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Note: 305

In-Vitro Metabolism StudiesUsing Data-Dependent™ LC/MSn Chromatography and Mass Spectrometry
Experimental Conditions
Key Words
Application Note
Microsomal fractions were prepared from rat, dog, • Metabolite
monkey, and human liver as described previously.3 characterization
Reaction mixtures (250 µL) with 5 or 50 M glyburide, W.H. Schaefer, D.M. Murphy, SmithKline Beecham 1 mg of microsomal protein/mL, 0.1 M potassium • FinniganLCQ
phosphate pH 7.25, 1 mM NADP, 10 mM glucose-6- phosphate, and 1 unit of glucose-6-phosphate dehydro- The data presented here can be acquired using the Finnigan LCQ
• Sensitivity
genase/mL were incubated at 37 °C for 30 min. They series of ion trap mass spectrometers.
were then quenched by addition of 250 µL of acetonitrile In-vitro
and the precipitate was removed by centrifugation.
The supernatant was diluted with 500 µL of 10 mM An integral part of the process by which a new drug ammonium acetate, pH 5.0, before analysis.
candidate is evaluated and characterized involves the Metabolic products were separated using a Prodigy™ investigation of its rates and routes of metabolism. Due 5 mM C8 150×2 mm column with a 10×2 mm guard to their convenience, relative simplicity and reliability, column. Solvent A was 10 mM ammonium acetate, pH in-vitro systems are used early in the drug discovery 5.0 and solvent B was acetonitrile. The metabolites were process to compare the biotransformation pathways eluted using the following linear gradient: 0 min, 30%B; across different species and to gain preliminary informa- 30 min, 30%B; 35 min, 60%B; 60 min, 100%B; flow 0.2 tion on the metabolic routes to be expected in humans.
mL/min. The mass spectrometer used was a Finnigan The current methodologies to characterize drug LCQ. The entire 0.2 mL/min flow was directed into the metabolites generally utilize LC/MS and LC/MS/MS, but source of the mass spectrometer without splitting, with frequently the data obtained is not sufficient to locate the the first 2.1 min diverted to waste using the built-in auto- site of metabolism on a candidate molecule. The Finnigan mated divert valve. The ion transfer tube was operated at LCQ series, with their ability to perform multi-stage MS 250 °C and sheath and auxiliary gases were set to 80 and fragmentation, offer MS3 and MS4 routinely during an 25, respectively. A relative collision energy of 25% was HPLC run. These second and third order product ion used for all MSn experiments with an isolation width of spectra afford data that allow metabolite identification 7.0 u to allow passage of 35Cl and 37Cl isotope peaks with greater specificity. An additional strength of the Finnigan LCQ series is their ability to perform auto- Strategy
mated Data-Dependent experiments. This means thatthe mass spectrometer makes real-time decisions about An effective strategy for metabolite characterization is to which MS experiment to perform based on the spectrum 1) obtain MSn data on the unmetabolized drug (used as a reference for following experiments), 2) perform a This approach will be illustrated with the example Data-Dependent experiment to screen the metabolites, of the analysis of metabolites derived from glyburide and 3) conduct selective multi-stage MSn experiments to (glibenclamide), a potent sulfonylurea drug.1,2 locate more specifically the site of metabolism. Since thesamples utilized microsomal preparations fortified with NADPH, only oxidative metabolism occurred. This made In this report, the application of benchtop ion trap API the analysis slightly simpler since the most likely meta- mass spectrometry to characterize in-vitro metabolites is bolic products were an unmodified parent, mono-oxy- discussed. The utility of Data Dependent MS/MS2/MS3 genated metabolites, and possibly di- or tri-oxygenated analyses, where the mass spectrometer makes “real-time” metabolites. A list of ions corresponding to the [M+H]+ decisions about the experiment to be performed, are for glyburide and its potential metabolites was entered in demonstrated using the characterization of glyburidemetabolites as an example.
the method setup for the analysis in order to prevent the graphic analysis, a simple LC/MS analysis is still fre- instrument from obtaining spectra on irrelevant, but quently valuable, especially when there are closely eluting potentially intense, ions in the samples. analytes with the same molecular weight. The data from The Finnigan LCQ was set up to perform the fol- an LC/MS analysis will have more data points (since it lowing Data-Dependent experiment: When one of the is not interrupted with MSn scans) to describe the chro- ions from the list was detected in MS (and above a user- matographic peaks better and reveal shoulders or minor defined threshold), the mass spectrometer automatically acquired a product ion mass spectrum (MS2) for this ion.
