posters[ Monday, March 9, 2009 ]

Pharmaceutical Applications of Liquid Chromatography

Session 600-3 • Poster 3Comparison of Fully- and Superficially-Porous Particles for the Analysis of Basic CompoundsKenneth J. Fountain, Waters Corporation

Abstract: HPLC columns containing 2.7 µm superficially-porous particles have gained increasing attention due to their ability to produce highly efficient separa-tions at low back pressure. Many of the studies performed with these materials utilize neutral compounds to measure column efficiency. However, a vast majority of the pharmaceutical drug compounds manufactured today are basic in chemical nature, so determining their chromatographic behavior on fully-porous and superficially-porous particle columns has practical implications. Unlike neutral compounds, bases are ionized at low pH, and secondary in-teractions can occur with the stationary phase, causing asymmetric peak shape and efficiency loss. A comparison of fully-porous silica and hybrid particles to superficially-porous particles was performed for the analysis of basic compounds. Peak capacity for these materials was compared using a standard mix-ture, as well as a forced degradation sample at low pH. In addition, hybrid materials were evaluated at high pH in order to establish a benefit of analyzing basic compounds in their neutral state in reversed-phase HPLC. Finally, a loading study was conducted to investigate the influence of mass load on column performance. Results indicate that 2.5 µm fully-porous particles give equal or better performance than superficially-porous particles for basic compounds analyzed at low pH. Further, when analyzed with high pH mobile phases on hybrid particle columns, peak shapes were dramatically improved, yielding much higher peak capacities (~30 %). Mass load capacity for fully-porous particles was about three-fold higher than superficially-porous particles at low pH, and up to 15-fold higher at high pH on hybrid particle columns.

Biomedical Analysis and Synthesis

Session 870-6 • Poster 6Application of UPLC Open Architecture System with FLR and MS Detection for the Determination of ß-Carboline Alkaloid Content in Ayahuasca Tea and Source Plants Banisteriopsis Caapi and Psychotria ViridisPatricia McConville, Waters Corporation

Abstract: Banisteriopsis caapi, also known as Ayahuasca, Caapi, or Yage, is a South American jungle vine of the family Malpighiaceae. B. caapi is most often used in combination with the Psychotria viridis plant to prepare Ayahuasca, a decoction considered a sacred medicine among the indigenous peoples of the Amazon Rainforest. The use of Ayahuasca and other “plant teachers” for visionary experiences and healing purposes is an ancient practice which has at-tracted the attention of modern psychiatry for its potential as a treatment for depression and other psychiatric disorders. Ayahuasca has also become part of a spiritual enlightenment movement as evidenced by the surge in “ayahuasca tourism” in the Amazon which attracts visitors from all over the world in search of healing, divination and worship. Ayahuasca, also known as Hoasca or “vine of the souls” by the Quechua Indians contains the beta-carboline harmala alkaloids and MAOIs harmine, harmaline, and tetrahydroharmine. The MAOIs in B. caapi allow the primary psychoactive compound, N,N-dimeth-yltryptamine (which is introduced from the Psychotria viridis plant), to be orally active. The chemical nature of its active constituents and the manner of its use makes Ayahuasca study relevant to contemporary issues in neuropharmacology, neurophysiology, and psychiatry. This interest supports continued investment and improvement in analytical methodology required for study of the active constituents of Ayahuasca teas. This poster describes the transfer and subsequent optimization of an HPLC/Fluorescence (FLR) method for analysis of harmala alkaloids to UltraPerformance LC® (UPLC®) with resultant increases in throughput, resolution and sensitivity. The UltraPerformance LC analysis employs Fluorescence and MS detection for the quantification of harmala alkaloids in Ayahuasca tea and samples of Banisteriopsis caapi and Psychotria viridis.

postersUV/VIS

Session 940-9 • Poster 9Fast Screening for Banned Food Colorings in Health Beverages and Powdered Fruit Drinks Using UltraPerformance LC Mark E. Benvenuti, Waters Corporation

