SUNDAY, MARCH 2nd Title: Introduction to Chromatography of Proteins, Peptides, and Related Molecules Name: Thomas Wheat Date: 08:30 AM - 05:00 PM Number: 39 Type: Short Course This full day program is an introduction to the separation techniques used to characterize biological macromolecules, with an emphasis on practical applications. The most useful modes for protein separations will be emphasized, and substantial description of peptide mapping will be presented. Topics include theory and principles of the separations. Rationale for choosing a technique will be systematically considered. Strategies for methods development will be presented. Analytical techniques that are often used in the same laboratories, including amino acid, glycan, and oligonucleotide analysis, will be introduced. The role of mass spectrometry in the analytical biochemistry laboratory will be described. Title: Lipidomic Profiling Using Sub-2µm Particle CO2 Based Supercritical Chromatography Mass Spectrometry Name: Giorgis Isaac Time: 1:30PM, Session 200, Location: S503b Type: Oral The typical chromatographic methods for analyzing fatty acids and neutral lipids are gas chromatography after derivitization and liquid chromatography-tandem mass spectrometry (LC/MS/MS). However, there are shortcomings associated with each of these methods. For example, GC methods require derivatization of the fatty acids to methyl esters (FAME), which is burdensome, time consuming, and there is a risk of re-arrangement of the fatty acids during derivitization which leaves doubt as to whether the esters formed are from free fatty acids or intact complex lipids. In LC/MS/MS methods, the runs typically involve labor intensive and time consuming sample preparation, and utilize toxic solvents, which are expensive to purchase and dispose. We have developed rapid (5min), high throughput and efficient method for the separation and analysis of free fatty acids and neutral lipids without derivitization using sub-2µm particle CO2 based supercritical chromatography. The organic extract from the matrix containing lipids is directly injected onto the system showing a significant saving in solvent, cost and sample preparation time. The separation mechanism is mainly based on the number of carbon chains and the number of double bonds on the acyl chain. The datasets were processed using TransOmics Informatics for Metabolomics and Lipidomics a new software tool that provides automatic peak detection followed a quantitative comparison and statistical analysis to differentiate those features that are significantly changing and finally identify those features from the mass spectrometry data. Title: Displacement Separations in SFC for Analytical and Prep Scale (Chiral and Non-Chiral) Name: John Whelan Time: 2:30 PM , Session #250, Location S503b,Type:Oral One of the advantages of using Supercritical Fluid Chromatography for large scale separations is the ability to quickly separate chiral compounds with low solvent use and faster dry down times[1]. In the past many chiral/non-chiral presentations have been done, but typically with only a baseline separation being used as an example in the area of SFC. Contained in this study we have demonstrated Displacement separations and the issues

involved. The body of work will include Retention Time RSD% and effects of slight co-solvent/modifier changes with regards to retention time correlated to the degree of displacement with comparison to recovery yield. Displacement chromatography has been discussed in the past [2] attempts at achieving practical true displacement SFC separation will be discussed. INTRODUCTION Chromatography is now a prominent tool at the forefront of chemical and biological developments to improve the human condition. Supercritical chromatography is recognized as a green technology impacting industries across the globe that enhances the preservation of natural resources, reduces waste generation and offers alternatives to fossil fuel-based infrastructures. Supercritical fluid chromatography (SFC) systems transforms CO₂ into a supercritical fluid. Originally developed in the 1960s, it regained popularity in the late 1990s. As a supercritical fluid, CO₂ demonstrates hydrocarbon-like properties under chromatographic conditions [4]. By substituting petroleum-based organic solvents with CO2 in chromatography, greenhouse gas emissions and overall solvent pollution are significantly reduced. CO2 is inert, safe and economical. Applications that have had a good record of success and visibility are chiral based separations that are used for large scale campaigns [5] and the throughput of SFC has been proven to be advantageous [6] elsewhere. Title: Method Development for Chiral Separations Using Analytical Scale Supercritical Fluid Chromatography Name: Thomas Swann Time: 3:05 PM, Session # 250, Location: S505a Type: Oral To comply with FDA mandates on developing chiral drugs, the pharmaceutical industry often investigates enantiomeric separations early in the drug discovery process. In addition, chiral analysis is important in the production of specialty chemicals such as flavors, fragrances and pesticides (agrochemicals). Developing chromatographic methods for chiral compounds is typically performed by either normal phase LC or supercritical fluid chromatography (SFC). In either case, stationary phases with chiral selectors are screened and the separation is optimized to separate the enantiomers. Due to the unpredictability of the separation, method development times can be time consuming and costly. The use of analytical scale SFC will be presented for the development of chiral separations. The use of a low dispersion SFC instrumentation in combination with small particle (≤ 3 µm) columns packed in smaller diameter columns (≤ 3 mm) facilitates rapid development of chiral separations. We will present the critical factors for resolution of enantiomeric compounds, including different co-solvents and co-solvent mixtures, additives to the mobile phase, column length, temperature, and pressure. In addition, we will demonstrate the use of analytical SFC with multiple detection methods, most notably mass spectrometry, to further speed up method development times for chiral compounds. Applications such as in vivo stereoselective metabolism studies, analysis of chiral pesticide formulations, and chemical synthesis reaction monitoring will be highlighted. Title: Development of a High Throughput Integrated, Multi-Disciplinary “Omics” Platform to Support Basic Research Into Disease Understanding and Patient Stratification Name: Rob Plumb Time: 3:25PM, Session # 200, Room S503b, Type: Oral Presentation

The MRC-NIHR National Phenome Centre,Imperial College London, is the first of its kind facility. Born out of the UK Olympic Legacy its mandate is to provide “high throughput, forensic quality, metabolic phenotyping to support large scale epidemiological studies as well as basic medical research into disease understanding and patient stratification”. As global life-styles change we are seeing increasing cases of obesity, diabetes, and mental health issues. This not only affects a person’s quality of life but also places increased strain on the health-care systems to provide the right treatment whilst managing costs closely. Metabolic Phenotyping offers a valuable and unique insight into the underlying biochemistry of diseases as well as the patients individual biochemistry “phenotype’, diet, health status, age and stress. To deliver this information the analytical data generated in processed via a variety of chemometric modelling and analysis methodologies to deliver the relevant biochemical information. These chemometric platforms employed vary from simple multivariant analysis to highly complex model based analysis and is presented in a format ready for interpretation by medics. This facility comprises of high field NMR instruments, accurate mass LC/MS instruments, tandem quadrupole LC/MS systems as well as dedicated training facility. In this presentation we will discuss the development of analytical platforms both LC/MS and NMR as well as a detailed discussion on the workflow, validation, reporting and decision making process. The presentation will cover the development and validation of the “discovery’ screening methods for polar, non polar metabolites and lipid profiling usingLC/MS methodology, as well asdescribe the use of proton NMR as an initial screen to eliminate contaminated samples. The quantitative targeted LC/MS assays will also be discussed the various compounds classes such as bile acids, amino acids, eicosanoids, and acyl cartanines. Title: SFC Modifier and Combined Stream Injection Modes, and Sample Diluent Effects Name: Steven Zulli Time: 4:05 PM, Session # 260, Location: S504bc Type: Oral Sample injectors for SFC systems can be characterized into two techniques, modifier (co-solvent) and combined stream injection modes. With modifier stream injections, only the co-solvent flows through the injector; whereas, in combined stream, the entire mobile phase flows through the injector. Each technique has potential advantages and disadvantages. Modifier stream mode often exhibits less of a sample diluent effect on a separation, the perturbation of the mobile phase strength that occurs with the introduction of the sample at the head of the column. Thus, it is often the preferred technique for semi-prep and prep scale purifications. A disadvantage of modifier stream mode is the slower sample loop sweep flow rate. Combined stream mode does not have this lower loop sweep flow rate disadvantage, and is often the preferred technique for analytical scale separations. For separations performed at very low co-solvent percentages, combined stream can be advantageous for semi-prep and prep scale purifications. A disadvantage of combined stream mode is that the sample diluent’s negative effect on a separation can be greater than that in modifier stream. Discussed are the implications in each injection mode of the sample diluent effect, including sample loop and column size, and loop sweep flow rate, and the potential effects on SFC stacked injections. This study was performed on SFC instruments capable of analytical,

