| Presenter |
Title |
Affiliation |
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Todd D. Maloney
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Very High-Pressure Liquid Chromatography-Mass
Spectrometry for Method Development and Impurity Identification in
Pharmaceutical Analysis |
Pfizer Global Research and Development, Ann Arbor, MIS |
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Essentially every drug on the market or being developed is analyzed
by high-performance liquid chromatography (HPLC). Although rugged and
robust, HPLC is often criticized as being the rate limiting step in drug
development where high-throughput analysis and high efficiency
separations are in demand. Reducing the particle diameter (dp) of the
packing material in liquid chromatography is the time proven method to
achieve increased separation power through faster analysis times and
higher separation efficiency. Recently, advances in instrument and
column technology have made it feasible to perform very high-pressure
liquid chromatography (VHPLC) utilizing small particle (< 2µm)
columns for routine analysis. The increased efficiency and resolution
from the small particles will ultimately yield faster method development
and higher throughput, and provide further cost reduction through
standardization of analytical methodologies. This could potentially lead
to a truly generic separation system where only small selectivity values
will be required to separate structurally similar compounds.
We have investigated VHPLC-MS for routine use in pharmaceutical
analysis. We have investigated the performance of chromatographic
columns tailored for VHPLC as well as characterized instrument
performance. Our presentation will focus on the performance of the
instrument for routine method development and discussion of practical
considerations for widespread implementation of VHPLC-MS.
|
Troy D. Wood
|
Detection of Gluten Exorphins B4 and B5 in Human Plasma
by LC-MS/MS |
University at Buffalo, The State University of New York,
Buffalo, NY
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Peptides derived from digestion of wheat gluten, called exorphins, have
been previously detected in urine samples from autistic subjects.
Because of their opioid activity, they have been suggested by some as
playing a role in the etiology of autism. Previous LC-MS results from
our laboratories indicated that exorphins degrade rapidly in biological
fluids in the absence of the protease inhibitor aprotinin. Here, we show
that two of the exorphins, B4 and B5, can be detected in blood plasma
samples of patients diagnosed with celiac disease (i.e. high intestinal
permeability) using LC-MS/MS. Experiments were performed on a Thermo
Electron Orbitrap mass spectrometer. The presence of exorphins in the
plasma was monitored from samples collected up to 2 hrs. after the
consumption of a pizza ("Pizza Test") by the subjects. The
exorphins were detected in three of four test subjects by LC-MS/MS;
MS/MS spectra prove conclusively that the peptides are indeed exorphins.
This is the first time exorphins have been detected in blood plasma
samples.
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Diana S. Aga
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Characterization of Glutathione Conjugation by
Pharmaceuticals Contaminants Using Liquid Chromatography/ Ion Trap Mass
Spectrometry |
University at Buffalo, The State University of New York,
Buffalo, NY
|
Soil contamination with non-metabolized antibiotics is an emerging
environmental concern, especially on agricultural croplands that receive
animal manure as fertilizer. In vitro conjugation reactions showed that
glutathione s-transferases (GST) induced in maize catalyzed the
conjugation of glutathione (GSH) with chlortetracycline (CTC), producing
stable conjugates that were structurally characterized using liquid
chromatography (LC) with ion-trap mass spectrometry (IT-MS). The
chromatogram of the GST-catalyzed reactions displayed two characteristic
features of the putative CTC-GSH conjugate: 1) the conjugate eluted
sooner than CTC and 2) the conjugate eluted in multiple peaks. The
removal of chlorine and the addition of a glutathione moeity result in a
highly water soluble CTC-GSH conjugate, thus a much shorter retention
time relative to the more hydrophobic CTC is expected. Several
fragmentation patterns in the mass spectrum of the CTC-GSH conjugate can
be used to verify the identity of the enzyme reaction product. The
expected molecular ion [M+H]+ of CTC-GSH conjugate (m/z 751) with
chlorine removal was not observed in the positive electrospray
ionization. Instead, a base peak of m/z 677, corresponding to the loss
of glycine (MW=75 Da) was observed. When m/z 677 was isolated in the ion
trap and subjected to further fragmentation, characteristic peaks
corresponding to the loss of glutamic acid (m/z=129) and water (m/z 18)
were observed in the MS/MS spectrum. Other pharmaceutical-GSH conjugates
were characterized by LC/MS and results will be included in the
presentation.