Abstract: Artificial food colorings are often added to health beverages and powdered fruit drinks for esthetic appeal, color stability and to eliminate the use of more costly natural products. These colorings, many of which are azo dye coal tar derivatives, have been linked to some cancers, attention deficit disorders, and hyperactivity in children, along with various other health problems. There is no international consensus as to which of these dyes should be banned making it difficult to monitor imports from various parts of the world. In the United States, three common dyes permitted in food are Yellow 5 (E102), Red 40 (E129) and Blue1 (E133). Examples of banned food dyes in the US include Amaranth (E123), Azorubine (E122), Ponceau 4R (E 124), and Quinoline Yellow (E104). All of these compounds go by various trade names. Traditional methods of analysis for these dyes include reverse phase HPLC with UV detection. Runtimes for these analyses can exceed 15 minutes. Our poster will illustrate a fast screening method for several dyes using UltraPerformance LC / PDA with a runtime of 5 minutes. Applicability to several food matrices with minimum sample preparation will also be described.

Bioanalytical MS, LC/MS, and GC/MS

Session 860-12 • Poster 12 Quantitative Analysis of Morphine and Morphine Metabolites in Plasma Using Mixed-Mode Solid Phase Extraction and UPLC-MS/MSZhe Yin, Waters Corporation

Abstract: Morphine is a highly potent drug used to treat severe pain. The major metabolites of morphine are morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G). In particular, M6G can have adverse effects including nausea and vomiting. Morphine can also form N-oxide, acetyl, and hydroxy metabolites, all of which must be quantified, as they may be toxic if accumulated in the body. A UPLC-MS/MS method was developed for the quantitative determination of morphine and five of its metabolites in plasma. Since mixed-mode solid phase extraction (SPE) is capable of minimizing matrix effects and producing very clean extracts, it has been widely used for analyzing drugs in biofluids. The traditional SPE protocol for basic compounds involves a high pH elution step, which is not suitable for some morphine metabolites due to degradation. Therefore, a weak cation exchange sorbent and optimized washing step was utilized on an Oasis® WCX plate, on which morphine and its five metabolites were extracted simultaneously from 250 µL of plasma. UPLC-MS/MS analysis was performed using an ACQUITY UPLC® System connected to a fast-scanning triple quadrupole mass spectrometer (TQD). The six compounds were separated on a 2.1 x 50 mm, 1.8 µm ACQUITY UPLC HSS T3 column with a 3-minute gradient using 0.1% formic acid and methanol as the mobile phases. Quantitation was performed by monitoring five MRM transitions in positive electrospray mode. The method was linear from 0.1 – 50 ng/mL, and recovery for each compound was greater than 85% with RSD values less than 5%. Matrix effects were less than 15%.

posters[ Tuesday, March 10, 2009 ]

Laboratory Informatics

Session 1280-5 • Poster 5 Intelligently Improving Procedures and Workflows Chris Stumpf, Waters Corporation

Abstract: A December 2006 article in Medical News Today estimated that the pharmaceutical industry loses approximately $50 Billion dollars per year to manufac-turing inefficiencies. Many of the analytical tests utilized during product testing employ paper forms in their workflows. In the QC laboratory, adherence to Good Manufacturing Practice (GMP) guidelines necessitates the need to maintain thorough documentation to ensure strict compliance with established procedures. Completing paper documents and ensuring their authenticity creates a burdensome bottleneck for the QC laboratory. The limitations of paper are well known and include transcription errors, legibility, high storage costs, record management, cumbersome GMP compliance, and difficulty in locating completed batch records. Hence, a logical step to improve manufacturing efficiency as well as boost efficiencies in industries that employ routine labora-tory procedures is to replace paper forms with electronic equivalents. However, moving to a paperless laboratory is not nearly enough. The electronic forms should be intelligent so that they not only emulate paper but serve to guide an analyst through a test procedure while interfacing with laboratory instruments and laboratory information systems, perform error correction, and reduce the burden to implement GMP. Therefore, this presentation will discuss strategies for using an electronic form based system to boost laboratory productivity.

