis of the “banded” regions indicate that the different compound classes. This rapid screening tool can be used for food testing PMRB is a manifestation resulting from the degradation of the nonkeratin intermac- by producers, food processors, food inspectors, and food suppliers (supermarkets) rofibrillar matrix (IMM) in the pre-keratin region of anagen hairs; PMRBs are also to screen for potential issues in foods from fresh produce, to processed products constrained to the cortex region with no observable damage to the cuticle layers. and nutritional supplements to give an assurance of food safety. PMRBs were also noted in hairs from several non-human species; studying PMRB formation in decomposing animals in controlled environments provided insight into 260 Use of Handheld Raman and Near-Infrared Spectroscopic the conditions in which PMRBs will form. Additional in-vitro studies include antemor- Techniques for Identifying Counterfeit Lifestyle and Life-Saving tem anagen hairs that were subjected to several conditions (e.g., pH series, prote- Medicines ase digestions) that may affect the IMM; the results indicate that some microscopic Sulaf Assi, Bournemouth University, Christchurch House, Poole BH12 characteristics of PMRBs can be replicated particularly with immersion of anagen 5BB, United Kingdom, Thomas Coombs, Jacob McEachran hairs in slightly alkaline aqueous ammonium salt solutions. NH4+/ NH3 are viable Medicine counterfeiting represent a public health threat affecting all countries world- causative agents because these compounds are produced in significant amounts wide. Its impact is huge not only on government economies and policies but also during autolytic and bacterial degradation of protein during decomposition. This re- on the public health where counterfeit medicines could result in toxic or lethal ef- search provides valuable insight into possible mechanisms of formation of PMRBs. fects. All classes of medicines could be counterfeit whether lifestyle or lifesaving medicines. Lifestyle medicines target to improve image, memory or performance; 257 Inspiring Microscopy whereas, lifesaving medicines include those intended for long-term conditions such Peter R. De Forest, John Jay College of Criminal Justice, 524 West 59th as cardiovascular or respiratory diseases. Both types of medicines could be coun- St., New York, NY 10019 terfeited in relation to the physical properties (color, particle size, formulation type) I am deeply honored to have been nominated for and to receive the prestigious Abbe or chemical constituents (active or inactive ingredients). Spectroscopic techniques Award of the New York Microscopical Society (NYMS). This award is appropriately including near-infrared and Raman have been reported as powerful tools for charac- named to honor Ernst Abbe whose accomplishments and contributions to science terizing the physicochemical properties of analytes of interest including medicines. and society were numerous. He was a superb scientist, successful businessman, Therefore, this work utilized handheld near-infrared and Raman spectroscopy for free thinker and a social reformer. Some of his contributions to optics and physics detection counterfeit lifestyle and lifesaving medicines. More specifically it focused will be discussed. Later in his life he found himself the sole owner of the Carl Zeiss on: 1) validating near-infrared and Raman spectroscopic methods for medicine au- Optical works. NYMS was established in 1877 and was one of the earliest American thentication, 2) identifying the active and non-active constituents present in authen- scientific organizations. I joined this society almost 50 years ago, and have always tic and counterfeit medicines, 3) quantifying the active ingredients in medicines, and recommended membership to my forensic science students. Many of my former 4) characterizing physical properties of different medicine formulations. This was students have been members and board members of the society for many years. In achieved by developing spectral libraries of medicines and constituents, unsuper- addition to the contributions of Ernst Abbe, this presentation will acknowledge the vised learning classification methods and multivariate regression models. The re- influence of my early mentors in criminalistics and microscopy. The first of these sults showed that the multivariate classification and regression approach was pow- mentors were my forensic laboratory directors Elliott Hensel (1960-61) and Thomas erful as a universal model for detecting counterfeit lifestyle and lifesaving medicines. Wieland (1961-62) from the Ventura County Sheriff’s Laboratory. These were fol- lowed in 1962 by my academic mentors at the University of California, Berkeley, Dr. 261 Handheld Visible Spectrometry: the Promise and the Limitations Paul Leland Kirk and Professor Jonas Ekblom Gullberg, for whom I also served as Alexander Scheeline, SpectroClick Inc., 60 Hazelwood Dr., Champaign, a graduate teaching assistant. IL 61820 Handheld spectrometry looks deceptively simple. Whether using cellular telephone 258 Use of Handheld Spectrometers as Screening Tools for Detection cameras as detectors or separate, self-contained spectrometers linked to a com- of Substandard and Falsified Medicines, Perceptions and Reality puter, tablet, or phone via USB, BlueTooth, or Wifi, all one seems to be doing is Mustapha Hajjou, United States Pharmacopeia, 12601 Twinbrook dispersing and detecting light using a compact, repackaged version of CCD cam- Parkway, Rockville, MD 20852, Stephen Kimatu eras attached to spectrographs and controllers as has been common laboratory The spread of substandard and falsified (SF) medical products constitutes a growing practice for 40 years. However, spectrometry is a systemic measurement approach, global public health concern with larger impact in low and medium income countries where the weakest link among instrument performance, calibration, stability, sample (LIMC). A number of screening devices/technologies have been developed to assist matrix, environment, and user skill can disrupt result validity. The presentation is in the rapid detection of SF medical products; these can be utilized in the field and in two parts. In the first, we describe an approach to wavelength dispersion and have been deployed with some success. It is recognized that accurate, reliable and spectral detection that is promising for using low dynamic range, consumer-grade portable screening tools can be of great assistance to regulators and also during cameras to perform quantitative spectrometry. In the second, we describe a more procurement processes. When used properly they can lighten the burden on quality global approach to measurement for users with limited measurement science train- control laboratories and reduce the overall cost of quality control testing. Evaluation ing. In summary, both parts seek to ensure that the main source of measurement of screening tools that provides unbiased information on their advantages and dis- variance is sample composition, and both parts note the difficulties of suppressing advantages is critical for medicine regulators to help them make informed decisions other variance sources. Obtaining a visible spectrum with any camera is reasonably on the selection of the tools that suit their needs. United States Pharmacopeia’s easy, but doing chemical analysis with adequate precision based on these spectra Global Pubic Health Technology Review Program has been evaluating some of the is not. Theory for how to proceed is largely in hand, but executing the steps required technologies to identify their advantages, disadvantages and limitations. To-date, by theory to give a valid result may make general hand-held measurements more evaluation of two technologies has been completed, Speedy Breedy (a portable expensive than is competitive with standard field sampling/laboratory determination respirometer) and CBex (a handheld Raman spectrophotometer), and the reviews’ approaches. Where field-portable instruments have proven most successful has results are available at both USP and World Health Organization websites. Current- been where sample preparation is minimal, a circumstance incompatible with minor ly three additional technologies are undergoing evaluation and the review reports and trace analysis in complex matrices. In both spectrometry and analysis, tacit are presented at the meeting. Case studies involving the use of handheld Raman knowledge is difficult to automate. 39 2018 EAS Abstracts November 2018

262 Innovative Techniques and Applications of Supercritical Fluid small peptides. Difficult separations of large peptides were also simplified using the Chromatography for Drug Discovery screening regimen developed. Observations suggested that known peptide prop- Yingru Zhang, Bristol-Myers Squibb, PO Box 4000, Princeton, NJ erties may effectively guide method selection without requiring a screen of multiple 08540, Chunlei Wang conditions. Additionally, the challenge of loading large peptides for purification was Supercritical fluid chromatography (SFC) is becoming a mainstream chromato- demonstrated to be greatly improved by choosing dimethylsulphoxide (DMSO) over graphic technique complimentary to reversed phase high-performance liquid chro- aqueous sample solutions. matography (HPLC) for analytical and preparative separations of both chiral and achiral molecules in the pharmaceutical industry. To enable SFC to solve more 265 Revolutionizing Pharmaceutical Compound Analysis by complex separation challenges, improved understanding and implementation of Implementation of 2D-LC-SFC-MS innovative techniques to maximize SFC advantages are required. Parallel-col- Mohammad A. Al-Sayah, Genentech, 1 DNA Way, South San Francisco, umn-screening and simulated-moving-column techniques have been used to maxi- CA 94080, Meenakshi Goel, Eli Larson, CJ Venkatramani mize separation and method development efficiencies. Here, we present our study Fast turnaround times have become a norm in the pharmaceutical industry neces- of tandem-column-SFC to resolve complex mixtures by serially coupling columns sitating the development of unconventional high-throughput analysis techniques. with different selectivity. Tandem column separation in SFC is more complex than Different researchers have focused on different areas to achieve high-throughput in HPLC due to the impact of column back pressure (CBP) on density and solva- analysis including sample preparation, analysis run times and data analysis times. tion power of the CO2–modifier (methanol) mobile phase. Conversely, tandem-col- This presentation will discuss the development of a two-dimensional reversed umn-SFC possesses unique opportunities for selectivity optimization. We studied phase liquid chromatography-supercritical fluid chromatography-mass spectrome- the effects of CBP on dead time (t0), solute retention, selectivity and resolution. Our try (2D LC-SF-MS) system to achieve simultaneous achiral and chiral analysis of results revealed that apparent t0 in SFC depends not only on the mobile phase den- pharmaceutical compounds. The peaks of interest from the first RPLC dimension sity, but also on the adsorption of the modifier into the stationary phase. We derived column were effectively trapped on small volume C-18 trapping columns and then the pressure-retention relationship in order to apply CBP as a unique separation injected onto the second normal phase chiral SFC dimension. With one sample parameter. Consequently, we can adjust retention and selectivity contributions from preparation, two simultaneous results can be obtained: the impurity profile from the individual columns for fast method development. Using the empirical mathematical RPLC dimension and the enantiomeric purity from the second SFC dimension. The equation, we calculated tandem-column-SFC separations based on a single reten- 2D LC-SFC-MS system was then optimized to assess the “in-vivo” inter-conversion tion time measurement on each column. The simulation compares well with experi- of chiral drug molecules. Such analysis can be quite challenging due to the low mental results and correctly predicts column order and back pressure effects on the abundance of the active pharmaceutical ingredient (API) and its potential metabo- separations. Furthermore, we illustrated continuous tuning of tandem-column-SFC lites in plasma. To achieve the desired sensitivity, different system parameters such selectivity through column back pressure adjustments of the upstream column. as 1D and 2D column dimensions, trapping column stationary phase, system tubing ID, and detection techniques (UV and MS) were optimized. The optimized system’s 263 Supercritical Fluid Chromatography-Mass Spectrometry for Use in limit of detection in the second dimension was determined to be 10ng/ml. The fully PK/PD Studies optimized system was successfully used to quantitate an API, its metabolite and Fangbiao Li, Merck & Co., 770 Sumneytown Pike, West Point, PA 19486 their corresponding enantiomers in a mouse hepatocyte treated sample without any Supercritical fluid chromatography (SFC) is an orthogonal technology to reversed desalting procedure. phase chromatography and is routinely used for separations supporting chemical synthesis. New developments in SFC hardware prompted the evaluation of SFC- 266 Neurotransmitter-Metabolite Detection with Fast Scan Cyclic tandem mass spectrometry (MS-MS) as a necessary tool for discovery bioanalytical Voltammetry labs focusing on small molecules drug discovery. In order to understand the general Alexander G. Zestos, American University, 4400 Massachusetts Ave, use and robustness of SFC-MS for routine bioanalysis, a generic SFC method using NW, Washington, DC 20016, Alexander Mendoza Waters UPC2 BEH column was developed to analyze 373 small molecules with A novel method will be developed to detect neurotransmitter metabolites. Tradition- diverse structures. The same compounds were analyzed using a generic ultra-per- ally, carbon-fiber microelectrodes (CFMEs) have been utilized to detect dopamine, formance liquid chromatography (UPLC)-MS method for comparison purposes. Of serotonin, and other important neurotransmitters, but this method is constrained by the compounds evaluated, 363 out of 373 were observed successfully using ei- the inability of these sensors to detect physiologically relevant nanomolar concentra- ther method; the remaining ten compounds could not be detected by either SFC or tions. Carbon nanotube microelectrodes will be utilized to detect physiologically lev- UPLC methods. SFC provided sharp and symmetrical peaks for a separate set of els of neurotransmitters with high temporal resolution in order to detect fast changes polar compounds which were not easily retained on a C18 column using the UPLC of neurotransmitter concentrations. Novel electrode coating and waveforms will also method. Sharp peaks and efficient separation of hydrophobic lipid compounds, cer- be used to detect several neurotransmitter metabolites, such as 3-methoxytyramine amide and glucoceramide, were also achieved using the SFC method. Comparable (3-MT), homovanillic acid (HVA), and 3,4 dihydroxyphenylacetic acid (DOPAC). Cur- limits of quantitation (LOQs) were observed between SFC and UPLC for the ma- rently, dopamine is thought to be an important neurotransmitter concerning several jority of compounds being evaluated in guinea pig and rat plasma. SFC tended to disease states, such as Parkinson’s disease, drug abuse (amphetamine, cocaine, provide better LOQs for polar compounds. The separation of four stereoisomers, etc.), and even for gambling and sex-disorders. However, dopamine is metabolized two pairs of enantiomers and epimers, of itraconazole (ITZ), keto-itraconazole (Ke- on a subsecond timescale, and studies have pointed to the importance of neu- to-ITZ) and N-desalkyl-itraconazole (ND-ITZ) was achieved on Waters UPC2 using rotransmitter metabolites in these disease states apart from dopamine. Presently, a Phenomenex Lux® 3m Cellulose-3 column and 12-min SFC gradient. there is no method to detect these neurotransmitter metabolites of dopamine utiliz- ing voltammetry. Through several waveform modifications, and polymer/electrode 264 Advantageous Use of SFC for Separations of Novel Therapeutic coatings, a method will be developed for detecting and separating several dopamine Peptides and Peptide Libraries metabolites utilizing fast scan cyclic voltammetry, which will help differentiate the Manuel Ventura, ChemPartner Corp., 280 Utah Ave., Ste. 100, South cyclic voltammograms of dopamine and dopamine metabolites. San Francisco, CA 94080 As peptide therapeutics gain momentum in the pharmaceutical discovery arena the 267 Naked-Eye Electrochemical E.coli. Detection challenge of high-performance liquid chromatography (HPLC) separation of target Kwok-Fan Chow, University of Massachusetts Lowell, 1 University Ave., compounds from automated synthesis remains a prevalent issue. Supercritical fluid Lowell, MA 01854, Sachintha Wijesinghe, Jungmin Oh chromatography (SFC) has become increasingly prominent in the pharmaceutical In this presentation, we report a naked-eye electrochemical E.coli. sensing platform industry as users have exploited its alternative selectivity and high efficiency for that is capable of detecting E.coli. on a working electrode and providing a visual small molecule chiral and achiral separations. SFC’s application to peptides has readout of the E.coli. concentration on a silver band counter electrode. The display long been reported but is not used routinely in their analysis and purification. This mechanism relies on the electrode oxidation of metallic Ag as a complementary work explores SFC for separations of peptides generated from automated synthe- reaction to the sensing reaction. The decrement of the silver band electrode, which sis along with strategies and methods to enhance performance. A minimal screen- is clearly measurable with the naked-eye, correlates linearly with the E.coli. concen- ing set of modifier additives and stationary phases to best tackle various peptide tration. We also demonstrated that this sensing platform can be operated without mixture separations using SFC/MS is described. Peptides evaluated include small, any electronic instrumentation. polar or nonpolar macrocycles along with intermediate to large, linear or cyclic se- quences (13-41 residues) with divergent hydrophilicities. Libraries of small peptides 268 Performance of Electrochemical Sensor of Nitrite, a Biomarker of with polar side chains subjected to the SFC column/mobile phase screen devised Oxidative Stress, in Exhaled Breath Condensate exhibited the greatest chromatographic performance diversity. In the majority of cas- Ashley Cole, Rutgers University, EOHSI, 170 Frelinghuysen Rd., es a modifier of methanol with ammonia plus Trifluoroacetic acid (TFA) produced Piscataway, NJ 08854, Azam Gholizadeh, Clifford Weisel, Mehdi performance advantages, and with a diol-based stationary phase retention diversity Javanmard, Vladimir Mishin was increased by >40% over pyridine-based phases for both polar and nonpolar A portable sensor was developed to measure nitrite in exhaled breath condensate 40 2018 EAS Abstracts November 2018