semi-prep and prep scale injections in both modifier and combined stream modes under otherwise identical conditions. Presented are separations exemplifying the topics discussed, demonstrating the advantages and disadvantages of each injection mode. MONDAY, MARCH 3rd Title: A Refractive Index Detector for UPLC Name: Patricia McConville Session 570, Location: Expo Floor, Back of aisles 1000-2500, Type: Poster UPLC is a separation technique that employs analytical LC columns packed with sub-2μm particles and an LC system which has been holistically designed to maximize the separating power of those columns. The UPLC technique has become increasingly popular due to the greatly enhanced separations power and shorter analysis times it can deliver. This has led to an interest in extending UPLC to those applications which employ alternative detection to the commonly used UV/PDA, MS and ELSD. Refractive Index (RI) is a robust device that approaches the ideal of universal detection. A chromophore is not required; detection of compounds eluted from a column is based upon the difference in refractive index between the analyte and the background mobile phase. In many laboratories, RI is the detector of choice when analyzing molecules lacking a chromophore because it is simple to use and needs no equilibration between injections. There is no need for a clean nitrogen supply as is required for ELSD, which provides an economic benefit and is ideal for a manufacturing facility. RI detection provides excellent repeatability and shows a linear response for quantification. RI detectors measure the differential refraction between a sample flow cell and a static reference cell filled with mobile phase and have the particular challenge in that anything that can create changes in mobile phase density, and thereby the refractive index of the mobile phase as it passes through the sample cell, must be carefully controlled. A Refractive Index Detector suitable for UPLC separations must maintain the fidelity of the chromatographic bands generated by the UPLC; this requires a significant reduction (X10) in both tubing and cell volume while maintaining thermal stability. HPLC/RI applications from food sugars, saccharides, pharmaceutical QC, and polymer analysis are transferred to UPLC/RI with significant gains in assay through put and resolution. Title: Investigations on Prep Supercritical Fluid Chromatography Concentrating on Overall System Performance and Its Correlation to CO2 Recycling Operation and Efficiency Name: John Whelan Time: 10:45 AM, Session #490, Location: S505a Type: Oral Supercritical Fluid Chromatography(SFC) is a useful tool for analytical and large scale separations. SFC is most often used with carbon dioxide as a mobile phase and an organic modifier such as some type of organic alcohol. It has some significant advantages over standard HPLC methods such as less pressure drop across the columns, faster column equilibration, faster method development, higher efficiency separations and significantly less generation of hazardous waste. Some of the main advantages for preparatory chromatography include solvent waste reduction, facilitated product recovery, lower solvent cost and the possibility for recycling. One major advantage of using Supercritical Fluid Chromatography for large scale separations is the ability to recover the CO2 and recycle it concurrently with executing the process. Contained in this study we have demonstrated CO2 recycling with multi-compound mixtures and Chiral compounds that yield two or more peaks for overnight periods. In using multi-compound and chiral mixtures a more accurate picture

of the capability of the Recycler being used is portrayed. The body or work will include Retention Time RSD% and effects of slight co-sovlent/modifier changes with regards to retention time. Recycle efficiency for the methods and conditions used in the multi-component tests will also be discussed. Our data will show the high recycling efficiency(RE) with the designed system and excellent chromatographic performance for the separations. Recovery data will be included as a validation of the results of the testing. Title: Analysis of Multiple Pesticides by Supercritical Fluid Chromatography/Tandem Mass Spectrometry with a Sub-2 Micron Particle Column - A Feasibility Study Name: Jinchuan Yang Time: Monday, Session #570, Location: Exposition Floor, Back of Aisles 1000-2500 Type: Poster GC-MS and LC-MS are common techniques for pesticide residue analysis and both are required due to the wide range of physiochemical properties of pesticides. It has been reported that SFC can be used as a single separation technique for the simultaneous analysis of LC-amenable and GC-amenable pesticides (1). This unique SFC approach provides a simple and convenient solution for multiple pesticide residue analysis. With the new development of UltraPerformance Convergence Chromatograpy (UPC2), its applicability and potential advantage in multiple pesticide residue analysis has been investigated. UPC2 is a separation technique that uses compressed carbon dioxide as the primary mobile phase. It takes advantage of the unique physical properties of compressed carbon dioxide (at or near supercritical state), sub-two micron particle chromatography columns and an advanced chromatography system design to achieve unique selectivity, high efficiencies and speed. In this work, we present a feasibility study using UPC2 with ultra sensitive tandem quadrupole MS/MS detection for 18 pesticides with a wide range of polarities (logPow=-4.6 to 7.1) and molecular weights (112-889). This study focused on UPC2 separation, interfacing with MS, and MS/MS detection. The sensitivity and repeatability of the multi-pesticide residue analysis in the presence of common food matrices, such as spinach, wheat flour, and apple juice will be presented. Title: Efficient Methods Development Combing Simultaneous Mass and UV Detection with Flexible Software for Mobile Phase Formulation Name: Paula Hong Session# 570, Location: - Exposition Floor, Back of Aisles 1000-2500 Type: Poster Methods development for reversed-phase liquid chromatographic (RPLC) separations typically requires many time-consuming steps, including manual preparation and pH adjustment of mobile phases, as well as extensive data processing. Also a single detection technique provides insufficient information for missed peaks and co-elutions: Isobaric compounds can be difficult to distinguish with a mass detector alone, while peak identification with UV is not possible for compounds that lack a chromophore. To address some of these challenges, multiple detectors can be used for analysis of a single sample with each detection technique dependent on a different physical or chemical property of the molecule. In this presentation, we will describe an efficient methods development approach that combines both dual detection and automated software for mobile formulation. This strategy will evaluate a variety of factors including mobile phase pH, organic solvent, temperature, stationary phase and physical parameters for a systematic method development approach.

Manipulation of mobile phase pH will be demonstrated through the use of flexible software, while a comprehensive software for simultaneous analysis of both mass and UV spectral data will allow for simplified data processing. The effect of these physical and chemical parameters will be illustrated with both mass and UV spectral data for peak identification. The benefits of peak tracking with combination of mass and UV spectra will be demonstrated for a variety of compounds including natural products and pharmaceuticals. Title: Optimizing Resolution in Reversed-Phase UPLC Methods Development with Automatic pH Selection Name: Aparna Chavali Session# 570, Location: - Exposition Floor, Back of Aisles 1000-2500 Type: Poster Retention in reversed-phase chromatography can show significant changes with slight variations in pH. The adjustment of pH can also or similarly serve as a very important and useful screening tool to adjust retention and selectivity. Choosing the optimum pH is difficult for a multi-component sample representing a wide range of properties because we cannot predict optimum separation pH. We have developed an efficient automation of this screening process by integrating the blending of stock buffers by the liquid chromatography system to deliver mobile phases of different pH and ionic strength. To extend the utility of automated blending of stock buffers by the system to compounds with different functional groups often requires selection of a different buffer system to cover a different pK range. We have developed a protocol that can be applied to unknown compounds exploiting combinations of buffers. Such a multi-buffer system can be used in different applications such as impurity profiling, stability testing and can be used with unknown compounds that have a mix of analytes sensitive to different ranges in the pH spectrum. We have developed and validated two buffer systems: one consisting of non-volatile buffer components for methods development of reversed-phase LC separations and one consisting of volatile buffer components for LC-MS methods development. These two buffer systems provide buffering capacity over a wide pH range. Using these two validated buffer systems, we demonstrate the effect of pH change on chromatographic properties of acidic, basic and neutral compound mixtures. This systematic and automated screening protocol using a single defined buffer system will prove to be useful for reversed-phase method development. Title: Pharmaceutical Applications of Sub-2-µm, Solid-Core Particle Columns Name: Kenneth Berthelette Session #840 Location: - Exposition Floor, Back of Aisles 1000-2500 Type: Poster Separations for pharmaceutical applications can always benefit from improved chromatographic performance. Whether impurity profiling a drug product, identifying a target compound in a complex sample matrix, separating components in a synthetic reaction mixture or finding a new pharmaceutical compound in a discovery setting, the ability to quickly resolve and identify peaks in a mixture is a powerful tool in advancing discovery or development workflows. New developments in column technology provide chromatographers with an added dimension to increase the peak capacity of their separations. Gains in column efficiency and thereby separation performance can enable chromatographers to resolve and identify desired components more quickly, potentially lessening the need for further method development. Additionally, the increased robustness of newly developed columns enable separations that currently have sufficient resolution to be run at higher flow rates while maintaining the performance of the original separation. Increased flow rates allow