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Hans F. Schmitthenner and Robert Saccente |
Micro-Prep LC and LC-MS of Imaging Materials |
Eastman Kodak Company, Rochester, NY
|
To facilitate the isolation of peaks from analytical LC or LC-MS, we
have developed a technique for automating the collection of separated
components in analytical LC or LC-MS and call this procedure
+IBw-micro-prep LC or LC-MS+IB0-. Micro-prep on the analytical scale
introduces unique challenges, which include issues focused on timing,
collection, and fraction verification. With the ability to run 50 to 100
sequential unattended runs (or more) during working hours (or overnight)
it is possible to isolate quantities of material sufficient for mass
spec infusion experiments and NMR experiments including various 2-D 1H
and 13C spectra. This presentation will illustrate how we used this
technique to solve characterization challenges in the area of imaging
materials including inkjet (IJ), organic light-emitting diode (OLED),
and optical molecular imaging (OMI).
|
| Joseph Fox |
Applications of a Novel Hybrid LCMS Ion Trap Time of
Flight Mass Spectrometer in Small Molecule Identification and Metabolite
Analysis |
Shimadzu
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This
talk will focus on application of a novel hybrid LC-ion trap, time of
flight mass spectrometer to several significant problems in the fields
of chemistry and biochemistry. We will review the identification
of unknown small molecule compounds – including both natural products
and synthetic pharmaceuticals - via a novel formula prediction software
that includes both MSn fragmentation data, as well as high
mass accuracy, to yield formula identifications of unknown compounds.
In addition, we will describe the use of the LCMS-IT-TOF to
accelerate metabolite identification, without compromising the
information needed for characterization. Here we have considered
an open strategy in metabolite detection that integrates the high mass
accuracy MSn analysis of the instrument with advanced data
filters to find both “expected” and “unexpected” molecules.
We will conclude with a brief description of the advantages of
this type of instrument in coupling with novel LC methodologies,
including UFLC (Ultra Fast Liquid Chromatography), for fast and accurate
compound analyses in complex samples and high-throughput environments.
|
|
| Jason A. Anspach |
Advancements in Packing Technologies for HPLC Columns |
Phenomenex
|
|
In the early days of
HPLC, much as was the case with GC, scientists performing the
experiments were expected to pack their own columns.
As the technology progressed, scientists have come to rely on
commercially packed HPLC columns to meet their separation needs.
As HPLC has matured the performance, reliability, and lifetime of
the columns expectations increased.
One area of recent development in HPLC columns is the
implementation of ever decreasing particle sizes.
Several commercial manufactures offer columns packed with
materials that are smaller than 3 µm in diameter.
These columns provide an increased number of theoretical plates
per unit length allowing the analyst to decrease their overall column
lengths, thereby decreasing the analysis times, without sacrificing the
overall separation power of the columns.
There are two drawbacks, however, to the use of smaller packing
materials. The first drawback is the pressure required to operate these
columns is significantly higher. .
Furthermore, the inner diameters of these columns are
significantly reduced, leading to lower peak volumes and subsequently
lower tolerances for band broadening or peak dispersion.
The higher backpressure and smaller dispersion requirements lead
to columns needing a more homogeneous bed that must tolerate
significantly high operational pressures.
Another area of column inadequacy has been for short preparative
columns, typically used to purify combinatorial libraries.
During the traditional slurry packing process, the column must be
depressurized in order to be fitted with the column end fitting.
Because of their large inner diameter (21.2 – 50 mm i.d.) and
short lengths (50-100 mm) the amount of wall support in these columns to
retain the packed bed is significantly decreased.
The decrease in wall support leads to a phenomena where a section
of the packed bed extrudes from the column while it is removed from the
packing reservoir to install the end fitting.
The media extrusion subsequently decreases the packing density in
the column, and subsequently reduces the column lifetime.
In this presentation we will outline packing technologies that
address the problems in these two areas of column technology. We will demonstrate that by optimizing the packing process
using a 2.5 µm material, columns efficiencies are obtained that are
comparable to columns packed with sub 2 µm materials that operate at
significantly lower backpressures.
This lower backpressure allows these highly efficient columns to
operate on a wide variety of HPLC instrumentation, not just newer higher
pressure HPLC instrumentation. We
will also show a revolutionary packing process to pack preparative
columns, where the pressure that is applied to the column during packing
is never released, which significantly increases the packing density,
and therefore the column efficiency and lifetime are improved.