(EBC) as a biomarker of oxidative stress in the respiratory system. A miniaturized electrochemical sensor was fabricated using reduced Graphene Oxide (rGO). The unique properties of rGO include its resistance to corrosion, rapid electron transfer 272 Improving RMID Results with Handheld Raman Instrument Control with electrolytes, high sensitivity, and minimal fouling. The rGO sensor housing is Parameters fabricated from polydimethyl siloxane (PDMS), which allows for analysis of <0.1 Adam Hopkins, Metrohm USA, 9250 Camden Field Parkway, Riverview, mL EBC and detection of nitrite at a low over-potential of 0.7 V with respect to the FL 33578, Mackenzie Speer Ag/AgCl reference electrode. The sensor’s performance was characterized using Handheld Raman spectroscopy holds the promise of eliminating sample quarantine standard nitrite solutions in acetate buffer and EBC, and by comparison to measure- and statistical sampling for raw material identification (RMID) applications and en- ments using a colometric Greiss method. The sensor responded linearly in buffer abling 100% container verification. However, the variety of packaging materials and between 1-100 μM nitrite concentrations with an R-squared value > 0.99 and re- types of analytes can cause difficulty for many handheld instruments. Some of the sponses among different sensors being within ± 12-34% across the concentration difficulties are very long acquisition or library building times, and sample or pack- range evaluated. To evaluate reproducibility and longevity of a sensor, 25 measure- aging material degradation. In this talk, we examine common excipients and active ments were made. The response for the last five measurements at 10 μM nitrite pharmaceutical ingredients (APIs) inside a variety of common packaging materials remained within ±5% indicating high reproducibility and utility for multiple measure- with the Mira handheld Raman spectrometer. We show that these difficulties can be ments from a single sensor. The Griess test was linear throughout the target nitrite eliminated through complete control over the data collection and analysis method concentration range in aqueous media and in EBC with a limit of quantification of combined with orbital raster scanning. 7 μM due to low optical densities (less than 0.1). Comparison of the EBC with and without spiked nitrite between the Griess test and the sensor, which has a lower limit 273 Investigation of Physical and Chemical Variability on Quantitative of detection, is discussed. Transmission Raman Models Caitlin N. Baldasano, GlaxoSmithKline, MS: UP9100, 1250 South 269 Zirconium-Iridium Mixed Oxide Electrocatalysts for the Oxygen Collegeville Rd., Collegeville, PA 19426, Benoit Igne, Christian Airiau Evolution Reaction Transmission Raman spectroscopy is increasingly becoming an applied analytical Edward Y. Zhang, Princeton University, Dept. of Chemistry, 5197 Frist tool in the pharmaceutical industry for oral solid dose analysis. It delivers rapid sam- Campus Ctr.. Princeton, NJ 08544, Rachel S. Selinsky, Xiaofang Yang, ple analysis with little to no sample preparation compared to typical analytical meth- Bruce E. Koel ods. This technique has the advantage over near infrared (NIR) spectroscopy to be The kinetically sluggish oxygen evolution reaction (OER) is the limiting factor for potentially more specific with sharper spectral features, as well as providing greater the efficiency of water electrolyzers, requiring highly active and expensive catalysts sensitivity for analysis of lower formulation dosage. To obtain valuable quantitative such as iridium oxide. In this study, we examine the properties of zirconium-iridium information from transmission Raman data, it is important to have in place an accu- mixed oxides as OER catalysts. Electrochemical techniques such as cyclic voltam- rate and robust multivariate model. There are many different factors that can impact metry and chronopotentiometry are applied to determine the catalytic activity and a quantitative model, such as tablet hardness, thickness and moisture content. If the stability of these materials in acidic conditions. To gain insights into the electronic, effects of these factors are understood, the variability can be accounted for during morphological, and structural changes at the catalyst surface, we use techniques model building, resulting in more robust predictions. This work investigates the ef- such as X-ray photoelectron, scanning electron microscope, and operando Raman fect of external factors onto the quantitative prediction of individual tablet content spectroscopy. These insights will speed up progress in the rational design of highly and content uniformity. Particularly, the effects of tablet thickness, weight and mois- efficient, cost-effective, and durable OER catalysts. ture content are considered. A comprehensive central composite design, including variability in active content, tablet weight, thickness and moisture was used to build 270 Application of 2D COS Raman Spectra to Structural Elucidation of multivariate models. These models were used to predict independent test sets with Polymers variability in these specific parameters. The impact of tablet thickness, weight and Fran Adar, HORIBA Scientific, 20 Knightsbridge Rd., Piscataway, NJ moisture onto the model performance was then assessed. Results present statisti- 08854 cal analysis of the impact of these physical and chemical sources of variability onto It is well known that interpretation of Raman spectra of polymers can yield valuable the robustness of quantitative models for the active ingredient using transmission information on their chemical and physical structure. However, this often requires Raman spectroscopy. extensive normal modes analysis and then band-fitting to separate contributions from similar states. By following spectral changes as a function of some systematic 274 Determination of Polycyclic Aromatic Compounds in SRM 1597a change, it becomes possible to visualize easily the correlations between structure via Normal-phase Liquid Chromatography and Gas and spectral changes. In particular, when there are overlapping bands between var- Chromatography Mass Spectrometry ious phases, contributions from these phases can be visualized without band-fit- Hugh V. Hayes, University of Central Florida, Physical Sciences Building, ting. In order to demonstrate these phenomena we show spectra of an amorphous Room 225, 4111 Libra Dr., Orlando, FL 32816, Walter B. Wilson, Lane C. sample of poly-ethylene terephthalate (PET) held at a temperature above the glass Sander, Stephen A. Wise, Andres D. Campiglia transition temperature and measured as a function of time, and polarized spectra of Polycyclic aromatic compounds (PACs), namely polycyclic aromatic hydrocarbons fibers held under tension and measured as a function of temperature. The goal is to (PAHs), polycyclic aromatic sulfur heterocycles (PASHs), and their methyl (Me-) reveal changes in conformation and changes in crystallinity, recognizing that certain derivatives, belong to a large class of environmental pollutants originating from nat- conformation changes are necessary before crystallization can occur. ural and anthropogenic sources. Identification of PACs in environmental samples is of extreme importance due to their potential carcinogenic properties. Traditional 271 Counterfeit Tablet Analysis Using a Handheld Raman Spectroscopy methods for identification utilize gas chromatography mass spectrometry (GC-MS). Stephen W. Hoag, University of Maryland-Baltimore, School of However, structural isomers having similar GC retention times along with identi- Pharmacy, 20 N. Pine St., Baltimore, MD 21201, Adam J. Hopkins cal mass fragmentation patterns pose difficulty in identifying specific isomers. To The globalization of the pharmaceutical supply chain has created a situation in help alleviate this issue, we investigated the normal-phase liquid chromatography which drugs can enter into the prescription marketplace from many different sourc- (NPLC) retention behavior of 333 PACs and determined their retention indices on es, which has led to a situation where counterfeit drugs are becoming increasingly an aminopropyl (NH2) stationary phase. The NPLC retention times of PAHs and common even among prescription drug products. In addition, highly skilled coun- PASHs correlated directly to the total number of aromatic carbons in the parent terfeiters can manufacture fake versions of common prescription drugs that require structures. Furthermore, methyl derivatives eluted slightly later than their respective laboratory analysis to tell from an authentic product. Given the problem protecting parent structures. The obtained data provided the foundation for a NPLC fraction- our drug supply, the goal of this study is to determine how well a hand held spec- ation procedure useful for complex environmental matrix cleanup, which was later trometer can distinguish fake from authentic drug products. When developing spec- used for PAC determination via GC/MS. This presentation discusses the application tral libraries and models for the identification of counterfeits one needs to know the of the NPLC fractionation – GC-MS methodology for the analysis of Standard Ref- sensitivity and specificity of the model, as you do not want to accept a counterfeit erence Material (SRM) 1597a. as real and conversely you do not want reject authentic product as counterfeit. To determine the sensitivity and specify of a product we use the United States Food & 275 Investigation of Photodegradation Products of High-Molecular Drug Administration (FDA) approved label for Viagra and used a compaction simula- Weight Polycyclic Aromatic Hydrocarbons in Seawater tor to manufacture a series of formulations (varying in drug and excipient concentra- Anthony F. T. Moore, University of Central Florida, 4111 Libra Dr., tion and manufacturing conditions) that closely resemble the approved Viagra, and Physical Sciences Bld. Rm. 255, Orlando, FL 32816, Sadia Arif, Andres then scanned these products using a hand held Raman spectrometer. By comparing D. Campiglia the spectrum for these series of products, we gained a better understanding of the The Deepwater Horizon oil spill was and industrial and environmental disaster Raman spectrometer’s robustness for determining counterfeits. that released several million barrels of petroleum into the Gulf of Mexico. A prima-

