P.R. Tiller, Thermo Finnigan
W.H. Schaefer, D.M. Murphy, SmithKline Beecham Pharmaceuticals
The data presented here can be acquired using the Thermo Finnigan LCQ
Series of ion trap mass spectrometers.
An integral part of the process by which a new drug candidate is evaluated
and characterized involves the investigation of its rates and routes of
metabolism. Due to their convenience, relative simplicity and reliability,
in-vitro systems are used early in the drug discovery process to compare
the biotransformation pathways across different species and to gain preliminary
information on the metabolic routes to be expected in humans.
The current methodologies to characterize drug metabolites generally utilize
LC/MS and LC/MS/MS, but frequently the data obtained is not sufficient to
locate the site of metabolism on a candidate molecule. The Thermo Finnigan
LCQ Series, with their ability to perform multi-stage MS fragmentation,
routinely during an HPLC run. These
second and third order product ion spectra afford data that allow metabolite
identification with greater specificity. An additional strength of the LCQ
Series is their ability to perform automated Data-Dependent experiments.
This means that the mass spectrometer makes real-time decisions about which
MS experiment to perform based on the spectrum just acquired. This approach
will be illustrated with the example of the analysis of metabolites derived
from glyburide (glibenclamide), a potent sulfonylurea drug.1,2
In this report, the appli
cation of benchtop ion trap API mass spectrometry
to characterize in-vitro metabolites is discussed. The utility of Data Dependent
analyses, where the mass spectrometer
makes real-time decisions about the experiment to be performed,
are demonstrated using the characterization of glyburide metabolites as
Microsomal fractions were prepared from rat,
dog, monkey, and human liver as described
Reaction mixtures (250 L) with 5 or
50 M glyburide, 1 mg of microsomal protein/mL,
0.1 M potassium phosphate pH 7.25, 1 mM
NADP, 10 mM glucose-6-phosphate, and 1 unit
of glucose-6-phosphate dehydrogenase/mL were
incubated at 37C for 30 min. They were then
quenched by addition of 250 L of acetonitrile
and the precipitate was removed by centrifugation.
The supernatant was diluted with 500 L of
10 mM ammonium acetate, pH 5.0 before analysis.
Metabolic products were separated using a
Prodigy 5 mM C8 1502 mm column with a
102 mm guard column. Solvent A was 10 mM
ammonium acetate, pH 5.0 and solvent B was
acetonitrile. The metabolites were eluted using
the following linear gradient: 0 min, 30%B;
30 min, 30%B; 35 min, 60%B; 60 min, 100%B;
flow 0.2 mL/min. The mass spectrometer used
was an LCQ. The entire 0.2 mL/min flow was
directed into the source of the mass spectrometer
without splitting, with the first 2.1 min diverted
to waste using the built-in automated divert
valve. The ion transfer tube was operated at
250C and sheath and auxiliary gases were set to
80 and 25, respectively. A relative collision energy
of 25% was used for all MSn
an isolation width of 7.0 u to allow passage of
35Cl and 37Cl isotope peaks within a single scan.
An effective strategy for metab
olite characterization is to 1) obtain MSn
data on the unmetabolized drug, (used as a reference for following experiments),
2) perform a Data-Dependent experiment to screen the metabolites, and 3)
conduct selective multi-stage MSn
experiments to locate more
specifically the site of metabolism. Since the samples utilized microsomal
preparations fortified with NADPH, only oxidative metabolism occurred. This
made the analysis slightly simpler since the most likely metabolic products
were an unmodified parent, mono-oxygenated metabolites, and possibly di-
or tri-oxygenated metabolites. A list of ions corresponding to the [M+H]+
for glyburide and its potential metabolites was entered in the method setup
for the analysis in order to prevent the instrument from obtaining spectra
on irrelevant, but potentially intense, ions in the samples.
The LCQ was set up to perform the following Data-Dependent experiment. When
one of the ions from the list was detected in MS1
a user-defined threshold), the mass spectrometer automatically acquired
a product ion mass spectrum (MS2
) for this ion. Next, a second
order product ion (MS3
) mass spectrum was collected for the base
peak from the MS2
spectrum. This MS1
sequence was repeated throughout the duration of the chromatographic peak.
