2015 CoSMoS Program

CoSMoS 2015 Technical Program and Conference Agenda
August 17 – 19, 2015
Sheraton San Diego Hotel & Marina, San Diego, CA

Day 1: Monday, August 17

7:30 – 8:30 am Breakfast (provided)
8:45 – 9:00 am Welcome / opening remarks
9:00 – 10:30 am Session 1 – Orals
Chairs:
Christine Aurigemma, Pfizer
Alan Marks, Shimadzu
Multidimensional Chromatographic Approaches for Highly Complex SamplesThe complexities of many natural or multi-step synthetic products pose significant challenges to qualitative and quantitative analyses due to the increased number of sample components. Multidimensional chromatography techniques such as 2D-LC provide enhanced separation power for components in complex samples and sample matrices where large numbers of co-eluting components are almost a guarantee. This session highlights the advantages of multi-dimensional chromatography, configuration considerations as well as real-world applications where 2D-LC can leverage your separation power when conventional techniques are not enough.

Separations of Small Molecules by Selective and Fully Comprehensive Two-Dimensional Liquid Chromatography – Recent Advances and Applications
Dwight Stoll
Gustavus Adolphus College
St. Peter, Minnesota

Since the late 1970’s interest in two-dimensional liquid chromatography (2D-LC) has blossomed because it is clearly the way to dramatically increase resolving power in a short period of time compared to conventional 1D-LC. 2D-LC has being used to good advantage over the past two decades in selected applications (e.g., proteomics) but in many areas (food science, forensics, polymer characterization) it is viewed as being of academic interest or as strictly a research tool but not for routine analytical purposes. There were a number of real limitations of the technique and barriers to its wider adoption; however, it is now our view that many of these limitations are now history and the barriers to wider use are much lower. Innovative developments in software, hardware, and understanding are leading to renewed interest in 2D-LC in the analytical community.In this presentation we will give an overview of some of the most important theoretical and practical developments in the past five years. We will discuss the central question – under what circumstances should one consider 2D-LC as an alternative to 1D-LC, and when should 2D-LC yield better analytical power? The peak capacity and speed of each second dimension separation is critical to the overall performance of 2D-LC, and its competitiveness with 1D-LC. With this in mind, we will discuss factors affecting the robustness of second dimension separations, as well as the influence of the properties of the first dimension mobile phase, as that fluid is injected into the second column, and approaches that can be used to improve the ‘compatibility’ of separations used in the two dimensions. Finally, we will use separations of moderately complex mixtures of small molecules in biological matrices to highlight the differences between selective and fully comprehensive 2D-LC separations, and the resolving power that reflects the state-of-the-art of these methodologies.

Is 2D-LC required for the analysis of complex mixtures of closely related species coming from C-H activation chemistry?
Erik L. Regalado
Merck Research Laboratories, New Jersey

The recent emergence of C-H activation is leading to exciting new synthetic chemistry reactions which can generate multitudes of closely related species (constitutional isomers, regioisomers, diastereomers, etc.) that often prove challenging for conventional chromatographic analysis and separation methods. The key question to be addressed in the pharmaceutical industry is whether existing ‘single dimensional’ separation tools can be used to address this emerging challenge, or whether next generation multidimensional separation tools will be required. In this presentation we describe our recent investigations into the separation and analysis of complex mixtures from modern C-H functionalization using a variety of single and multi-dimensional chromatography approaches. Strategies and approaches for application of chiral or achiral stationary phases, MS deconvolution and the use of offline/online 2D liquid chromatography are discussed, and several examples of the practical use of multidimensional chromatography methods for resolution of multicomponent mixtures are presented.

Harnessing Multi-Dimensional Chromatography in Pharmaceutical Industry
C. J. Venkatramani
Genentech, CA

There is a general perception in the scientific community that multi-dimensional chromatography is pivotal to the analysis of complex samples. Although it’s true, there are several applications in small molecules pharmaceutical industry where multi-dimensional separation techniques like 2D-LC and 2D LC-SFC can be used to address simple yet challenging problems.
In pharmaceutical industry, assessing stability indicating methods for potential co-elution can be challenging, as chemical components eluting in and around the main component are usually similar and or isomers of main component limiting the use of DAD and or MS.   Further, levels of co-eluting impurities are usually several orders magnitude lower than the main component adding to complexity.   The industry relies on manufacturing controls and extensive method development strategy to mitigate potential co-elution issues.   Although it might be adequate, lack of co-elution cannot be guaranteed by conventional HPLC.
Similarly, assessing chiral purity of active pharmaceutical ingredient (API) can be challenging especially in compounds with multiple chiral centers. Genotoxic impurity analysis is another area of concern in pharmaceutical industry.   2D-LC and 2D LC-SFC can play a pivotal role in assessing chiral purity and genotoxic impurity analysis my presenting a much simpler sample to complementary secondary column.
The presentation will cover several real world examples of LC x LC and LC x SFC in assessing stability-indicating methods for potential co-elution, analysis of residual genotoxic impurities in the midst of main component and simultaneous achiral-chiral analysis in a single run.