Process Chemical Analysis

Session 1330-1 • Poster 1 On-Line UPLC Method for the Support of Cleaning ValidationTanya Jenkins, Waters Corporation

Abstract: During the manufacturing and packaging of active pharmaceutical ingredients (APIs), the removal of drug residues from the equipment is usually per-formed by a series of cleaning procedures. It is imperative that the production equipment used in this process be properly cleaned in order to avoid cross-contamination of drug products. The safety acceptance criteria for API residues vary with drug substance. More potent compounds will require a lower acceptance limit. In general, most processes aim to have a lower safety limit in the 10 ppb – 1 ppm range (10 ng/mL – 1 µg/mL). In order to achieve these limits, sensitive analytical techniques are required. Typically these samples (either swabs or rinse solvents) are taken to an off-line QC laboratory for analysis. The time it takes to receive results from the off-line laboratory can range from hours to days. During this time, the production equipment must sit idle until it is determined to be clean. If laboratory results are positive for API residues, the cleaning process and testing must be repeated. This paper describes a fast, on-line, UltraPerformance Liquid Chromatography (UPLC) method which monitors the rinse solvents at the outlet of the production equipment. By monitoring the rinse solvents on-line, the point at which the API has been removed from the production equipment can be determined. This reduces the volume of wash solvent required and substantially reduces the time that the equipment must be taken off-line before a new batch can be initi-ated. The results from the on-line method are compared to those obtained by testing swabs and rinse solvents with an off-line UPLC System.

postersPharmaceutical Applications of GC, LC, MS

Session 1650-4 • Poster 4 Increasing Sensitivity and Throughput of Alkyl Sulfonic Acid Alkylating Agents and Ester Analysis in Genotoxic Testing Peter G. Alden, Waters Corporation

Abstract: Alkyl sulfonic acids, particularly methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid, are a common class of alkylating agents used in the pharmaceutical industry as alkylating reagents, catalysts, and in purification steps in the chemical synthesis of an API. In addition, these sulfonic acids are often used as the final salt form of the drug due to improved chemical properties or bioavailability. The presence of any residual alcohols from synthetic reaction or recrystalization steps may result in the formation of alkyl esters of the sulfonic acids. Many of these mesylate, besylate, or tosylate esters are known to be genotoxic while others are potentially genotoxic requiring monitoring in the drug substance and drug product. The most common analytical techniques for monitoring alkyl sulfonate esters has been GC/MS or HPLC/UV/MS with derivatization using pentafluorothiophenol. More recently, HPLC/MS has been shown to give good results without the need for a complicated derivatization step, however, runtimes on the order of 20-30 minutes were required to achieve sufficient resolution from API. This poster demonstrates the improvements obtained using sub-2 µm LC column packing materials with UPLC/MS for the analysis of methane, benzene, and p-toluene sulfonic acids and their alkyl esters. Analysis times of less than 5 minutes with improved sensitivity are demonstrated without the need for pre- or post-column derivatization.

Session 1650-11 • Poster 11 A Workflow Approach for the Identification and Structural Elucidation of Impurities in Quetiapine Fumarate Drug SubstanceMichael Jones, Waters Corporation

Abstract: ICH guidelines state that the analysis and understanding of pharmaceutical impurities are essential for any final product. It is a business objective to un-derstand as many impurities as possible and for that reason, the purpose of this presentation was to explore a multidimensional approach with a workflow concept capable of highly specific and highly sensitive detection and determination of impurities present in quetiapine fumarate drug substance as a pri-mary test case. The workflow approach demonstrated the ability to evaluate known and unknown impurities in a pharmaceutical substance. UPLC/MS was used to detect numerous related impurities in the antipsychotic drug substance Quetiapine fumarate. The use of UltraPerformance Liquid Chromatography (UPLC), inline with Mass Spectrometry, has offered real improvements in throughput of analysis. Accurate mass and MS/MS fragment data was acquired and evaluated automatically using software solutions to propose structures of the found impurities present in a pre-developed method for the impurity profile of the Quetiapine. The information generated from exact mass and MS fragmentation experiments was rich and important, not only for the ability to detect very low levels of impurities, but also in the characterization of these compounds and subsequent identification of their origin. Accurate mass results were gathered and elemental compositions of the impurity peaks are automatically assigned to each detected compound. Using various software solutions within a central chromatographic data system, results were reported via a data browser in which chromatographic and exact mass spectroscopic data proposed the structures of the impurity compounds. This workflow approach provided a rapid systematic set of comprehensive results that decreased decision making time and provided greater confidence in the direction to improve the quality of the drug synthetic process.

postersSession 1650-12 • Poster 12 A Method Development Approach to Achieve an Impurity Profile for Impurities Present in A Bulk Drug Substance Using Sub-2 µm Porous Particle LC Combined with UV-MS Michael Jones, Waters Corporation

