Northeast Regional
Chromatography
Discussion Group
Fall 2000 Meeting
October 25, 2000 MEETING
Summary of Accepted Papers
(click on the title to view the Abstract)
| Title | Author | Affiliation |
| Keynote Presentation Increasing Speed in Capillary Separation Techniques |
Milton L. Lee | Department of Chemistry, Brigham Young University |
| Monolithic Silica HPLC Columns - A New Era in Chromatography | Fred Rabel |
EM Science |
| Safe Handling of Compressed Gases | Stephen P. Chubon | Air Products and Chemicals, Inc. and Mills Welding |
| Scott Caprio | NuGenesis Technologies Corporation | |
It Really Can Be That Simple!! Introduction to Gerstel Twister (Stir Bar Sorptive Extraction) |
John Thorp | Gerstel Inc. |
| Practical Aspects of Developing LC/MS and CE/MS Methods | Dr. Alex Schilling | Agilent Technologies |
| TLC - New Uses for a Classical Analytical Tool | F. Rabel | EM Science |
Post Column On-Line Reaction as a Sensitive Detection Technique in HPLC |
Minoti Sharma, Ph.D | Department of Molecular & Cellular Biophysics, Roswell Park Cancer Institute |
Applications of Ion Chromatography Coupled with Mass Spectrometric Detection |
William Schnute | Dionex Corporation |
The Importance of Client Server in the Chromatography Laboratory |
Jonathan McSayles and Maureen Stone | Scientific Software, Inc. |
Evaluating Supercritical CO2 as a Pseudo Dry-Packing Method for CEC Columns |
Todd D. Maloney and Luis A. Colón | State University of New York at Buffalo, Department of Chemistry |
Reversed-Phase Analytical Chromatography at Extended pH Ranges (1-12)Why and How? |
Joe Solon | Waters Corporation |
| Terrence S. Reid | Restek Corporation | |
| Robin DiFrancesco | The University at Buffalo, School of Pharmacy | |
| Kevin Turnbull, Michael McBrien, Antony Williams, and Eduard Kolovanov | Advanced Chemistry Development Inc. | |
| Poster Session | ||
| Larry G. West | Dionex Corporation | |
| Richard A. Henry, Richard L. Leathers and Todd A. Shaffer | Keystone Scientific, Inc. | |
| A General SFC Method: Conquering the Requirements | Joan M. Stevens, and Alan E. Hamstra | Gilson Inc |
| New Chiral Detection in HPLC and SFC | Muneo Saito | JASCO Incorporated |
| M. Young, J. Krol, D. Phillips, P. Iraneta, and U. Neue | Waters Corporation | |
Novel Polymeric Mixed-Mode Sorbents for the Extraction and Analysis of Drugs of Abuse |
(Yung-Fong Cheng, Ziling Lu, Uwe Neue, Pamela
Iraneta, and Debra Laviolette)1 (Michael A. Evans, James Kraner, and Evan Holzberg)2 |
1-Waters Corporation and 2-AIT Laboratories |
High Throughput Analyses Using 96-Well SPE Plate and Novel Hybrid Technology with LC/MS(/MS) |
Yung-Fong Cheng, Ziling Lu, and Uwe D. Neue | Waters Corporation |
| Yung-Fong Cheng, Pam Iraneta, Ziling Lu, Uwe D. Neue, Dorothy Phillips, and Mark Banyham | Waters Corporation | |
Using
LCMS for Developing Methods to Analyze |
Valerie A. Frerichs and Robin DiFrancesco |
Laboratory for Antiviral Research SUNY at Buffalo |
| Kevin Turnbull, Michael McBrien, Antony Williams, and Eduard Kolovanov | Advanced Chemistry Development Inc | |
| Thiophilic Interaction Chromatography of Human Transferrins | James T. MacKenzie and Thamarapu Srikrishnan |
Molecular & Cellular Biophysics, Roswell Park Cancer Institute |
ABSTRACTS
Keynote Presentation
Milton L. Lee
Miniaturization of the analytical column, particularly with the utilization of very small diameter packing materials, requires the use of pressures much higher than used in conventional gas and liquid chromatography to generate high mobile phase linear velocities. For the condensed-phase chromatographic techniques, pressures higher than provided by typical pumping systems are needed. At the same time, columns that have higher permeability (i.e., lower pressure drop) and mobile phases that have lower viscosity are desirable.