10:30 – 11:00 am Break
11:00 am – 12:30 pm Session 2 – Workshop
Chairs:
William Farrell, Pfizer
Karen Alsante, Pfizer
Jeffrey Kiplinger, Averica Discovery
4th Annual Method Development OlympicsThe organizers of the CoSMoS Conference recognize the value of diversification when engaging in new analytical challenges. More exposure to a variety of techniques and approaches usually leads to a successful outcome. In order to highlight this type of thinking, CoSMoS has designed an analytical challenge to help stimulate healthy discussion on the different approaches to analytical method development.   Conference participants will apply their method development strategy to an analytical problem defined by the CoSMoS organizing committee.Participants review the analytical challenge and submit their application to participate on the CoSMoS website. The top 3 finalists are invited to present their method session and the audience votes to determine the Bronze, Silver, and Gold Medalists awarded at the Tuesday evening dinner.
12:30 – 1:30 pm
Presented by:
Brian C. Trinque, PhD
Lathrop & Gage
Lunch (provided)Tutorial and workshop: The Small-Molecule Legal Landscape: A Path to a Successful Patent Strategy

Brian C. Trinque is a partner in the Boston office of Lathrop & Gage with a Ph.D. in organic chemistry. Brian focuses his practice on IP law in the pharmaceutical and chemical arts, and works with his clients to create and manage portfolios related to all aspects of pharmaceutical development.

1:30 – 3:00 pm Session 3 – Orals
Chairs:
Christine Aurigemma, Pfizer
Elaine Ricicki, Agilent
Alan Marks, Shimadzu
small Molecule, BIG Science: Role and Impact of Analytical Chemistry Techniques for Ensuring Clean WaterSafe drinking water is fundamental to sustaining life, and clean water is vital for our science, biology, and ecology. The efforts that go into analyzing a simple molecule and generating high purity water are remarkable. California is in the midst of its worst drought in history, and compounded by the impact of climate changes, deteriorating infrastructure and exhaustion of groundwater sources, traditional water supplies are getting dirtier every year. Stormwater and wastewater all introduce chemical and bacterial contaminants which pollute our water supply. Additionally, pipelines are aging and resulting in breaks and leaks that release untreated wastewater. This session will feature presentations on current research and innovative efforts for analyzing and purifying our water supply, ultimately protecting our most valuable resource.

Toilet to Tap: Is it Safe?
Shane A. Snyder
Co-Director, Chemical and Environmental Engineering
Arizona Laboratory for Emerging Contaminants, U of Arizona

Around the globe, human population continues to grow and urbanize. Population density in major urban zones is often growing at rates that demand water supplies which natural recharge cannot fulfill. This water deficit can be met by three primary means, water importation, desalination, and/or water reuse. Historically, water importation was the dominant means for augmenting dwindling freshwater supplies. However, water transmission across vast distances and elevations is often exceedingly energy and infrastructure intensive. Moreover, political and environmental concerns are often limiting factors in water importation projects. While ocean desalination also is energy intensive, it can provide a reliable source of water that is drought resistant and produces water of generally high purity. For both coastal and inland communities, reuse of municipal wastewater is a viable option for extending potable water supplies. However, wastewater by definition contains a highly complex mixture of chemicals and pathogens. Thus, potable water reuse systems have unique challenges in terms of water quality constituents as well as public perception. These challenges present novel opportunities for interdisciplinary teams of experts spanning engineering, toxicology, microbiology, public health, and social sciences, among others. While modern analytical techniques can selectively and sensitively identify trace levels of contaminants in water, the procedures utilized are generally time-consuming, labour-intensive, and technically-sophisticated. Considering the rapidly growing interest in direct potable reuse, which connects directly wastewater treatment plants to drinking water systems, monitoring for water quality and ensuring process reliability must be instantaneous and fail-safe. Thus, a great need exists for on-line real-time sensor systems and robust software networks that can monitor water quality and autonomously control water systems, shutting down or diverting water if a problem is detected. In addition, most communities will rely on a diverse portfolio of water resources, which can be better managed through smart water grid networks that aid in optimization of blending, water age calculations, and energy optimization. This presentation will share the latest discoveries in the move towards potable water reuse and the opportunities for collaborative cross-cutting research for the imminent water paradigm of the future.

Occurrence of contaminants of emerging concern (CECs) and some associated disinfection byproducts in southern California treated wastewater
Eric D. Nelson
Sanitation Districts of Los Angeles County

Contaminants of Emerging Concern (CECs) is a term used to describes a wide range of chemicals that may impact environmental and human health. These chemicals are generally not new, but their human and environmental health impacts are still not well understood. Compounds such as human hormones, pharmaceuticals, personal care products, flame retardants, natural products and formulation chemical make up some of the CECs that can enter the environment through municipal wastewater treatment plants.   Environmental risk from CECs may be dependent upon pharmacokinetic properties, wastewater removal efficiencies, species impacted and non-lethal toxicological endpoints. In addition to these parent CECs, disinfection byproducts may be inadvertently created by the treatment processes that may also have significant toxicities. We have been able to use sensitive and selective analytical techniques to study some CECs and their disinfection byproducts (DBPs) in wastewater effluents over the last eight years. Analytical strategies try to include as many CECs into one analysis as possible, but sometimes a particularly important but uncooperative analyte may need a separate analytical scheme. With optimized HPLC/MS/MS methods and extraction techniques, CEC behavior may be quantified/characterized and the environmental risks posed by these chemical contaminants may be better understood.