Abstract: Impurity profiling of pharmaceutical drug substances or dosage forms require methods involving high sensitivity and specificity as well as desirable analy-sis times. Liquid chromatography and mass spectrometry have become essential tools in the analysis and characterization of drug impurities, with analysis in the 30 minute to 1 hour timeframe. While throughput can be increased, it is usually at the expense of chromatographic resolution, thus resulting in the failure to detect all the impurities in the sample. In this presentation an efficient method development screening process was employed utilizing short UPLC Columns and a generic gradient to fast track the method development screening time. The process takes advantage of the high chromatographic efficiency of sub-2 µm particle (UPLC) technology to deliver rapid method scouting. The use of short columns allowed many column chemistries to be screened quickly in an automated manner using a column manager capable of addressing four columns. Once the best column chemistry was selected, further optimization for resolution was achieved by varying gradient slope and temperature on the longer column dimension. Employing photodiode array detection coupled with single quadrupole MS facilitated the peak tracking as the conditions were varied. The use of specific labeling custom fields in the chromatographic data system allowed for the creation of custom reports to help expedite the mining of the resulting data. Additionally, multiple vendors of a pharmaceutical drug substance were obtained and evaluated by comparing the impurity profiles using the newly developed UPLC impurity method.

Bioanalytical Applications of Liquid Chromatography

Session 1590-20 • Poster 20 Utilization of Liquid Chromatography at Elevated pH and Elevated Pressures in Quantitative Bioanalysis Paul Rainville, Waters Corporation

Abstract: Maximizing the sensitivity of bioanalytical assays for the quantification of potential new medicines and their metabolites is often required during develop-ment of new therapeutics for disease. This is primarily due to the fact that many new medicines have become more potent enabling smaller doses to be administered thus reducing the circulating levels in patient biofluids. Furthermore, the detection of metabolites associated with medicines is of particular importance due to the toxicity potential that may be associated with their formation in humans. Sensitivity gains can be accomplished for bioanalytical assays in a variety of ways including: the use of tandem quadrupole mass spectrometry, reduction in chromatographic column diameter and increased amount of sample injected onto the analytical system. Additionally, the introduction of chromatographic columns packed with sub-2 µm particles has dem-onstrated the potential for increasing sensitivity in quantitative bioanalytical applications due to the inherent increased chromatographic efficacy offered by the technique. The approach of mobile phase modification relative to pH is a simple but often overlooked approach to increased sensitivity. Increases in selectivity, improved peak shape and increased MS response are often the result of operating under high pH conditions. Here we present the utiliza-tion of chromatographic columns packed with sub-2 µm particles and elevated pH mobile phases as a means to increase sensitivity and lowering LLOQ in quantitative bioanalytical applications.

posters[ Wednesday, March 11, 2009 ]

General Applications for Liquid Chromatography

Session 1990-12 • Poster 12 Fast Analysis for Aflatoxins Without DerivitizationMark E Benvenuti, Waters Corporation

Abstract:Aflatoxins are toxic metabolites of the molds Aspergillus Flavus and Aspergillus Parasiticus. They are designated as G2, G1, B2, and B1 with B1 being the most toxic and abundant. They occur in a wide range of food stuffs including nuts, seeds dried fruits, spices, and grains. Due to their toxicity, most countries along with the European Union have established permissible levels in foodstuffs and require testing for compliance. Traditional methods of analysis for aflatoxins incorporate reverse phase HPLC with fluorescence detection. However the fluorescence of aflatoxins G1 and B1 is quenched in aque-ous solvents and must be enhanced. Methods for this enhancement include pre column derivitization with trifluoroacetic acid (TFA) or post column with Iodine, electrolytically generated bromine (Kobra Cell) or UV light (PHRED). Our poster will show that with UltraPerformance LC-FLD and a modified flow cell, required detection limits can be achieved without resorting to any pre or post column derivitization step. The applicability of this method to several food matrices will be demonstrated.