significantly faster runtimes and higher sample throughput, resulting in efficient use of instrument time and overall resource cost savings in the lab. In this poster, we demonstrate the performance gains of sub-2-µm, solid-core particle columns for pharmaceutical applications, including impurity profiling, forced degradation, and natural product analysis. Title: Applications of Sub-2-[micro]m Solid-Core Particle Columns Name: Kenneth Fountain Time: 2:05 PM, Session # 700, Location: S503b Type: Workshops Advances in chromatographic packing materials and instrumentation have led to the use of smaller particles packed into smaller diameter (2.1 and 3.0 mm) columns to achieve faster and more efficient separations. In order to realize the full benefit of these columns, it is critical to understand the role that extra-column dispersion plays to provide the highest separation performance per unit time. In addition, new generations of solid-core particles have allowed unprecedented increases in throughput, thus expanding the number of applications possible in all areas of separations science. In this presentation, we will briefly discuss the impact of LC instrumentation on the performance of these new sub-2-µm, solid-core packing materials. A majority of the presentation will be spent on the types of applications that benefit from these advances, most notably in drug discovery and development, bioanalysis, food and environmental applications, and forensic toxicology. Title: The Science Behind a New Generation of SFC Stationary Phases Name: Jacob Fairchild Time: 3:05 PM, Session # 790, Location: S504bc Type: Oral In pursuit of wider selectivity envelopes and increased retention for Supercritical Fluid Chromatography (SFC) separations, we have developed a series of new bonding chemistries. These next generation SFC stationary phases are based upon functionalizing hybrid particles with novel bondings containing judiciously chosen selectors, which significantly improve peak shapes, reducing the need for mobile phase additives. In addition, these bondings significantly increase retention and limit unwanted interaction between analytes, mobile phase and the base particle. Such interactions have led to observed retention losses of >15% over several weeks for the previous generation of chromatographic materials, significantly reducing reproducibility in SFC separations. In the past, this was attributed to instrumentation, but high performance SFC systems have shed new light on the source of this variation. Our next generation SFC stationary phases effectively eliminate these retention losses. Over a twenty day period, a 2-ethylpyridine bonded hybrid material had an average retention loss of 13.2% for five probes (Fig. 1). This was reduced to a 0.3% average loss in retention for the new materials over a similar time frame. By reducing retention variance over time and maximizing selectivity with simple co-solvents, our new generation of stationary phases will facilitate robust analytical methods. Attributes and mechanisms of these novel stationary phases will be discussed and explained regarding selectivity, retention and peak shape for basic compounds. Title: Investigating Triple Detection Combined with Ultra Performance Convergence Chromatography for Profiling of Natural Products Name: Paula Hong Time: 3:25 PM, Session # 790, Location: S504bc Type: Oral Convergence chromatography (CC) is a separation technique that utilizes compressed carbon dioxide (CO2) as the primary mobile phase component to achieve unique selectivity,

low solvent usage and high efficiencies. The use of supercritical fluid as a mobile phase provides higher diffusivity and lower viscosity than liquid mobile phases, thereby providing higher throughput and chromatographic efficiencies as compared to liquid chromatography. Convergence Chromatography provides high efficiency separations which are beneficial for the analysis of complex samples, such as natural products. However, identification and confirmation of the numerous components found in natural products can be challenging. To address this challenge by LC, multiple detectors are typically used during a single analysis whereas each detection technique is based on a different physical or chemical property of the molecule. For example, mass detectors and PDA are commonly combined to obtain both mass and UV-spectral information. Evaporative light scattering, a more universal technique, addresses compounds without ultraviolet absorbance (no chromophore) and poor ionization by MS. The combination of these three detection techniques allows for analysis of a wide range of compounds. In this presentation, we will investigate the analysis of a number of natural products using triple detection in combination with CC. Identification and quantitation of compounds will also be illustrated. Guidance combining triple detection with CC will be provided based on the observations attained throughout the analysis. This approach when combined with sub-2 µm column chemistries will allow for a the analysis of a wide range of compounds by convergence chromatography. TUESDAY, MARCH 4th Title: Ultra-Sensitive Simultaneous LC-MS/MS Quantification of Human Insulin, Glargine, Lispro, Aspart, Detemir and Glulisine in Human Plasma Using 2D-LC and a Novel High Efficiency Column Name: Erin Chambers Time: 8:30 AM, Session# 1030, Location: S502b Type: Oral For quantification of biotherapeutics LC-MS/MS has advantages in short development times, high accuracy and precision, multiplexing, no cross-reactivity, and facile distinction between closely related analogs. Intact insulins are difficult to analyze by LC-MS/MS, as MS sensitivity is low and insulin and its analogs suffer from non-specific binding and poor solubility, making LC and sample preparation method development difficult. A few LC-MS/MS methods exist. Most involve time-consuming and laborious immunoaffinity purification or nano-flow LC. This work provides a simple method for the simultaneous quantification of intact human insulin and 5 critical analogs in human plasma, achieving LODs of 50-100 pg/mL for each. Average accuracy and precision for standard curve and QC’s samples were 90-95%. Matrix factors for all analogs were calculated in 6 sources of human plasma and CV’s were <15% in all cases, supporting the selectivity of the method. A specific challenge when quantifying insulins is the ability to distinguish between human insulin and insulin Lispro, as they differ by a reversal in the position of 2 amino acids. Only a single low molecular weight fragment differentiates the two, making selective sample prep and chromatography critical. This methodology takes advantage of mixed-mode SPE to eliminate interferences and a novel ultra-high efficiency charged surface column which produces narrow peak widths and improved mass transfer to facilitate high sensitivity quantification. Selectivity studies show that the presence of high levels of human insulin (i.e., type II diabetes), does not interfere with quantification of lispro or any of the other analogs. Title: Development of and Applications for a Ceramic Microfluidic UHPLC System Name: James Murphy

Time: 9:10 AM, Session # 970, Location: S503a Type: Symposia Microfluidic technology has made significant inroads in analytical chemistry, with systems for analyzing a wide variety of samples and compounds being reported. Sample concentration and other sample preparation steps, reaction monitoring, immunoassays, sensor technologies and separations methods have all been reduced to practice in microfluidic formats. With regards to separation technologies, however, the bulk of these have been based on capillary electrophoresis, with the more widely applicable HPLC systems comprising a greater challenge for designing high performing devices. Over the past several years we have been developing an ultra high pressure capable microfluidic HPLC system based on high temperature co-fired ceramics with performance equivalent to standard packed capillary columns, yet offering the advantages of a microfluidics format. The optimized system operates at pressures up to 10 kpsi, and can be packed with a variety of stationary phases. Sample loading can be via direct injection or with a custom designed trapping system. Device cartridges can be readily changed without using tools and have built-in electronics for temperature control and other functions. Prototype devices have been particularly useful for high sensitivity peptide analysis including biomarker identification, analysis of bioactive peptides in biofluids and protein bioanalysis via signature peptide quantification. Lipidomics and other metabolomics analyses have also proven to be highly advantageous with the microfluidic system. These assays and other small and large molecule applications will be described to illustrate key system attributes. Title: Bioanalysis of Teriparatide Using a Prototype 150 µm ID Micro-Fluidic Device Name: Erin Chambers Time: 10:25 AM, Session # 1030, Location: S502b Type: Oral Improving sensitivity using small sample volumes is particularly important in the bioanalysis of peptides and proteins. Historically this work was done by various ligand binding assays (LBAs) which are characterized by very high sensitivity (low fmol/mL or lower) and utilize only a small volume of sample, several µL or less. LBAs suffer some significant disadvantages though, including lack of standardization, poor linear dynamic range, variability and availability of reagents, and long development times. These disadvantages led to the desire to use LC/MS in this research area. However, obtaining equivalent sensitivity and sample usage to LBAs has been challenging. Nano and microscale LC/MS offers significant sensitivity advantages in supporting microsampling, biomarker discovery, and bioanalysis of biomolecules. In this work, prototype 150 µm i.d., 50 mm microfluidic devices packed with charged-surface 1.7µm (both fully porous and solid core particles) were evaluated for the high sensitivity analysis of the peptide teriparatide. Teriparatide is a 4118 MW fragment of human parathyroid hormone, used to treat osteoporosis. A multi-step sample preparation method using protein precipitation followed by solid phase extraction selectively separates teriparatide from closely related endogenous peptides and reduces interferences. Samples were concentrated 6X, without evaporation, minimizing the risk of adsorptive losses. Samples were run with injection volumes from 1 to 5 uL (approximately 200 to 1000 µL on a 2.1 mm i.d. column) under linear gradient conditions after trap and back elute on a 300 µm x 25 mm C18 trapping column. The resultant LC/MS method is equivalent to LBAs without its disadvantages. Title: Determination of Paraquat and Diquat in Environmental Samples Using a Sub-2-µm, Solid-core Particle HILIC Column Name: Kenneth Fountain Session # 1380, Location: - Exposition Floor, Back of Aisles 1000-2500 Type: Poster

