Northeast Regional
Chromatography
Discussion Group
Fall 2002 Meeting

October 9, 2002 MEETING

Summary of Accepted Papers
(click on the title to view the Abstract)

Dr. Troy Wood, Associate Professor
Department of Chemistry and Structural Biology,
State University of New York at Buffalo

"Miniaturization of Electrospray:  A Driving force for Proteomics."

With the completion of the Human Genome Project, biochemists have increasingly turned their attention to proteomics.  Proteomics essentially is the determination of protein primary structure (including post-translational modifications) and/or a specific protein quantity as a function of cellular state, which is key in identifying molecular mechanisms of disease states.  Mass spectrometry has emerged as one of the
key tools in proteomics research.  In the last several years, one focus of our research group has been the development of miniaturized electrospray ionization, or nanospray, as a tool in protein structure determination by mass spectrometry.
Here, results from a special type of polyaniline-coated nanospray emitter developed in our laboratory will be presented.  One of the key features of this type of emitter is its stability, which facilitates analysis of proteins in the negative ion mode.  Examples of differences between positive ion and negative ion nanospray mass spectrometry, and the advantages of negative ion mode analysis will bediscussed.

Biographical Information

I was born February 11, 1967, in Elkhart, Indiana, and grew up in St. Joseph Co., Indiana. After graduating from Penn High School in Mishawaka, I attended college at Indiana University in Bloomington, where I majored in chemistry. I received my B.S. degree in chemistry with Honors in 1989. I worked one summer at Miles, Inc. (now Bayer) before getting married and starting graduate school at the Ohio State University. I did my Ph.D. work with Professor Alan G. Marshall in the area of Laser Desorption Fourier Transform Mass Spectrometry (FTMS). After graduating in 1993, I took a postdoctoral position with Professor Fred W. McLafferty at Cornell University. My research area at Cornell was Electrospray Ionization Fourier Transform Mass Spectrometry (ESI-FTMS) of proteins. In 1995 I left Cornell and started my career as an Assistant Professor in the Department of Chemistry at the University at Buffalo, State University of New York.  I founded Nanogenesys, Inc., in 2000 to develop and commercialize conductive polymer-coated nanoelectrospray emitters.

My wife and I have one son born in 1997 and a daughter born in 1999. I am the past FTMS Interest Group Coordinator for the American Society for Mass Spectrometry, and former Chair of the Western New York Section of the American Chemical Society. I enjoy baseball, playing softball, science fiction, and genealogical research. I am a long-time Chicago Cubs fan, and newer Buffalo Sabres fan. I enjoy genealogy very much, and there are a few famous cousins.  My Grandmother Wood's cousin, Allan "Rocky" Lane, was a Western Film Star and was also the voice of Mr. Ed. You can view a video clip of one of Rocky's movies at cowboypal. I am a distant cousin to Ulysses S. Grant, Union War Hero and 18th President of the United States through our mutual ancestor Walter Palmer.  My wife and son are direct descendants of Mayflower passengers John Howland and Elizabeth Tilley, from whom three US Presidents are also descended (Franklin Delano Roosevelt, George H. W. Bush, and George W. Bush).

Peter T. Papagelis
Eastman Kodak Company
Analytical Technology Division, Materials Separation & Characterization

"Gas Chromatography in the Research Laboratories:
 Going with the flow"

Gas chromatography is a mature technique used to separate volatile materials for subsequent qualitative and quantitative analysis.  Modern chromatographs are more thermally stable and include options such as electronic pressure control and programmable temperature injection ports.  Capillary columns today are more thermally stable and inert.  Equipment manufactures and column vendors provide an array of products to serve a broad range of analytical needs.  For the materials we were asked to analyze, traditional chromatography has served us well in the past.  Current chemistries at Kodak include materials in excess of 600 molecular weights and have challenged us to go beyond “typical” instrument conditions.  This talk will describe methodologies developed to meet those chromatographic challenges; in many cases without sacrificing separation of low molecular weight compounds.  Examples of how the new methods have provided more information in the areas of OLED and gelatin analysis will be shown.  