Session 1990-13 • Poster 13 A Modular HPLC System for Routine Analysis of Capsaicin from Hot SaucesTanya Jenkins, Waters Corporation

Abstract:Capsaicin, and several other related compounds such as dihydrocapsaicin, are called capsaicinoids and are produced as secondary metabolites by chili peppers. Because of the burning sensation caused by capsaicin when it comes in contact with the mucous membranes found in the mouth, it is frequently used in food products to give them added spice or “heat” (pungency). Capsaicin can also be found in topical lotions where it acts as a pain reliever for minor aches and pains that may be associated with backaches, strains, sprains and arthritis. The degree of heat found within a food can be measured on the Scoville scale, however high performance liquid chromatography (HPLC) is a more accurate method of calculating heat pungency caused by capsaicin content in a food product. This paper describes the analysis of capsaicin and dihydrocapsaicin by HPLC utilizing a simple-to-use, modular chromatographic system. The resulting method possesses excellent area count reproducibility, retention time reproducibility, reliable quantification, and low carryover performance, demonstrating the excellent performance of this modular system.

postersApplications of LC/MS

Session 2290-23 • Poster 23Analysis of Drug Residues in Milk by ON-line SPE/LC/MS/MS Claude Mallet, Waters Corporation

Abstract:Countries worldwide are under constant vigilance to ensure the health safety of their citizens. In current agricultural practice, raising animals for food depends heavily on the use of drugs, which is essential to the animal’s health and for the economics of the industry. Veterinary drugs or residues can enter human food and increase the risk of ill health in persons who consume products from treated animals. As a consequence, many government agencies have established maximum residue levels (MRLs) for a variety of veterinary drugs in animal tissues, milk and other food products. Unlike drinking water, the analysis of drug residue in food products requires elaborate sample extraction and clean up protocol to minimize matrix effects. However, one major drawback is the high amount of manual labor required to produce a clean extract. A typical extraction protocol usually starts with a sample homogeniza-tion in an aqueous or organic buffer. The homogenate is then centrifuged, concentrated and cleaned up with a wide range of extraction techniques, such as liquid-liquid, dispersive or by SPE, just to name a few. With recent advances in turn-key systems, many steps can be automated and provide an immedi-ate benefit, a reduction of manual labor needed to produce a final extract. The coarse extract can then be further refined with an ON-line SPE/LC/MS/MS system. This research will discuss the performance of ON-line SPE/LC/MS/MS for the analysis of drug residues in milk. The milk samples were subjected to a protein precipitation with acetonitrile (1:1 ratio), followed by a centrifugation at 3500 rpm for 15 minutes. The supernatant was collected and diluted with 3 mL of water. The dilution is intended to reduce the organic percentage at 20% to avoid breakthrough during the loading phase. The entire volume (5 mL) was injected onto an ON-line SPE/LC/MS/MS system for enrichment, cleanup and analysis. The results showed good signal-to-noise ratio at low ppb levels.

Session 2290-31 • Poster 31 An Automated Approach to Isolation of Impurities of a Pharmaceutical Drug SubstanceMichael Jones, Waters Corporation

Abstract:Many pharmaceutical analytical applications are focused on the identification and quantification of the Active Pharmaceutical Ingredient (API) and related impurities. This activity requires highly resolving and highly sensitive liquid chromatography methodology. Once the methodology is set, any unknowns that appear within the API batches that are above 0.1% UV area must be characterized and identified utilizing orthogonal techniques such as accurate mass, MS/MS and NMR. In the case of NMR, enough sample (~25 mg) must be isolated and collected for the analysis. This presentation demonstrates the process of scaling from UltraPerformance LC to Preparatory Chromatography to maintain the chromatographic resolution of the impurity(s) of interest. We discuss the step-by-step approach for optimizing efficient fraction collection utilizing a batch of Quetiapine Hemifumarate as an example. The UPLC impurity profile developed to isolate the impurities of interest will be used for the rapid confirmation of the purified fractions.

postersAnalysis of Food and Related Products I

Session 2280-19 • Poster 19Development of UPLC Columns and Methods for Carbohydrates: Applications to Saccharide AnalysisChristopher J. Hudalla, Waters Corporation