41 2018 EAS Abstracts November 2018

ry component of petroleum is polycyclic aromatic hydrocarbons (PAHs), a class the electrochemistry of extreme environments is challenging and performing this of hydrocarbon compounds consisting of two or more fused benzene rings. The task in real time allows the researcher a more complete understanding of the en- United States Environmental Protection Agency (US EPA) lists 16 PAHs as priority vironment under study. Electrochemical data from hydrothermal vent areas on the pollutants. However, of particular interest are high-molecular weight PAHs (HMW- ocean floor using Alvin, DSV at 9N EPR and other extreme environments on Earth PAHs), PAHs with a molecular weight greater than or equal to 302 g/mol. Diben- is presented. zo[a,l]pyrene, a HMW-PAH, is significantly more toxic than the most carcinogenic PAH listed by the US EPA, benzo[a]pyrene. Determining the short- and long-term 279 Screening of Pesticides in Bat Guano fate and bioavailability of the PAHs released into the environment is important to Katherine Harms, Kutztown University of Pennsylvania, 15200 Kutztown understanding the overall and long-term impact of these disasters on the ecosys- Rd., Kutztown, PA 19530, Archie Covely, Serena Mang, Joe Benton, tem. One possible pathway significant to the fate of PAHs is photodegradation due Amanda Vangieri, Kasandra Cruz, Thomas Betts to exposure to the sun. While HMW-PAHs generally exhibit relatively lower solubility Bat populations across the United States are dwindling due to habitat loss, wind tur- in water, photodegradation processes are expected to oxidize the PAHs into prod- bines, and, more tragically, disease. For example, white-nose syndrome, a fungus ucts that exhibit greater solubility in water, and hence, greater bioavailability in the that attacks hibernating bats, has killed over six million bats. Concern over declining ecosystem. Herein, the photodegradation of PAHs and the formation of products is bat populations has prompted researchers to question whether exposure to pesti- investigated by fluorescence excitation–emission matrices (EEMs) and liquid chro- cides disrupts bodily systems in these insectivorous mammals. This study utilizes a matography–mass spectrometry (LC-MS). Fluorescence EEMs allows for observing noninvasive approach to assessing bat exposure to pesticides by examining gua- the total fluorescence of the photodegraded samples, while LC-MS will assist in the no droppings from various bat roosts, thereby increasing access to test subjects separation and elucidation of the photodegradation products. in a non-stressful manner. Guano samples from collection sites located primarily in Pennsylvania, with one site in Indiana, were analyzed for both herbicides and 276 Determination of High Molecular Weight Polycyclic Aromatic insecticides. Target analytes were chosen based upon their likelihood of applica- Hydrocarbons via Fluorescence Wavelength-Time Matrices and tion within the study areas. QuEChERS (quick, easy, cheap, effective, rugged, and Time-Resolved Excitation Emission Matrices in Environmental safe) extraction followed by high-performance liquid chromatography (HPLC) with Extracts tandem mass spectral detection was used to screen for approximately twenty-five Khang D. Trieu, University of Central Florida, 4000 Central Florida Blvd., pesticides including various triazine, chloroacetamide and dinitroaniline herbicides, Orlando, FL 32816, Stacy M. Wise, Anthony Santana, Andres Campiglia along with several organophosphate and neonicotinoid insecticides in bat guano. Due to the ubiquitous occurrence and extreme eco-toxicological relevance of poly- The analysis of guano samples is ongoing, but several target compounds have been cyclic aromatic hydrocarbons (PAHs), the United States Environmental Protection detected in each of the guano samples analyzed to date. Agency (EPA) and the European Community include sixteen PAHs in their “Prior- ity Pollutants List” with molecular weights (MW) ranging from 128 (naphthalene) 280 Abstract withdrawn by the author to 278 (dibenz[a,h]anthracene). Unfortunately, the environmental monitoring of 16 EPA-PAHs is no longer sufficient. Eco-toxicological studies attribute a significant portion of the biological activity of PAH contaminated samples to the presence of 281 Design and Synthesis of Functionalized Magnetic Nanoadsorbents high molecular weight PAHs (HMW-PAHs), i.e., PAHs with MW greater than 300. for the Selective Removal of Metal Ions This presentation focuses on the analysis of PAH isomers with MW 302. Similar Kanika Solanki, Green Chemistry Network Centre, Dep. of Chemistry, chromatographic behaviors and almost identical mass fragmentation patterns make University of Delhi, 105 Block B, First Floor, New Delhi 110007, India, separation and identification by gas chromatography mass spectrometry (GC-MS) Rakesh K. Sharma and high-performance liquid chromatography (HPLC)-MS difficult. We present Rapid industrialization and urbanization without safeguard mechanisms have given laser-based instrumentation for the collection of fluorescence wavelength-time rise to some grave issues such as water pollution. One of the prime reasons of water matrices (WTMs) and time-resolved excitation emission matrices (TREEMs) for pollution is the disposal of noxious metal ions in to water resources. The magnetic multi-way calibration (Parallel Factor Analysis) of commercially available isomers in nanoadsorbents have been considered as one of the most promising recourses in complex environmental extracts. WTMs and TREEMs combine spectral and lifetime present era for the removal of these metal ions from water. Here we report, synthe- information that allows unambiguous determination of PAH isomers in HPLC frac- sis and functionalization of magnetic nanoadsorbents by a facile covalent immo- tions at the parts-per-billion to parts-per-trillion concentration levels. bilization approach in which metal selective chelators have been anchored on the surface of amine functionalized silica encapsulated ferrite nanoparticles. The struc- 277 New Simultaneous Optical Technique for Monitoring Organic tural exploration and characterization have been carried out by employing several Pollutants in Source Water sophisticated techniques such as Fourier transform infrared spectroscopy, X-ray Linxi Chen, HORIBA Scientific, 20 Knightsbridge Rd., Piscataway, NJ diffraction, energy dispersive X-ray fluorescence, energy-dispersive X-ray, scanning 08854, Adam Gilmore, Reiji Kojima, Karoly Csatorday electron microscope, transmission electron microscopy, field emission- scanning Rapid, reliable analysis is important for early detection of contaminants in source electron microscope and vibrating sample magnetometer. Various critical adsorp- water used for drinking water treatment. This paper describes the unique, sensitive tion parameters like amount of nanoadsorbents, effect of pH, adsorption time, non-destructive method of simultaneous absorbance-transmittance and fluores- type of eluting agents and their volume, adsorption isotherms and reusability have cence excitation-emission mapping (A-TEEM) to fingerprint contaminants. Automat- been investigated thoroughly. Experimental studies revealed that these magnetic ed modeling facilitates recognition of changes in source water composition including nanoadsorbents displayed ease of separation, better adsorption capacity, greater contamination events. We investigated occurrence of various oil and poly-aromatic reusability, smaller sorption-desorption cycle and required mild elution conditions. hydrocarbon (PAH) in the presence of high natural organic matter (NOM) concentra- tions. The A-TEEM molecular fingerprinting method corrects fluorescence inner-fil- 282 New Reversed-Phase SPP Columns with Alternate Selectivity for ter effects to yield quantitative data largely independent of NOM concentration. Small Molecules Classical least squares (CLS) regression established individual calibration models Richard A. Henry, Independent Consultant, 983 Greenbriar Dr., State for various oil and PAH compounds in pure water, and models were then validated College, PA 16801, Stephanie A. Schuster, Conner McHale, William with oil and PAH compounds spiked in raw water. This A-TEEM and CLS analysis Johnson, Joseph J. DeStefano approach can serve as an effective source water screening tool for oil contamination Several common high-performance liquid chromatography (HPLC) phase chemis- because of:(1) rapid data collection time (2- 3 minutes per sample); (2) high sensi- tries (C18 or C8, RP-Amide, Phenyl or Phenyl Hexyl, and PFP) have proven to be tivity in parts-per-billion (ppb) for oil and PAH compounds; and (3) minimal sample highly useful to separate pharmaceuticals or other small-molecule formulations in preparation compared to conventional solid-phase extraction (SPE) and liquid or the reversed-phase (RP) mode; however, there continues to be a need for novel RP gas-chromatography (LC/GC) analytical methods. In this paper, we discuss the use phases for cases when traditional phases do not separate all major components, of A-TEEM molecular fingerprinting and CLS methods as effective early warning trace impurities and degradants. A common approach to development of a complete systems for source water monitoring. and rugged HPLC separation is to screen the traditional column phases, organic modifiers, and other parameters to determine whether those conditions provide ad- 278 Application of Electrochemistry in Extreme Environments equate resolution. As samples increase in complexity, novel phases and conditions Donald B. Nuzzio, Analytical Instrument Systems, Inc., PO Box 458, may be needed for resolution changes that lead to more accurate quantitative anal- Flemington, NJ 08822 ysis. Newer HPLC phases can provide added selectivity toward isomers and other Chemical measurements obtained in any environment rely largely on sampling and closely-related compounds and operate across a wider pH and mobile phase range ex-situ analyses of water and sediment samples. To improve our understanding that may include high-aqueous solvents. In this presentation, the performance and of the biogeochemical processes regulating the distribution and flux of elements properties of some newer RP stationary phases, including AQ-C18 and Biphenyl, between reservoirs, it is necessary to monitor the geochemical composition of these on superficially porous silica will be described with examples of how their separa- environments continuously with a high spatial and temporal resolution. Monitoring tion performance complements traditional RP column phases and each other. The 42 2018 EAS Abstracts November 2018

new phases in this study are compatible with highly aqueous mobile phases which predict optimized gradient profile and separation temperature. Finally, the in-silico differentiate them from the very hydrophobic straight-chain alkyl phases such as C8 predictions were verified experimentally. The separate case studies discussed in and C18. Separations of polar compounds on these phases will demonstrate their this work demonstrates that factors such as stationary phase composition, organic extended separation range and alternate selectivity. modifiers, pH and separation temperature have complex effects on chromatograph- ic conditions. Therefore, it is critical to include in-silico optimization as an integral 283 Streamlined Reversed Phase HPLC and UHPLC Method component of reverse-phase chromatographic method development to evaluate the Development Using a Combined Column Screening and Software interplay of these factors, there by greatly enhancing the robustness and rugged- Modeling Approach ness of the method. Geoffrey M. Faden, MAC-MOD Analytical, 103 Commons Ct., Chadds Ford, PA 19317, Alan P. Mckeown 286 A Multicompartment Transfer System: Understanding a Non-Linear The process (and success) of reversed-phase method development can vary de- In-Vivo Behavior of Compound A Amorphous Solid Dispersion pending upon experience, resources and available time. The adoption of structured Sanjaykumar Patel, Merck & Co., MS: RY 80T-A168, 126 E. Lincoln approaches to method development has the potential to provide significant savings Ave., Rahway, NJ 07065, Andre Hermans, Hanmi XI, James Ormes, in both development time and costs. Column screening can help identify a suit- Wei Zhu, Binfeng Xia, Filippos Kesisoglou, Wei Xu, Justin Pennington able stationary phase and is a popular strategy. Using this approach, the influence Solubility, dissolution, and precipitation in the gastrointestinal (GI) tract can be criti- of liquid chromatography (LC) stationary phase on retention and separation is ex- cal for the oral bioavailability of a drug. To understand dissolution/precipitation of the plored by screening the sample on multiple stationary phases designed to maximize drug during the transfer out of the stomach and into the intestine, a multi-compart- chromatographic selectivity. Combinations of different mobile phase conditions may ment transfer system was developed and evaluated. This transfer system includes also be used to expand the selectivity ‘space’ and provide a more comprehensive a gastric compartment, an intestinal compartment, a sink/supersaturation compart- dataset. Once a suitable stationary/mobile phase has been selected, the chromato- ment, and a reservoir compartment. The novel transfer system was applied to un- graphic conditions are then optimized to provide the final separation. The use of LC derstand the pharmacokinetic (PK) behavior of a solubility enhancing amorphous modeling software has the potential to further refine method development at the solid dispersion formulation. The amorphous solid dispersion formulation was devel- optimization stage and provide a more informed and robust final method. In this talk oped for Compound A to mitigate solubility challenges. Non-linear PK performance a six column screen using six alternative column chemistries, together with subse- was observed at higher doses of Compound A amorphous solid dispersion formu- quent retention time software modeling is presented. Initially, gradient scouting with lation during single ascending dose study. The transfer model experiments were six ACE reversed-phase columns is used to identify the optimum stationary phase conducted at nominal dose and two higher doses to evaluate non-linearity at higher and mobile phase conditions. Once the optimum combination is identified, additional doses. The transfer model results explained that precipitation in absence of polymer gradient runs are performed and the data entered into ChromSword software to dissolution in gastric pH led to lower drug dissolution later at intestinal pH for higher model the separation. Simulation experiments are then used to rapidly determine doses. Gastric emptying time varied by adding 15 min of lag time compare to stan- a final separation. dard transfer time which indicated further reduction of dissolution at later intestinal pH. Results from scanning electron microscope (SEM) further confirmed crystalline 284 A Bio-Inert, Durable, and Reliable Surface for HPLC and UHPLC API presence on surface of amorphous solid dispersion. The dissolution data from Columns and Components Used in the Analysis of Proteins and transfer model correlated very well with in vivo data. A multi-compartment transfer Other Difficult Molecules system successfully investigated the in vivo non-linear behavior of Compound A and Jesse Bischof, SilcoTek Corporation, 225 PennTech Dr., Bellefonte, PA the data showed promising results to support the transfer system as an alternative 16823, David Smith, Gary Barone, Luke Patterson way to assess supersaturating formulations. As high-performance liquid chromatography (HPLC) components use higher pres- sures and smaller diameter columns for analysis, interactions between the sample 287 All You Wanted to Know about HPLC Method Development and and the walls of the flow path are no longer negligible and the need for an inert Transfer, but were Afraid to Ask flow path to minimize the loss of analytes has become more critical. The loss of Stephanie A. Schuster, Advanced Materials Technology, Inc., 3521 analytes caused by surface activity has been a common issue with small sample Silverside Road, Ste. 1-K, Quillen Building, Wilmington, DE 19810, sizes in gas chromatography (GC) for years, and SilcoTek has been at the forefront Conner W. McHale, Thomas J. Waeghe of minimizing that loss with their inert coatings. Now there is a solution for the liquid When you are developing new ultra-high-performance liquid chromatography (UH- chromatography (LC) community and the analysis of low concentration analytes, PLC) methods, it is important to be aware of key instrument parameters that may proteins, and highly reactive molecules: Dursan. Dursan provides a robust, bio-inert have significant impact on the performance of your column. The relationship be- surface for the entirety of the flow path. Everything from the pump to the detector tween the instrument and column performance is even more critical if a method including frits, columns, and tubing can be coated with Dursan for ultimate inertness. is transferred to a different laboratory with different instrumentation. For example, Traditional materials such as stainless steel cannot compete with Dursan’s bio-inert instrument characteristics such as maximum allowable pressure, extracolumn vol- properties, and other inert materials like PEEK suffer from corrosion and swelling ume and dispersion, gradient delay volume, and flow cell volume may affect best when exposed to solvents, leading to high back pressures. This presentation com- achievable performance with a particular column geometry, particle size and particle pares stainless steel, PEEK, and Dursan-coated stainless steel with respect to chro- platform. We review some of the ways that one can measure these key instrument matographic performance of difficult-to-analyze compounds. Discussion also center parameters, and we demonstrate how these measurements can guide your choice on the application flexibility of the coating, particularly to all parts of the flow path of column when developing new methods and transferring existing ones. The use- from frit to pump. Finally, to demonstrate durability, data on exposure to both caustic fulness of a rugged, reliable 2-µm SPP (superficially porous particle) column for and acidic environments is presented. method development and improved resolution is presented. Examples of transfer of an isocratic and a gradient separation from an HPLC system, using a 5-µm HPLC 285 Retention Modeling and In-Silico Optimization: An Integral column, to a UHPLC system using a 2-µm SPP UHPLC column are shown. Finally, Component of Reverse-Phase Liquid Chromatography Method transfer of a challenging UHPLC separation to an HPLC system using a 5-µm SPP Development column of comparable resolving power is shown. Karthik Jayaraman, Bristol-Myers Squibb - Biocon Research Center, Syngene International Limited, Plot No.2 & 3, Bommasandra IV Phase, 288 The Effect of Alkaline Earth Cations on Amlodipine Besylate and Jigani Link Rd., Bangalore 560099, India, Ashok Kumar Rajendran, Croscarmellose Sodium Drug-Excipient Interaction in a Sample Saravanan Natarajan, Santosh Gandhi, Mallikarjun Narayanam, Solution Hemant Bhutani Prasad Panzade, Apotex Inc., 150 Signet Dr., Toronto, ON M9L1T9, This study reports the use of computer assisted method development and reten- Canada, Yuliya Yarkho tion modeling during reverse phase liquid chromatographic method development of Drug-excipient interaction between primary amine drug substances and Croscar- various small molecule drugs (across various early stage programs) using Drylab® mellose sodium is well known fact that described in different literature sources. Such software. Prior to the experiments required for retention modeling, a set of stationary interaction impacts recovery of the active compound from the matrix of the excipi- phase, organic modifiers and method parameters were chosen based on a platform ents during sample preparation for determination of assay or degradation products. method or based on preliminary screening experiments. Depending on the com- This presentation describes a technique that helps to achieve a complete extraction plexity of the target analyte, one or more of the following DryLab® models: 1) tG-T of the active from the placebo matrix containing Croscarmellose sodium during (gradient time – temperature) with four input runs, 2) tG-T-pH (gradient time-tem- sample preparation for high-performance liquid chromatography (HPLC) analysis perature-pH) with 12 input runs and 3) tG-T-tC (gradient time-temperature-ternary by adding alkaline earth cations in to the sample solvent as well as potential mecha- eluent composition) with 12 input runs was used to build the model for achieving nism of interaction between amine compound, croscarmellose sodium and alkaline optimized separation. Modeling of reverse phase separations by DryLab® is based earth cations at samples’ concentrations and empirical determination of the alkaline on the measurement of both retention times and peak areas. DryLab® was used to earth cations’ concentration that will provide complete recovery of active. 43 2018 EAS Abstracts November 2018