Recent developments in orthogonal acceleration and high speed data acquisition/processing have made time-of-flight mass spectrometry the method of choice for mass spectrometry detection where high speed and high sensitivity are required. Off-axis (orthogonal) acceleration of ions from an ion beam into the flight tube allows for both improved resolution and duty cycle. New processors with high speed memory make it possible to acquire and process data at the speeds required. Up to 100 spectra per second can be produced in real-time operation from summing of spectra that are acquired at rates as high as 10,000 spectra per second.
In this presentation, efforts in the authors' laboratory to increase the speed of analysis of the packed capillary formats of liquid chromatography, electrochromatography, and supercritical fluid chromatography are described. Specific topics include the use of nonporous particles, large-pore particles, continuous-bed columns, high pressures or voltages, and time-of-flight mass spectrometry.
"Monolithic Silica HPLC Columns - A New Era in Chromatography"
Author: Fred Rabel
Affiliation: EM Science
ABSTRACT
HPLC columns packed with particulate packings have been the mainstay of almost all of the HPLC work done in the past 35 years. For over 20 years, there has been an interest in making a continuous rod of silica gel, thereby avoiding many of the problems associated with particulate columns. Researchers, however, were successful in making only smaller ID monolithic columns. Recently standard 4.6mm ID columns have been successfully manufactured and are now available in North America. They are called Chromolith TM columns. Because of the unique structure of these columns, they can be flow programmed with little loss in efficiency compared to particulate columns. Thus late eluting peaks can be quickly eluted by simply increasing the flow rate, rather than introducing a gradient for speeding up the separation.The details of the manufacture of these products will be outlined, and examples of separations shown.
.
Presenter:
Stephen P. Chubon
Affiliation: Air Products and
Chemicals, Inc.
ABSTRACT
A one hour slide presentation and discussion of safety guidelines for the handling and
storage of compressed gases.
The presentation addresses the general properties and hazards of gases and also discusses
valves and regulators..
"Getting the Signals Right in Data Management"
Author: Scott Caprio
Affiliation: NuGenesis Technologies Corporation
ABSTRACT
New technologies are producing an ocean of data, but acting on that information is more than a few steps behind. Certainly, scientific information has value, but having a lot of data does not mean it is worth a lot. Within the pharmaceutical industry alone, investments in data generation technologies (high throughput screening, combinatorial and parallel synthesis) have resulted in an unintended consequence - decreased value of data through information overload.
In theory, electronic record keeping is easy. In practice, advanced technologies and lab applications rapidly generate a torrent of bytes - all products of proprietary sources - each stored in its own proprietary "language," none able to communicate with another. In the ultimate irony of laboratory automation, the output for each sophisticated instrument is a printed report. Converting data into usable information requires manually piecing together a puzzle of paper.
This paper will discuss a set of technologies that systematize the collection, storage and sharing of scientific data by unifying it into a common electronic format. Specific examples of record maintenance in an application independent environment within the pharmaceutical industry will be discussed. The technology provides for storage and maintenance of machine readable data in its native file structure and for the capture of human readable report data which can be viewed independent of the original processing environment. Scientists can capture data from a variety of lab and business application sources and store it in a central database using context sensitive tags that allow the data to be located easily using simple search tools, while maintaining its graphic integrity. Scientists anywhere in the organization can view the assembled data using a web browser.
This technology satisfies both business and regulatory needs and presents a comprehensive strategy for managing scientific data generated during the drug development and manufacturing processes.
"It Really Can Be That Simple!! Introduction to Gerstel Twister (Stir Bar Sorptive Extraction)"
Author: John Thorp
Affiliation: Gerstel Inc.
ABSTRACT
Gerstel Twister is an exciting new sample extraction technique expanding on the principles of SPME. Solvent less extraction of organic molecules by partioning into the phase coating of a magnetic stir bar. Basic theory, qualitative and quantitative results are discussed.