Both column-packing techniques raise the performance, reliability
and ruggedness of the HPLC column to meet the scientist’s needs.
|
|
| Jeannette
Karczmarski |
Achieving High Definition in Complex Mixture
Analysis with SYNAPT “High Definition” MS/IMS/MS, with MSE
and TAP Fragmentation |
Waters Corporation
|
|
The presentation will
focus on application of a unique quadrupole-ion mobility-TOF mass
spectrometer for the analysis of a complex biological samples, such as
drug metabolite ID in biological matrices, intact proteins, protein
complexes, protein conformations, Top-Down protein sequencing, and
structural identification using MS/IMS/MS analysis.
The talk will explain how this instrument leverages the proven benefits
of ion mobility to combine fast gas-phase separations with the high
resolution and mass accuracy of TOF MS to provide an additional
dimension of separation, and enabling the analysis of samples
differentiated by size, shape, charge, as well as accurate mass.
Some typical mobility experiments will be used to demonstrate how
MS/IMS/MS delivers specificity beyond that achievable by conventional
mass spectrometers.
We will also highlight more empowering features of the system including
the Driftscope and TriWAVE technology, MSE, and TAP
fragmentation and how these features, combined with Ultra-performance
liquid chromatography, are being used to deliver the highest level of
definition in sample analysis. The following will be provided:
1) Explanations of UPLC/MSE as a novel “data independent”
method for obtaining molecular and structural information for every
detectable component in an LC separation and,
2) How the deconvolution function in MSE can provide an
alternative to “perfect” chromatography
3) The utility of the Driftscope display for both data processing and
performing selective background subtraction.
4) The power of incorporating Time-Aligned-Parallel fragmentation for
solving complex structural elucidation problems.
We
will conclude the presentation by describing how the T-Wave technology
within our MS instrumentation has been specifically engineered to couple
with Ultra Performance LC systems (ACQUITY and nanoACQUITY) to maximize
overall system performance then review how this combined system can
improve productivity and enhance scientific discovery. |
|
| Tamara Marchincin |
Utilizing Temperature to Achieve HPLC Separations on
Various Columns |
Eastman Kodak Company
|
The interest in the use of temperature as a tool to enhance separations
has increased. In our analytical laboratories at Eastman Kodak Company,
we have investigated the role that temperature can play in our
separations. This presentation will show the gains achieved by both
increasing and decreasing temperature. The improvements discussed will
include enhanced separations, sharper peak shape, and resolution gains.
The implementation of sub-2 micron particles with temperature increase
will also be discussed.
|
|
| Randall Clouette and Matt
Clabaugh |
Use of LC/MS/MS for the forensic
determination of drugs of misuse |
Applied Biosystems Inc., Foster City, CA
|
|
Typically, the forensic determination of drugs of abuse or misuse
has been done by an assortment of analytical techniques. These
techniques include Gas Chromatography (GC), High Pressure Liquid
Chromatography (HPLC), and Gas Chromatography Mass Spectrometry
(GC/MS). With the introduction of high pressure liquid
chromatography tandem mass spectrometry (HPLC/MS/MS) it has become
routinely possible to decrease sample size, improve reliability,
decrease detection limits and increase laboratory through-put.
This work describes the development and use of
HPLC/MS/MS as an analytical technique to determine the concentrations of
multiple drugs of abuse or misuse in human urine samples as a means of
detecting the diversion of misused pharmaceuticals from retail pharmacy
stores. The initial screening for these drugs consisted of the
monitoring a single transition (SRM) of precursor ion to product ion and
comparison to established retention time. Initial positive screens
were subjected to repeat testing using multiple reaction monitoring
(MRM) to monitor the transitions from the precursor ion to two product
ions and comparison to established retention time. Limits of
detection and upper limits of linearity were established relative to an
industry established cutoff value. Some of the potential
interferences and associated instrumental expenses are also
investigated.
|
| Lee Marotta |
Sample
Introduction Techniques in Gas Chromatography: Which technique is
most appropriate for my sample type |
PerkinElmer LAS, Shelton, CT
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Gas
Chromatography is a very useful tool to the analytical laboratory.
Not only does it provide universal and selective information with
the choice of its many detectors, gas chromatography offers the ability
to provide information of analytes in many sample matrices taking
advantage of its various sample introduction techniques.
Using
such techniques, the chemist can enjoy the ability of reporting
solutions with ease of use, with enhanced efficiency and robustness,
with improved detection limits and with excellent precision and
accuracy.
This
presentation will discuss which technique is appropriate to the sample
matrix and to the carbon range being investigated. The techniques
will be headspace, thermal desorption, purge and trap, split, splitless
and solvent purge injections. Applications of various different
types of samples, typically with MS detection, will be discussed.
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