289 Correlated Chemical and Morphology Imaging to Investigate herb and spice adulteration studies using NIR spectroscopy demonstrates the use Formulation Dissolution of Adulterant Screen as a rapid screening method for suspect materials. Slobodan Sasic, Renishaw, 1001 Wesemann Dr., West Dundee, IL 60118, Kenneth J. Smith, Tim Prusnick, George Butcher, Hazel 293 Testing the Next Generation of Quality Assurance Devices for High Garvie-Cook Commodity Products Understanding dissolution processes is critical in the development of pharmaceuti- Mei-Ling Shotts, Ohio State University, 2015 Fyffe Rd., Columbus, OH cal formulations. Chemical composition, particle size, and particle distribution play 43210 vital roles in establishing dissolution profiles, and need to be quantified to ensure No abstract submitted by the author. products are optimized and repeatable. Relatively little is known about the relation- ship between chemical composition and morphology of material surfaces, and how 294 GenX - Analytical Environmental Chemistry Illustrates the Failures this affects dissolution and bioavailability. Here we apply a combined chemical/mor- of our Regulatory System phology confocal Raman imaging system to a formulation which has been partially Lawrence Cahoon, University of North Carolina-Wilmington, Dept. dissolved, to better understand the mechanisms at play during this process. Chemi- Biology & Marine Biology, Wilmington, NC 28403 cal and morphological images are acquired at the same time, providing full chemical The discovery of “GenX”, a mono-ether perfluorinated carboxylic acid, along with composition, particle sizing and particle distribution information, all in relation to numerous other perfluorinated alkylated substances (PFASs) in the Cape Fear morphological changes occurring as the formulation dissolves. River, North Carolina, and that these compounds were not removed by standard drinking water treatment processes has prompted scientific, regulatory, and legal 290 Photonics Devices - Do We have Everything We Need to put these responses. GenX replaced perfluorooctanoic acid (PFOA) as an intermediate in Devices into the Hands of an Untrained User production of fluorinated materials. PFOA production in the US was phased out Ellen V. Miseo, TeakOrigin, 39 Blacksmith Dr., Needham, MA 02492 beginning in 2006, owing to toxicological and epidemiological concerns. The history The food supply chain has grown more complicated over the past 50 years. The of PFOA production and regulation and the alarms raised by discovery of GenX in consumer may believe that the “farm fresh” sign in the market is true. But with the public drinking water highlight the weaknesses in the legal and regulatory systems number of steps in the process to get the food from the farm to the customer, both in managing the challenges of GenX, an “emerging contaminant.” This presenta- the retailer and the consumer have no way of knowing. Karl Norris at United States tion examines the critical importance of major advances in analytical environmental Department of Agriculture (USDA) in the 1960’s investigated near-infrared methods. chemistry as a remedy for these weaknesses. Commercial applications most notably from Foss and Perten Instruments are used in the laboratory. A food retailer can’t afford the 1-5 day turn-around if a distribution 295 Green Chemistry Solutions to Water Pollution center is getting truckload after truckload of produce every 5 minutes. A much fast- Rakesh K. Sharma, University of Delhi, Green Chemistry Network er more robust method is needed. And with the increased attention to consumer Centre, Dept. of Chemistry, Delhi 110007 India, Satinder Ahuja spectroscopic devices there is more need to understand what impacts the analyses. Conventional water treatment processes are generally used for removing many of This presentation discusses the drivers for a new approach, how the revolution in the chemical as well as microbial contaminants of concern to public health. Unfor- miniaturized optics is an enabling technology, and how traditional analytical chem- tunately the effectiveness of these processes is hampered by three major factors: istry coupled with miniaturized optical platforms and cloud based data analysis can expansion in the scope of regulated contaminants, rapid population growth, and change the landscape. But only if you understand the chemistry! industrial development. As a result, ensuring reliable access to clean and afford- able water remains one of the greatest global challenges of the present century. 291 Lies, Damned Lies and Statistics - How Metrics Make the Model Fortunately, green chemistry offers a variety of solutions to address this globally Suzanne Schreyer, Rigaku Analytical Devices, 30 Upton Dr., Ste. 2, important water pollution problem. A variety of methods based on green chemistry Wilmington, MA 01887 can be used to remediate water pollution problems.[1-2] Discussion focuses on some The methodology behind developing a robust application for material identification selective methods that can be used for socio-economic solutions for the problems or quantitative analysis is often poorly understood or poorly implemented. This is associated with metal contaminated wastewater. The integration of sustainability especially evident in direct to consumer products that give unrealistic claims about and solid phase extraction methodology has already been implemented in the form the utility of models and consumer spectroscopic instruments. However a properly of silica based organic-inorganic hybrid materials for the recovery of heavy metals designed chemometric approach to data analysis is required in order to develop from wastewaters. A newly designed reactor has been developed for large scale, models that can be consistently applied and will stand the test of time. Along with online, efficient and fast extraction of chromium from tannery waste using metal properly designed sampling approaches, an analytical approach for model develop- specific chelating polymer. Such technologies can certainly minimize water pollution ment and the correct use of chemometric tools is essential to both identification, and and allow selective recovery of useful materials. especially so for quantitative analyses. This discussion uses examples to illustrate References: some of the approaches required to develop robust models for both identification [1] S. Ahuja, Green Chemistry and Other Novel Solutions to Water Pollution in Nov- and also quantitative analysis. The examples focus on quantitative applications in el Solutions to Water Pollution, S. Ahuja and K. Hristovski, Editors, American food/agricultural and also identification and library development in portable spectro- Chemical Society, Washington DC, 1-14 ( 2013). scopic instruments. [2] R. K. Sharma, Green Chemistry Solutions to Water Pollution in Water recla- 292 Detecting Herb and Spice Adulteration Using Near-Infrared mation and Sustainability, S. Ahuja, Editor, Elsevier, Amsterdam, 57-77 (2014). Spectroscopy Ariel Bohman-Paolo, PerkinElmer, 710 Bridgeport Ave., Shelton, CT 296 Challenges to Mitigating the Risks Posed by Cyanobacterial Toxins 06484, Kathryn Lawson-Wood in the 21st Century Food fraud is a subject which has become increasingly prominent over recent years James S. Metcalf, Brain Chemistry Labs, Box 3464, Jackson, WY 83001 and is sometimes referred to as economically motivated adulteration (EMA). Com- Cyanobacteria, ancient photosynthetic aquatic organisms are increasing in range pared with the often trace levels involved in accidental contamination, adulterants and frequency of occurrence. Often aesthetically unpleasant, cyanobacteria have are usually added in substantial quantities in order to ensure greatest profit gain. been associated with a number of human and animal adverse health events and Herb and spice trading incorporates a wide range of products from diverse origins are capable of producing a wide range of acute and chronic toxins. With increasing around the globe, with some countries at higher risk of EMA. Adulterants – anything human pressures on waters through agriculture and population increases, problems from cheaper plant material of similar appearance, to natural and synthetic dyes, to associated with cyanobacteria are predicted to increase. Challenges to addressing chemicals such as talc – not only negatively impact consumer confidence, but can cyanobacterial blooms include detection, remediation, risk assessment and com- also be detrimental to public health. Near-infrared (NIR) spectroscopy is an already munication. A major challenge to identifying the risk(s) posed by cyanobacteria and widely used technique in the food industry for quantitative analysis of nutritional and their toxins is appropriate analytical chemistry. In addition to basic research needs quality parameters. For dried materials like herbs and spices, NIR spectroscopy is and appropriate communication, policies and legislation are required to protect hu- the ideal solution as it is a non-destructive technique and requires no sample prepa- man health from exposure to cyanobacterial toxins through drinking and recreation- ration, allowing the analyzed samples to be retained. Existing targeted approach- al waters. Ultimately, good stewardship of pollution and population pressures, in es for adulterant screening require a quantitative calibration to be developed for addition to point and diffuse nutrient sources will aid in minimizing the risks posed by each potential adulterant. Non-targeted screening approaches can determine when cyanobacteria and their toxins. This review aims to address the challenges posed by there is a potential adulteration problem but can neither identify nor quantify the cyanobacteria and their toxins in drinking and recreational waters. adulterant. Adulterant ScreenTM, a semi-targeted screening method, combines the advantages of both targeted and non-targeted approaches, allowing easy detection and quantitative estimation of adulteration at relevant levels. A range of examples of

44 2018 EAS Abstracts November 2018

297 Distribution of Microplastics in Waters Around the New York larger amounts of starting material. We expect the combination of CE-MS for limited Metropolitan Area and Assessment of their Role as Potential samples and LC-MS to raise investigative opportunities to better understand metab- Vectors of Toxic Compounds olism in the developing embryo using X. laevis. Beizhan Yan, Columbia University, Lamont-Doherty Earth Observatory, 61 Rt. 9W, Palisades Parkway, NY 10946 301 Cannabinoid Monitoring in Dried Cannabis Flower and Edibles by No abstract submitted by the author. HPLC-PDA Wilhad Reuter, PerkinElmer, 710 Bridgeport Ave., Shelton, CT 06484 298 UPLC-MS-MS Analysis of Cortisol in Hair and Saliva Collected from Over 20 states in the United States have legalized the use of cannabis for both Human Subjects medicinal and recreational purposes. To assure the quality, safety and potency of Jake E. Cortigiano, University of Connecticut, Center for Environmental marijuana products, reliable analytical procedures are pivotal for the quantitative Sciences and Engineering, Unit 4210, 3107 Horsebarn Hill Rd., Storrs, analysis of the cannabinoids and terpenes, as well as any pesticides that may be CT 06269, Sarah Anderson, Patrick Kaplita, James D. Stuart, Anthony absorbed during cultivation. This presentation discusses the sample preparation A. Provatas, Christopher R. Perkins and analytical methodology for the chromatographic separation and quantitation of Cortisol, a well-known organic compound belonging to the steroid family, is called seven cannabinoids in dried cannabis flower and 12 cannabinoids in several food the “stress hormone” as humans under significant stress produce cortisol from the matrices using high-performance liquid chromatography (HPLC) with photodiode adrenal cortex. The analysis of cortisol in hair gives a measure of chronic stress array detection (PDA). The utilization of a superficially porous particle (SPP) column while its analysis in saliva measures a short-term stress level. In the past, to test allowed for the rapid separation of twelve cannabinoids in four minutes while staying for cortisol levels field researchers would collect human saliva in test tubes and well within the pressure constraints of a conventional HPLC pump. Cannabinoids submit them to a laboratory for analysis. In this study, we used a novel approach of were detected using a wavelength of 228 nm. The method provides exceptional blotting saliva onto Whatman FTA blood spot cards. The saliva spots were cut out chromatographic reproducibility with cannabinoid limits of quantification ranging and then extracted with methanol. Hair samples were mechanically minced and from 0.013 to 0.036 ug/mL, well below the current concentration limits of interest. pulverized prior to the addition of methanol as the extraction solvent. Extensive This method presents a robust, cost-effective solution for high-throughput cannabi- sample sonication and vortexing followed and the extracts were evaporated to dry- noid monitoring in a variety of matrices. ness in an automated evaporator. For both extracts, a sensitive method utilizing ultra-high-performance liquid chromatography-tandem mass spectrometry was de- 302 Direct Thermal Extraction Analysis of Food Packaging Using the veloped to measure cortisol levels in both hair and saliva. Method detection limit, GERSTEL MPS Robotic Sampler accuracy and precision studies were performed for method validation followed by Laurel A. Vernarelli, GERSTEL, Inc., 701 Digital Dr., Ste. J, Linthicum, sample screening of hair and saliva collected human subjects. MD 21090, John Stuff, Jackie Whitecavage Direct thermal extraction (DTE) is a thermal desorption technique in which a small 299 Structural Characterization of a Galactoglucomannan from the amount of sample, typically 10-50 mg, is placed in an empty thermal desorption Eremurus Hisaricus Roots Grown in Tajikistan tube. The sample is heated in the thermal desorption unit under of flow of inert Gary D. Strahan, United States Drug Administration, ARS/ERRC, 600 E. gas, in order to release volatile and semi-volatile compounds from the sample. The Mermaid Lane, Wyndmoor, PA 19038, Jamshed T. Bobokalonov, Ikrom analytes are trapped and then analyzed by gas chromatography mass spectrometry B. Ismoilov, Hoa K. Chau, Arland T. Hotchkiss, Zayniddin K. Muhidinov, (GC-MS). Scalping is the loss of components from a product to its packaging. Migra- LinShu D. Liu tion is the transfer of unwanted compounds from the packaging to the product. Both Traditionally important medicinal plants are often examined as potential sources processes are undesirable and can cause off-odors or off-flavors in a product. DTE of bioactive compounds, including polysaccharides. We have isolated a new ga- requires little sample preparation and can be used for trace analysis of packaging lactoglucomannan (GGM) derived from the tuber roots of Eremurus hissaricus (E. material. This technique was used to analyze the packaging of the three brands of hissaricus) grown in Tajikistan and have structurally characterized it for the first time crème filled chocolate sandwich cookies, peanut butter filled sandwich crackers and by using chromatographic methods and nuclear magnetic resonance (NMR) spec- soft and chewy candy. The compound benzaldehyde was quantified in one brand of troscopy. The monosaccharide composition was found to consist of D-galactose, soft and chewy candy packaging and found to be 79 ± 6 ƞg. D-mannose and D-glucose in a 1.8:1.6:1.0 molar ratio. Using high-performance size exclusion liquid chromatography- multi-angle laser light scattering-V-refractive 303 Surface-Enhanced Raman Spectroscopic Surveillance of Bacteria index-ultraviolet (HPSEC-MALLS-V-RI-UV) with TSK GMPW columns, the mo- within Row Crops In-Situ lecular weight of the acid-soluble fraction (ASP) was found to be 179 kDa, with Michael E. Hickey, University of Massachusetts, 102 Holdsworth Way, a polydispersity of 3.69. The anomeric region of the NMR two-dimensional (2D) Amherst, MA 01003, Lili He 1H-13C- heteronuclear single quantum coherence (HSQC) indicates the presence A real-time method to surveil mass bacterial communities directly in-situ is reported. of four primary sugar units (one Gal, two Glc, one Man), and one much weaker one Surface-enhanced Raman spectra were collated en masse to generate panoramic (presumed to also be Man). The JCH coupling constants of all of these dominant chemical images of bacteria populations. Bacteria cells were coated in 3-mercap- sugar units were found to be less than 160 Hz, indicating that each has their H-1 in tophenylboronic acid for complexation with gold (Au) nanoparticles. This molecular the axial orientation, and thus they were each assigned as being in the beta-con- architecture enhanced the detection of scattered Raman photon frequencies which figuration. The NMR 2D- heteronuclear multiple bond coherence (HMBC) analyses were indicative of bacteria cells. The approach was successfully employed to indis- provided heteronuclear through-bond evidence indicating that the main chain of the criminately monitor mass bacteria populations directly among edible plant tissue. GGM consisted of beta-D-Glcp (1→4) beta-Manp units, consistent with other known galactoglucomannans. 304 A Rapid and Sensitive Method for the Determination of Acrylamide and Related Compounds in Food and Beverages 300 Comparative Metabolic Analysis of Embryonic (frog) Cells by CE Carl M. Zimmerman, MAC-MOD Analytical, 103 Commons Ct., Chadds and HPLC MS Ford, PA 18960, Geoffrey M. Faden, Edward A. Faden, Alan Mckeown Aleena J. Andrews, University of Maryland, 8051 Regents Dr., College Acrylamide is a naturally occurring compound which can be formed in food from the Park, MD 20742, Erika P. Portero, Jie Li, Peter Nemes reaction of amino acids (primarily asparagine) and sugars during the Maillard reac- Knowledge of the metabolome would allow us to deepen our understanding of mo- tion. This occurs when starchy foods are heated above 120 °C during cooking pro- lecular mechanisms of healthy and impaired tissue development. However, this goal cesses such as frying, baking and roasting. In addition, acrylamide can form in non- depends on the development of bioanalytical methodologies capable of addressing starchy foods through heating by the oxidation of fats to substances such as acrylic broad physical dimensions in developing embryos, ranging from single cells and acid and acrolein and subsequent reaction with asparagine. Acrylamide has been limited populations of cells to entire tissues. Here, we characterize metabolic com- identified as a potential human carcinogen following tests on rats, with high level position of cells and tissues in early developing embryos of the frog Xenopus laevis, exposure reportedly resulting in the formation of cancercausing cells. As of April a powerful model in cell and developmental biology. We dissected single identified 2018, the European Food Safety Authority introduced benchmark levels for acryl- cells and tissues from 16-cell embryos and extracted small polar metabolites in amide in foods with guidelines for the food industry on how to reduce the acrylamide 40% methanol and 40% acetonitrile. The resulting extracts were measured using a content in foods. The determination and quantification of acrylamide in foodstuffs is microanalytical capillary electrophoresis (CE) electrospray ionization (ESI) platform therefore of increasing interest. This poster details the reversed-phase separation and microflow high-performance liquid chromatograph (HPLC) and detected by of acrylamide, methacrylamide and methacrylic acid. A range of stationary phases electrospray ionization high-resolution mass spectrometry (MS). CE-MS revealed were assessed to maximise the retention of acrylamide and reduce the potential for ~250 molecular features, ~70 of which were assigned to metabolites based on accu- interfering matrix effects. A novel C18 based phase featuring enhanced ligand cov- rate mass, tandem mass spectrometry, and comparison of separation time against erage was found to provide the strongest retention for acrylamide. Full separation of data available in MS databases or measured for chemical standards. HPLC-MS is the three analytes was successfully achieved using gradient elution. The method is anticipated to provide complementary coverage of the metabolome, albeit requiring suitable for the determination of acrylamide in foodstuffs, using methacrylamide as 45 2018 EAS Abstracts November 2018