"Practical Aspects of Developing LC/MS and CE/MS Methods"
Authors: Dr. Alex Schilling
Affiliation: Agilent Technologies
ABSTRACT
This presentation will focus on practical aspects of selecting mobile phases and modifications in LC and CE that are compatible with MS analysis. Aspects of mobile phase modifications such as the choice of buffers, the use of ion pairing agents, and LC mobile phase modifiers/modifications will be discussed. Considerations will be given to mobile phase volatility, ionic strength and other characteristics. Selection criteria for mobile phase additives and examples from some applications will be discussed.
Author: F. Rabel
Affiliation: EM Science
ABSTRACT
TLC is still a widely used analytical tool. However, it is used only in about 50% of the laboratories where it could be a valuable, time saving addition. This is due mostly to the fact that it is still a manual technique. Only a few parts of the entire process have been automated. In spite of this, it is ideal for mass screening of many samples. These could be results from combichem experiments or eluted fractions from SPE tubes. Even HPLC labs can use it simply to scout solvents and to see what does or does not move through a column, and help with its cleaning should impurities collect on it.
New applications and products will be shown to demonstrate the continuing uniqueness and simplicity of TLC in everyday applications
"Post Column On-Line Reaction as a
Sensitive Detection Technique in HPLC"
Author: Minoti Sharma, Ph.D.
Affiliation: Department of Molecular & Cellular Biophysics, Roswell Park Cancer
Institute
ABSTRACT
On-line postcolumn reaction in HPLC offers highly sensitive
detection following simple spectrophotometric measurement. Bromate, a disinfection
by-product of the ozonation of bromide-containing waters, is carcinogenic. Postcolumn
on-line derivatization of bromate with fuchsin reagent in ion chromatography offers a
detection limit 0.1 (g/ L without the chloride interference observed with conductivity
measurement. Tamoxifen, an antiestrogen used in breast cancer therapy and as a
prophylactic agent, is inherently nonfluorescent On-line post column UV irradiation in
HPLC converts tamoxifen and its metabolites to highly fluorescent agents. The power of the
technique to assay DNA damage induced by the activated metabolite will be presented.
"Applications of Ion Chromatography Coupled with Mass Spectrometric Detection"
Author: William Schnute
Affiliation: Dionex Corporation
ABSTRACT
Electrospray mass spectrometry (ESI-MS) is a powerful detection technique that is
particularly appropriate for the analysis of permanently ionized or polar compounds. As
such, I has found wide spread use in conventional LC/MS for biotechnological and
pharmaceutical applications. To date, applications of IC coupled with ESI-MS have been
limited. This paper reports on using IC/ESI-MS for the confirmation and quantitation of
important trace contaminants, such as perchlorate, bromate, and haloacetic acids, in
environmental water samples. The use of IC/ESI-MS for metal speciation, as well as for the
identification of previously unknown compounds in wastewaters, will also be discussed.
"The Importance of Client Server in the Chromatography Laboratory"
Authors: Jonathan McSayles and Maureen Stone
Affiliation: Scientific Software, Inc.
ABSTRACT
Recently, everyone seems to be talking about client server software applications. Also, there seems to be different ideas as to what exactly is meant by the term "client/server". This paper defines a client server application. A basic definition of what a network is and the components used in building a network are given. In addition to the definitions and explanations, the benefits of operating in the client server environment are addressed. Finally, an overview of the type of knowledge management which can be achieved using the C/S environment is shown.
"Evaluating Supercritical CO2 as a
Pseudo Dry-Packing Method for CEC Columns"
Authors: Todd D. Maloney and Luis A. Colón
Affiliation: The State University of New York at Buffalo, Department of Chemistry
ABSTRACT
We are currently evaluating packing protocols used in capillary electrochromatography (CEC) in order to maximize column performance. Of particular interest to us is the incorporation of a drying-step in the packing protocol. We have demonstrated previously that incorporating a drying-step in columns packed by centripetal forces can increase column performance compared to columns that were not dried (Maloney, T.D.; Colón, L.A. Electrophoresis 1999, 20, 2360-2365). Supercritical CO2 packing, in essence, can be considered a dry-packing technique since the packed bed is dry after packing. In the work presented here, we will present CEC column performance of capillary columns packed by supercritical CO2, which have been fritted prior to solvating the packed bed with the mobile phase. These capillary columns will be compared to columns that have been fritted after solvating with the mobile phase.
"Reversed-Phase
Analytical Chromatography at Extended pH Ranges (1-12)
Why and How?"