William Smith
Eastman Kodak Company
Contributor: Dr. Mike Martin, Eastman Kodak Company

"Applications of Ion Chromatography - Mass Spectrometry"


Ion chromatography is a technique that is widely employed to analyze anions and cations.  The current suppressor technology, which greatly increases analyte sensitivity to conductivity detectors by exchanging certain highly conductive species for less conductive ones, also enables the marriage of ion chromatography to mass spectrometry (IC/MS).  This hybrid technique is a powerful problem-solving tool, enhancing our ability to identify unknowns in IC analysis and allowing heretofore "mass spec unfriendly" chromatography eluents to be run into the mass spectrometer.  The process of ion suppression, in its different modes of operation and how this facilitates interfacing with mass spectrometry, will be discussed.  Several examples of the use of IC/MS chemistry will be presented, including small and large organic molecules and inorganic molecules.

Julie Marr 
Agilent Technologies

“Atmospheric Pressure Photoionization:  A Complementary Interface for LC-MS” 

Atmospheric pressure photoionization (APPI) is a relatively new ionization technique that is complementary to electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI).  APPI works well for nonpolar molecules and other compounds that are not amenable to ESI or APCI. 

The PhotoMate APPI source, developed by Syagen Technology Inc, in cooperation with Agilent Technologies Inc., creates ions by using a high fluence gas discharge lamp that produces 10 eV energy photons.  They are high enough to ionize many classes of organic compounds but low enough to minimize the ionization of air and common HPLC solvents such as water, methanol, and acetonitrile.  Because it features direct photoionization of analytes, APPI is not limited by gas-phase, acid-base chemistry.  Positive ions can be produced by the capture of a photon and subsequent loss of an electron.  Negative ions can be generated in the presence of a modifier such as acetone or toluene.  This presentation will describe the features of the APPI source and provide examples using the Agilent LCMSD and the ion trap.

Douglas Stevens
Micromass/Waters Corp.

“Considerations in Selecting the Most Suitable Type of Mass Spectrometer for Your GC/MS Applications”

Many different types of mass spectrometers are now available as highly sensitive and selective detectors for GC. Selecting the type of mass spectrometer best suited to a given application can be challenging.

This presentation will outline some of the important considerations in deciding which type of mass spectrometer is best suited for a given GC application by outlining the advantages and disadvantages of each instrument type.

Dr. Kevin G. Owens, Associate Professor
Department of Chemistry, Drexel University

"Quantitative Analysis of Peptides and Proteins by MALDI TOFMS: Prospects and Problems"

Since it's introduction in the late 1980's, matrix-assisted laser desorption/ionization (MALDI) has become a widely used soft ionization technique for the mass analysis of a broad range of biological (including peptides, proteins, oligonucleotides, carbohydrates) and synthetic polymer materials. While recognized for its qualitative strengths, the ability to perform quantitative analysis by MALDI is generally questioned. 

Work in our laboratory on the basic mechanisms of the MALDI technique indicated that problems encountered in the initial sample preparation step are often responsible for the lack of quantitative capability. We have investigated the use of electrospray deposition to overcome many of the problems in quantitative sample preparation.

 This seminar will review the basic issues involved with MALDI sample preparation and illustrate some of our recent results on the quantitative analysis of small proteins from biological matrices.
The importance of sample pretreatment, such as the use of solid-phase extraction techniques for sample clean-up on the quantitative ability of the technique is also discussed.

Biography

Kevin Owens received a B.S. in Chemistry from the University of New Hampshire in 1982. He received a Ph.D. in Analytical Chemistry from Indiana University in 1989, working under the direction of Dr. James P. Reilly using molecular beam techniques to study the gas phase spectroscopy of small aromatic molecules. Detection of these species involved both laser induced fluorescence and multiphoton ionization followed by time-of-flight mass spectrometry (TOFMS) detection. He joined the faculty of the Chemistry
Department of Drexel University as an Instructor in the fall of 1987; he is currently an Associate Professor with tenure.
The research interests of his group include studying the mechanism of the matrix-assisted laser
desorption/ionization (MALD/I) process, investigating the application of MALD/I to the analysis of synthetic polymer systems, and extending the quantitative ability of MALD/I by use of electrospray sample deposition techniques. Other work involves improvement of the TOFMS system (particularly the means for improving the detection efficiency of high mass ions) and the development of automated data processing techniques based on correlation analysis.