Environmental Applications of GC-ICP-MS
Armando Durazo
Laboratory Manager, Chemical and Environmental Engineering
U of Arizona

Chromatographic separation with inductively coupled plasma mass spectrometry (ICP-MS) detection is an analytical tool used widely in environmental analysis to differentiate and quantitate various forms (species) of elements in a given sample, i.e., As(III) and As(V), Br and BrO3, and iodinated contrast media (ICMs) as examples.   Typically this analysis is performed with non-volatile analytes using either an HPLC or an IC interfaced directly with the liquid sample interface on the ICP-MS. Despite the fact that many elements can be quantitated at low concentrations under these “wet plasma” conditions, there are limitations that can come about due to the presence of water introduced into the plasma by way of traditional sample delivery. These limitations include the formation of oxide and hydride interferences, as well as very large signals due to gas-phase atomic argon ions given the typical hot plasma conditions used for this analysis.

Our laboratory became motivated to explore the use of GC-ICP-MS to study the formation of halogenated disinfection byproducts (DBPs) in environmental waters upon chloramination. Our experiments revealed that GC-ICP-MS is a very powerful tool for screening total levels of volatile halogens and features elemental specificity (which is not afforded with traditional GC-ECD analysis). We found that performing ICP-MS under “dry plasma” conditions allows one to perform essentially “interference-free” analysis without the use of a collision/reaction cell, and that the use of lower RF-power discriminates against the formation of interferences that arise from the ionization of non-easily-ionized elements (i.e. Ar, O2, N2).

Based on our work performed with the halogenated DBPs, we sought to explore what other analytical opportunities GC-ICP-MS would present. recent work utilizing GC-ICP-MS found that dry plasma conditions greatly facilitated 32/34S analysis, as revealed by analysis performed on oil-contaminated waters. Given our successes with both 35Cl and 32/34S analysis, we decided that 19F would represent a challenging element to quantitate by ICP-MS, given its poor ionization properties and the interferences on m/z 19 due to oxygen-based (16OH3+ and 18OH+) and argon-based species (38Ar2+). We found that 19F can be quantitated using GC-ICP-MS, despite many works that describe it as an element that is non-amenable to plasma phase analysis. The results of this work focusing on 19F quantitation will also be presented.

3:00 – 3:30 pm Break
3:30 – 5:00 pm Session 4 – Orals
Chairs:
Wolfgang Goetzinger, Amgen
William Farrell, Pfizer
Second-tier Chiral Separation Approaches & ApplicationsMany chiral compounds can be separated using a first tier screen of most broadly applicable columns/solvent combinations. This type of screen usually includes the popular carbohydrate phases (AD, OD, AS, OJ, etc…) and a Pirkle-type phase (e.g. Whelk-O) while using an alcohol (IPA, MeOH, EtOH, etc.) to effect a separation. However, if the initial screen is unsuccessful, what are the best approaches for achieving that elusive separation in the second-tier screen?   This session will focus on alternative column chemistries and non-traditional approaches for successful chiral separations.

Enantiomeric Resolution of Primary Amine Racemates Using Crown Ethers
Wesley W. Barnhart
Amgen, Thousand Oaks, CA

While many chiral compounds can be separated using the traditional polysaccharide and Pirkle type columns, there are cases where samples containing primary amino moieties are not as easily separable using those standard chiral stationary phases (CSPs). Crown ethers, developed in the late 1960s, provide a way to separate primary amino racemates by taking advantage of the complex formed with alkylammonium moieties and the crown ether. Chiral crown ether columns such as Crownpak and ChiroSil have allowed for chromatographic separation of many primary amino racemates, including amino acids, as well as aminophosphonic acids. Other non-chromatographic separations involve diastereomeric salt formation, thus allowing for enantioenrichment via recrystallization.

In this presentation, more detailed background of crown ethers will be discussed.   In addition, the application of crown ether CSPs in our chiral separation method development/practices will also be expanded upon. Lastly, the more modern cyclofructan based CSPs for the separation of primary amino racemates will be mentioned.

Non-chromatographic approaches to chiral resolution and purification
Neal Sach
Pfizer, La Jolla, CA

Whilst the field of chiral chromatography has developed in leaps and bounds over the past decade, there remains occasions where the analytical separations are problematic or inefficient on larger scales, and alternatives methods may be valuable. This presentation will focus on such methods, specifically classical resolutions and enzymatic resolutions. Examples will be presented where, in collaboration with the embedded analytical team, resolving methods have been developed to fund kilograms of enantiopure intermediates. The methods and processes used in identifying these resolutions will be detailed with examples.

Wisdom of the Crowd
William Farrell – Moderator

Since approaches chiral chromatographic separations are as varied as the compounds themselves, this workshop will explore suggestions by attendees in an open forum discussion format.

6:00 pm Dinner (on your own)

Day 2: Tuesday, August 18

8:00 – 9:00 am Breakfast (provided)
9:00 – 10:30 am Session 5 – Orals
Chairs:
Douglas Kiehl, Lilly
David Stranz, Sierra Analytics
Extractables and Leachables: Analytical Approaches, Challenges and Case StudiesThe detection and identification of impurities in drug products is essential to the establishment of safety, purity and compliance of pharmaceutical products. Extractables and leachables (E&L) may be present in pharmaceutical products as a result of direct or indirect contact with components associated with packaging, storage, container/closure systems, processing equipment and/or drug delivery devices. Leachables may represent a subset of extractables, and comprehensive characterization and profiling of extractable compounds is necessary to fully understand the potential impact on the patient and drug product quality. The evaluation of E&L may involve analysis of samples representing highly complex and diverse mixtures of organic and inorganic compounds, and can present significant analytical challenges.   This session will explore approaches to evaluating E&L while examining some case studies illustrating analytical challenges.