an internal standard, and may also be readily translated to ultra-high-performance translation and validation of the forced degradation workflow from the PAL2 to the liquid chromatography to reduce analysis time. CTC PAL3 and demonstrate the new PAL3 features and its validation. 305 Analysis of Lactose in Lactose-Free Labelled Products by HPAEC- 308 Protein Mobility at a Food Emulsion Interface by Confocal Laser PAD Scanning Microscopy and Fluorescence Recovery after Nicholas Santiago, Antec Scientific, One Boston Pl., 26th Fl., Boston, Photobleaching MA 02108 Matthew Sillick, Ingredion Inc., 10 Finderne Ave., Bridgewater, NJ Lactose is a disaccharide that occurs naturally in the milk of mammals and is gen- 08807, Will Borchert erally found to be around 5% (w/w). Other dairy products (like yoghurt and cream), Surface active proteins can be used to create food emulsions and are essential and processed foods like sausages and cookies often contain lactose in detectable for controlling their long term storage stability. Assessing the interactions between amounts. Lactose intolerance is a condition where a person cannot digest the nor- proteins and interfaces is therefore broadly important for food product design with mal levels of lactose present in the dairy/food products due to low levels of lactase alternative proteins such as faba bean. Confocal laser scanning microscopy (CLSM) in their intestine. Lactase deficiency results in various degrees of abdominal discom- was applied to a model mayonnaise to visualize fats, starches and covalently la- fort after consuming the products, depending on the amount of intake and lactase belled molecular proteins both in the bulk and at oil/water interfaces. Beyond just levels. As a result, the food industry has started producing various ‘lactose-free’ la- imaging, this work applies the CLSM as a spatially resolved spectrofluorometer to beled products which contain decreased levels of lactose for consumers who would compare bulk concentration and interfacial excess of the labelled protein to calcu- otherwise suffer from their intolerance. In the past ‘lactose-free’ labeled products late the partitioning coefficient. Faba bean protein concentrate was indeed surface had levels of lactose below 100 mg/100g product (0.1%), but recent product label- active with an apparent partitioning coefficient of at least 15 to 1. Fluorescence ling indicates a lactose level below 10 mg/100g product. Current methods to detect recovery after photo bleaching (FRAP) experiments demonstrate that while bulk lactose as described by the standardization agencies International Organization for diffusion was rapid, bleached regions recovered in less than 0.5 seconds, replen- Standardization (method 22662:2007; HPLC-RI) and Association of Official Agricul- ishment of protein at the interface was adsorption controlled and occurred over tens tural Chemists (method 984.15; enzymatic/VIS) do not have the required sensitivity of seconds. These CLSM techniques are useful in that they allow specific types to meet the new product labelling standards. High-performance anion exchange molecular probes to be followed as they adsorb and desorb from the interface. chromatography (HPAEC) with pulsed amperometric detection (PAD) is a cost-ef- fective and sensitive technique for routine analysis of lactose in lactose-free labeled 309 Application of USP Apparatus 5 and USP Apparatus 7 to In-Vitro products. Detection limits obtained from this study fall well below recent product Drug Release for Nicotine Transdermal System labelling amounts of Lactose in lactose-free foods. Furthermore, results indicate Ming Li, Logan Instruments, 19C Schoolhouse Rd., Somerset, NJ 08873 that two additional lactose isomers, i.e., Allolactose, Epilactose and Lactulose can To monitor in-vitro drug release in Nicotine transdermal systems, the USP Appa- be easily separated and quantified. ratus 5 and USP Apparatus 7 was selected. In this work, we studied the in-vitro Nicotine drug release test using a USP Apparatus 7 from Logan Instruments, and 306 Utilizing the Andrew Alliance for Automated Liquid Handling during compared it with a USP Apparatus 5 system made by Logan Instruments. Two dis- Early Development solution devices marketed in United States for the controlled release of 7.0 mg of Alexandra Andrews, Merck & Co., MS: RY80T-B164, 126 E. Lincoln Nicotine in a 72-hour period were evaluated. The results demonstrate that the drug Ave., Rahway, NJ 07065, Edward Mularz, Erin Hein release profiles obtained using the USP Apparatus 5 and USP Apparatus 7 systems The Andrew Alliance is a pipetting robot that can be used in a variety of different are equivalent. The drug release profiles obtained using the two 7-sample of the Lo- aspects throughout the sample preparation process. The Andrew Alliance can per- gan USP Apparatus are also equivalent. Repeatability was measured on the Logan form dilutions for sterile liquids dosage forms, tablet dosage forms, and stabilizing instrument and found acceptable. dilutions for poorly soluble compounds. It can be extremely beneficial during early phase method development, where quick timelines and/or limited supplies frequent- 310 Improvement of Separation of Monoclonal Antibodies Using Core- ly occur. The Andrew Alliance can also be used throughout the development pro- Shell Column cess and easily transferred to more complex instrumentation such as the SOTAX Norikazu Nagae, ChromaNik Technologies Inc., 6-3-1 Namiyoke, Tablet Processing Workstation (TPW), which requires more involvement for method Minato-ku, Osaka 552-0001, Japan, Tomoyasu Tsukamoto, Makoto development. In any robotic laboratory, it is critical to have a variety of platforms for Sato different stages of a project or the different skill levels of the users. Implementation Following low-molecular medicine, the antibody medicine group is attracting at- of a robotic platform can eliminate the variability associated with glassware or from tention and recognized as the most important medicine due to its effectiveness. It scientist to scientist. The instrument is highly customizable with different vial Dom- is well known that size exclusion chromatography (SEC), hydrophobic Interaction inos for a variety of vials and dilution schemes. The Andrew Alliance is a powerful chromatography (HIC) and reversed phase chromatography are used as a typi- stand-alone tool as well as an orthogonal instrument to the TPW. The Andrew Alli- cal separation analysis method for monoclonal antibodies. In this study, silica base ance platform is highly customizable with user-friendly software and can function as material, pore diameter and stationary phase were compared in reversed phase a walk-up robotic platform for liquid dilutions or sample preparation. chromatography. Although it is known that the core-shell silica column has a higher number of plate and higher efficiency than the totally porous silica column, the same 307 New Generation CTC PAL 3 Walk-UP Automated Forced result was obtained for the separation of the monoclonal antibodies. Although a Degradation Workflows to Accelerate Drug Development packing material with 30 nm of pores has been frequently and satisfactorily used for Lina Liu, Merck & Co., MS: RY801-B207, 126 E. Lincoln Ave, PO Box a separation of proteins whose molecular weight was 100,000 or less, 100 nm of 2000, Rahway, NJ 07065, Timothy Rhodes, Robert Hartman, David pore leaded a better separation of monoclonal antibodies whose molecular weight Schenk was around 150,000 to compare with 30 nm of pore. As a result, core-shell silica Forced degradation studies are an important element in assessing a compound’s with 100 nm pore bonded with butyl group (C4) showed the best separation for drug-like properties and understanding the chemical liabilities that will need to be monoclonal antibodies (IgG) which was obtained by cell culture and purified on a addressed in preclinical development. Forced degradation studies help in early de- protein G affinity column. velopment to devise and to validate stability-indicating analytical methods. The new generation autosampler CTC PAL 3 Platform was purchased in 2016 to replace 311 A Green Triton X-114 Extraction Protocol for the Separation of the ten year old benchtop automation platform developed by LEAP Technologies Virus-Like Particles from Lipid-Based Preparations for running an automated forced degradation workflow. The new platform makes Douglas B. Vieira, IMV, 130 Eillen Stubbs Ave., Ste. 19, Dartmouth, NS it possible to enhance our current workflow and increase throughput by reducing B3B 2C4, Canada, Andrea Penwell, Arthvan Sharma, Annie Tanguay, run time with the integrated ultra-performance liquid chromatography (UPLC) con- Amy Noe, David Whitacre nection and allows us to expand to new workflows, such as solubility or dissolution Virus-like particle (VLP) vaccines have emerged as an excellent alternative strate- measurements with real time sample preparation and analysis. Three syringe siz- gy to prevent infectious diseases such as malaria. However, the development and es provide us flexibility for conducting a wide dynamic range of sample dilutions manufacturing of safe and effective VLP vaccines relies on the availability of ac- while three integrated independent temperature stations allows expansion to new curate, precise, and robust analytical methods, without damaging the VLP during workflows such as sterile formulation stability studies or solution stability. Overall, procedures that involve pelleting or precipitating the particles. A gentle and green the CTC PAL3 is a walk-up friendly benchtop automation solution which can be extraction method was developed to isolate VLP162 (P. falciparum vaccine can- applied to address multiple applications including, but not limited to the evolution didate) from a lipid-based formulation, followed by monitoring the VLP yield and of the forced degradation workflow, which has been used widely for understanding stability with analytical methods such as size-exclusion chromatography high-per- the chemical stability liabilities and to devise and validate stability-indicating ana- formance liquid chromatography (SEC-HPLC) and polyacrylamide gel electropho- lytical methods. The workflows can save anywhere from hours to days of analyst resis (PAGE). Phase separation using Triton X-114, a non-ionic detergent with a time, especially for kinetic forced degradation. This poster will discuss the evolution, low clouding point at 22 °C, allowed the VLP to be sequestered into an aqueous 46 2018 EAS Abstracts November 2018