"ESI LC/MS/MS Analysis of Gluten Exorphins"

 Authors: Christopher L. Pennington & Troy D. Wood

Affiliation: University at Buffalo, The State University of  New York

Department of Chemistry

             Gluten Exorphins are biologically active peptides, similar in structure to Enkephalins, that are derived from wheat gluten¹. Gluten Exorphins have been implicated as possible causative agents in neurological disorders such as Autism². In an effort to understand the pathology of Autism, and the possible role that Gluten Exorphins play in the causation of that disorder, our group is working toward developing an LC/MS method for the quantification of Gluten Exorphins, and their metabolites, in human blood plasma. 

            Additionaly, we are beginning to explore the interaction of Gluten Exorphins with various enzymes, present in human blood plasma, that are responsible for the degradation of endogenous opioid peptides. Our poster will present some of the preliminary results obtained from our work with Gluten Exorphins including the utilization of LC/MS to detect subtle modifications in synthetic Gluten Exorphin peptides.

 1) Fukudome, Shin-ichi & Yoshikawa, Masaaki, FEBS Letters, 296(1), January 1992; 108

2) Whiteley, P., Rodgers, J., Savery, D., Shattock, P., Autism: The International Journal of Research and Practice,  3(1), March 1, 1999; 45-65

“Characterization of Placental Opioid-Enhancing Factor by Liquid
Chromatography and Tandem Mass Spectrometry”

Authors: William L. Wood, Alexis C. Thompson, Mark B. Kristal, Troy D. Wood

Affiliation: University at Buffalo, The State University of New York, Buffalo, NY 

            Most mammals, except some semi-aquatic and fully aquatic species, camels and humans, engage in placentophagia, ingestion of placenta and amniotic fluid. It has been hypothesized that placentophagia enhances opioid-mediated antinociception at parturition, enhancing the effectiveness of endogenous opiates. This is important because high opiate levels can interfere with the onset of parental care.  The active substance(s) responsible for this effect has been named POEF, for Placental Opioid-Enhancing Factor.

            Because placenta and amniotic fluid contain a complex mixture of biomolecules, isolation is needed before MS characterization can be achieved. 

            We report here initial liquid chromatography separation of components in amniotic fluid.

“The Analysis of the Various Distributions of Polyaniline” 

Authors: Anthony R. Dolan & Troy D. Wood

Affiliation: University at Buffalo, The State University of New York Department of Chemistry

            Polyaniline is one of several conducting polymers that has seen considerable attention in the literature over the past decade.  The ease of tuning both the conductivity and the oxidation state of polyaniline with acid-base chemistry has made this polymer one of the most commercially utilized conducting polymers.  As an example, polyaniline has recently been used for the conductive coating on nanospray emitters that are easier to use than conventional emitters, especially when coupling to low-flow separation techniques.

            One of the main drawbacks in using polyaniline commercially is that the exact composition of the polymeric mixture is unknown.  When analyzing low molecular weight oligomers that are soluble in acetonitrile, several different distributions can be detected.  Not only are there varying percentages of the different oxidation states of polyaniline present, but oligomers with different end group types, and also oligomers that are the products of tail-to-tail synthesis and head-to-head synthesis are also present.  This poster shows evidence of these products through tandem MS studies on an API 3000 triple quadrupole.