Examining Relevant Roles for Informatics and Software Technologies in Extractable and Leachables Studies
Patrick Wheeler
ACD Labs

Safe and proper performance of drug containers and medical devices relies ultimately on adequate chemical comprehension. Strategic in this regard is the identification, elucidation, and characterization of extractables and leachables (E&L) and the determination of hazards and consequent risk they pose. Controlled extraction studies provide analytical data designed to permit recognition of compounds and materials that may generate concerns. Nevertheless, managing, analyzing, and interpreting various sets of hyphenated GC or LC and mass spectrometry data in an effective, both thorough and robust manner, to find extractables signals, especially when using different instruments and software can be a major challenge. One role of software is therefore facilitating manual and automated analysis workflows. A related role is creating and searching a knowledge base of identified compounds. Collection of interpreted data and results also allows simplified reviewing of results and creation of summary reports and data packages. Cumulatively, these represent core aspects of unified laboratory intelligence software technologies, which support transforming chemical data into actionable knowledge. This work will consider some key challenges and opportunities currently facing software technologies for E&L studies.

Extractables and Leachables from a Toxicologist’s Perspective
Courtney Callis
Eli Lilly & Company

Polymers in direct contact with parenteral drug product during manufacturing, storage, or delivery are considered critical and must be qualified, controlled and maintained appropriately for their designated use. Characterization of the polymer’s suitability for use is part of Quality by Design and includes an extractables/leachables assessment.   A toxicologist’s input during material characterization can help minimize safety risks to the patient.   The toxicologist’s role includes recommendations for exposure limits for extractables and leachables that would result in no appreciable risk to human health. However, guidance on how to derive science-based limits for extractables/leachables for parenteral applications has not been published. In silico structural assessment and the threshold of toxicological concern, a science-based limit used for food safety and for contaminants in pharmaceuticals, is also useful for assessment of extractables and leachables.   This presentation describes a risk assessment process aligned with well-established methodologies and agency guidelines that can be used to derive consistent, health-based limits for extractables and leachables from polymers in contact with parenteral drug substances.

What is the Future of Leachables and Extractables Assessment?
Daniel L. Norwood
SCĪO Analytical Consulting, LLC
Presentation sponsored by Sierra Analytics

Leachables and extractables have been of concern to pharmaceutical development scientists and container/closure system suppliers since the 1980s. Over the approximately two and a half decades since that time, we have learned to apply laboratory techniques and advanced analytical technologies to isolate, separate, identify and quantitate complex mixtures of both drug product leachables at trace levels, and container/closure system component extractables. Throughout the 1990s there was little regulatory guidance on the issue, only a few contract research organizations with any measure of knowledge or capability, and insufficient analytical technology (even though in the 1980s trace organic analysis was a well-developed field). This presentation will discuss the future of the leachables/extractables field, with emphasis on future analytical technologies, the regulatory environment, and what it will take to actually get the work done.

10:30 – 11:00 am Break
11:00 am – 12:30 pm Session 6 – Orals
Guest chairs:
Giorgis Isaac
Jimmy Yuk
Session Sponsored by Waters Corporation
Perspectives in natural product research: “The good, the bad and the ugly” A major challenge in natural product research is the analytical complexity of the samples. To achieve a thorough understanding of the known and identify unknown components in these rich and diverse samples, innovative tools/methods/technologies are needed. This session features leading scientists in natural products presenting innovative and cutting-edge analytical approaches to understand the complexity of their samples. From creative techniques for sample preparation, unique hardware modifications/innovations and informatics platforms for complex data analysis, the analytical workflow offers a diversity demanded by the natural product research itself.

Biome mining for natural product discovery using structural mass spectrometry and big data strategies
John A. McLean
Vanderbilt University

In comparison to combinatorially synthesized compounds, natural products demonstrate increased chemical and structural complexity that exhibits a strong correlation to current drug molecules. We consider hypogean organisms an interesting source of natural products, as these extremophiles survive in resource scarce environments. One of the most daunting tasks in natural product discovery is the determination, isolation, and dereplication of potential therapeutics from the massive molecular inventory present in extracts. Our work merges microorganism manipulation (e.g. homologous expression, chemical challenge, etc.) with high dimensional structural mass spectrometry data and processing techniques directly aimed at streamlining lead compound prioritization. We have developed a natural product discovery workflow using hypogean environments as an organism source. We interrogate microbial secondary metabolomes through multi-dimensional separations including UPLC, SFC integrated with ion mobility-mass spectrometry (IM-MS). Using UPLC-IM-MS and SFC-IM-MS we investigate conditional manipulation as a means of organism interrogation to isolate new lead compounds. To enhance the relevance of our measurements, we have demonstrated structural discrimination IM-MS as a means of crude extract prioritization Natural products possess unique structural characteristics, which we hypothesized would result in measurable deviations, resulting from cyclization, halogenation, metal chelation, and glycosylation, among others. Using these strategies, we have demonstrated the structural consequences of peptide cyclization on IM separations and consequently isolated a cadre of new compounds from these bacterial sources. Furthermore we have discovered previously unobserved natural products by virtue of utilizing a gas-phase structural metric to metabolite screening. Finally, we have applied self-organizing map based analyses to further evaluate the effects of homologous expression. Combined these approaches have increased our understanding of metabolic consequences of clinically-relevant antibiotic resistance on the microbial metabolome. This report describes this workflow using a variety of instrumental and bioinformatic strategies to facilitate faster prioritization of lead compounds of interest in drug discovery efforts.