At the end of the peak, the mass spectrometer returned to MS1
mode until another ion from the mass list was detected and the cycle was
repeated for this new ion.
Having obtained MS1
the retention time, molecular weight and significant structural information
were obtained in t
his one analysis. Additional structural data were collected
from subsequent LC/MSn
analyses designed to collect MSn
data for specific ions of interest. Although the Data Dependent analyses
provide a tremendous amount of data from a single chromatographic analysis,
a simple LC/MS analysis is still frequently valuable, especially when there
are closely eluting analytes with the same molecular weight. The data from
an LC/MS analysis will have more data points (since it is not interrupted
scans) to describe the chromatographic peaks better
and reveal shoulders or minor peaks more clearly.
Results and Discussion
Glyburide was characterized using MS2
experiments in positive ion mode during an
infusion of a 1 g/mL stock solution. A 7 isolation
width was used to collect both 35Cl- and 37Cl-containing
ions. Thus, the product ions included the
Cl isotope pattern. These data are summarized in
Scheme 1. These spectra were used as references
to aid in interpretation of the spectra of metabolites.
Shifts in masses observed in spectra for
metabolites relative to spectra for glyburide, as
well as differing fragmentation patterns facilitated
characterization of the metabolite structures.
experiment was performed on a microsomal sample. The
Data-Dependent analysis afforded MS1
data providing retention time, molecular weight and structural information.
The reconstructed ion chromatogram (RIC) reproduced in Figure 1 indicated
that there were seven metabolites that resulted from the incorporation of
a single oxygen molecule (at 8.72, 9.92, 10.65, 11.89, 13.01, 16.79 and
23.87 min.). The MS1
spectra of all these metabolites were
(see Figure 2) affording an [M+H]+
ion at m/z 510. The MS2
spectra for the first six metabolites (see Figure 3) afforded ions at m/z
369, 395, 492, 352 and 169. With the exception of the ion at m/z 492 (elimination
O) these ions were identical to those observed in the MS2
spectra of glyburide. This indicates that the site of metabolism was the
cyclohexyl ring, since the loss of the cyclohexyl moiety resulted in an
identical spectrum. Additionally the MS3
spectra for these metabolites
were identical to the MS3
spectrum derived from glyburide.
spectrum (see Figure 4) obtained from the metabolite
at 23.87 min afforded ions at m/z 385, 367, 411, 492 and 169, indicating
that the site of hydroxylation was not the cyclohexyl moiety. The data from
spectrum in conjunction with the data from the MS3
spectrum (see Figure 5) allowed the fragmentation pathway to be delineated
(see Scheme 2).
Figure 1. RIC m/z 510.
Figure 2. MS1 spectra.
Figure 3. MS2 spectra of early eluting metabolites.
Figure 4. MS2 spectrum of metabolite at 23.87 min.
Figure 5. MS2 spectrum of metabolites at 23.87 min.
The data obtained thus far were compatible with three different metabolite
structures (see Figure 6). To enable a mor
e specific structural assignment,
a further LC/MSn
experiment was performed using negative ion
mode. These data are summarized in Scheme 3. The ions observed at m/z 323,
198 and 134 indicate that structure C in Figure 6 was incorrect. Thus only
experiments enabled the novel metabolite at 23.87
min to be identified as hydroxylation of the ethyl chain at either the benzylic
position, or alpha to the amide nitrogen (structures A and B in Figure 6).
Figure 6. Possible structures of metabolite at 23.87 min.
With the use of Data-Dependent MS1
analyses seven metabolites
of glyburide were structurally characterized within two LC/MS analyses. This
approach not only afforded molecular weight, retention time, and structural
information with greater specificity than LC/MS and LC/MS/MS using a triple
quadrupole, but reduced the analysis time.
1. Glibenclamide, Therapeutic Drugs, Ed. Collin Dollery, Churchill Livingstone,
New York, NY (1991) G21-G26.
2. D.G. Kaiser and A.A. Forist, A review of Glyburide metabolism in man
and laboratory animals, Micronase: Pharmacological and Clinical Evaluation,
Ed. H. Rifkin et. al., Excerpta Medica Foundation International Congress
Series No. 382, Princeton, NJ. (1975) 31-41W.
3. Clarke, SE, Ayrton, AD and Chenery, RJ., Xenobiotica, 24 (1994) 517-526.
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