phase at 37 °C, while the lipids were extracted into a detergent phase. The method that of the plasma of 95.35 ± 2.77 μm. The blood flow-rate was 7.14 μL/min and was executed by diluting the VLP162-lipid sample 1:1 with Triton X-114 followed the flow-rate of the dilution buffer solution (0.01 M phosphate solution, pH 7.4) was by centrifugation at 37 °C. This step was repeated up to four times to completely 42.86 μL/min, giving a final dilution flow-rate of 50 μL/min. Under these conditions remove the lipids. Analysis of the aqueous phase by SEC-HPLC showed greater the plasma separation efficiency was 98.0% ± 0.87%. The dilution was estimated than 97% recovery of VLP162 with a relative standard deviation of 2.2%. The purity according to the output hematocrit of blood before the bifurcation, so, for an initial and ID of the VLP were confirmed by PAGE analysis. This study demonstrated that hematocrit of 47.65 ± 0.61%, the hematocrit measured, once the blood was diluted a VLP can be successfully purified from a lipid preparation and analyzed by different in the microchip, 7.00 ± 0.06%. analytical techniques without lipid interference (SEC-HPLC and PAGE). Because of its non-specific nature, this protocol may also be suited to the separation of other 315 Selectivity Manipulation for LC-MS Analysis of Intact Protein hydrophilic proteins from lipid- or liposome-based preparations. Variants Geoffrey M. Faden, MAC-MOD Analytical, 103 Commons Ct., Chadds 312 Analytical Method Exploration in Transmetalation of MnCl2 with Ford, PA 19317, Ben Libert, Stephanie Schuster, Brian Wagner, William Grignard Miles, Barry Boyes Weidong Tong, Merck & Co., MS: RY818-B215, 126 E. Lincoln Ave., Superficially porous particles (SPP) are well demonstrated to provide superior Rahway, NJ 07065, George Zhou performance capabilities as packed columns for liquid chromatography (LC) sep- Methylation by Grignard reagent is a typical chemical reaction. Introducing MnCl2, arations. We have previously demonstrated that to use the advantages of SPP a non-toxic and cheap salt, to facilitate the transmetalation, provides the unique for separations of large biomolecules (>50,000 MW), very large pore sizes are re- reactivity and stereo selectivity under mild reaction conditions. However, The MnCl2 quired. The present work demonstrates highly efficient protein separations using is insoluble in reaction solvent, anisole/THF, the transmetalation takes place very 1000 Å pore size SPP materials, and further describes the use of multiple surface slow with Grignard reagent, and the chemistry of Manganese and the synergism modified reversed phase to demonstrate manipulation of selectivity for high resolu- are unclear. To monitor this transmetalation, we don’t have any existing literature tion LC mass spectrometry (MS) analysis of intact protein variants, particularly for method to follow. In this work, we discuss the analytical exploration by Raman, monoclonal IgG1, IgG2 and other biotherapeutic proteins. The improved resolution inductively coupled plasma (ICP) and high-performance liquid chromatography - and high separations performance for these large biomolecules leads to confident charged aerosol detection (HPLC-CAD). We have discovered the unique Raman assignment of protein structures, including description of post translational and spectra for reaction species, and their quantification method. This analytical study, chemical modifications. Monoclonal antibodies (mAbs) immunoglobulin G (IgGs) helped to understand the reaction rate, and provided a useful IPC method. Further- and standard proteins were dissolved at 1-2 mg/mL in either 0.1% formic acid (FA), more, it may benefit on other IPC method for this type air and moisture sensitive trifluoroacetic acid (TFA) or difluoroacetic acid (DFA). Columns of HALO® Fused- transmetalation. core 1000 Å pore size, 2.7 µm diameter silica particles (AMT, Inc., Wilmington, DE) with C4, C18 and diphenyl bonded phases were compared for MS spectral quality, 313 XPS of Copper-Organic Interactions Related to Cultural Heritage retention, peak widths and tailing factors, to fully porous particles (FPPs) of 300 Å Collections pore size silica or 4 µm diameter 1500 Å pore PSDV material. 2.1 mm or 0.2 mm ID Emma Heath, University of Delaware, Dept. of Chemistry and columns were eluted using increasing organic solvent contents (acetonitrile, isopro- Biochemistry & Dept. of Art Conservation, 206 Amstel Way, Newark, DE panol, n-propanol and mixtures thereof), modified with FA, DFA or TFA. Electrospray 19711, Marcie Wiggins, Thomas P. Beebe Jr., Karl S. Booksh, Jocelyn ionization mass spectrometry detection used the Thermo Velos Pro LTQ Orbitrap Alcántara-García hybrid instrument. Copper-containing compounds are some of the oldest colorants of dyes and water- colors, even though they were known causes of corrosion when applied to organic 316 Dr. Richard Saferstein’s Contributions to Forensic Science in New materials. This project investigated the role of copper-organic interactions within Jersey the degradation of historic materials via X-ray photoelectron spectroscopy (XPS), Tom Brettell, Cedar Crest College, 100 College Dr., Allentown, PA 18104 supplemented with fourier transform infrared spectroscopy (FTIR) and time-of-flight Dr. Richard Saferstein served as President of EAS in 1989 and served on the EAS secondary ion mass spectrometry (TOF-SIMS). Multiple historic-based samples dis- Board for many years. Dr. Saferstein retired in 1991 from the New Jersey State playing different interactions between copper and cellulose were analyzed: 1) quer- Police as Chief Forensic Scientist after heading the NJSP Forensic Science Bureau cetin extract (C15H10O7) on Whatman filter No. 1 paper where copper ions acted for over two decades. He was a leading national expert and author in the field of as a mordant; 2) two samples of verdigris pigment (copper (II) acetate) similarly forensic science and was a highly sought-after consultant, participating in a multi- applied as a direct dye, dissolved in water, and as a watercolor. Preliminary results tude of high profile cases throughout the country. Dr. Saferstein served as an expert from XPS and TOF-SIMS suggest initial alteration of the cellulose chains coinciding witness over 2000 times in nearly 150 federal and state courts involving a variety of with the reduction of the mordant from copper (II) to copper (I). This Fenton-like forensic issues. His areas of expertise encompassed breath and blood testing for activity is further demonstrated within basic [Cu(CH3COO)2 · Cu(OH)2 · 5H2O] and alcohol, pharmacological effects of alcohol and drugs, detection and identification of neutral [Cu(CH3COO)2 · H2O] verdigris watercolors, which show the formation of drugs in biological fluids and many other areas. His expert opinions influenced court copper (I) oxide, catalyzed by the presence of polysaccharides such as gum Arabic, decisions throughout the State of NJ particularly in the area of breath and blood historically used as a binder in watercolors. The oxidation of verdigris and copper alcohol analysis. During his 21 years with the NJSP Forensic Science Bureau, Dr. (I) oxide was subsequently followed with accelerated degradation. Results from this Saferstein had a profound influence on forensic science in the State of New Jersey. ongoing investigation will be applied to historic collections and the development of He shaped and developed policies for the crime laboratory, which grew at an expo- conservation treatments for cultural heritage materials. nential rate under his leadership. The laboratory expanded from a small laboratory at the NJSP Headquarters in West Trenton, NJ to a regional laboratory system in- 314 Microdevice Based on Centrifugal Effect and Bifurcation Law for cluding three regional laboratories and an equine testing laboratory in the Meadow- Separation of Plasma from Whole Blood lands. He also served as an adjunct professor at The College of New Jersey where María del Pilar Cañizares-Macías; National Autonomous University of he taught an Introduction to Forensic Science for over 10 years. México, Dept. of Analytical Chemistry, México D.F., 04510, México, Kenia Chávez-Ramos 317 Forensic Mass Spectrometry: The Past, Present and Future - A The separation of whole blood is considered an important and decisive preliminary Discussion Honoring the Immeasurable Contributions of Dr. step for further biological analysis. Centrifugation is the most commonly used con- Richard Saferstein to the Field of Forensic Science ventional technique for blood plasma separation in clinical laboratories. A a great Adam B. Hall, Northeastern University, 360 Huntington Avenue, 140 The variety of works have achieved the separation of blood plasma in microdevices lab- Fenway, Rm. 421, Boston, MA 02115 on-a-chip reaching high values in the efficiency of plasma extraction, but it is neces- On July 17, 1941, Richard Saferstein was the first-born child to Sadie and Milton sary previous dilution of the blood sample of up to 100x. Saferstein. Throughout his humble beginnings in Brooklyn, NY he was a precocious In this work, a microdevice in PDMS was constructed for the separation of plasma and intelligent child who had a mind of his own. He became very interested in sci- in human blood sample diluting it in the same microdevice, taking advantage of two ence around the age of nine but truly aspired to be a dentist, not a forensic scientist effects: (1) centrifugal effect, when implementing a spiral with microchannels with as we all knew him to become. Dr. Saferstein’s life was remarkable. He had a fam- rectangular cross section, with the objective of concentrating the greater number of ily whom he loved. He had a long professional career as the Director of the New blood cells in one of the walls of the microchannel and (2) effect of Zweifach-Fung, Jersey State Police Crime Laboratory. He was a highly sought after expert witness by varying the widths of the two output microchannels for the modification of their and educator all while producing some of the most notable textbooks in the field of flow rates. In Figure 1 is shown the manifold and the microchip design to separate forensic science which continue on. During this two-part discussion we walk through plasma from whole blood; on the right of the figure the separation of plasma is the life and immeasurable contributions of Dr. Richard Saferstein followed by a more shown. The channels depth was 98.05 ± 7.70 μm, the spiral channel width was technical discussion on the past, present and future of forensic mass spectrometry. 388.70 ± 5.84 μm, that of the output of the whole blood of 778.65 ± 4.02 μm and From numerous and immeasurable contributions to the field of forensics to modern 47 2018 EAS Abstracts November 2018

day precision in measurement science and mass spectrometry, the irony and impact SERS combined Raman spectroscopy and nanotechnology. The uses of certain of this lecture will not go unnoticed. Our discussion includes early developments nanostructure can enhance the inherently weak Raman signal tremendously, which in mass spectrometry following WWII to the production of routine, bench top, unit advances the technique from characterization to detection. Various SERS based mass resolution systems and robust, high-resolution systems today. Where will the methods, especially the imaging based methods, have been developed for rapid science drive us and how will mass spectrometers continue to assist investigations detection of bacteria, pesticides, and silver and titanium dioxide nanoparticles in into the latter part of the 21st century? complex food matrices. The capability of rapid and sensitive fingerprint acquisition is a highlight for this technique. 318 Dick Saferstein: Leader, Mentor, Friend Neil D. Jespersen, St. John’s University, 8000 Utopia Parkway, Queens, 322 Pharmaceutical Case Studies Using Transmission Raman NY 11439 Julia A. Griffen, Agilent Technologies, 174 Brook Dr., Milton Park, Oxford In the fall of 1998 while doing sabbatical research in Dave Locke’s lab at Queens OX14 4SD, United Kingdom College, Dave asked me to consider becoming the New York American Chemical This presentation covers a variety of case studies demonstrating how transmission Society Section’s delegate to the Eastern Analytical Symposium. Joining the EAS Raman spectroscopy (TRS) is used in the pharmaceutical industry. TRS is predom- started me on a thirty-year adventure with this very special organization. My presen- inantly used for quantitative content uniformity (CU) testing. Examples presented tation covers my first seven years where Dick Saferstein had a large influence over include the non-destructive quantification of multiple active pharmaceutical ingredi- the organization and provided excellent mentorship and friendship that is warmly ents (APIs) and excipients in intact tablets, which is done in a single measurement in remembered. a matter of seconds. These quantitative Raman results are compared with primary analytical methods and the method development process is also discussed. Addi- 319 Remembering Dick Saferstein: The Man Who Saved EAS from tional examples show how the technology is progressing towards the analysis of low Extinction dose formulations containing <1%w/w drug substance. Another major application Stephen Scypinski, Daiichi-Sankyo Inc., 520 Mount Airy Rd., Basking area for TRS is solid state analysis, crystallinity and polymorphism. Examples in- Ridge, NJ 07920 vestigate a low dose formulation and comparisons are made to traditional solid state It was the fall of 1988. I received a phone call from Dick Saferstein, the upcoming analytical methods. The final example discusses Warfarin tablets analysed by TRS Chairman of the 1989 Eastern Analytical Symposium (heads of the meeting were quantify API and a degradation product. called Chairmen in those days). Dick asked me to be his Treasurer for the 1989 meeting, thereby placing me into succession to be Chairman in 1991. I graciously 323 What are the Physical Limits to Field Amplification and Intensity accepted. EAS had moved from its very successful and profitable venue at the NY Enhancement in Surface Enhanced Raman Spectroscopy? Penta Hotel in 1986 to the NY Hilton in midtown, also shifting its date from mid-No- Stefan Franzen, North Carolina State University, 363D Partners III, 2620 vember into October. Although 1986 was the 25th anniversary of EAS and many Yarbrough Dr., Raleigh, NC 27695 special events and sessions were offered, attendance was low. Over the next sev- Surface-enhanced spectroscopies remain one of the most controversial topics in eral years, EAS spiraled downward as costs rose and numbers of both exhibitors science. The reported enhancement facts have ranged from 10^3 to 10^15. Al- and attendees dwindled. EAS began to rely on its cash reserves until only a small though these values are reported under different conditions, the notion that the amount remained. EAS was heading for bankruptcy. To add to the crisis, Pittsburgh effect can have such widely varying possible intensity enhancements presents us Conference was to be held in New York City in March 1990. A financial analysis pre- with the scientific conundrum. Are these differences due to different physical condi- dicted that if EAS were held in New York in October 1990, the organization would not tions, such as hot spots? Or is it possible the chemical mechanism involving strong be able to pay its hotel bill. Something had to be done. Enter Dick Saferstein. In a interactions of molecules with surfaces, including bonding to the surface, are not true display of brilliance and sound leadership, Dick orchestrated a course of action properly being separated from electromagnetic effects? The speaker has worked that set EAS on a new path toward growth and financial success. This is the story on several aspects of these phenomena from a unique perspective using tunable of that strategy, one that many (including newer EAS board members) do not know. conducting metal oxides (CMOs) as the conductor for study. The difference between surface plasmon resonance (SPR), localized surface plasmon resonance (LSPR) 320 Directional Raman Scattering: A Tool for Measuring Adsorption and epsilon near zero mode (ENZ) is defined precisely using the example of CMOs and Chemical Content at Smooth Interfaces and the interaction of SPR with molecular resonances is demonstrated experimen- Emily A. Smith, Iowa State University, 2415 Osborn Drive, 1605 Gilman tally in a way that is unique in the field. Hall, Ames, IA 50011, Charles K.A. Nyamekye, Stephen C. Weibel There is an increasing demand for nondestructive in-situ techniques that measure 324 Accelerating Pharmaceutical Development from pre-Candidate chemical content, total thickness, and interface locations for thin multilayer films. Selection to First-Time-in-Human Clinical Studies Using Automated Directional surface-plasmon-coupled-emission (SPCE) Raman spectroscopy in Platforms combination with appropriate data models can provide this information. Directional Kaitlin M. Grinias, GlaxoSmithKline, MS: UP1100, 1250 S. Collegeville Raman spectroscopy has been used to determine the chemical composition and Rd., Collegeville, PA 19426 thickness of nanometer to micron thick polymer films, biological cells, and self-as- Intensifying the deployment of laboratory automation can accelerate the chemistry, sembled monolayers on smooth planar gold surfaces. The technique is performed manufacturing, and control (CMC) activities and provide a deeper understanding by optically coupling a sample/gold substrate to a Weierstrass-type prism and scan- of the pharmaceutical compounds in development. Laboratory automation, such ning the angle of the incident laser while collecting data. The simultaneous collec- as the Freeslate Core Module 3 (CM3), increases the throughput by automating tion and quantitation of the SPCE cone (which encodes information similar to a sample preparation through liquid and solid dispensing (with integrated balance) as surface plasmon resonance experiment) and the full directional Raman scattering well as heating, cooling, stirring, and vortexing in a single platform. The system is signal radiating from the SPCE cone are measured using a single instrument. This programmable through the LEA software suite enabling the scientist to customize provides a multidimensional dataset for extracting sample properties using simple each experimental design. The platform interfaces with analytical instrumentation angle-dependent models. As the sample thickness increases, there is an increase (e.g., high-performance liquid chromatography and pH electrodes) to automate data in the incident angles that produce the SPCE cone and Raman scattering as well handling by pulling the data from an entire study into a single database. These as an increase in the SPCE cone diameter. For waveguide samples, the number laboratory automation tools facilitated the design and implementation of solubility, of SPCE cones and their angular intensity pattern also encode useful information formulation development and forced degradation screens to accelerate the develop- to extract sample properties. Directional Raman spectroscopy is a viable non-de- ment of an investigational new drug. structive method capable of determining chemical composition and thicknesses of nanometer to micron thick films with self-assembled monolayer sensitivity. 325 Peptide Mapping: Best Practices for Generating Reliable and Robust Liquid Chromatography Methods 321 Surface Enhanced Raman Spectroscopy for Food Safety Jennifer Simeone, Waters Corporation, 34 Maple St., Milford, MA Applications 01757, Paula Hong Lili He, University of Massachusetts-Amherst, 102 Holdsworth Way, The retention of peptides on a chromatographic column is highly sensitive to small Dept. of Food Science, Amherst, MA 01003, Tianxi Yang changes in solvent strength or solvent composition. For this reason, peptide map- Our food system can be contaminated intentionally and unintentionally by many ping methods typically consist of gradients which impart a small percent change in agents, such as bacteria, pesticides, and engineered nanomaterials as emerging organic solvent per unit time to facilitate maximum separation. In addition, peptides food contaminates. Early-on detection of these contaminants in food system is crit- have a low optimal linear velocity due to the low diffusion rates of peptides. The ically important to keep our food safe. Currently, all three types of contaminants result of this is that peptide mapping methods are typically run at flow rates which use different detection methods, which all have limitations in terms of speed and are significantly lower than what is used for traditional small molecules. The combi- cost-effectiveness. Surface enhanced Raman spectroscopy (SERS) is perhaps the nation of low flow rates and very shallow gradients results in method conditions that only technique that has been explored for detecting all classes of contaminants. can often be challenging for a liquid chromatography (LC) system to deliver reliably. 48 2018 EAS Abstracts November 2018