Stevia Glycosides: What Tastes Best?
James McChesney
Ironstone Separations
Etta, MS

An increased interest in non-caloric natural sweeteners has generated significant attention to glycosides from Stevia. The GRAS acceptance for Rebaudioside A and Stevioside has led to their utilization in foods, beverages, etc. However these major glycosides have an undesirable lingering aftertaste which has raised interest for characterization of new and/or minor glycosides from Stevia which may have a “cleaner” sweet taste. The major interest has been in glycosides found in the polar region because of their expected better taste profiles. The most commonly accepted analytical HPLC method, established by Joint FAO/WHO Expert Committee on Food Additives (JECFA), is appropriate for the major glycosides but does not provide good resolution for the minor glycosides that co-elute with the major glycosides present. The JECFA analytical method is particularly poor for resolution in the polar region of the Stevia extract. The complexity of the commercially available extract is illustrative of most plant natural product preparations. We’ve established alternative HPLC methods to the JECFA method which better resolve different regions of the Stevia extract. The complexity of the Stevia extract and the similarity of the glycosides in physical and chemical properties are such that no single method is suitable to resolve all compounds found in the extract. Use of these methods has enabled Ironstone to purify and characterize numerous novel glycosides.

Application of novel analytical techniques and data analysis tools for comprehensive metabolomics analysis from complex samples
Vladimir Shulaev
University of North Texas

Metabolomics analysis of complex mixtures using a single separation technique is challenging due to the diversity of metabolites polarity, volatility and the large range of concentrations in biological samples. We examined two novel analytical approaches for comprehensive analysis of lipids and polar metabolites in various biological matrices. First approach is based on Ultra Performance Convergence Chromatography (UPC2), a chromatographic system that utilizes liquid CO2 as primary solvent and co-solvents such as methanol as a mobile phase to leverage the chromatographic principles and selectivity of normal phase chromatography while providing the ease-of-use of reversed-phase LC. We utilized the sub-2µm particle supercritical chromatography for the separation of free fatty acids, neutral and polar lipids in a single lipid extract. The sub-2µm particle supercritical CO2 system was coupled to a time of flight MS for accurate mass multivariate analysis and a triple quadrupole MS for targeted analysis. Second approach utilizes Atmospheric pressure GC (APGC) is a ‘soft‘ chemical ionization technique that generates a spectrum conserving the molecular ion species with minimal fragmentation, which differentiates it from traditional vacuum source GC-MS based on electron ionization. We combined APGC with ion mobility (IM) to enhance peak capacity and improve selectivity and specificity of analysis. Ion mobility (IM) is a gas-phase separation of ions based on shape, size and charge. IM mobility separation occurs in milliseconds and thus it can be placed between GC separation (seconds) and TOF detection (microseconds) to provide an additional dimension of resolution. We demonstrated the utility of APGC-IM-TOF-MS for metabolomics analysis of various mutant plant genotypes. Raw data were analyzed using Progenesis QI 2.0 that adopts an intuitive workflow approach to performing comparative data analysis. The workflow starts with raw data file loading, then retention time alignment and deconvolution, followed by analysis that creates a list of features. The features are then identified with compound searches and explored using multivariate statistical methods.

12:30 pm – 1:30 pm Lunch (provided)
1:30 – 3:00 pm Session 7 – Workshop
Chairs:
Jeffrey Standish, ACS
Alan Marks, Shimadzu
Sabra Botch-Jones, Boston University
Inside the Matrix: Improving workflow via innovative sample preparationAnalyzing small molecules is typically less complicated when they are neat solids or standalone solutions. But in reality, these analytes of interest are usually surrounded by and embedded in “The Matrix”… an all-encompassing collection of endogenous materials that invariably interfere with the identification and quantitation of small molecules. Although these matrices come in many forms, depending on the application with some differences found in pharmaceuticals, foods, and environmental industries for instance. The issue of extraction efficiency is the common thread that must be optimized for a successful outcome in terms of isolating target compounds of interest in an optimal time period. This session will explore the current state-of-the-art approaches being applied in the struggle against the oppression of the Matrix…

Novel 3D Micro Extraction Technique with Multi-Dimensional Chromatography for Forensic Toxicological Analysis
Claude Mallet
Waters Corporation

The field of Forensic Toxicology encompasses several disciplines, such as analytical chemistry, pharmacology and clinical chemistry with the main objective of assisting medical and legal investigation of death, poisoning and drug use. The use of spot testing is perhaps the most known technique in forensic analysis for rapid results. For a complete legal identification, a more robust methodology is required and the current trend in forensic laboratories is the use of liquid chromatography coupled to mass spectrometry (LC/MS or LC/MS/MS). However, to achieve satisfactory results most often with a trace level detection requirements, extensive and time consuming sample preparation protocols are required to reach sub ppb levels. In recent years, advances in analytical capabilities with hyphenated instrumentation platforms have enabled sensitivity and efficiency to detect trace level analytes. However, the bottleneck resides with the same sample preparation techniques, often time created several decades ago. Although these techniques are “tried and true”, there is much room for improvement. Three Dimensional (3D) Micro Extraction with Multi-Dimensional chromatography can decrease sample preparation time without sacrificing the quality seen with current single dimension chromatography techniques. The extraction process was performed using a reversed-phase sorbent using several solid phase extraction techniques such as 1D, 2D optimized, 3D sequential and cumulative elution. The concept of sequential 3D extraction was designed to capture the retention behaviour of a target analyte in response to various extraction parameters (sorbent strength, elution polarity, solubility … etc). Therefore, optimized conditions can be selected to excise a region of interest during extraction. In this application, 12 illicit drugs were spiked in human urine and extracted using three extraction protocols for performance evaluation. The chosen 2D chromatography conditions used in this application was identified using a 6×6 automated methods development protocol (144 methods total). The manual extraction of urine samples were completed in less than 20 minutes. The analysis was performed using 200 uL of the final organic solvent (MeOH) extracts. The LOD for all drugs was measured at 100 ppt from a 1 mL sample volume. Several analytes showed excellent signal at 10 ppt.