The doubly charged quaternary ammonium compounds paraquat and diquat (quats) are common herbicides used worldwide. These compounds are commonly determined in environmental water samples by ion-pairing reversed-phase liquid chromatography with UV detection. However, the ion-pairing reagents used are not usually suitable for LC-MS analysis. In recent years, HILIC-based LC methods have been demonstrated that require no ion-pairing, and thus are more suitable for LC-MS analysis. However, the HILIC approach has not been widely adopted for UV detection because baseline separation of paraquat and diquat is difficult. In this poster, we demonstrate the unique selectivity and efficiency of a sub-2-µm, solid-core particle HILIC column for baseline separation of diquat and paraquat. This column can be used for LC with either UV or mass spectral detection. In this study, both means of detection were used independently or in series. With the mass spectrometer, detection limits well below 100 ng/L (ppt) were easily achieved for tap-water samples. Using UV detection, detection limits below 500 ng/L were achieved. The samples (10 mL) were processed using a mixed mode weak cation exchange SPE cartridge prior to LC analysis. Title: Fast Quantitative Analysis of Astaxanthin in Dietary Supplements Derived from Haematococcus Pluvailis by UPC2 – UV Name: Jacquelyn Runco Session 1100, Location: - Exposition Floor, Back of Aisles 1000-2500 Type: Poster Astaxanthin is a xanthophyll carotenoid found abundantly in Haematococcus pluvialis algae. Due to its high antioxidant activity, it is widely used in food, cosmetics and dietary supplements. Currently, quantitative analysis of astaxanthin employs spectrophometric or HPLC methods. The spectrophotometric method, however, often suffers from low accuracy; while the HPLC methods require the use of complex solvent systems and long analysis time due to the low polarity of the analyte. With a growing industry-wide focus on monitoring micro-nutrients in fortified foods and supplements for regulatory compliance, there is an increasing demand for rapid and reliable analytical methods for the quantitative analysis of astaxanthin. Due to non-polar CO2 being the primary constituent of the mobile phase, UltraPerformance Convergence ChromatographyTM (UPC2) becomes a logical choice for improving analysis time. In this poster, a systematic study on developing a fast and reliable UPC2 method for astaxanthin quantitation will be presented. With the proper choice of column, mobile phase, and temperature, a 2-min UPC2 method was developed for the quantitative analyses of different astaxanthin supplements. The UPC2 method was also compared to a HPLC method in efficiency, speed, accuracy and reproducibility. Our results suggest that UPC2 shows great promise in becoming a routine analytical technique for quantitative analysis of astaxanthin and fat-soluble carotenoids in general. Title: Fraction Collection Using Sub 2 [micro]m UHPLC Separations: Challenges and Solutions Name: Andrew Aubin Session # 1370, Location: - Exposition Floor, Back of Aisles 1000-2500 Type: Poster Liquid chromatography has benefited from the increased separation performance of sub-2-µm column technology along with low dispersion instrumentation that produces sharp, narrow, and more concentrated peaks. When there is a desire to collect these small pure peaks, especially from complex mixtures, traditional fraction collection instrumentation designed for preparative HPLC conditions does not provide an adequate solution. Issues with excessive internal volumes, which can cause peak dispersion, slow collection to collection vessel movement, and collection to collection carry over are not uncommon.

To overcome these traditional limitations, an ideal solution to this problem is a purposefully built UHPLC scale fraction collector. In this poster, we will discuss some of the challenges of fraction collection at this scale along with some innovative solutions for collection of peaks that are often between 1-5 seconds wide with peak volumes that can range from 2 µL to 200 µL, and larger using column flow rates that can range from 100 µL/min to 2 mL/min that must be accommodated. Solutions for very fast collector movement to minimize fraction loss, reduced carryover, maintenance of peak integrity between the detector and collection vessel to better manage low volume peaks will also be discussed. Title: Isolation of a Bioactive Compound from Tillandsia recurvata Plant Extract Using Supercritical Fluid Extraction and Mass Directed Preparative Liquid and Supercritical Fluid Chromatography Name: John McCauley Session # 1370, Location: - Exposition Floor, Back of Aisles 1000-2500 Type: Poster Natural products are a rich source of diverse chemical entities and widely used in nutraceutical, pharmaceutical and related industries. The challenges in isolating the bioactives from natural products often arise from the overwhelming complexity of the sample matrix and the wide dynamic range of the components. In this poster, we present a case study of using supercritical fluid extraction, mass directed preparative high performance liquid chromatography (HPLC), and mass directed supercritical fluid chromatography (SFC) to isolate a bioactive compound from Tillandsia recurvata plant extract. The Tillandsia recurvata plant material was first extracted by SFE. The analytical profiling of the plant SFE extract by UPLC/MS and UPC2/MS revealed a desirable orthogonality between the two chromatographic techniques in selectivity, manifested in both resolution and elution order for the peaks of interest. Leveraging the orthogonality between the two techniques, a two-step purification process using mass-directed prep LC followed by mass-directed prep SFC, was subsequently implemented, resulting in our obtaining the pristine compound in a form suitable for further bioanalysis. The purified target was also subjected to HRMS, MSE and NMR analyses, and the complete structure of the compound was successfully elucidated. The proposed workflow is generally applicable for high-efficiency natural product isolation and purification in the pharmaceutical, nutraceutical and related industries. Title: Matrix Specific Sample Preparation Strategies for Opioid Analysis Name: Jonathan Danaceau Session # 1090, Location: - Exposition Floor, Back of Aisles 1000-2500 Type: Poster The analysis of natural and synthetic opioid drugs continues to be an important area of clinical research. Depending on the application, these drugs may be analyzed in urine, blood or, increasingly, oral fluid. These matrices each have individual challenges which need to be overcome. The methods presented herein describe strategies for sample preparation in each of these three matrices followed by revered-phase UHPLC/MS/MS analysis. Using a silica-hybrid UHPLC column, all analytes eluted in less than 5.5 minutes, and even the most polar compounds were adequately retained. This allowed for direct analysis of glucuronide metabolites, eliminating the need for enzymatic hydrolysis. Urine and oral fluid samples were both prepared using mixed-mode strong cation exchange solid phase extraction (SPE). Whole blood samples were prepared using phospholipid removal plates which allowed for cell lysis and protein precipitation within a single well, followed by vacuum elution into a 96-well collection plate. For urine samples, mixed-mode SPE prepared samples demonstrated reduced matrix effects, and improved sensitivity, linearity, accuracy and precision compared to samples prepared by a simple dilution