Abstract: The chromatographic analysis of carbohydrates has become a valuable tool in many areas of research. For the food industry, the identification and quanti-tation of mono- and disaccharides present in a sample is crucial. The addition of other additives, more complex sugars, sugar alcohols, artificial sweeteners and salts often result in complex mixtures which are often difficult to analyze. For the pharmaceutical industry, the identification of simple or complex mixtures of sugars or sugar derivatives, used as pharmaceutical incipients, is critical for regulatory compliance. In the field of energy production, ethanol is one the fastest growing energy sources in the world. Cellulose and hemicellulose, which are frequently used as a feed stock for ethanol production, are complex polymers of sugar which comprises greater than 75 percent of all plant material. Yeast is used to ferment the hydrolysates of these complex polymers into ethanol. Analysis of the intermediate cellulosic hydrolysate provides information critical to the development and evaluation of the process. The application of chromatographic separations for these fields of research is often hindered by multiple problems, including the mutarotation of sugar anomers, on column formation of Schiff bases, chromatographic interferences from complex sample matrices, and shortened column lifetime. Here we pres-ent our research on Amide bonded 1.7 µm Ethylene Bridged Hybrid (BEH) particles. The versatility and robust nature of this prototype material enables fast, efficient separation of many simple and complex carbohydrates, with extended column lifetimes, gradient compatibility, and removal of salt interfer-ences. As an added benefit, the use of a low dispersion UPLC System capable of 15,000 psi permits the use of 2.1 mm ID columns which leads to reduced solvent consumption (reduced cost and waste).

Session 2280-24 • Poster 24 Analysis of Seed Oil Adulteration Using a Rapid and Sensitive UPLC MethodPeter J. Lee, Waters Corporation

Abstract:Seed oil is an important component of food, cosmetic and personal care products. Oil quality, price, and suitability for human consumption depend mainly on the source and quality of raw materials, extraction methods, processing and storage conditions. The frequent occurrence of edible oil adulteration has raised safety concerns; therefore, seed oil analysis is of great importance for protecting public health, satisfying legislation and fair trade. Non-aqueous reversed-phase and silver-ion chromatography methods have been used to analyze seed oil components. However, those methods require a long runtime and use halogenated solvents that are known carcinogens, restricted, and sometimes prohibited in laboratories. In addition, since those methods lack sen-sitivity and resolution, they are not able to effectively distinguish different seed oils and detect adulteration. This presentation describes a fast, sensitive, and high resolution analysis method using UPLC/PDA/MS technology to fingerprint seed oils, analyze blended seed oils, examine seed oil oxidation, and identify TAG components with low toxicity mobile phases, acetonitrile and 2-propanol. Various seed oil samples including olive, hazelnut, walnut, tea seed, soybean, canola, peanut, corn, palm, and sunflower were analyzed. The UPLC method reveals their unique TAG composition and chromatographic patterns. The method can easily identify adulterated olive oil unambiguously, even when only 1% of the other oil is present. In addition, it can also detect oxidized and decomposed TAG components. This fast and sensitive testing method can be used to ensure seed oil quality and authenticity to protect consumer and public health worldwide.

postersSample Preparation

Session 2360-14 • Poster 14 Developing Faster, More Sensitive, More Robust Bioanalytical Methods with Innovations in SPEErin E. Chambers, Waters Corporation

Abstract: Evolving FDA regulations require that scientists develop more selective and reliable bioanalytical methods than ever before. In addition, the number of samples to be run continues to increase due to additional validation criteria such as matrix effects and new recommendations for incurred sample reanaly-sis. Both of these needs place additional burden on method development scientists and on laboratory productivity. An innovative solid phase extraction (SPE) format has been developed to meet these challenges. This new 96-well plate SPE technology combines patented design, proven sorbent chemistries, and streamlined method protocols in a low elution volume (as low as 25 µL), high capacity device. Scientists can easily perform SPE cleanup and con-centrate analytes without time-consuming evaporation and reconstitution. Use of this technology also facilitates significant increases in sensitivity over traditional format SPE. Two different examples are given: increasing sensitivity using the same sample size, and using increased sensitivity when sample limited. Recovery for a variety of basic analytes will be calculated at increasing sample load levels to demonstrate capacity of the device. Resultant matrix effects will also be calculated. A method development strategy is described and implemented using this novel SPE technology to develop a fully validated bioanalytical method for ropinirole which easily achieves an LLOQ of 5 pg/mL.