Multipath Liquid Chromatography – Mass Spectrometry: On-Line Preparation and Multipath Separation for Simultaneous Small and Large Molecule Analysis
Kevin Schug
University of Texas at Arlington
Presentation sponsored by Shimadzu

Many precious biological samples may contain multiple analytes of interest; these may span the range from small molecule metabolites to large proteins. Typically, sample preparation, analytical separation, and quantitative determination schemes for different classes of chemical compounds can vary substantially – so much so that they require completely separate analyses. We have developed a multipath liquid chromatography – mass spectrometry system, which enables sample preparation and analysis of multiple chemical compound classes from a single injection. Through integrated restricted access media, multiple valves, high efficiency stationary phases, and flow-paths, it is possible to segregate the components of a complex biological mixture and facilitate their separation, followed by ultra-trace detection on a triple quadrupole mass spectrometer. This could be desirable to streamline analysis of, for example, multiple biomarkers or small and large molecule conversion products of biotherapeutics, simultaneously in one analytical run. Our work includes development of ultra-trace determination of endogenous estrogens using bulk derivatization and restricted access media. We have further optimized multiple reaction monitoring schemes for intact protein analysis on the triple quadruple and achieved detection limited in the ng/mL regime. We utilize automated method scouting to evaluate the selectivity of current generation phases for analytes with wide variation in physicochemical properties and molecular weight. In a multipath system, these components have been integrated for segregation and simultaneous separation and determination of small and large molecule analytes from a single injection. Effectiveness of the system has been demonstrated for combined analysis of small and large molecules from biological fluids such as urine and plasma.

Tunable Synthetic SLE
Matthew Brusius
Phenomenex

Simplified Liquid Extraction (SLE), also known as Supported Liquid Extraction, has been developed as an alternative sample preparation
method to liquid-liquid extraction (LLE) to remedy some of the drawbacks including the formation of emulsions, difficulty in
automating, and the use of large amounts of hazardous solvents. The particle of the solid support in an SLE material mimics
the shaking event in a LLE workflow and creates micrometer scale containers for an aqueous sample that allow for a highly efficient
liquid-liquid extraction. Novum, a synthetic SLE sorbent, yields results that are identical in most cases to contemporary diatomaceous
earth SLE products, but is not restricted by limitations that are associated with natural resources such as availability and variation
in composition. By investigating a corticosteroid panel case study, it is shown that Novum is capable of achieving equivalent recoveries,
while also offering the analyst the added option of additional clean up and phospholipid removal that cannot be achieved on traditional
diatomaceous earth SLE products.

3:00 – 3:30 pm Break
3:30 – 5:00 pm Session 8 – Orals
Chairs:
Sabra Botch-Jones, Boston University
Elaine Ricicki, Agilent
Investigations: Making the Unknown KnownToxicological analysis, be it forensic, pharmaceutical, sports doing, or research focused, can at times be like looking for a ‘needle in a haystack’. As scientists we face similar analytical challenges regardless of the type of compounds we are looking for. This session will focus on both traditional and novel techniques utilized for identifying unknowns.
(illegal substances / drugs of abuse)

Biological Assays and Effect-Based Analyses: The Next Generation of Sports Doping Testing
Amy Cadwallader
Aegis Sciences Corporation

The detection of drugs of abuse and especially sports doping is moving away from identifying exposure to prohibited substances and is evolving into detecting effects of prohibited substances. Cell-based biological assays are the next generation of assays for forensic toxicology and anti-doping laboratories. These methods do not require knowledge of a compound’s structure, but instead use biological endpoints of the drug’s action as opposed to detecting drug and/or metabolite(s) present in urine or other biological fluids. Bioassays detect all compounds interacting with a chosen endpoint regardless of their structure. For example, in an androgen receptor bioassay, all ligands including designer steroids and non-steroidal androgen receptor modulators (SARMS) that bind to the androgen receptor will be detected in a biological sample or dietary supplement. As another example, a bioassay employing cannabinoid receptors, can be incredibly useful in identifying forensic samples containing frequently evolving cannabimimetic compounds. Bioassays using living cells represent the in vivo drug actions of receptor activation, compound uptake into cell, and eventual protein production or biological response. There are applications of bioassays beyond their ability to detect the presence of banned substances in samples. These include use in conjunction with mass spectrometers to determine the structure of new ligands, the capability to determine the relative biological activity of ligands, and coupling with other molecular biology techniques such as microsomal metabolism for additional studies on newly identified compounds. With all of the benefits involved in using bioassays, their use in forensic toxicology is increasing.