“Towards Online Micro-LC/CE MS Using Tapered Silica Emitters”

 Authors: Douglas R. Smith

 Affiliation: University at Buffalo, The State University of New York Department of Chemistry

            Coupling capillary-based separation techniques with mass spectrometry has presented numerous difficulties and/or adverse effects upon the separation.  The most common ionization method for these techniques is electrospray ionization (ESI).  Most coupling approaches utilize either a sheath-flow to both generate a voltage supply source and to increase the flow rate to be more consistent with ESI, or a liquid junction in order to generate a voltage supply source while maintaining the low flow rates of micro- or nano-ESI.   Addition of make-up flow results in loss of chromatographic resolution, as well as dilution of the sample decreasing the mass spectral response.  The development of online separation columns/emitters has been hindered by emitter failure during the chromatographic run.

            This poster will present preliminary work on the development of a durable, low-flow ESI emitter made of a tapered length of fused silica capillary.  This capillary can be used for low-flow LC, CE, or CEC.  There is no liquid junction or sheath-flow to detract from the chromatography, while the polyaniline coated silica is highly resistive to electrical discharge, both in the positive and negative ionization mode.

“Reverse Phase High Performance Liquid Chromatography Method for the Analysis of Lopinavir and Ritonavir in Heparinized Human Plasma”

Authors: Keil, K.; Brewer, J.; Morse, G

Affiliation: Laboratory for Antiviral Research, Department of Pharmacy Practice, State University of New York at Buffalo, Buffalo, NY

            There has been a continued interest in developing methods for the analysis of a variety of antiviral drugs used in the treatment of AIDS.  Since many patients are treated with multiple combinations of antiviral drugs at one time, analysis of multiple species is desirable.   A method for the determination of Lopinavir and Ritonavir, including one internal standard has been developed.  The method uses reversed phase high performance liquid chromatography (RP-HPLC) for sample analysis and was validated using a liquid-liquid extraction process.  Drug species are chromatographed using a Waters Symmetry^(TM) shield RP8, 4.6 X 50-mm column and detected using a 486 Tunable Absorbance Detector, scanning at a 215 nm wavelength.  The method is currently being used in the laboratory in support of clinical studies.

“Method Development of a Dr. Curtis Haas Project” 

Authors:  V. Frerichs,  C. Zaranek

 Affiliation: University at Buffalo Department of Pharmacy Practice

               A method has been developed for the separation of the following analytes:  midazolam, hydroxy-midazolam, omeprazole, hydroxy-omeprazole and the internal standard, flurazepam.  The developed method uses HPLC coupled to tandem mass spectrometry.    The method was experimented upon using a Liquid-liquid extraction for preparation of plasma samples.  The separation of substances was performed on a Symmetry Shield RP-8 column (3.0x150 mm, 5mm) column with a mobile phase consisting of methanol and 5mM ammonium hydroxide, adjusted to pH 8.2 with formic acid.  An Applied Biosystems API 3000 tandem mass spectrometer equipped with an electrospray ion source operated in the positive mode was used for detection.  Presented here will be the intricacies in developing a method for the simultaneous analysis of these species.

"Capillary Electrophoresis for the Determination of Unbound Protease Inhibitors"

 Authors:  Valerie A. Frerichs, Jennifer Herrman, Luis A. Colón¹, Gene D. Morse²

Affiliation: 1. Department of Chemistry 2. Department of Pharmacy Practice, School of Pharmacy
                    University at Buffalo, The State University of New York, Buffalo, NY 14260

         Protease inhibitors (PIs) are a class of compounds used in the treatment of human immunodeficiency virus (HIV) infection. This class of drugs inhibits the HIV-1 protease, which acts to process viral proteins essential for the completion of the viral life cycle and subsequent infection of other cells.   The necessary concentration of PIs that provides antiviral activity depends on the amount of protein binding of these drugs within plasma.  Plasma protein binding is currently estimated using equilibrium dialysis, which is somewhat labor intensive.  Unbound drug is also quatitated by preparing samples using ultrafiltration to exclude protein-bound species.  The effects of the membrane, such as adsorption of unbound species, is assumed to be negligible.  It is in our interest to determine the amount of unbound drug in plasma samples without using any sample preparation procedure.  Capillary electrophoresis (CE) has been shown to be useful in the determination of analytes within biological matrices with minimal or no pre-preparation.  We are developing a method based on CE to observe the amount of bound and free amprenavir, indinavir and lopinavir in samples of these drugs mixed with protein.  Presented here will be the details and progress to date of this research.