Authors: Joe Solon
Affiliation: Waters Corporation
Successful method development requires the chromatographer to make the most effective use of analyte manipulation to create the optimum separation of the peaks of interest. In the past this has been achieved by exploiting the minor different selectivity properties of silica-based C18 columns and more recently by the advent of a different selectivity achieved through the use of ‘shielded chemistries’ (embedded polar groups). The drive for faster method development, however, has re-focussed attention onto the more powerful selectivity tool of pH manipulation. Traditionally, this approach has been hampered by the limited pH range of silica-based C18 materials (pH’s 2-8). With newer materials now available we can take a fresh look at developing methods faster and in a manner which tackles some of today’s most difficult problem separations.
In this seminar, we explore how to take advantage of the properties of these newer materials in the development of new HPLC methods.
- Investigation of the relationship of retention with pH vs. solvent composition for acidic, basic and zwitterionic analytes
- Discussion of analyte pKa shifts in different solvent mixtures and how this affects method validation and robustness
- Benefits of high pH analysis with respect to peak shape, detectability, sensitivity and selectivity
- How to compensate for the changes of retention due to pH manipulation
- New buffer choices for high pH separations
"Reversed Phase HPLC with Highly Aqueous Mobile Phase"
Author: Terrence S. Reid
Affiliation: Restek Corporation
Reversed phase HPLC analyses of highly polar compounds are a desirable alternative to ion
exchange and reversed phase-ion pair methods, which are generally less robust.
Retaining highly polar compounds by reversed phase requires weak or highly
aqueous mobile phases. Conventional straight chain alkyl (i.e., C18) stationary
phases sometimes show a reversible loss of
retention when using 100% aqueous mobile phases, often referred to as chain folding.
This paper will demonstrate some of the factors that control chain folding,
including bonded phase chemistry, silica pore size, mobile phase additives, and column
pressure. By considering these factors, you can achieve highly reproducible
retention with reversed phase separations using 100% aqueous mobile phases.
"A Molecular Structure Based Chromatography
Applications Database with
Retention Time Prediction Capability"
Authors: Kevin Turnbull, Michael
McBrien, Antony Williams, and Eduard Kolovanov
Affiliation: Advanced Chemistry Development Inc.
ACD/ChromManager is a unique Chromatography Data System (CDS) due to its structural capability as well as links to other ACD software such as physicochemical and naming prediction and structure-based method development software.
The structural search capabilities of this CDS enable users to retrieve chromatographic methods based on similar functionality. The program is capable of searching multiple databases simultaneously; this means that the user can search an Internal Applications Database composed of contributed applications from various chromatographic vendors at the same time as their own data. Once retrieved, data from archived chromatograms can be used to predict retention times for new compounds using ACD/LC Simulator.
"From Traditional Ultraviolet or
Fluorescence Detection to state-of-the-art Mass Spectrometry:
Early Experiences at The University at Buffalo LCMS Facility"
Author: Robin DiFrancesco, Facility Administrator
Affiliation: The University at Buffalo, School of Pharmacy
ABSTRACT
After many years of utilizing liquid chromatography (LC) assays using
ultraviolet or fluorescence for detection to measure pharmaceuticals in an academic
research environment, the predominance of mass spectrometry detection in the industrial
environment has created a must-convert challenge to academic curriculum. A
successful NIH shared equipment grant application afforded the opportunity for The
University at Buffalo to take on a commitment to LCMS methods development. We have
learned: all mass spectrometers (MSs) are not created equal! As such, this
presentation will focus solely on the use of an Applied Biosystem's API 3000 using a
turbospray ion source. Basic topics covered will include: how to determine the
best way detect your compound in the MS, MS plus LC/advantages,
limitations and changes when converting from traditional LC, sample preparation
considerations, and some snags we've experienced along the way.
If you have not yet braved the challenges of this scientific resource, are just beginning
to, or are already deeplyentrenched in it, please attend this presentation.
In addition to this presentation, The University at Buffalo will have a poster
highlighting some of the problems the new facility has experienced and a round table
discussion chaired by both authors. Please join us!
Posters
"Gas Chromatography Automation of Multi-vendors"
Author: Larry G. West
Affiliation: Dionex Corporation
ABSTRACT
There have been three areas that the analytical instrument industry has always cited in
making multi-vendor control so elusive. The first is the "instrument drivers";
these are used for the communication with the myriad of different analytical instruments.