Results and Discussion
Next, a second order product ion (MS3) mass spectrumwas collected for the base peak from the MS2 spectrum.
Glyburide was characterized using MS2, MS3, and MS4 This MS/MS2/MS3 sequence was repeated throughout the experiments in positive ion mode during an infusion of a duration of the chromatographic peak. At the end of the 1 g/mL stock solution. A 7 amu isolation width was used peak, the mass spectrometer returned to MS mode until to collect both 35Cl- and 37Cl-containing ions. Thus, the another ion from the mass list was detected and the cycle product ions included the Cl isotope pattern. These data are summarized in Scheme 1. These spectra were used Having obtained MS, MS2 and MS3 data, the retention as references to aid in interpretation of the spectra of time, molecular weight and significant structural infor- metabolites. Shifts in masses observed in spectra for mation were obtained in this one analysis. Additional metabolites relative to spectra for glyburide, as well as structural data were collected from subsequent LC/MSn differing fragmentation patterns facilitated characteriza- analyses designed to collect MSn data for specific ions of interest. Although the Data Dependent analyses provide An LC/MSn experiment was performed on a micro- a tremendous amount of data from a single chromato- somal sample. The Data-Dependent analysis afforded glyburide
MS, MS2 and MS3 data–providing retention time, molec- The MS spectra of all these metabolites were identical ular weight and structural information. The reconstructed (see Figure 2) affording an [M+H]+ ion at m/z 510. The ion chromatogram (RIC) reproduced in Figure 1 indi- MS2 spectra for the first six metabolites (see Figure 3) cated that there were seven metabolites that resulted from afforded ions at m/z 369, 395, 492, 352 and 169. With the incorporation of a single oxygen molecule (at 8.72, the exception of the ion at m/z 492 (elimination of H2O) 9.92, 10.65, 11.89, 13.01, 16.79 and 23.87 min). these ions were identical to those observed in the MS2 spectra of glyburide. This indicates that the site of The MS2 spectrum (see Figure 4) obtained from the metabolism was the cyclohexyl ring, since the loss of metabolite at 23.87 min afforded ions at m/z 385, 367, the cyclohexyl moiety resulted in an identical spectrum.
411, 492 and 169, indicating that the site of hydroxyla- Additionally the MS3 spectra for these metabolites were tion was not the cyclohexyl moiety. The data from the identical to the MS3 spectrum derived from glyburide.
MS2 spectrum in conjunction with the data from theMS3 spectrum (see Figure 5) allowed the fragmentationpathway to be delineated (see Scheme 2).
Figure 3. MS2 spectra of early eluting metabolites Figure 4. MS2 spectrum of metabolite at 23.87 min The data obtained thus far were compatible with Thus only two LC/MSn experiments enabled the three different metabolite structures (see Figure 6). To novel metabolite at 23.87 min to be identified as hydrox- enable a more specific structural assignment, a further ylation of the ethyl chain at either the benzylic position, LC/MSn experiment was performed using negative ion or alpha to the amide nitrogen (structures A and B in mode. These data are summarized in Scheme 3. The ions observed at m/z 323, 198 and 134 indicate that structureC in Figure 6 was incorrect.
Figure 5. MS3 spectrum of metabolites at 23.87 min Figure 6. Possible structures of metabolite at 23.87 min m/z 510
m/z 411
m/z 385
m/z 393
m/z 367
m/z 350
m/z 169
m/z 286
m/z 302
m/z 508
m/z 323
m/z 383
m/z 198
m/z 134
With the use of Data-Dependent MS/MS2/MS3 analysesseven metabolites of glyburide were structurally character-ized within two LC/MS analyses. This approach not onlyafforded molecular weight, retention time, and structuralinformation with greater specificity than LC/MS andLC/MS/MS using a triple quadrupole, but reduced thelength of 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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