Clinical LC-MS/MS Assays and False Positives: Detection and Elucidation
David Masters-Moore
Millennium Health

Clinical LC-MS/MS Assays have become increasingly employed for medical reconciliation providing physicians and mental health professionals with information to support improved clinical decision making. With millions of specimens analyzed each year, LCMS assays are thoroughly validated and stress-tested to ensure clinical robustness. However, for a few analytes, rare cases of false positive results have been detected and elucidated. Assays are subsequently revised and incorporate new features aimed at eliminating the incidence of false positive results.

Solid Phase Extraction of Forensically Relevant Compounds
Jeffrey Hackett
Forensic Laboratory Division-Office of Chief Medical Examiner
San Francisco
Presentation sponsored by CoSMoS

The aim of this oral presentation is to inform scientists, analysts, and forensic toxicologists, involved in whole blood, urine or tissue testing, about how a diverse range of forensically significant compounds may be best extracted employing both solid phase extraction and gas/liquid chromatography coupled with mass spectrometry. This is a typical set up found in many forensic, clinical, and bio-pharmaceutical laboratories. The information presented in this presentation should assist those scientists in understanding how powerful a tool solid phase extraction is in the extraction of drugs from whole blood samples, and the limitations that exist with gas/liquid chromatography mass spectrometry as an analytical tool. This presentation will explain the use of aqueous buffers, hydrolyzing agents as well as the use of mixed mode solid phase sorbents to extract a wide range of compounds such as barbiturates, benzodiazepines, cannabinoids opiates, and other drugs both prescribed and illicit.

5:00 – 6:00 pm Poster session and reception
7:00 – 9:30 pmBay Tower Garden Terrace Conference banquet and guest speakerMethod Development Olympics awards
Stories from Research with African species
Dr. Matt Anderson, Dir, Behavioral Ecology, San Diego ZooBased on his passion for conservation research with rare and endangered species Dr. Matt Anderson, Director of Behavioral Ecology at the San Diego Zoo Institute for Conservation Research brings us insights to such topics as the secret language of elephants and the intriguing story of the African cheetah.

Day 3: Wednesday, August 19

8:00 – 9:00 am Breakfast (provided)
9:00 – 10:30 am Session 9 – Orals
Chairs:
Justin Stroh, Pfizer
Oliver McConnell, Merck
Structure ElucidationModern structure elucidation of naturally occurring and synthetic compounds draws upon a myriad of NMR and MS techniques using an impressive array of sophisticated instrumentation, and is most effectively carried out on samples where compound purity has typically been assessed in part by chromatography.   However, structure elucidation of compounds in mixtures or absolute configuration of enantiomers requires more specialized techniques and instrumentation. If difficult structure elucidation is an issue for you, then don’t miss this session where you will hear speakers who will share their current state-of-the structure elucidation approaches of natural products and synthetic compounds that broadly span applications and markets.

Social Networks For Molecular Analysis
Pieter Dorrestein
UCSD

The chemical makeup of biology is incredibly complex. It is already difficult to analyze the molecular makeup of one organism, let alone 100s or 1000s of different organisms? How can we look at the molecular makeup of all such organisms at once? How many molecules do they have in common? How many are different? How do we retain the collective mass spectrometry knowledge of the community? As mass spectrometers are becoming faster and more sensitive we can detect a lot of molecular information. There are now mass spectrometers that can analyze 10,000 samples a day. However, there is no infrastructure to analyze this amount of molecular information or to correlate this information to other Big Data generating approaches such as sequencing. On average, however, only 1-5% of all the molecular information that is collected by mass spectrometry can be annotated. It is simply too much information for one person or lab to analyze this information with the existing tools that are available. In this lecture we will explore the strategies for organizing and visualizing the massive amount of information. We have launched a social networking tool to annotate molecular information. Currently our social molecular analysis platform has nearly a billion spectra uploaded and thousands of users from 65 countries.

The applications of these tools, including genome mining, will be demonstrated through answering specific questions. Some of the questions we may address in this lecture include: How can we uncover new chemistries using molecular networking? How can crowd source analysis be used for dereplication and structural analysis? Can we use this to tease apart metabolism of molecules in a host or the environment?
3 learning objectives.
1) Defining the magnitude of the problem. Although the tools exists to detect a lot of molecules real samples from nature (e.g. soil, microbiota and plant, human or animal tissues) there are no good approaches to deal with the complexity of this molecular information. This is especially true for the majority of metabolites that can be detected. The fundamental question is how do we annotate this molecular information or otherwise will we continue to only look under the light post?
2) We will introduce the topics such as MS based genome mining, molecular cartography and molecular networking.
3) How social and crowd source molecular analysis is accelerating our understanding of molecular complexities. How can the molecular analysis of a coral invasion and bleaching be translational to understand exacerbation in a CF patient?

Chiroptical Spectroscopy for Three Dimensional Molecular Structure Determination
Prasad L Polavarapu
Vanderbilt University
Presentation sponsored by CoSMoS

The determination of absolute configuration and predominant conformations of chiral molecules is an area of active interest in chemical sciences, especially in pharmaceutical industry. Chiroptical spectroscopy is well suited for this purpose. Four different methods, namely Electronic circular dichroism (ECD), optical rotatory dispersion (ORD), vibrational circular dichroism (VCD) and Raman optical activity (ROA), provide the needed information for elucidating chiral molecular structure. Facilitated by recent developments in chiroptical spectroscopic instrumentation and quantum chemical predictions of ECD, ORD, VCD and ROA properties, it is now becoming more or less routine to use combined experimental and computational studies for structural characterization of chiral molecules. These methods are well suited for characterizing small chiral molecules at an unprecedented level of reliability, although larger chiral molecules can also be characterized provided more than one method can be simultaneously applied. In this talk I will provide a review of these methods, citing successful examples as well as potential pitfalls that one should avoid.

