Anal. Chem. 2000, 72, 3342-3348
Application of Atmospheric Pressure Ionization Time-of-Flight Mass Spectrometry Coupled with Liquid Chromatography for the Characterization of in Vitro Drug Metabolites Hongwei Zhang and Jack Henion*
Analytical Toxicology, Cornell University, 927 Warren Drive, Ithaca, New York 14850 Yi Yang and Neil Spooner
DMPK, SmithKline Beecham Pharmaceuticals, 709 Swedeland Road, King of Prussia, Pennsylvania 19406
Atmospheric pressure ionization time-of-flight mass spectrometry coupled with high-performance liquid chromatography was used to characterize the in vitro metabolites of glyburide. Metabolic products formed in vitro by human microsomes were separated using a C18 column with gradient elution at a flow rate of 200 µL/min without postcolumn splitting. In-source collision-induced dissociation (CID) by automated nozzle potential switching was employed to obtain both abundant protonated molecules and characteristic fragments whose accurate masses were measured simultaneously by internal mass calibration, performed by continuous postcolumn infusion of two reference standards. The mass errors were within 9 ppm for all ions measured, whose abundance was greater than 5%, relative to the most abundant isotopic “A” ion. Exact mass differences between the parent drug and metabolite(s) were determined and these values corresponded to a unique elemental composition. The elemental compositions of all metabolite fragment ions were generated based upon the known compositional elements of the protonated molecule. The structures of metabolites and their fragment ions were proposed based on the determined elemental composition and in-source CID spectra. The elemental composition and fragmentation pathways of four cyclohexyl hydroxylation metabolites and one ethylhydroxy metabolite are discussed. In vitro drug metabolism studies using microsomes are becoming increasingly important for the characterization of new drug candidates at the discovery stage. Presently, most of the analytical characterization associated with this work is carried out by atmospheric pressure ionization- liquid chromatography/mass spectrometry/mass spectrometry (API-LC/MS/MS) using triplequadrupole or ion trap mass spectrometers, because of their capability for performing MS/MS or MSn experiments.1-3 APItime-of-flight mass spectrometry (API-TOF) has recently received * Corresponding author: (e-mail)
[email protected]. (1) Bu, H. Z.; Poglod, M.; Micetich, R. G.; Khan, J. K. J. Mass Spectrom. 1999, 34, 1185-1194.
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renewed interest, because of advancements in the technology. This includes fast mass spectral acquisition speed with high fullscan sensitivity, owing to a much higher duty cycle. In addition, enhanced mass resolution and accurate mass measurement capabilities allow for the determination of elemental composition. These analytical attributes provide the potential for LC-API-TOF to play an important role in the future of drug metabolism studies. The current commercial API-TOF technologies usually provide between 5000 and 8000 resolving power using the definition of full width at half-maximum (fwhm). The number of possible elemental compositions increases with a decrease in the mass accuracy of a system, an increase in mass of the ion, and an increase in the number of different elements. The number of possible elemental compositions is also related to the mass defect.4 With 10 ppm of mass tolerance (mass accuracy), the number of possible elemental compositions increases sharply as the mass increases. It can be calculated that with 10 ppm of mass tolerance, there would be 2, 28, and 100 possible elemental compositions for masses 160, 350, and 500 Da, respectively. This assumes that only C, H, N, O, and S are involved and the nitrogen rule is applied.5 If additional elements are considered, even more compositions are possible. This situation places an increased demand upon LC/MS experiments designed to rapidly obtain elemental composition information for in vitro metabolite studies. Compounds of pharmaceutical interest typically have molecular masses in the range of 200-1000 Da. To date, it has been difficult to rapidly determine the elemental composition of an unknown drug and its metabolites. Some constraints are needed to obtain a unique elemental composition. Russell et al.6 determined the elemental composition of a naturally occurring substituted flavin (2) Yu, X.; Cui, D. H.; Davis, M. R. J. Am Soc Mass Spectrom. 1999, 10, 175183. (3) Lopez, L. L.; Yu, X.; Cui, D. H.; Davis, M. R. Rapid Commun. Mass Spectrom. 1998, 12, 1756-1760. (4) Grange, A. H.; Donnelly, J. R.; Sovocool, G. W.; Brumley, W. C. Anal. Chem. 1996, 68, 553-560. (5) McLafferty, F. W.; Turacek, F. Interpretation of Mass Spectra, 4th ed.; University Science Books: Mill Valley, CA, 1993. (6) Edmondson, R. D.; Gadda, G.; Fitzpatrick, P. F.; Russell, D. H. Anal. Chem. 1997, 69, 2862-2865. 10.1021/ac000089r CCC: $19.00
© 2000 American Chemical Society Published on Web 06/14/2000