Session 2360-15P • Poster 15Automated Solid Phase Extraction of Steroids and Hormones in Drinking WaterMichael Ebitson, Horizon Technology, Inc. Abstract: Steroids and hormones in drinking water are a subject of global concern as they have been categorized as endocrine disruptors. Infiltration into our drink-ing water supplies has occurred via our sewage treatment plants, as once the water has been through the treatment process, it is returned back into public drinking water. This paper will focus on a recent study whereby a method to determine steroids and hormones in drinking water by SPE is explored. The SPE extractors passed 500 mL of water, spiked with standards, through a disk to collect the compounds. The compounds were eluted into a VOA vial – all in one automated procedure, and then the extract was dried and concentrated, also utilizing an automated procedure. The techniques for ensuring consis-tent extractions and establishing proper extract handling procedures to optimize recovery data using SPE will be discussed. Also demonstrated will be the automation of the process which allowed increased levels of productivity and precision, proving the SPE disk to be an efficient alternative to liquid-liquid extraction, as well other SPE methods. The cost advantages of implementing an automated system will also be demonstrated.

postersBioanalytical Sample Preparation

Session 2300-3 • Poster 3 Improving Sample Handling for Amino Acid AnalysisThomas E. Wheat, Waters Corporation

Abstract:Amino acid analysis provides fundamental information for many applications and research endeavors. These include protein structure characterization, determining the nutritional value of foods and feeds, monitoring cell cultures and other processes, and clinical research applications involving metabolic pathways. In all cases, the measurements must be accurate and precise with the highest throughput consistent with reliable results. We have previously de-scribed development of a robust protocol for derivatization of amino acid samples followed by chromatographic analysis. This UPLC Amino Acid Analysis Solution has been applied across the range of applications with increased analytical throughput. With faster and more robust analysis, however, the limit-ing steps in the analysis process are now in sample handling. Two opportunities exist for improving throughput. One is preparation of sample hydrolysates, and the other is the derivatization. Microwave hydrolysis has been suggested to reduce the hydrolysis process from many hours to several minutes. We have optimized that technique for samples of pure and for complex samples represented by animal feeds. The comparison of these tests with conventional oven-based hydrolyses will be reported. The factors in optimization will be described in detail. Secondly, a robotic sample preparation instrument has been optimized for the amino acid derivatization. Results will be compared to the established manual process. The specific variables requiring optimization will be described in detail. The total workflow of optimum rapid hydrolysis, robotic sample derivatization, and a robust analytical solution leads to higher throughput for accurate and precise amino acid analysis.

Applications of LC-MS Session 2290-30 • Poster 30 Techniques for Improving Isolation and Purification of Synthetic PeptidesJo-Ann M. Jablonski, Waters Corporation

Abstract:Peptides play important roles in the physiology of organisms. Pure peptides are required to effectively study biochemical interactions in organisms as well as to understand structure-activity relationships in the development of peptide therapeutics. Modern research increasingly requires peptides that present several difficulties in assessing purity and in recovering sufficiently pure product. These challenges include detection, dissolution for purification of hy-drophobic peptides, and recovery from the chromatographic column. Traditional purification schemes typically employ UV detection, but some biologically important peptides have very low extinction coefficients. An additional mode of detection, such as mass spectrometry, can be useful. Examples are shown of peptides with better UV signal and others with better MS signal. The combination of the two detectors is shown in assessing the quality of raw synthetic product, in planning a purification, and directing the collection points for maximum purity and recovery. Hydrophobic peptides that exhibit limited solubil-ity are among the most difficult samples. Liquids that dissolve the crude product often compromise the chromatographic isolation. The steps in optimizing at-column dilution for these samples have now been defined. When these hydrophobic peptides are successfully applied to the column, they may still pres-ent problems in the elution because of poor solubility in the mobile phase. Temperature, focused gradients, and at-column dilution may be used separately or in combination to more efficiently handle peptides with large numbers of nonpolar amino acid residues. In this study, we illustrate the application of these three techniques that vastly improve the isolation of hydrophobic peptides.

postersSession 2290-34 • Poster 34 Simultaneous Acquisition of Qualitative and Quantitative MS Data Coupled with UPLC as a Tool for Bioanalytical Method DevelopmentPaul Rainville, Waters Corporation