“Comparison of Positive and Negative Ionization Modes in Mass Spectrometry For Proteomics Applications”

Authors:  Paul M. Bigwarfe Jr. and Troy D. Wood

Affiliation: University at Buffalo, The State University of New York Department of Chemistry

        Traditionally, protein identification in proteomics is accomplished by the bottom-up approach.  This entails enzymatic digestion of the protein mixture and using mass spectrometry to create a peptide mass fingerprint that is database searched to identify the proteins.  This process typically gives 30-40% sequence coverage for each protein in the mixture.  The mass spectrometric analysis, however, is almost always performed in the positive ion mode, which may not be suited for all the resulting peptides.

In this report, both the positive and negative modes of electrospray (ESI) and matrix-assisted laser desorption ionization (MALDI) were investigated to see if any additional information is gained.  It was found that both modes produced unique peaks and their appearance in one spectrum versus the other is dependent on the residue types contained in each tryptic peptide.  By using this approach sequence coverage can increase by an average of 10% and database search scores can be significantly improved.

“Charge Reduction in Electrospray/Nanospray Mass Spectrometry”

Authors:  Cheng Zhao, Troy D.Wood & Stanley Bruckenstein

 Affiliation: University at Buffalo, The State University of New York Department of Chemistry 

        Electrospray/nanospray is an important ionization methods used in biomolecular analysis such as protein and DNA. However there are several problems in electrosray/nanospray ionization. The multiple charged ions are formed in electrospray/nanospray, but the charge determination of them in the high mass range is extremely difficult to achieve. If the sample is a mixture, the multiple charged states for each single analyte in mixtures yield too many overlapping peaks to permit effective discrimination of the various species present.

        One effective method reported recently to solve these problems is to use a neutralization chamber to reduce charge of the ions. The advantage of this method is to improve the sensitivity of electrospray and make the peaks in spectra simpler so that they are easier to be analyzed, especially when it is used in large biomolecules analysis such as proteins and DNA. This poster shows another methods to reduce the charge states,  that involved adding reduction reagents of TCNQ or hydroquinone to the protein sample to reduce charge states.

“Improving MS Dynamic Application Range Through Carryover
Elimination”

Affiliation: JM Science, Inc 

The demand made on drug discovery screening and DMPK laboratories for increased production of new drug candidates has significantly augmented the requirement for mass spectrometers.  MS speed and sensitivity demands fast chromatography (HPLC and SFC) and sample presentation.  The result is a current unmet need for high throughput sample presentation to tandem mass spectometers.  

        Sample capacity and speed of presentation drive this need.  Improvements in sample presentation speed have been negated by the failure of current generation LC/MS autosamplers to eliminate carryover.  Fast mechanical movement and capacity are negated by the necessity to run multiple blanks before reaching sufficiently low carryover to prevent compromising the integrity of the mass spectral data.

        This poster describes an autosampler capable of presenting sample concentrations over a dynamic range of greater than four orders of magnitude with no detectable carryover.

“Packing of Nanospray Emitters for Direct Detection of Capillary Separations”

 Authors:  Lisa Jean Baird, Luis A. Colón, and Troy D. Wood.

 Affiliation: University at Buffalo, The State University of New York, Department of Chemistry

         Low-flow electrospray ionization or nanospray emitters have been developed in our lab with a durable and stable conductive polymer coating.  This development can permit stable coupling of capillary separation techniques, such as capillary liquid chromatography or capillary electrochromatography, directly to nanospray mass spectrometry.  This coupling allows for high sensitivity detection and characterization of biological molecules directly from complex mixtures of biological fluids.  To fully integrate the separation process, packing procedures are currently being investigated to pack the actual fused silica emitters to avoid problems, which may arise during the connection of a separation column to a separate emitter.  Silica based particles, with the ability to facilitate protein separations, are currently being studied and characterized.  The packed emitters can then be used to provide nanospray emitters capable of functioning throughout the course of a capillary separations technique.