protocol. Oral fluid samples prepared by mixed-mode SPE also had excellent linearity and quantitative performance. In addition, the μElution format was ideal for the limited sample volumes often encountered with oral fluid samples. The phospholipid removal plates used for whole blood processing enabled rapid cleanup and processing with minimal need for method development. This represents a comprehensive sample preparation and analysis strategy for this important group of compounds in three important matrices. Disclaimer: This method is intended for clinical research use only, not for use in diagnostic procedures Title: Information Rich Orthogonal Detection to Provide More Complete Characterization of an USP Assay Name: Aparna Chavali Time: 1:30 PM, Session # 1330, Location: S502b Type: Oral HPLC is one of the most widely applied analytical techniques. It can serve as a powerful separation technique for chemical mixtures consisting of a wide range of compounds including isomers. HPLC with its choice of detection techniques can separate the analytes but provides little information about what a compound might be. The photodiode array can provide some information about peak purity or presence of coeluting peaks but can only provide identification by comparing to standards in the same mobile phase. Adding a mass detector to a HPLC-PDA system can provide information for peak identification, for recognizing coeluting chromatographic peaks and for confirming peak purity. A mass detector, however, cannot provide complete sample characterization by itself. It may not prove useful in distinguishing isomers or compounds that either poorly ionize or do not ionize under the selected conditions. Components present in trace levels may go undetected against a more abundant background and ion suppression may affect detection of coeluting peaks. We have employed orthogonal detection techniques to help fully characterize a USP tablet following the monograph guidelines for sample preparation. The combination of the chromatographic configurations including the HPLC system with a diode array detector, a mass detector and software tools to combine data from these orthogonal detectors provides information to enable a more thorough characterization of the sample. Title: Automated Multimodal Chromatographic Method Development Integrating Complementary Optical and Mass Spectral Detection Name: Daniel Root Time: 2:30 PM, Session # 1330, Location: S502b Type: Oral The characteristics of the components within a sample determine the complexity of chromatographic methods development. Often, two of the largest problems are managing the range of chromatographic behavior found in a typical formulation and, second, ensuring peak identification and quantitation for a range of components with different detection properties. The analysis of a common nutraceutical supplement containing glucosamine HCl and vitamin D3 serves as an example. This formulated product has active ingredients that are exceedingly polar and exceedingly non-polar. Additionally, these ingredients span the range from strong UV absorbance to complete transparency. Highly polar sample components, like glucosamine, will not retain well in reversed phase (RP) mode. Hydrophilic interaction chromatography (HILIC) has been found to be a more effective approach. The exclusive nature of the mobile phases, wash solvents and columns associated with HILIC makes running both modes a challenge to methods development. When components have no UV response, sample detection also becomes a challenge. Most often, a mass spectrometer (MS) is added to augment UV detection. Adding a MS, as well as managing the different data acquisition and processing needs of the detectors, further complicates

method development. A chromatographic system performing both RP and HILIC modes in a single set-up, with integrated optical and mass spectral detection is used to develop methods for the analysis of glucosamine and vitamin D3 from a nutraceutical supplement. The system software allowed automation of instrument operation and harmonized the acquisition and processing of both the optical and mass spectral data. Title: Analyses of Fat-Soluble Vitamins, Carotenoids and Lipids by Supercritical Fluid Chromatography with Sub-2µm Particle Columns Name: Jinchuan Yang Time: 3:05 PM, Session # 1360, Location: S505a Type: Oral UltraPerformance Convergence Chromatography (UPC2) is a separation technique that uses compressed carbon dioxide as the primary mobile phase. It takes advantage of the unique physical properties of compressed carbon dioxide (at or near supercritical state), sub-two micron particle chromatography columns and advanced chromatography system design to achieve fast and reproducible separation with high efficiencies and unique selectivity. These improvements lead to new interest in applying this technology to various industrial analytical areas, especially those areas where normal-phase liquid chromatography (NP LC) has been commonly used, such as fat-soluble vitamins (FSV), carotenoids, and lipids. Nine representative FSV and carotenoids have been successfully separated simultaneously by UPC2 within four minutes on a single C18 column. These FSV and carotenoids include vitamin A acetate and palmitate, alpha-tecopherol and its acetate, vitamin D2, vitamin K1 and K2 (MK4), beta-carotene and lycopene. The repeatability (n=6) of all the nine compounds was less than 0.25% in retention times (RT) and less than 2.6% in peak areas. The investigation of lipids separation by UPC2 showed that Bridged Ethylene Hybrid (BEHTM) silica columns provided the best separation of lipid classes among the Flouro-Phenyl, 2-EP, and BEH UPC2columns. The lipid classes investigated include ceramides, sphingomyelin, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, lyso-phosphatidylcholine and lyso-phosphatidylethanolamine. The UPC2 has been applied to biological samples and showed successful separation of lipid classes. Separation and analysis of free fatty acids and neutral lipids was also developed. These results indicate that UPC2 is a promising chromatographic technique for FSV, carotenoids and lipids analyses. Title: Orthogonal Detection Techniques for the Identification and Confirmation of Impurities Using an USP Chromatographic Method Name: Aparna Chavali Time: 3:05 PM, Session # 1330, Location: S502b Type: Oral When developing methods for an unknown sample, long separation methods with high resolution are developed to ensure that all components of the sample are separated. This approach has two desired outcomes. First, all the components are separated with no coelutions, and, second, all important impurities are detected. In reality, neither objective is met with certainty. We have approached this complication by combining two orthogonal detectors that are sensitive to different physical principles. This facilitates recognizing coelutions and for more completely detecting the components of the sample. This system includes the tools necessary for adjusting the separations if any coelutions are recognized. With complete separation and orthogonal detection, it is possible to construct libraries describing all the sample components and impurities by their retention time and spectral properties. In the course of analyzing an authentic sample, any peak can then be confirmed as a member of this library and the amount can be compared to specified limits. This workflow will be applied to an assay, based on an USP method of extracted tablet where many excipients and related compounds are present. The library will be used to confirm or

exclude the presence of impurities. This will be based on confirming the mass, retention time and UV spectra of these impurities. Title: Development and Evaluation of a Chromatographic System Combining UV and MS Detection Used in Separation Development Name: Thomas Wheat Time: 3:45 PM, Session # 1330, Location: S502b Type: Oral Liquid chromatography is used as an analytical technique to provide qualitative and quantitative sample composition. Development of a suitable chromatographic method can be a complex process that exploits many physical and chemical parameters. With changes in chromatographic variables, the sample components may change their elution order. This adds uncertainty to systematic method development in that comparisons of different conditions may not accurately track each sample component and coelutions may go unrecognized. A chromatographic system for separations development should provide both efficient exploration of separation space and complete detection for assessment of the results. For the separation, the system includes an automated screening of different columns, as has been routine for some time. Selectivity changes derived from mobile phase manipulation have been harder to define and automate. It is cumbersome and time-consuming to prepare the combinations of solvents and pH/ionic strength modifiers. Instrumental functions have been combined with solvent management algorithms to permit continuous adjustment of pH while using different organic solvents. These functions are combined with column screening and variations in physical operating parameters to fully explore separation space. These experiments, however, require a way to track the individual components within the chromatogram. Here, the combination of photodiode array and mass detectors is considered as an approach to complete sample characterization. The complementary data sets from the two detectors are combined using new software tools that can aid peak tracking and the assessment of peak homogeneity. The combination of mobile phase manipulation with information-rich component identification and peak tracking in a unified UPLC system makes chromatographic method development more complete and efficient. Title: Screening Environmental Samples for a Diverse Range of Compound Classes and Structures with Accurate Mass LC-MS and an Integrated Scientific Information System Name: Kenneth Rosnack Session # 1300, Location: - Exposition Floor, Back of Aisles 1000-2500 Type: Poster The presence of an increasingly complex array of pharmaceuticals and personal care products (PPCPs) in water bodies throughout the world is placing a greater demand on techniques used to screen for these compounds. Using a high resolution accurate mass ToF screening technique employing MSE where high and low energy switching enables the acquisition of precursor and fragments ion in a single injection, provides information rich data that can be used to reduce the large number of false detects that arise from the use of accurate mass alone. In combination with an integrated scientific information system, the ability to screen for the presence of PPCPs, their adducts and potential metabolites in a single injection is now possible in a routine laboratory. Owing to the wide chemical diversity of the compounds described as PPCPs, the extraction and separation of the many classes and structures pose a major analytical challenge. Depending on detection level required, two alternative approaches could be taken for