Abstract: The process of developing LC/MS methodology for the purpose of quantitative assays for medicines and their associated metabolites is a daunting challenge. Human and animal biofluids such as urine, plasma and bile vary in components and complexity. Additionally, endogenous analytes generated by a subject’s age, gender and medication regimen can further interfere, contributing to the complexity in quantitative bioassay development and valida-tion. Techniques such as UPLC have shown significant contributions to the area of quantitative bioanalysis. This is due to the increased chromatographic efficiency and subsequent generation of sharp chromatographic peaks, often 2–3 seconds at base, resolving analytes of interest from interfering matrix components. However, even under the best chromatographic performance co-elution can occur. Therefore implementing an approach whereby qualita-tive MS scan data obtained from the matrix is simultaneously acquired with quantitative MRM MS data can aid in the monitoring of potential interfering compounds ensuring assay robustness and reproducibility. Furthermore, the fact that scan data is obtained simultaneously with the MRM data means data mining can be performed at a later time if questions arise as to the presence of analytes not targeted in the original analysis. This leads to significant time and money saving because repeat analysis is less likely to be required. It also helps maximize information where sample amount may be limited such as the case as with analysis of pediatric samples. Here we present the utilization of simultaneous acquisition of MS scan and MRM data coupled with UltraPerformance LC as a method development tool for quantitative bioanalytical applications.

Organic Analysis for Environmental Samples

Session 2340-5 • Poster 5 Trace Level Analysis of Emerging Contaminants in River Water by ON-line SPE/LC/MS/MS Claude Mallet, Waters Corporation

Abstract: The analysis of “emerging contaminants” (ECs) has become an area of significant interest in the field of environmental research. ECs comprise a large selection of chemicals such as pharmaceucticals, veterinary drugs, nanomaterials, personal care products and household chemicals. In the early 90’s, European scientists identified trace amount of pharmaceutical in surface and ground waters. This prompted national wide surveys in Europe and America. The results showed over 100 contaminants at unexpected ppb levels. ECs are defined as pollutants that are not currently registered in a routine monitoring program. In order to achieve trace level analysis, large sample volumes are usually extracted using various OFF-Line extraction methods and concentrated into a smaller volume. As an example, a typical extraction method usually starts with a 500 mL sample and ends up with a final volume of 100 µL (5000:1 enrichment ratio). If higher sensitivity is required, the only alternative is to process a larger sample volume. A few years ago, SPE cartridges were reformatted into extraction columns. By combining the extraction column, LC, analytical separation column, MS/MS and software, a system is created known as “ON-line SPE/LC/MS/MS”. A major advantage of this integrated platform is the elimination of the labor intense evaporation and reconstitution steps in favor of a direct elution into the mass spectrometer. This research will discuss the performance of ON-line SPE/LC/MS/MS for the analysis of emerging contaminants in river water. One major advantage is the reduction of on-site sampling volume needed from 1 liter to a 20 mL sample. With a 1000:1 enrichment factor, the limit of detection (LOD) was measured at 10 ppt. In practical terms, manual labor required to process one sample was limited to simply filling a 20 mL vial. Overall, the extraction protocol was reduced from 3–5 hours to less than 5 minutes. River waters samples were screened for various ECs.

posters[ Thursday, March 12, 2009 ]

Pharmacology/Drug/Applications for Liquid Chromatography

Session 2730-35 • Poster 35 Preparatory Chromatography of Natural Product Extracts Utilizing an UV Based Open Access Walk-up Purification Strategy Andrew Aubin, Waters Corporation Abstract: Extraction of potential active medicinal compounds from plant materials is accomplished in many diverse ways. Ultimately the chemist is left with a raw extract that contains the potentially active compounds along with many other non-active constituents. Following extraction, the next logical step is purification or isolation of those compounds of interest. Preparatory liquid chromatography is often utilized to accomplish this. The diversity of compounds that may be extracted is large and an adequate purification requires access to different chromatographic methods for effective isolation. An Open Access liquid chromatography system that may have many methods available for use by walkup chemists is therefore desirable. Methods can vary in column size, run time, gradient conditions, solvents, modifiers, and other variables. These options can make it difficult for a chemist to choose the best method for their extract and delay the isolation and development of the compounds of interest. To simplify the choosing of an appropriate first try method for each chem-ist’s extract, an Open Access system using method filtering software is described. As well, strategies for method selection criteria will also be discussed. By using separation methods tailored to their extracts, chemists have a better ability to obtain higher purity compounds by picking a suitable method (as opposed to a single generic method for all extracts), essentially reducing the number of re-analysis and re-purification steps ultimately speeding up the entire process.