“Pharmacokinetics and Metabolism of Phenethylisothiocyanate in Humans Following Watercress Ingestion”

 Authors:  Yan Ji, Lisa M. Predko, and Dr. Marilyn E. Morris 

Affiliation: Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences University at Buffalo, Buffalo NY

Purpose: Organic isothiocyanates (and glucosinolates, the biosynthetic precursors of isothiocyanates in plants), also known as mustard oils, are widely distributed in edible plants including cruciferous vegetables, with human consumption at mg quantities daily.  More than 20 natural and synthetic isothiocyanates and several glucosinolates have been shown to block chemical carcinogenesis in animal models and phenethylisothiocyanate (PEITC) is being investigated in clinical studies as a cancer preventive agent.  GSTM1 is the major enzyme involved in ITC metabolism and since approximately 50% of the population lack this enzyme, this may significantly impact the availability of PEITC and other ITCs. 

Objective: To determine the bioavailability of PEITC after watercress ingestion for subjects that have been genotyped for GSTM1. 

Methods:  Seven healthy subjects were recruited and informed consent was obtained.  Each subject consumed approximately 100g of fresh watercress obtained from a local supermarket.  All subjects were genotyped for GSTM1 using either whole blood or buccal cells by PCR.  Urine samples were collected at 0, 0-1, 1-2, 2-4, 4-6, 6-8, 8-12, and 12-24 hours after watercress ingestion and analyzed for PEITC and the N-acetylcysteine conjugate of PEITC using HPLC.

Results:  All subjects excreted the N-acetylcysteine conjugate of PEITC with greatest amount appearing between 2-4 or 4-6 hours.  No unchanged PEITC was determined in urine samples.  PCR amplified a 273bp product corresponding to GSTM1spanning a single intron.  Lack of band indicates the homozygous for the deletion.  Two subjects exhibited PCR product.  The minimum value for bioavailability (based on urinary recovery of the mercapturic acid conjugate of PEITC) was variable was a geometric mean of  62.2% and a median value of 54% (range of 9 to 185%). High values, obtained in 2 subjects, may reflect the fact that the content of the PEITC glucosinolate (gluconasturtiin) present in watercress was estimated based on literature values for content, or interferences with the HPLC assay. The half life for elimination of the PEITC N-acetylcysteine conjugate, based on urinary excretion rate plots, varied from 2-3 hours.

Conclusions:  Our results indicate that some subjects possess a genetic deletion for the enzyme GSTM1 and therefore produce no product on PCR.  The minimum value for bioavailability, based on urinary recovery of the mercapturic acid conjugate of PEITC, was variable with a geometric mean of 62.2%.  This value is consistent with previous estimates by Chung et al. (1992).

Future studies will determine the pharmacokinetics of PEITC and its N-acetylcysteine conjugate in blood following watercress consumption in subjects genotyped for GSTM1.  Additionally, watercress samples will be analyzed for gluconasturtiin content. 

"Characterization of Analyte Interactions In Reverse Phase HPLC Using
Perfluorinated Stationary Phases"

Authors: Matthew Przybycial; Michael A. Santangelo

Affiliation: ES Industries

        Hydrocarbon based stationary phases such as n-octadecyl and n-octyl are widely utilized in reverse phase HPLC.  The interaction of many analytes, including classes of analytes with these hydrocarbon phases are well characterized.  However, the interaction of analytes with Perfluorinated stationary phases is not well characterized.  It is the goal of this study to better understand the analyte interaction with these perfluorinated phases.
        Using chromatographic test probes and mathematical modeling techniques will be able to investigate the specific analyte interactions.Perfluorinated stationary phases based on phenyl and n-octyl templates will be used for this study.  The effects of mobile phase solvents, buffer composition and pH effects will also be investigated.

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