This study examines the performance of a binary high-pressure mixing LC system 330 New Zinc Oxide Assay as per USP 591 Using Ion Chromatography for the development of reproducible peptide mapping methods. with PCR and UV-Vis Detection Shibu Paul, Metrohm USA, 9250 Camden Field Parkway, Riverview, FL 326 A New Route to Automation of Experimentation 33578, Jay Sheffer Scot Abbott, Phoenix, 1419 Alverado St., Pittsburgh, PA 15216 Zinc oxide has long been used in a wide range of cosmetics and personal care prod- A serious limitation in many laboratories is the cost and inflexibility of commercial ucts including makeup, topical sunscreens and creams, ophthalmic solutions, and instrumentation and robotics for automation of experiments. In many cases, a ma- foot powders. Since it is used in many over-the-counter and doctor-prescribed prod- jority of the instrumentation is available, but automation is not easily added because ucts, the quality of zinc oxide in both raw materials and pharmaceutical products is of electronic or programming barriers. We had that problem in our labs and avoided important for patient safety. Ion chromatography has emerged as a useful tool for the high cost of automation by using a simple system integration tool which makes measuring active ingredients, excipients, and impurities in a number of pharmaceu- control and automation possible directly from an Microsoft Excel® workbook. Sever- ticals. Ion chromatography (IC) is uniquely suited for separation of charged ions, al examples are provided and discussed which show its use integrating instruments reducing most interferences caused by complex sample matrices and simplifying and adding robotic type automation. sample preparation. IC may be coupled with several modes of detection including conductivity, ultraviolet-visible (UV-Vis) spectroscopy, and amperometry. To improve 327 Improvement of a Current EP Monograph Related Substances sensitivity and further reduce matrix interferences, transition metals may be ana- Method for a Widely Used API Using a 100% QbD-Aligned Approach lyzed by post-column reaction followed by UV-Vis detection at visible wavelengths. and the Fusion QbD® Software Platform Recent updates have been completed in 2018 for the General Chapter 591 Zinc De- Richard Verseput, S-Matrix Corporation, 1594 Myrtle Ave., Eureka, CA termination monograph of the US Pharmacopeia to include ion chromatography as 95501, Joseph Turpin, Ravi Ravichandran a method for assay of zinc oxide. This method utilizes cation exchange chromatog- Using a 100% quality-by-design (QbD)-aligned approach and the Fusion QbD soft- raphy for separation of Zn+2 ion followed by post column reaction with PAR reagent ware profoundly improved a current EP Monograph method. The software enabled and UV-Vis detection. In this work, a zinc oxide assay using the new United States rapid development of both ultra-high-performance liquid chromatography (UHPLC) Pharmacopeia -approved methodology is demonstrated using a fully-automated ion and HPLC versions of a final method with full robustness characterization. This chromatograph equipped with post-column reaction and UV-Vis detection. enabled achieving all five critical method development goals stated in the Analytical Target Profile for the method: 1) obtain a robust method in which all compounds 331 Determination of Polyphenols, Glycoalkaloids and Saponins in have resolutions ≥ 2.00 and tailing ≤ 1.50; 2) establish a fully characterized robust Solanum scabrum Fruits Using LC/UV/MS final design space and proven operating ranges for Resolution, Tailing, and active Bo Yuan, Rutgers University, 65 Dudley Rd., New Brunswick, NJ 08901, pharmaceutical ingredient (API) area % relative standard deviation (RSD); 3) in- James Simon, Qingli Wu crease the temperature to ≥ 30 ° C to increase method repeatability and transfer- Solanum scabrum is an economically important crop in sub-Saharan Africa whose ability; 4) eliminate the need for the additive; and 5) reduce the multi-step gradient leaves are consumed as leafy vegetables. The blueberry-like berries despite prolific to a single gradient method. production, however, are generally discarded due to conceptual toxicity and remain as neglected agricultural waste. Rediscovery of such underutilized berry resources 328 Increasing Sample Throughput Using Parallel Column depends on insights of the berry chemical composition and particularly the phyto- Regeneration chemical profile. The aim of this study was thus to systematically determine the phy- Zhimin Li, Waters Corp., 34 Maple St., Milford, MA 01757, Paula Hong, tochemicals of S. scabrum berries for discovery of new application potential. Berries Patricia McConville of different maturity from a total of eight different genetic sources were cultivated In various industries, liquid chromatography (LC) laboratories are constantly chal- and collected in Rutgers research farms in 2016 and the berry extract was prepared lenged to analyze more samples in less time. Some common practices to shorten for analysis by Agilent 1100 series HPLC-MSD ion trap mass spectrometry. Phy- LC analysis time include increasing flow rate, using a shorter column and reducing tochemical identification and structural elucidation was achieved using UV/visible column re-equilibration time. However, there are limits to the extent that these pa- detection, MS/MS and acid-assisted hydrolysis. A total of fifty-four phytochemicals rameters can be modified without impacting chromatographic performance. Parallel were identified in the berries including chlorogenic acid and its isomers, flavonol gly- column regeneration is a solution where the total analysis time is significantly re- cosides of quercetin and isorhamnetin, largely acylated anthocyanins of petunidin, duced with no impact to the methods being run. By adding an additional pump and delphinidin and malvidin, saponins of diosgenin and tigogenin, and glycoalkaloids utilizing the switching valves of the column compartment, a standard LC system of solasodine and novel hydroxylated and methylated and/or methoxylated coun- can be configured to perform parallel column regeneration. In this study, we will use terparts. Significant diversity in berry phytochemical profile was noted across dif- a 2D (two-dimensional) LC system which is equipped with two binary pumps and ferent genetic sources. Berries from a few of the genetic sources were high in ben- switching valves within the column manager. A food application will be used. Sample eficial polyphenols with limited and safe content of antinutritive glycoalkaloids and analysis is alternated between two identical columns with identical flow paths. For therefore evaluated as potentially new food supply in sub-Saharan Africa, whereas example, one sample is injected and separated on the first column, while the second berries with enriched and inedible level of glycoalkaloids could be developed into column undergoes washing and re-equilibration. Once the separation is complete, glycoalkaloid-based natural antimicrobial reagents. the valves switch positions, and the subsequent injection is directed to the second column for analysis while the first column undergoes washing and re-equilibration. 332 Functional Cannabis: Pharmacological Foundations of Cannabis With this configuration, the washing and re-equilibration steps can be excluded from Chemovars the cycle time. Depending on the particular LC method conditions, including column Mark Lewis, Napro Research, 8521 Younger Creek Dr., Sacramento, dimensions, programmed gradient time and flow rate, the overall analysis time can CA 95828 be reduced up to 50%. With reduced analysis time, increases in productivity or Cannabis is divided into several chemical phenotypes defined by principal cannabi- throughput are realized. noid. Type I THC-predominant cannabis is the most common type in both medical and recreational marketplaces in North America. Type II cannabis, much less com- 329 Reviving and Repurposing Laboratory Equipment mon until recently, produces both THC and CBD, while CBD-predominant cannabis Scot Abbott, Phoenix, 1419 Alverado St., Pittsburgh, PA 15216 is Type III. Certain Type I chemovars have achieved considerable market success Many labs have a lot of equipment which was expensive but which has been put in the United States based upon their unique essential oil (terpene) expression. Pre- out of service for various reasons, mostly lost or outdated software, electronic fail- dominant terpenes commonly produced by popular Type I cannabis cultivars include ures, or expense of upgrades. Until recently, we have had this problem in our labs. myrcene, limonene, beta-caryophyllene and terpinolene. There is a large body of We have developed a system integration tool which has let us put hundreds of popular claims that variations in terpene content are responsible for the varying thousands of dollars’ worth of equipment back into use. We developed an easy to sedative, stimulating or other mind-altering effects noted among these cultivars by use system integration tool which let us control instrument modules, take and post consumers. It has been posited by Russo, Watson, Clarke and others that pharma- process data, and add automation at modest cost and without the need for elec- cological synergies are evident between cannabinoids and terpenes that may ex- trical engineering or a programmer. The system has two key elements: 1) It uses plain the difference in effects. Some of these specific chemovars have common and Microsoft Excel® as the user interface so that no new programming language was often colorful names like Blue Dream, OG Kush, Trainwreck, Purps and a myriad needed. 2) Excel speaks directly to the electronics for data taking and for instrument of others. This presentation examines and attempt to codify these synergies, while control. Several examples are provided. extending beyond Type I, high-THC chemovars, in hopes to create a vocabulary for the cannabis industry to communicate effects of alternative chemotypes.

49 2018 EAS Abstracts November 2018

333 Terpenes and Residual Solvents in Cannabis by Headspace GCMS tryptophan, tyrosine, and phenylalanine. The strongest contribution to the fluores- Thomas Mancuso, PerkinElmer Inc., 710 Bridgeport Ave., Shelton, CT cence of proteins comes from the tryptophan emission. The tryptophan emission 06484, David Scott, Lee Marotta properties (fluorescence lifetime, quantum yield, emission maximum) are very A dual analysis has been developed where both terpenes and residual solvents are sensitive to the local microenvironment, such as solvent accessibility, polarity of analyzed in the same run streamlining the workflow for cannabis laboratories. The the solvent, solvent pH, concentration of salt, energy transfer and quenching. The analysis of residual solvents is critical for processed cannabis products since sol- modification in the tryptophan microenvironment due to conformational changes of vents are used widely in their manufacture. Over 100 terpenes are known to contrib- proteins can be detected by the intrinsic fluorescence. To evaluate sensitivity of ute to the unique aroma of many different cannabis strains that range from fruity to this method, tryptophan fluorescence was measured as function of pH and polarity woody and finally, skunky. The terpene profiles, including their relative abundances of the solvents studied. Time-resolved fluorescence decays from model proteins are useful in strain identification/validation but also useful in determining the thera- (albumins, myoglobin) were collected to estimate changes in higher order structure peutic effect for medical purposes. It has been said that the terpene profile makes using energy transfer phenomenon and other cooperative effects. Finally, fluores- a significant contribution to both the recreational and medical effects of cannabis. cence decays from monoclonal antibodies were acquired and analyzed. The data Fifteen different terpenes are responsible for the aroma/therapeutic properties of the obtained is discussed in relation to identity and changes of higher order structure of cannabis volatile fraction however; there is an increasing interest in the identification monoclonal antibodies. of the minor components as well. The increase in analysis time needed to complete- ly separate the volatile fraction is a major stumbling block to its common use in the 337 Proteomic Analysis of the Lake Trout (Salvelinus Namaycush) laboratory, since the lab is usually backlogged with samples and sample throughput Emmalyn J. Dupree, Clarkson University, 8 Clarkson Ave., Box 5810, is paramount in the analytical test lab. The focus of this study is to target up to 42 Potsdam, NY 13699, Bernard Crimmins, Thomas Holsen, James terpenes found in cannabis with attention to the speed of analysis and robustness, Pagano, Costel C. Darie while maintaining excellent chromatographic separation and using mass spectrom- The Great Lakes Fish Monitoring and Surveillance Program (GLFMSP) has placed etry to assist with compound identification. Pressure Balance headspace was used great importance on the species Salvelinus namaycush (lake trout) in monitoring due to its ease of sample preparation and accurate delivery of a precise aliquot to of toxic compounds. Legacy chemicals such as polychlorinated biphenyls and or- the gas chromatography mass spectrometry system for analysis. Data results such ganochloride pesticides are shown to reside in the Great Lakes for decades and as chromatograms, spectra, calibration curves and repeatability are shown. the chemical structure of these compounds allows them to bioaccumulate in organ- sims, such as fish. Bioaccumulation of these compounds can lead to changes in 334 Cannabinoid Monitoring in Dried Cannabis Flower and Edibles by transcribed genes, translated mRNAs, and thus, proteins produced and post-trans- HPLC-PDA lational modifications of these proteins. Although lake trout is used widely as a con- Jamie Foss, PerkinElmer, 710 Bridgeport Ave., Shelton, CT 06484 taminant biomonitor, there is currently little information available about the proteome Over 20 states in the United States have legalized the use of cannabis for both of this species. In this study, samples from the liver and blood of Salvelinus namay- medicinal and recreational purposes. To assure the quality, safety and potency of cush were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis marijuana products, reliable analytical procedures are pivotal for the quantitative (SDS-PAGE), followed by in-gel trypsin digestion and finally analyzed by nano-liquid analysis of the cannabinoids and terpenes, as well as any pesticides that may be chromatography tandem mass spectrometry (LC-MS/MS). The data was searched absorbed during cultivation. This presentation discusses the sample preparation against different National Center for Biotechnology Information (NCBI) and Swis- and analytical methodology for the chromatographic separation and quantitation sprot databases in Mascot Daemon. This output was then analyzed by Scaffold of 7 cannabinoids in dried cannabis flower and 12 cannabinoids in several food 4.3 software. Analysis with a nanoAcuity UPLC coupled to a QTOF Xevo G2 (both matrices using high-performance liquid chromatography (HPLC) with photodiode from Waters) revealed many novel proteins for the Salvelinus genus. These proteins array (PDA) detection. The utilization of a superficially porous particle (SPP) column were found in more developed databases from NCBI and Swissprot, like Actinopte- allowed for the rapid separation of twelve cannabinoids in four minutes while staying rygii, Salmonidae, and the highly studied Danio rerio. This preliminary study using well within the pressure constraints of a conventional HPLC pump. Cannabinoids a 150 minute gradient gave rise to 8330 proteins in the liver and 782 proteins in the were detected using a wavelength of 228 nm. The method provides exceptional blood. Further analysis with longer gradients and longer reversed-phase chroma- chromatographic reproducibility with cannabinoid limits of quantitation (LOQs) rang- tography columns will potentially give rise to even more novel proteins. This ongoing ing from 0.013 to 0.036 ug/mL, well below the current concentration limits of interest. project will potentially lead to a more developed, comprehensive proteome data- This method presents a robust, cost-effective solution for high-throughput cannabi- base for lake trout that can be used for further proteomic studies on the effects of noid monitoring in a variety of matrices. legacy chemicals in the Great Lakes ecosystem. 335 Wide Pore Superficially Porous Particles with Various Bonded 338 Mass Spectrometry Based Proteomics Investigation of Induced Phases for High Resolution Protein Chromatography Obstructive Sleep Apnea (OSA) in Rat Atria William Miles, Advanced Materials Technology, 3521 Silverside Rd., Costel C. Darie, Clarkson University, 8 Clarkson Ave., Potsdam, NY Quillen Building STE 1-K, Wilmington, DE 19810, Barry Boyes, 13699, Brian Panama, Devika Channaveerappa, Meredith McLerie, Benjamin Libert, Stephanie Schuster, Brian Wagner, Conner McHale Jacob Lux, Kelly L. Wormwood The use of superficially porous particles (SPP) in packed columns for liquid chro- Obstructive sleep apnea (OSA) is associated with several atrial diseases. The spe- matography (LC) separations achieves high resolution ranging from small mol- cific molecular mechanisms by which OSA causes atrial disease remain elusive. To ecules to proteins. The present work highlights examples of highly efficient and study the OSA-induced cardiac changes, we have initiated a proteomics study on useful separations of intact monoclonal IgG1, IgG2, and their variants, as well as OSA rat models and matched controls. To identify dysregulated proteins in apnea other biotherapeutic proteins and conjugates, using 1000Å pore size SPP materi- vs. control rat models, atrial tissues were homogenized and subjected to sodium do- als. Initially, efforts focused on familiar short chain alkyl (C4) bonded phase 1000Å decyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), digested by trypsin SPP to explore efficiency and column load effects. Comparisons of the C4 phase and the resulted peptide mixtures were analyzed by a Nano Acquity UPLC coupled to long chain alkyl (C18) and aromatic (Diphenyl) phases demonstrated similarly with Xevo G2 Mass Spectrometer. Data analysis was done using ProteinLynx Glob- high resolution protein separations, while enabling selectivity manipulation of highly al Server (PLGS 2.4), Mascot server and Scaffold 4.1 software. Rats were given similar protein variants. Chromatographic characterization of these different phases transient apneas for two durations: 13 and 23 s, we termed ‘moderate’ and ‘severe,’ through separations of both small and large molecules exhibits retention and selec- respectively. These two apnea durations are within the range of those experienced tivity effects unique to compound classes. Manipulation of selectivity in biomolecule by people with OSA. Electrocardiogram recordings showed that the P wave dura- separations is achieved by the combination of these distinct 1000Å SPP bonded tions (associated with left and right atrial depolarization) and T wave amplitudes phases with gradient elution variables such as acidic mobile phase additives, or- (associated with ventricular repolarization) were increased by 2 weeks of chronic ganic solvent, and temperature. This combination of SPP technology and gradient OSA. This was important since OSA patients show atrial electrical remodeling. The elution variables leads to selectivity manipulation and high resolution separations of apnea model recapitulated the pathophysiological characteristics of OSA. The pro- large biomolecules, favoring correct structure assignment by LC-mass spectrome- teomics analysis revealed that 3 of the 9 enzymes in glycolysis and 2 proteins re- try, including description of post translational and chemical modifications. lated to oxidative phosphorylation were down-regulated in the severe apnea group. In contrast, several structural and pro-hypertrophic proteins were up-regulated with 336 Application of Time-Resolved Fluorescence Spectroscopy to chronic OSA. The data suggests the chronic OSA causes proteins changes which Monitor Protein Higher Order Structure Changes lead to cessation of glycolysis, a diminished capacity to generate reducing equiva- Sergey Arzhantsev, United States Food and Drug Administration, 645 S. lents (i.e., NADH) as well as promotion of cardiac hypertrophy. Newstead Ave., Saint Louis, MO 63110 Time-resolved fluorescence spectroscopy is a powerful tool in biochemical research due to its high sensitivity and the simplicity of the method. The intrinsic fluorescence of proteins originates from the fluorescence of the aromatic amino acid residues: 50 2018 EAS Abstracts November 2018