sample preparation. The first involves simple filtration followed by large volume injection of the water sample. If lower levels of detection were required, a method employing mixed mode solid phase extraction was used. UPLC separation on a 2.1 x 100 mm HSS T3 analytical column (1.7um) was found to provide optimum chromatographic conditions to resolve the range of chemically diverse PPCPs. Title: Selective Analysis of Patulin in Apple Juice Using the Acquity UPLC H-Class with the Acquity QDA Detector Name: Kenneth Rosnack Session #: 1420, Location: - Exposition Floor, Back of Aisles 1000-2500 Type: Poster Patulin is a mycotoxin produced by several Penicillium, Aspergillus and Byssochlamys mold species and is commonly found on apples. Apples which have been damaged or bruised prior to processing are more susceptible to contamination by patulin-producing molds. When patulin contaminated apples are processed into juice, high levels of patulin are possible. The thermal stability of patulin prevents its decomposition during pasteurization. The U.S. FDA has set a maximum residue level (MRL) of 50 ug/kg of patulin in apple juice and apple juice concentrates. Other countries including China, Japan and the EU have also set the maximum contamination of patulin to 50 ug/kg in apple juice products. The EU also has lower limits for patulin in solid apple products (25 ug/kg), such as apple puree, and products designed for infants and young children (10 ug/kg). To protect both the producer and consumer, accurate testing is required to prevent the contamination of apple juice with patulin. To improve the selectivity and reduce limits of quantification, mass detection is desirable in the analysis of patulin. In order to offer laboratories the opportunity to capture the benefits of mass detection without the challenges associated with the adoption of mass spectrometers, the ACQUITY QDa Detector has been developed. Single ion recording (SIR) was used to monitor patulin and RADAR technology allowed for the simultaneously acquisition of full spectrum data. Patulin was successfully detected in apple juice and mass detection with SIR provided high selectivity for this analysis. Patulin was detected down to spiked concentrations of 1 ug/L. The lowest spiked concentration that resulted in a signal-to-noise (S/N) ratio above 10 (using the peak-to-peak method) was 5 ug/L. This level is ten times lower than required by the EU and FDA regulations for apple juice and half of the strictest EU level for baby food. Title: Selective and Sensitive Detection and Quantification of Stockholm Convention POPs Including Dioxins, Using Atmospheric Pressure Gas Chromatography MS/MS Name: Douglas Stevens Session 1380, Location: - Exposition Floor, Back of Aisles 1000-2500 Type: Poster Atmospheric pressure GC (APGC), first developed in the 1970s, has recently made a comeback as an alternative to high resolution EI-GC/MS and EI-GC/MS/MS. For many of the POPs on the Stockholm Convention, only the molecular ion is formed under APGC ionization conditions. By avoiding fragmentation, the sensitivity in MRM or SIR mode is enhanced. In this work, APGC with a high sensitivity tandem quadrupole mass spectrometer was used in MRM mode under "dry" N2 conditions. Standards for dibenzo-p-dioxins and furans were obtained from Wellington Labs. Data were acquired with two transitions for each of the

native compounds and their 13C analogues. The results demonstrated that the sensitivity achieved using APGC-MS/MS is comparable with high resolution GC/MS. Injection of a 1/10 dilution of calibration standard CSL of concentration range 10-100 fg/ul resulted in S/N from 10 to 65. A calibration curve was generated by analyzing 1/10 CSL up to CS4 standard. Coefficients of determination (r2) for all compounds over the calibration range were all > 0.998. To test the sensitivity of the system, the lowest calibration point was diluted 1/10. The S/N for the first or second MRM ranged from 10 (10 fg TCDD/TCDF) to over 50 (other congeners 50-100 fg). Repeatability for n=10 injections of the CSL standard were all less than 10% RSD. The relative standard deviations ranged from 2.5 to 9.6%. In addition, ion ratio deviations were all less than 20% across the calibration range. These results meet the specification often set for high resolution GC/MS systems. WEDNESDAY, MARCH 5th Title: A Novel Approach to the Reduction of False Positive and Negative Identifications in Screening of Pesticide Residues in Food Analysis Name: Kenneth Rosnack Time: 8:50 AM, Session # 1590, Location: S503a Type: Oral Current trends indicate that more than 500 compounds are routinely used under strict regulation on a global basis. With increasing global trade there is a requirement for multi-analyte screening strategies capable of efficiently detecting residue violations to protect consumer safety. Benefits of full spectra acquisition and the specificity of accurate mass measurement is well characterized and is used in combination with, time tolerances, isotope fits, fragment ions/ratios and response thresholds to reduce false positive/negative identifications in screening assays. Nonetheless, it is a challenge to identify targeted compounds present in the sample with a large number of co-extracted matrix components. The application of ion mobility to remove false positive identifications and importantly false negative identifications will be presented. The assay is based on the analysis of sample extracts and matrix matched calibrants of pear, ginger, leek and mandarin, as well as quality control samples generated for an EU-RL proficiency test. UPLC HDMSE drift times generated from the solvent standards and the matrix matched calibrants were shown to statistically belong to the same population. Hence it can be shown that the drift time of the residues is independent of the matrix and can be utilized as a confirmatory parameter to increase confidence in identification. The drift time data generated was entered into a scientific library within a new scientific information system. This allowed the expected and determined drift times to be utilized to reduce false identifications in the proficiency test samples and matrix matched calibrant series analyzed. Title: Impurity Isolation from Synthetic Dyes Using Mass-Directed Preparative Liquid Chromatography Name: Rui Chen Time: 10:05 AM, Session # 1590, Location: S503a Type: Oral Synthetic dyes are widely used in food, cosmetic, pharmaceutical and related industries. Due to the structural variation of the starting material and synthetic process, many commercial dyes are often presented as mixtures of isomers and subsidiary colors. It is, therefore, imperative to efficiently isolate the impurities for subsequent identification and characterization. The challenge in chromatographic resolution of the constituents in a dye mixture mainly arises from their structural similarity. The issue is further compounded by the dynamic range of the impurities with respect to the main compound. In this poster, we

present a case study using mass-directed preparative LC to simultaneously isolate multiple impurities from a commercially available synthetic dye. Column chemistry, proper scale-up from the analytical to preparative scale, and the impact of MS detection on overall purification efficiency will be discussed. Title: Rapid Separation of Hexabromocyclododecane Diastereomers and Tetrabromobisphenol-A Using a Novel Method Combining Convergence Chromatography and MS/MS Detection Name: Douglas Stevens Time: 11:05 AM, Session # 1580, Location: S502b Type: Oral The brominated flame retardants hexabromocyclododecane (HBCD) and tetrabromobisphenol-A (TBBPA) are compounds which are monitored for their presence in the human population & the environment. The ability to resolve the various HBCD isomers from one another is important due to the differences in isomeric distribution in biota, abiotic systems and technical formulations. Separation of the three α-, β- and U- diastereomers of HBCD and TBBPA can be achieved using reversed phase liquid chromatography (RP-LC) and this is currently the method of choice. Convergence chromatography (CC) is a technique based on the use of a supercritical fluid and has shown to have enhanced efficiency due to higher diffusivity and lower viscosity of supercritical fluid compared to liquids, making it well suited to isomeric separation. The development of a CC method that analyzes these two BFR’s also offers the advantage of lower solvent usage, as well as the ability to inject a variety of solvents that are not typically compatible with RP-LC analysis. We describe a method using supercritical CO2 and methanol to baseline separate the three most abundant HBCD diastereomers and TBBPA within a three minute run time. A tandem quadrupole mass spectrometer using negative mode ESI was used for detection, operating in multiple reaction monitoring (MRM) mode. Ionization was enhanced by the addition of a make-up flow, which is introduced to the post-column effluent. Method limit of detection (LOD) and limit of quantification (LOQ) for α-, β-, and U- HBCD were based on peak-to-peak signal to noise ratios of greater than 3 or 10, respectively. The LOD for all HBCD diastereomers was 100 fg/ul, and LOQs 500 fg/ul for α- and U- HBCD and 250 fg/ul for β- HBCD. In order to test the efficacy of this method, a subset of human serum extracts were analyzed, and the detection of a sub-pg/ul concentration of α- HBCD was made. Title: Automated On-Line UHPLC Analysis Enabled by a Novel Process Sample Manager Name: Aaron Phoebe Session # 1660, Location: - Exposition Floor, Back of Aisles 1000-2500 Type: Poster Determining the optimal reaction conditions and the endpoint of a reaction is crucial to ensure the highest quality yield of the compound of interest and to minimize the formation of secondary products and process impurities. HPLC is recognized as an indispensable quantitative tool supporting Design of Experiments (DoE) to map out the optimal reaction conditions. However, it is also recognized that HPLC is too slow of a technique to provide real-time analysis of reactions. UHPLC provides real-time analytical speed combined with enhanced resolution, sensitivity, and dynamic range but the manual process of extracting and preparing samples significantly delays results. Using a novel process sample manager as part of the UHPLC system, samples are automatically extracted from the reaction vessel and prepared for analysis providing real-time results and superior assay fidelity with improved sample stability, sample solubility and the safe handling of potentially hazardous samples. In this presentation we will discuss the use of UHPLC for quantitative online reaction monitoring of a multi-step 1,4-substituted-1,2,3-triazole synthesis. Continuous flow