While some instrument companies have chosen to vigorously protect these from third party
use; others have made them available. The second is the communications protocol. Some have
chosen IEEE, RS232, and IP address, etc. Dionex utilizes all three in third party
communications. Lastly is the notion of having a configurable front end that will address
multi-vendor instruments. Dionex has addressed this in the use of "panels". This
presentation will discuss interface capability to Varian and Agilent series Gas
Chromatographs.
"Ultra-Fast LC-MS Column Design"
Author: Richard A.
Henry, Richard L. Leathers and Todd A. Shaffer
Affiliation: Keystone
Scientific, Inc.
ABSTRACT
The emergence of mass spectrometry (MS) as a rugged, sensitive detector for HPLC has generated new interest in column design. The speed and selectivity of MS has opened up the opportunity to use very short columns which normally would not be suitable if used with UV and other detectors. Much recent work has focused on the use of very short columns which act as a selective interface between the HPLC system and the MS detection system.The pharmaceutical industry has developed combinatorial methods to speed up new drug discovery. Fast screening techniques are needed to provide analytical information about these samples, which can contain a wide variety of compound types. LC-MS with short columns and high flow rates seems to be ideal for this purpose. This paper will compare the performance of very short columns, with various geometries and packings, under high linear velocity LC-MS conditions.
Author: Joan M.
Stevens, and Alan E. Hamstra
Affiliation: Gilson Inc
ABSTRACT
Supercritical fluid chromatography is a method of analysis that holds many advantages over HPLC; namely, less solvent consumption, faster column equilibration, and pressure gradients. In semi-preparative mode (flow rates up to 25 mls/min), SFC also offers non-aqueous fraction collection in a substantially smaller volume. Compared to RP-HPLC under the same running conditions we have a drastic decrease in the time required for fraction dry down. Although SFC seems to be a viable alternative or complementary to RP-HPLC, many chromatographers are unfamiliar with SFC and the conditions to employ as a general SFC method.
The purpose of this application is to present to the chromatographer a table of results based on a mixture of three compounds (neutral, basic, and bifunctional) in which the mixture was chromatographed under various conditions. The conditions that were used consist of various chromatographic media, organic solvents, addition of modifier, pressure and temperature. Based on the results presented in the SFC table conditions will be offered that can be employed as a general chromatography method for SFC.
Author: Muneo Saito
Affiliation: JASCO Incorporated
ABSTRACT
There are two chiral detection methods in HPLC. One is based on
conventional optical rotation (polarimetric detector) and the other on circular dichroism
(CD detector). CD is popular in protein analysis. However, usefulness in detection
of small compounds such as pharmaceutical compounds has not been highlighted until
recently. We will present operation principles and applications of a CD detector in
HPLC as well as SFC applications.
"A New Mixed-Mode Solid-Phase Extraction Sorbent; Extraction and
Cleanup Strategies
for the LC/MS Determination of Fungicides in Fruit Juices"
Authors: M. Young, J. Krol, D. Phillips, P.
Iraneta, and U. Neue
Affiliation: Waters Corporation
Benzimidazoles, such as thiabendazole, are commonly used as fungicides for protection of fruit and vegetable crops. Although reliable methods have been developed for LC or GC determination of fungicide residues in fruit matrices, these methods require multiple clean-up steps using either liquid-liquid extraction (LLE) or solid-phase extraction (SPE).
This paper discusses a novel, mixed-mode (reversed-phase and cation exchange) sorbent. Because of the unique nature of the sorbent, the retention mechanism for polar, basic analytes can be predominantly cation exchange at low to neutral pH, or reversed-phase at high pH. Therefore, by employing pH and solvent adjustments, the analyst can develop extraction and cleanup steps using only one SPE cartridge where previously, two or more different sorbents were required.
Using logical method development strategies, SPE protocols were developed for the LC/MS determination of thiabendazole and related compounds in various fruit juices. These SPE protocols employ a single Oasisâ MCX cartridge. Extraction and cleanup steps were accomplished using all the possible retention modes available from this unique sorbent.