339 Preventing Separation Anxiety: Strategies and Techniques for as available. Several non-selective methods were replaced by selective and sensi- Improved Biomacromolecule Separations tive technologies. Ion chromatography offers selectivity and sensitivity, for various Cory E. Muraco, MilliporeSigma, 595 North Harrison Rd., Bellefonte, PA chemical medicine analysis, for identification, assay and impurity. A good example is 16823, Gary Oden Jr., Michael Ye the utilization of ion chromatography (IC) for the determination of active ingredients, Separating proteins and antibodies is a unique analytical challenge owing itself to excipients, and traces of impurities. Formulations, especially in over-the-counter the complexity of the analytes. This complexity is a result of the myriad number of (OTC) drugs, come in many different forms and require different ion chromatograph- modifications to these analytes in a given sample including, but not limited to, phos- ic approaches. With a broad range of separation columns and detection methods, IC phorylation, methylation, and glycosylation. Both heterogeneity and non-specific covers a diverse field of applications in the pharmaceutical industry. Latest update binding to the silica surface often result in extensive peak broadening and tailing. on USP monograph modernization with Ion chromatography application for fluoride, This seminar focuses on several different chromatographic strategies that the an- zinc, potassium, magnesium family and other relevant applications are presented. alytical scientist can employ to resolve and quantitate various biomacromolecules, including mAbs and antibody–drug conjugates. Selected aspects of size-exclusion, 343 Strategy and Troubleshooting for Analytical Method Transfers to hydrophobic interaction, ion-exchange, hydrophilic interaction, and reversed-phase Global Manufacturing Sites in Support of a Small Molecule chromatography are discussed. Finally, some aspects of method development are Multistep Synthesis presented to aid in the development of robust and reproducible analytical chromato- Peter I. Tattersall, Bristol Myers Squibb, MS: NB 105 1162, One Squibb graphic schemes for the separations of protein and antibody mixtures. Dr., New Brunswick, NJ 08903, Xuejun Xu, Xin Bu, Lydia Breckenridge, Mohan Kanthasamy, Adrian Doggett, Diarmuid Scanlon, Morgan 340 Probing Peptide-Peptide/Peptide-Excipient Interactions Using O’Sullivan Native Mass Spectrometry Analytical method transfer is required during late stage development to afford Yuejie Zhao, Merck & Co., Research Laboratories, 126 E. Lincoln Ave., quality control (QC) support of manufacturing campaigns and registrational stabil- Rahway, NJ 07065, Pengyi Zhao, Jameson Bothe, Alexandra Andrew, ity studies. Here we review approaches towards the transfer of analytical method Hao Chen, Yong Liu, Andreas Abend, Peter Wuelfing knowledge with specific consideration on timelines and challenges encountered A deep understanding of the physicochemical stability of therapeutic proteins and from a highly accelerated (within half the normal timeframe) project with two starting peptides formulations is important to ensure successful development of marketable materials, four intermediates onto the drug substance. The methods we discuss formulations. Mass spectrometry (MS), an emerging biophysical characterization were successfully transferred to four sites at different geographical locations. The tool for biomolecules, affords higher resolution, better sensitivity and rapid mea- discussion focuses on: 1) instrumentation differences, challenging chromatography, surement of samples with minimum sample consumption comparing to traditional and compendial methods; 2) validation, co-validation, and verification as transfer techniques such as dynamic light scattering (DLS) and vibrational circular dichroism approaches; 3) method interpretation / documentation, communication and time- (VCD). However, MS characterization of peptide-peptide and peptide-excipient in- lines; 4) expectations vs. outcomes as lessons learned. Specific examples of chal- teractions in pharmaceutical formulations is challenging due to the relatively weak lenges and troubleshooting we encountered will be described and include: 1) Instru- peptide-peptide and peptide-excipient interactions and the non-volatility of excipi- mentation / geography effects on KF, GC and trace metal testing compared to the ents and buffers in formulations. Herein, we report the development of native MS expert laboratory experiences and data. 2) Investigation into problems with impurity coupled with different ionization techniques to address these challenges. Firstly, profiles and sample stabilization during preparation followed by the subsequent root aqueous buffers at a neutral pH and native MS parameters are used to preserve cause determination. 3) Troubleshooting an in-process control’s accuracy concerns weak noncovalent interactions. Secondly, liquid desorption electrospray ionization and sample stability where an incidental instrument setting had an impact on meth- (DESI) is introduced for its higher tolerance towards MS incompatible components, od performance. 4) Trace impurity testing of specific impurities where validation allowing formulations to be directly sprayed without buffer exchange. Multiple in- failure at the vendor required a change in method and analytical control strategy. Ex- trinsically soft ionization techniques including electrosonic spray ionization (ESSI), perience with vendor to vendor method transfers is also discussed with examples. Small emitter ESI and Nano ESI have also been examined. Native MS was applied to study oligomerization of GLP-1 type peptide Liraglutide. Our preliminary results 344 Multiple Techniques for Determination of Amorphous Content of show that peptide oligomers and peptide-excipient complexes formed due to weak, Crystalline Pharmaceutical Materials non-covalent interactions in solution can be preserved and detected by MS. The Charles Potter, TA Instruments, 159 Lukens Dr., New Castle, DE 19720 effects of key excipients on peptide oligomerization were also investigated. Amorphous content affects the physical properties and stability of a crystalline material such as a pharmaceutical, confection, or semicrystalline polymer. Many 341 USP-FDA Collaboration: Development of a Documentary Public techniques are used to detect and quantitate amorphous content with X-ray diffrac- Standard Diphenhydramine and Phenylephrine Hydrochlorides tion (XRD), differential scanning calorimetry (DSC), and gravimetric dynamic vapor Oral Solution sorption (DVS) being the common techniques. Several isothermal microcalorimetry Clydewyn M. Anthony, United States Pharmacopeial Convention, 12601 (IMC) approaches are also used which include: calorimetric-DVS and heats of solu- Twinbrook Parkway, Rockville, MD 20852, Douglas Kirkpatrick, Jan tion calorimetry. Humidity adsorption isotherms, humidity ramp, and humidity jump Yang, Margaret Fein, Leonel M. Santos DVS methods are used on both gravimetric and calorimetric DVS instruments. All One of the major resolutions reached at the United States Pharmacopeia (USP) approaches for amorphous content determination require some-kind of calibration 2015 - 2020 Convention was to update documentary standards in the USP com- with 1) a completely amorphous standard, 2) completely amorphous and completely pendium to maintain their scientific and regulatory relevance. In a joint collaboration crystalline standards, 3) binary mixtures of highly crystalline and highly amorphous with the Food and Drug Administration (FDA), USP identified the need to modernize standards, or 4) reference standards validated by a referee technique. DSC can and develop monographs related to over-the-counter (OTC) products. Concerted be calibrated with a single completely amorphous standard, but other techniques efforts and focus have been dedicated to both OTC drug products for which there require also require a completely crystalline standard (or standard of known crys- are currently no monographs in the USP compendium; and for those that have in- tallinity). In most cases, obtaining a completely crystalline standard is not practical, adequate testing protocols and/or outdated analytical methods. It has been a major and having a standard of known crystallinity can be problematic. priority to modernize these standards to better protect OTC formulations from eco- nomically motivated adulteration. One such OTC drug product is Diphenhydramine 345 The Karl Fischer Titration Process and Phenylephrine Hydrochlorides Oral Solution, a combination antihistamine and Bruce C. Herzig, MilliporeSigma, 2909 Highland Ave., Norwood, OH decongestant formulation, for which USP does not currently possess a documentary 45212 standard. A detailed overview of the scientific approach and collaborative efforts Karl Fischer titrations are used throughout the chemical and pharmaceutical indus- between USP and the FDA to develop a public standard for Diphenhydramine and tries for evaluating moisture content of products and raw materials. This presenta- Phenylephrine Hydrochlorides Oral Solution will be discussed. The advantages and tion covers system setup and choice of proper reagents and standards. Techniques benefits of the chromatographic procedure utilized will also be presented including such as titer determinations and the standards that should be used to insure quality the possibility of adopting this approach to develop documentary standards for more results of Karl Fischer titrations are discussed. Data demonstrating choosing the complex multi-active OTC formulations currently on the market. correct titrant and standard for your analysis to ensure accurate and reproducible results. We also discuss the validation of titration results using standards to ensure 342 USP Monograph Modernization - Ion Chromatography Applications that a complete and accurate titration was performed. Harihara Subramanian Narayanan, Metrohm USA, 9250 Camden Field Pkwy, Riverview, FL 33578 346 Accurate Moisture Determination in Lyophilized Products Pharmaceutical laboratories build their quality assurance/quality control processes Kerri-Ann Blake, Metrohm USA, 9250 Camden Field Parkway, based mainly on the United States Pharmacopeia and National Formulary (USP- Riverview, FL 33578 NF). USP has embarked on a global initiative to modernize monographs across The quality, efficacy, and shelf life of lyophilized pharmaceutical products and in- all of its compendia to include more selective and more sensitive methodologies gredients depends on water content. Routine Karl Fischer titration is the prescribed 51 2018 EAS Abstracts November 2018

technique for measuring residual moisture content to monitor manufacturing, in- ical or physical interactions with active pharmaceutical ingredients or additional for- sure samples are within specification, and optimize freeze-drying processes for ly- mulation components, resulting in degradation or reduction in overall performance ophilized products. Attend this session to hear applications experts explain special of drug products. Understanding these types of interactions remains critical to devel- techniques and reagent considerations when testing pharmaceutical samples for oping a robust and stable pharmaceutical formulation. The goal of this study involves moisture. Additionally, learn how to properly use standards and unique extraction the analytical characterization of common pharmaceutical excipients, sucrose and techniques to greatly improve the repeatability and accuracy of Karl Fischer titra- histidine, used in sterile solution formulations under thermal and light stress con- tions. ditions. Sucrose is a non-reducing sugar, but under high temperature conditions, it can decompose into reducing sugars, glucose and fructose. Sucrose-containing 347 Impact of Pharmaceutical Excipients on Chemical Stability of Drug formulations were evaluated under thermal stress conditions, including autoclave Product Formulations Under Thermal Stress and Light Exposure cycling, in order to monitor the generation of decomposition products using liquid Margaret Brunell, Merck & Co., MS: RY80T-B164, 126 E. Lincoln Ave., chromatography coupled to mass spectrometry (LC-MS). Upon exposure to light, Rahway, NJ 07065, Elizabeth Pierson, Paul Walsh histidine undergoes photo-oxidation reactions to form reactive side-chain peroxides Drug-excipient and excipient-excipient interactions play a major role in the stability that can impact chemical stability of drug products. Photo-oxidation of histidine was and effectiveness of pharmaceutical formulations. These types of interactions are monitored by high-performance liquid chromatography (HPLC) and LC-MS to de- of special concern for sterile solution formulations since drug products in the liquid tect histidine oxidation products as a function of pH. Additionally, the interaction of phase are more susceptible to degradation than solid dosage forms. Additionally, histidine and sucrose decomposition products was evaluated in order to identify po- manufacturing processes for solution formulations, such as terminal sterilization, tential reaction products that may enhance the degradation of solution formulations. significantly impact the stability of drug products. Excipients can participate in chem-

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