reactions have many well documented advantages over traditional batch reactions including safety, automation, throughput, and yield. But, direct analysis of continuous flow reactors in real-time has proven to be very challenging. The ability to simultaneously detect and quantitate all components of the reaction (raw materials, intermediates, process impurities, and product) has been elusive. Data will be presented for a DoE optimizing the continuous flow synthesis conditions and monitoring the reaction mixture. Title: Chiral and Achiral Reaction Monitoring with Ultra-Performance Chromatography and Mass Detection Name: Sean McCarthy Session # 2020, Location: - Exposition Floor, Back of Aisles 1000-2500 Type: Poster Once a chemical hit is found through a library screening process and verified, optimization of the synthetic route and the compounds desired properties takes place. This step involves an iterative process of synthesis and reactivity measurement of the new compounds to further develop drug candidates into the lead phase. Reactions may proceed along a course of minutes, hours, or days. During this process, chemists need to know as soon as possible if their syntheses are proceeding as desired. This means utilizing measurement capabilities that require minimal sample preparation with a fast response giving low detection limits. In addition, it is often necessary to monitor multiple parameters or parallel reactions simultaneously. In this presentation we present the use of Ultra Performance Chromatography coupled with robust mass detection for monitoring the synthesis of several active pharmaceutical ingredients. Our results will include an optimized screening approach to monitor chiral and achiral components with a single chromatographic system when coupled with a mass detector. In addition, we will discuss workflows that demonstrate the utility of mass detection during analysis, purification, and post-purification analysis that The benefits of these approach will show improvements in analysis time via a streamlined decision making process as well. Title: Development of a Quantitative LC-MS/MS Assay for the Simultaneous Quantitation of Acetylcholine, Histamine, and Their Metabolites in Human Cerebrospinal Fluid (CSF) Using sub 2µm HILIC UPLC Name: Mary Lame Session # 2010, Location: - Exposition Floor, Back of Aisles 1000-2500 Type: Poster Biochemical biomarkers, derived from biological fluids, are often used in drug discovery and development as a way of identifying disease or effectiveness of drug treatment. Acetylcholine (ACh) and Histamine (HA) are highly polar neurotransmitters that act in the peripheral and central nervous system. Given the physiological importance of ACh, HA, and their metabolites in regards to memory and cognition, the ability to measure changes in their physiological concentrations as a function of disease progression or drug treatment make them highly advantageous as potential biomarkers, and therefore, the simultaneous measurement of these biomarkers is a valuable tool that may lead to insight into mechanisms and/or therapeutic treatment of cognitive impairments. While there are a number of analytical methodologies for quantifying these analytes (GC/MS, HPLC-EC, and LC/MS) there are few in which ACh, HA, and their metabolites have been quantified simultaneously. Additionally, quantitative analysis of ACh, HA, and their respective metabolites, is a persistent challenge due to the demand for high sensitivity, selectivity and the need for fast sample analysis times.

The method described herein demonstrates the simultaneous quantification of ACh, HA, and their metabolites in human CSF in a 96-well format. This application uses a single step sample preparation using a small sample volume, based on a 5X sample dilution with isotopically labeled internal standards, followed by HILIC UPLC-MS/MS analysis using an ultra high efficiency sub 2µm HILIC column to increase analyte sensitivity achieving analyte resolution from endogenous matrices in an analysis time of 2.5 minutes. Title: Development of a Strategy for Scaling SFC Methods Name: Kenneth Fountain Session # 2050, Location: - Exposition Floor, Back of Aisles 1000-2500 Type: Poster It is well known that for separations using CO2 as the principal component of a mobile phase, analyte retention factors are influenced largely by the mobile phase density and temperature. Because of the high compressibility of CO2 under standard operating conditions, the density can change significantly with changes in pressure (under isothermal conditions) with retention factors decreasing with increasing mobile phase density (pressure). In addition, the selectivity and resolution of the analytes may be impacted as they respond differently to the same changes in mobile phase density. This can present a challenge when attempting to transfer a method between different column configurations that involve changes in column length or stationary phase particle size, which in turn alters the pressure (density) profile for the separation. This is best exemplified when analytical scale separations are developed using sub-2-µm stationary phases, and then need to be scaled up to preparative SFC using 5 µm particle size stationary phases. The difference in the density profiles across the column, between the analytical and the preparative system, will lead to very different chromatography unless the scale-up procedure is guided by a systematic approach. Here, we present a strategy for scaling SFC separations between various column configurations. This will be demonstrated for the transfer of methods between analytical columns of different configurations, and more interestingly, for a method developed under analytical SFC conditions with subsequent scale up to preparative SFC conditions. This ability enables the rapid screening of methods on the faster analytical scale, with the direct transfer of the final method to preparative chromatography, resulting in significant savings in time and mobile phases. Title: Improving Efficiency in Polar Compound Isolation from Herbal Extracts Using Preparative HILIC Chromatography Name: Jo-Ann Jablonski Session # 1670, Location: - Exposition Floor, Back of Aisles 1000-2500 Type: Poster Traditional medicine (TM) represents an active research area that often starts with isolation and identification of the bioactive compounds from complex extracts. Since many TMs are ingested in the form of decoction, one area of particular interest in TM research is the highly water-soluble and hydrophilic constituents in herbal extracts. These compounds, however, present a challenge for reversed phase liquid chromatography (RPLC) because they retain poorly on classic RP stationary phases. As a result, the chemical identities of many hydrophilic compounds in herbs remain largely unknown. In recent years, hydrophilic interaction liquid chromatography (HILIC) has emerged as an enabling chromatographic technique for polar compound separations. In this poster, we present a case study using mass-directed preparative HILIC to isolate Catalpol, a polar biomarker extracted from Radix Rehmannia, one of the fundamental traditional Chinese medicines. The detector choice, chromatographic efficiency, loading capacity and their

contributions to the overall process efficiency are discussed. The methodology has general applicability for polar constituent isolation and purification from natural products. Title: Operating Considerations in Migrating Separation Methods Among Narrowbore and Microscale UPLC System Name: Daniel Root Session # 2050, Location: - Exposition Floor, Back of Aisles 1000-2500 Type: Poster One important trend in modern chromatographic science is the reduction of size of the complete separation system. Such developments may be important for several reasons, including enhanced sensitivity, reduction in the amount of sample required, minimizing the amount of solvents used and waste generated. The limiting factor, however, is often the volume of the column. Modern chromatographic analyses employ 2.1mm columns. The present efforts focus on extending the routinely usable range to 1.0mm and 300µm columns to obtain 77% and 98% reductions, respectively, in sample amount requirements. The benefits of size reduction can be further enhanced by using UPLC technology employing columns packed with sub-2µm particles. The effective application of these dimensions of columns requires migrating methods among sizes while preserving chromatographic quality. Three main instrument characteristics must be optimized, specifically, solvent delivery, sample introduction, and detection. The solvent manager was evaluated at flow rates appropriate for the three UPLC column diameters. A simple translation of flow rates was developed to give constant linear velocity that preserves resolution. This translation was extended to account gradient operation including the effect of system volumes and time offsets. The sample manager has been tested for the interaction among mass loaded, volume loaded, sample diluent, and retentivity. This interaction has been thoroughly measured. Finally, TUV detector cell effects on the small volume peaks has been determined and optimized. These instrument characterization experiments facilitate the use of different dimension columns while preserving resolution and selectivity in UPLC analyses. Title: A Simplified Load-Wash-Elute Solid Phase Extraction Procedure for the Reversed Phase Micro Elution Plate Name: Xin Zhang Time: 3:05 PM, Session # 1950, Location: S505a Type: Oral Solid-phase extraction (SPE) is a sample clean-up technique that can be used prior to the analysis of drugs in biological matrices such as plasma or urine. Drugs and/or their metabolites are isolated from endogenous matrix components that interfere with the precise and accurate quantitation of the target analytes. There is an increasing desire to work with smaller sample amounts (less than 100µL) for both humane and monetary reasons. We are able to work with a micro elution plate designed specifically to handle µL-volumes while still maintaining the goals of the SPE technique: clean extracts with high analyte recoveries. This plate format produces concentrated extracts in 50 µL elution volumes that can be directly injected into your LC/MS/MS, eliminating the need for the time-consuming evaporation and reconstitution steps. In this study, a simplified Load-Wash-Elute procedure, eliminating the conditioning and equilibration steps, was evaluated using the reversed phase micro elution plate. To demonstrate the efficacy of this approach, typical samples used in drug discovery and development were analyzed. Therapeutics such as antidepressant, antiretroviral and non-steroidal anti-inflammatory drugs were used to evaluate this simplified procedure in plasma and urine. Long-term method robustness and batch-to-batch consistency of the product