"Novel Polymeric Mixed-Mode Sorbents for the Extraction and Analysis of Drugs of Abuse"
Authors: (Yung-Fong Cheng,
Ziling Lu, Uwe Neue, Pamela Iraneta, and Debra Laviolette)1
(Michael A. Evans, James Kraner, and Evan Holzberg)2
Affiliation: 1-Waters Corporation and 2-AIT Laboratories
Drug abuse is a profound social issue. A key to addressing this social issue is to be assured that the best drug testing technology is available. The most commonly analyzed drugs of abuse are the NIDA 5. The NIDA 5 include benzoylecgonine (amphoteric), amphetamines (amphetamine, base, methamphetamine, base), opiates (6-monoacetylmorphine, base; morphine, amphoteric), phencyclidine (neutral), and THC (acid). Based upon the nature of these analytes, it is necessary to have several sample preparation methods and derivatization steps for their positive identification and quantification. Therefore, it would be desirable to have simplified methodologies to determine these drugs.
In this paper, we will demonstrate straightforward Oasis® mixed-mode methods for the extraction of the NIDA 5 from human urine. The extracts are quantitated either by GC/MS or by LC/MS analyses. For the GC/MS analysis, the extracts are dried and then derivatized following the current existing GC/MS protocol. For the LC/MS analysis, the extracts are directly injected onto a XTerra™ column containing a bonded hybrid particles, 2.1 mm x 20-mm. These seven components of the NIDA 5 drugs are well separated in less than three minutes and quantitated in either SRM or SIM mode using a Micromass™ mass spectrometer.
In routine work, these streamlined methodologies facilitate the fast throughput, robust, and efficient positive identification of NIDA 5 drugs of abuse.
"High Throughput Analyses Using 96-Well SPE Plate and Novel Hybrid Technology with LC/MS(/MS)"
Authors: Yung-Fong Cheng, Ziling
Lu, and Uwe D. Neue
Affiliation: Waters Corporation
Sample preparation is required to maximize the sensitivity of the analytical results and to minimize the downtime of expensive analytical tools such as LC/MS/MS systems. High throughput sample preparation is frequently achieved by solid-phase extraction in a 96-well format due to its compatibility for automation. The enabling technology for 96-well plates is a single reversed-phase sorbent that can be used to extract all drug classes from biological fluids. A generic sorbent and a simple solid phase extraction (SPE) method coupled with fast chromatographic LC/MS(/MS) are the keys to automating the analyses for pharmacokinetics studies in drug discovery and the clinical trial samples in drug development. This paper shows the utility of Oasis ® 96-well plates, which contain a polymer based water-wettable sorbent, for the extraction of drugs in biological fluids. A wide range of drugs and metabolites (including acids, neutrals, and bases) in biological fluids (plasma, serum, and urine) are extracted using logical and simple SPE methodologies. After the Oasis® sample cleanup, the extracted sample solutions are separated with 2.1 mm x 20 mm packed with Symmetry® and XTerra™ packings. All the chromatographic separations are achieved in less than three minutes. The identification and quantitation of parent drugs and their metabolites are performed in either selected reaction monitoring (SRM) mode or selected ion monitoring (SIM) mode on a Micromass™ mass spectrometer, which is equipped with a ZSpray™ ion source. The results demonstrate that the use of the 96-well extraction plate and fast LC/MS(/MS) analysis facilitate today's high throughput analysis. Recovery greater than 85% and variability less than 6% are achieved for a wide range of drugs from biological matrices.
"Strategies
for Developing Sensitive and Selective Solid-Phase Extraction Methods
for High Throughput Bioanalysis"
Authors: Yung-Fong Cheng, Pam
Iraneta, Ziling Lu, Uwe D. Neue, Dorothy Phillips, and Mark Banyham
Affiliation: Waters Corporation
The demands for increased sensitivity and selectivity in bioanalysis are elevating the need for improvements in sample preparation. We will demonstrate how well-designed solid phase extraction (SPE) sorbents (Oasis® HLB, Oasis® MCX and Oasis® MAX) and intuitive SPE methods accelerate and simplify sample preparation. All Oasis® sorbents are water-wettable and pH stable (0-14) polymers. There are no silanol groups to complicate method development and the mode of retention.