Elucidating the Components of Hemp Oil
Cathy Moore
Exova, Santa Fe Springs

Interest in nutraceuticals is rising and the methods to test constituent identification claims are under development (DNA barcoding) or already exist in the USP Dietary Supplement monographs (currently using TLC and HPLC with certified reference materials). These methods do not include structural information. But materials derived from natural sources provide multiple challenges for traditional structural methods: a complex matrix and difficult structures that often include stereochemical centers that need to be determined. This talk will discuss recent studies of hemp oil refined to 24% cannabidiol: what are the other components and their concentrations? Using a combination of LC-MS and NMR, some of these components will be identified and the experiments to elucidate the structures presented.

10:30 – 11:00 am Break
11:00 am – 12:30 pm Session 10 – Orals
Chairs:
Karen Alsante, Pfizer
Douglas Kiehl, Lilly
William Farrell, Pfizer
The Allotrope Framework: An innovative approach to improve data interchange, increase research efficiency & realize the full value of your dataAnalytical labs create lots of data, stored in proprietary files. The metadata needed to document experiments is often incomplete, incorrect due to manual entry, or distributed among multiple applications.   This introduces complexity into data sharing and reuse and prevents the realization of its full value. The Allotrope Framework provides a permanent solution that fixes this problem at its root. The Framework is comprised of: open, standards-based, highly performing, extensible file format; metadata definitions & repository to provide accurate, complete & consistent metadata; and a software toolkit to facilitate a consistent adoption of the Framework. The first year of development demonstrated the technical feasibility of the core concepts of the Allotrope Framework through the creation and delivery of proof of concept framework components and applications. The project has moved towards implementation of the Framework via integration in member companies. Attendees will join a discussion of our findings and how they are directing construction of the Framework and the underlying taxonomies for analytical techniques.

Gang Xue
Amgen Inc.
Cambridge, MA

12:30 pm – 1:30 pm Lunch (provided)
1:30 – 3:00 pm Session 11 – Workshop
Chairs:
Karen Alsante, Pfizer
Douglas Kiehl, Lilly
William Farrell, Pfizer
Analytical Quality By DesignQuality by Design (QbD) is well established in the development and manufacture of pharmaceutical drug substance and drug product processes. The aim of QbD is to design a quality product that consistently delivers the intended performance. These same principles and concepts have been applied to the development of methods and termed Analytical QbD (AQbD). The outcome of AQbD is the design of a quality, robust method that consistently delivers the intended performance. The knowledge obtained during method development, optimization and performance verification help justify the establishment of the method operable design region (MODR), the “design space” of the analytical method. As a result of AQbD methods development, enhanced method understanding and robustness will result in fewer method failures and transfer issues over the lifecycle of the method. This session will discuss AQbD concepts and case studies.

Chromatographic Method Development in an Analytical Quality by Design (AQbD) Framework
James Morgado
Pfizer

Analytical methods provide critical data in support of the understanding and control of pharmaceutical materials. There are a multitude of approaches to methods development from utilization of the “favorite” column, stationary phase or vendor to systematic method screens employing software optimization and nuances in between. The demonstration that the method is suitable for its intended purpose is shown from the method development (ensuring the method performs as required) through method validation and subsequent use. The method needs (e.g., purpose, specificity, sensitivity, cycle time, accuracy, precision) must be well understood, and these requirements used to build a method that meets the needs of the program. Quality by Design (QbD) is well established in the development and manufacture of pharmaceutical drug substance and drug product processes. The aim of QbD is to design a quality product that consistently delivers the intended performance. These same principles and concepts have been applied to the development of methods and termed Analytical QbD (AQbD). The outcome of AQbD is the design of a quality, robust method that consistently delivers the intended performance. The knowledge obtained during method development, optimization and performance verification help justify the establishment of the method operable design region (MODR), the “design space” of the analytical method. As a result of AQbD methods development, enhanced method understanding and robustness will result in fewer method failures and transfer issues over the lifecycle of the method. This presentation will discuss AQbD concepts, a typical reversed-phase LC workflow and a case study. The chromatographic method development occurs in a series of experiments meant to roughly understand robustness and select draft conditions. Based upon the project specific method development knowledge gained and experience, a risk assessment is performed to identify risk factors that should be experimentally evaluated in a design of experiment (DOE). An assessment of the DOE data provides a method operable design region and center point/control strategy (defined method parameter set points and operating space) for the method.

Application of Quality by Design (QbD): Pharmaceuticals to Containment and Delivery Systems
Diane Paskiet
West Pharmaceutical Services

The foundation for utilizing QbD to enhance and modernize the regulation of pharmaceutical manufacturing and product quality was introduced in a 2002 FDA report entitled; Pharmaceutical cGMPs for the 21st Century – A Risk Based Approach. Dr. Woodcock, FDA Director of the Office Pharmaceutical Quality (OPQ), describes high quality drug products as those that consistently deliver clinical performance, are free from contamination and available to patients. Focus on patient outcomes and QbD has been evolving for over thirteen years and has become the standard approach for pharmaceutical development and manufacture. Positive experience for patients will depend upon the protection and delivery of the drug product which extends QbD practices to the development and manufacture of containment and delivery systems.

3:00 pm Closing remarks / Conference adjourns

 

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