were demonstrated by the use of sorbent lots manufactured between 2005 and 2013. For most applications, the simplified Load-Wash-Elute procedure gives the same high analyte recoveries, low variability, and reduced matrix effects as the standard SPE procedure while reducing processing times by one third. Title: Process Analytical Technology (PAT) Improving Efficiency and Workflows in the Laboratory Name: Ernie Hillier Time: 3:05 PM, Session # 1940, Location: S503b Type: Oral In today’s laboratory environment, new technology is one of the key drivers for improving overall efficiency in the workflow. With the introduction of UHPLC with process sample management, chromatographic analysis speed, resolution, and sensitivity are a reality in a fully automated system that gives the laboratory a quantum leap in obtaining process information. This technological advancement allows for significant improvements in laboratory workflow through richer information in reduced time. As a result of these capabilities, combined with full system automation of sampling and dilution, variability is reduced. The system configurability also allows for significant flexibility that further enhances the laboratory's capabilities from development through transfer directly to manufacturing. Process reaction optimization is demonstrated through baseline characterization and targeted parameter adjustments and measurements. As the data is processed and understood, additional parameters can be added and evaluated for a more complete understanding of the process and the conditions that affect the outcome of the final product. Ideally suited for the task, the UHPLC technology provides faster information, resolution of closely related compounds, and detection of very low level impurities or contaminants in the presence of the target compound. The chromatographic data provided from these experiments gives the information and understanding of those key areas and decision points that are most critical and important about the process. The combination of all these capabilities provide reliable quantitative answers which other techniques cannot. Title: Developing a Workflow for Development of a Continuous Process with Online UHPLC Monitoring Name: Charles Phoebe Time: 4:05 PM, Session # 1940, Location: S503b Type: Oral Integration of online reaction monitoring into continuous processes has been recently gaining attention with the popularity of flow chemistry beginning to take hold. Integration of UPLC offers unique advantages in the control and understanding of continuous processes. Described here will be a process research perspective on developing a monitoring process for continuous multistep synthesis in a plug flow reactor. A two-step triazole synthesis strategy, involving explosive and toxic azides along with isolation will be described along with the integration of low level monitoring of potentially genotoxic impurities. This set of experiments was monitored by an online UHPLC system that allowed for the high speed qualitative and quantitative evaluation of the reaction conditions. This data was then incorporated into a response surface model to allow for understanding of a safe window of operations to produce material of high quality. Title: Use of Mass Detection in Method Development for Components with No UV Absorbance Name: Sean McCarthy Session # 2020, Location: - Exposition Floor, Back of Aisles 1000-2500 Type: Poster

The development of chromatographic methods for pharmaceutical products can be challenging for many reasons. These challenges can include changes in selectivity under different elution conditions, poor or no UV absorbance, or similar UV profiles between components among others. To monitor changes in selectivity practitioners often utilize standards, analyzed separately, under each of the separation conditions to efficiently track components. For samples which contain components that exhibit poor or no UV absorbance, practitioners may need to utilize several modes of detection such as UV and evaporative light scattering (ELS). We will present the development of a method for analyzing the active pharmaceutical ingredient memantine and its associated metabolites. Memantine is commonly used for the treatment of dementia which is often associated with the of the onset of Alzheimer's disease. Each of these components exhibits no UV absorbance, and currently their detection is accomplished with ELS or by post column derivitization to allow indirect UV detection. While these approaches have shown utility, they lack the specificitiy often desired in assays. Our presentation will demonstrate the use of a robust mass detector for efficiently tracking theses components during the method development process. We will demonstrate the linearity, reproducibility, and specificity achievable with mass detection, which would allow this technology to be deployed for routine assays. We will also discuss how an easy to use mass detector can provide reliable data to guide decision making during the development of chromatographic methods and their deployment for routine use. THURSDAY, MARCH 6 Title: Recent Progress in the Development of Gram Scale Preparative SFC Instrumentation Name: Rui Chen Time: 9:10 AM, Session # 2140, Location: S404d Type: Symposia Preparative SFC has been widely used in gram to kilogram scale purifications of intermediates, active ingredients, and other compounds in pharmaceutical industry, primarily in the chiral realm. Recent years have witnessed the expansion of prep SFC applications in areas such as natural products, agrochemical and chemical material. As a result, there has been an increasing need for instrumentation advancement to support a more efficient and cost-effective purification effort. In this presentation, the evolution of the preparative SFC in fundamental principles, instrument design, and practices will be reviewed. In particular, the hardware advancement to accommodate a more user-friendly, more versatile, and more robust prep SFC instrument will be discussed. Title: A Comparison of the Effect of System Dispersion on 2.1 and 3.0 mm i.d. Columns Packed with Sub-2- [micro]m Solid-Core Particles Name: Jonathan Turner Time: 11:05 AM, Session # 2210, Location: S502b Type: Oral

The past decade has seen substantial improvements in both chromatographic packing materials and instrumentation, resulting in faster and more efficient chromatographic separations. The once common 4.6 x 150 mm column, packed with 5 µm particles, has been replaced by smaller dimension columns packed with highly efficient materials. However, even today, the use of sub-2- µm particles packed into 2.1 x 50 mm columns remains a challenge. Often the actual performance of these highly efficient materials is lower than what is achievable for these materials. The key is to understand the role that the chromatographic system plays on the ultimate column performance achievable from the sub-2 µm packings. To maximize the performance from highly efficient materials packed into 2.1 mm i.d. columns, it is imperative that the chromatographic system also be optimized with the lowest possible system volume. The objective of this investigation is to characterize the dispersion of three chromatographic systems and demonstrate their impact on demanding applications using 2.1 mm and 3.0 mm i.d. columns packed with sub-2- µm solid-core materials. Chromatographic examples will be shown that illustrate the performance that is achievable with a chromatographic system that has been optimized and one that has not been optimized for use with 2.1 mm i.d. columns. Title: Two-Dimensional LC-SRM Bioanalytical Assays for Small Molecules and Peptides Name: Catalin Doneanu Time: 11:10 AM, Session # 2100, Location: S402b Type: Symposia Multidimensional chromatography has gained significant interest in the bioanalysis community in recent years. The major driving force behind its adoption is related to the ability of 2D-chromatographic methods to improve the separation of the analyte of interest from other sample components (matrix) in order to reduce suppression of the analyte signal. In this way, 2D-chromatographic techniques are responsible for increased sensitivities when compared to one dimensional LC methods using the same amount of sample. Two bioanalytical applications will be described covering identification and quantification of host-cell protein (HCP) impurities in protein biopharmaceuticals and a universal methodology for quantification of therapeutic mAbs in human serum. In the case of the HCP assay, the therapeutic biopharmaceutical (a mAb) is reduced, alkylated and digested with trypsin. Peptides were separated by comprehensive two-dimensional RP/RP chromatography with high pH in the first dimension and low pH in the second dimension. Peptides from the second-dimension RP separation were detected and fragmented by a quadrupole time-of-flight mass spectrometer using alternating collision energy (low/high CE) MS scans. The data is searched against a database to identify HCPs in the mixture and to provide target peptides (and MRM transitions) for HCP quantification. The development of a generic, fast and automated LC-SRM method for quantification of therapeutic mAbs in human serum using two dimensional 2.1 mm ID LC-SRM with heart cutting will be discussed. Peptides were separated by RP chromatography at pH 10 in the first chromatographic dimension. Only peptides used for trastuzumab quantification were transferred to the second chromatographic dimension undergoing an orthogonal separation at pH 2.5. After 2D chromatographic separation, the sensitivity of the LC-SRM assay was restored to 75% of the sensitivity observed in the absence of the serum digest matrix.