Bioanalysis with the Oasis® HLB sorbent employing a generic reversed-phase SPE method gives high and consistent recoveries for a wide range of compounds including drugs and their metabolites. This generic method is ideal for fast throughput analysis on 96-well plates or cartridges with LC/MS/MS detection. A more selective 2-D reversed-phase SPE method simultaneously isolates the analytes of interest from complex sample matrices and eliminates interferences. The 2-D method has been shown to enable lower detection limits when using UV detectors. Even higher selectivity and sensitivity can be achieved by using the mixed-mode sorbents to extract basic or acidic analytes from biological fluids. The Oasis® MCX and Oasis® MAX sorbents in low elution volume 96-well plates detect analytes at ng/mL and pg/mL levels.
The demands for selectivity and high throughput can also be achieved with on-line SPE methods using the Oasis® HLB extraction column and mass spectrometry detection. This method allows direct injection of plasma samples with analysis times can be as short as 1.2 minutes. This presentation will cover the above four methods development strategies in great detail and gives numerous application examples from biological matrices. High throughput analyses and enhanced sensitivity detection with LC/MS(MS) will be demonstrated as well.
"A Molecular Structure Based System for LC Simulation"
Authors: Kevin Turnbull, Michael
McBrien, Antony Williams, and Eduard Kolovanov
Affiliation: Advanced Chemistry Development Inc.
ACD/LC Simulator predicts retention times and optimal experimental conditions using structures and/or experimental data.
LogD, the octanol/water partition coefficient for dissociative systems, is the crucial component in the prediction of the retention times of different compounds in HPLC analyses. Additionally, in some cases the values of molar volume and MW are also exploited. Utilizing LogD prediction, the user can predict optimal pH levels for separations based purely on molecular structures. Other parameters, such as temperature, solvent composition, and gradient can also be optimized using experimental data. ACD/LC Simulator can also utilize any experimental pKa, LogP, or LogD information that might be available for compounds in order to increase the accuracy of prediction for related species.
"Using LCMS for Developing Methods
to Analyze
Antiviral Drug Species of Current Interest"
Authors: Valerie A. Frerichs and Robin
DiFrancesco
Affiliation: Laboratory for Antiviral Research SUNY at Buffalo
It is in our interest to develop better methods for the analysis of antiviral drugs used
in the treatment of AIDS. Traditionally, development of techniques for
pharmaceutical drug analysis has relied heavily on liquid chromatography (LC) coupled to
optical detection formats. However, evolution of mass spectrometric instrumentation
that is increasingly reliable and easily coupled to LC provides advantages over the
traditional methods. For example, using LC with mass spectrometry (LCMS) affords the
analysis of a greater number of drug species in a decreased amount of time due to the
ability of the detector to deconvolute coeluting compounds. This leads to higher
sample processing efficiency. In addition, due to the increased sensitivity of MS
versus ultra-violet absorbance detection, smaller volumes of sample are necessary.
Because of these inherent advantages, we are in the process of validating LCMS methods for
drug species of current interest. One of these validations results from a merging of
several LC methods to one LCMS method. Presented here will be a comparison of
the two techniques with respect to these drugs. Also, we will
review the requirements LC to LCMS method conversion, the advantages and disadvantages,
and the challenges we have encountered.
"Thiophilic Interaction Chromatography of Human Transferrins"
Authors: James T. MacKenzie and Thamarapu Srikrishnan
Affiliation: Molecular & Cellular Biophysics, Roswell Park Cancer
Institute
Transferrins are a class of iron binding proteins widely distributed in biological fluids which includes lactoferrin and serum transferrin. All transferrins possess two metal binding sites, each of which can bind a ferric iron. Transferrins play a major role in plasma iron transport and have additional anti – bacterial, anti – inflammatory, and immunological functions.
Our results indicate, for the first time, that human transferrins, as well as numerous other species' transferrins, do bind to T – gels. As has been hypothesized, the strength of the affinity for a T – gel correlates with the presence of the clusters of aromatic residues that are present on the surface of the transferrins. We have been able to demonstrate that thiophilic interaction chromatography (TIC) can be used as a method for additional purification of commercial products. With the use of TIC, we have devised a simple and accurate method of monitoring transferrin levels in human serum. This ability to monitor serum levels may prove to be a very valuable medical application since serum transferrin levels decline with the advance of Alzheimer's disease.
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