adenine dinucleotide (FAD) of nitroalkane oxidase by obtaining the exact mass difference between the FAD substituent and FAD using accurate mass matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Unreasonable C/H ratios or the nitrogen rule has also been used as constraints to eliminate unlikely elemental compositions. The isotopic pattern7,8 has long been used to constrain the number of possible elemental composition. Grange et al.4 uniquely assigned elemental composition from exact mass based on mass peak profiles of the molecular ion (A) and the A + 1 and A + 2 ions using 20 000 mass resolution. The “basket in a basket” approach recently reported by Wu9 is a good example for determining the elemental composition of an unknown compound from combinatorial synthesis using multistage accurate mass spectrometry. The unique elemental composition of the smallest mass fragment was first determined based on built-in chemical information from the synthesis, including the kinds of elements involved and chemicals used for synthesis. These data were then used to establish the lower limit for determination of the elemental composition of its parent ion under MSn conditions using FTMS. Since the precursor/product relationship between fragments is clear for multistage accurate mass spectrometry technology, the determined elemental composition of a product ion in each stage can be used to set the lower limit for determination of elemental composition of its parent ion. With this information, the elemental composition of the unknown compound can be determined. Although not as sophisticated, another approach presented here provides an efficient means of obtaining elemental compositions for precursor ions as well as fragment ions from relatively complex mixtures employing routine LC/MS technologies. This alternative strategy uses upper limits for the number of elements as a constraint to characterize the in vitro metabolites of glyburide, the target drug of this study. LC/API-TOF/MS is employed to obtain accurate mass measurement for each precursor ion and the fragments resulting from “up-front” collision-induced dissociation (CID).10 Glyburide (glibenclaimide) is a sulfonylurea drug that has been used for treatment of non-insulin-dependent diabetes mellitus.11 A previous investigation12 demonstrated that glyburide was extensively metabolized by in vitro microsomal systems via oxidative pathways. In the present study, the drug was incubated with human or rat liver microsomes as previously12 and the in vitro samples were then analyzed by LC/API-TOF/MS under the experimental conditions that provided accurate mass measurement for the precursor and product ions of the parent drug and several of its oxidative metabolites. EXPERIMENTAL SECTION Reagents and Materials. Glyburide was obtained from Research Biochemical International (Natick, MA). Its chemical structure and relative molecular mass (Mr) are shown in Figure (7) Hogenboon, A. C.; Niessen, W. M. A.; Little, D.; Brinkman, U. A. Th. Rapid Commun. Mass Spectrom. 1999, 13, 125-128. (8) Michelsen, P.; Karlsson, A. Rapid Commun. Mass Spectrom. 1999, 13, 21462150. (9) Wu, Q. Anal. Chem. 1998, 70, 865-872. (10) Duffin, K. L.; Wachs, T.; Henion, J. Anal. Chem. 1992, 64, 61-68. (11) Dollery, C., Ed. Therapeutic Drugs; Churchill Living-stone: New York, 1991; pp G21-G26. (12) Tiller, P. R.; Land, A. P.; Jardine, I.; Murphy, D. M.; Sozio, R.; Ayrton, A.; Schaefer, W. H. J. Chromatogr., A 1998, 794A, 15-25.
Figure 1. Structure of glyburide.
1. A 1 mg/mL stock solution was prepared in dimethyl sulfoxide (DMSO). The mass calibration standards used were N,N-bis(hydroxyethyl)-2-amino-2-methylpropanol hydrochloride (BisAMP) and reserpine, which both were obtained from Sigma Chemical Co. (St. Louis, MO). Acetonitrile and DMSO were purchased from J. T.Baker (Phillipsburg, NJ). Glacial acetic acid was obtained from Fisher Scientific (Fair Lawn, NJ). Ammonium acetate (98% pure) was obtained from Sigmal Chemical Co., and deionized water was generated in-house with a Barnstead Nanopure II filtration system (Boston, MA), Microsomal Incubation Samples. The human microsome sample was prepared in-house from human liver, which was purchased from Anatomic Gift Foundation (White Oak, GA, Federal I.D. No. 52-1853905). The incubations were performed in a final volume of 1.0 mL in a shaking water bath at a temperature of ∼37 °C. Each incubation contained ∼1.0 mg/mL microsomal protein and 10 µM glyburide. The volume was adjusted to 0.8 mL by the addition of 50 mM potassium phosphate buffer (pH 7.4). Following a 5-min preincubation at ∼37 °C, the reaction was initiated by the addition of 0.2 mL of prewarmed cofactor solution. The cofactor solution was composed of ∼1.7 mg of nicotinamide adenine dinucleotide phosphate (NADP), 7.8 mg of glucose 6-phosphate, and 6 units of glucose 6-phosphate dehydrogenase (G-6-PDH) per milliliter of 2% (W/V) sodium bicarbonate. The reaction was terminated after ∼30 min of incubation by adding 1.0 mL of acetonitrile, vortex mixing, and freezing at ∼-70 °C until analysis by LC/MS. A “no-cofactor” and a “no-glyburide” control incubation were performed by adding 0.2 mL of 2% (w/v) sodium bicarbonate in place of cofactor solution or DMSO in place of 10 µM glyburide and incubating as above for ∼30 min. The samples were diluted 1:1 with 10 mM ammonium acetate, pH 5.0, and centrifuged before injection and analysis by LC/MS. LC/MS Instrumentation. Two Shimadzu LC-10AS pumps (Shimazdu Corp., Kyoto, Japan) were used for gradient elution. The samples were analyzed using a Keystone C18 2 mm × 100 mm column packed with 5-µm particles and an Opti 1-mm C18 guard column. Gradient elution was performed at a flow rate of 200 µL/min. Sovent A was a mixture of 10 mM ammonium acetate, pH 5, and acetonitrile in a 90:10 ratio. Solvent B was acetonitrile. Acetic acid at 17 and 1.7 M in water was used to adjust the pH of the 10 mM ammonium acetate aqueous solutions of mobile phase. The following linear gradient was used to elute the metabolites: 0.01 min, 0% B; 3 min, 22% B; 10 min, 22% B; 25 min, 100% B. An Endurance autosampler (Spark-Holland, AJ Emmen, The Netherlands) was used for injection of samples. The injection valve was fitted with a 100-µL sample loop for a 50-µL partial loop injection. Analytical Chemistry, Vol. 72, No. 14, July 15, 2000
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The total effluent from the column was transferred directly to the Turbo IonSpray LC/MS interface without postcolumn splitting. A PE Biosystems Mariner Biospectrometry workstation APITOF mass spectrometer equipped with a Turbo IonSpray source operated in the positive ionization mode was used for the work described herein. The TOF-mass spectrometer is composed of an orthogonal pulsed source operated at a rate of 20 000 Hz and a reflectron with an effective flight path of 1.3 m. Mass resolution was maintained at greater than 5000 (fwhm). The mass range was set at 100-1000 amu, and the acquisition rate was 1 s/spectrum. Nitrogen gas supplied from a liquid nitrogen dewar boil-off at 80 psi was used for nebulizer, drying, and curtain gases. Drying gas was set at 10 L/min and the Turbo IonSpray probe temperature was maintained at 350 °C. Nebulizer gas was set at 0.8 L/min and the curtain gas was set at 1.5 L/min. The Turbo IonSpray voltage was maintained at 5500 V. The detector voltage was set at 2200 V. Nozzle-Switching In-Source CID. Structural characterization of the parent drug and its metabolites was facilitated by in-source CID and the availability of accurate mass measurement information on the precursor and product ions. The nozzle potential was automatically switched between three voltages to perform insource CID to obtain abundant ions throughout mass range. The three nozzle voltages used were 110 V for the protonated molecule, 160 V for the mid mass range fragment ions, and 230 V for the lower mass fragment ions. Internal Mass Calibration. Two-point internal mass calibration was performed by continuous infusion of reference standards postcolumn through a polyetheretherketone (PEEK) tee (a “T” union made from PEEK material). The reference masses used were m/z 178.1438 from Bis-AMP and m/z 609.2807 from reserpine, which covered the mass range of interest. The concentration of mass reference standards was 2 µg/mL for BisAMP and 1 µg/mL for reserpine dissolved in 90% methanol and 10% water. The flow rate of the infusion pump was 10 µL/min. The mass spectra were averaged across a chromatographic peak to determine accurate mass. The signal intensities of both reference ions and investigated ions should be high enough to ensure adequate peak statistics and signal-to-noise ratio and should be comparable, which are needed for accurate assignment of peak position. For nozzle-switching in-source CID, where low nozzle voltage is also involved, the signal intensities of ions at m/z 178.1438 and 609.2807 are still high, and so these two ions are still used as reference ions. The maximum mass error was 9 ppm after mass calibration with the reference standards, and in most of instances, the mass error was less than 3 ppm. Nine ppm of mass tolerance was used for elemental composition calculation. Data Processing. The calculations of monoisotopic masses, isotopic ratios, and elemental compositions were performed on Data Explorer software (3.2.1.3 version) provided by PE Biosystems. RESULTS AND DISCUSSION Strategy. Metabolites of a targeted drug can usually be detected by observing shifts in their masses relative to drugs. A shift of +16, +32, +48, +30, +80, +192, -14, -28, and -16 may indicate hydroxylation, dihydroxylation, trihydroxylation, carboxylic acid formation, O-sulfate conjugate, O-glucuronide conjugate, demethylation, 2× demethylation, and deoxygenation, respec3344 Analytical Chemistry, Vol. 72, No. 14, July 15, 2000
Figure 2. LC/MS traces of human microsomal incubation samples: (A) TIC for 0-min control sample, (B) TIC of 30-min incubation sample, and (C) extracted ion chromatogram at m/z 510.2 ( 0.5 of the incubation sample. M1-M5 are isomeric metabolites of glyburide. Acquisition range: 100-1000 amu. Control sample: 10 µM glyburide incubated with cofactors and microsomes for 0 min. Incubation conditions: glyburide at 10 µM was incubated with cofactors in human microsomes for 30 min. See chromatographic gradient elution conditions in Experimental Section. The nozzle voltage was fixed in these experiments.
tively. In this study, the exact mass shift between an unknown metabolite and an otherwise well-characterized drug is determined to predict the elemental composition of the metabolite. The expected elemental composition of the metabolite can be used as an upper element limit to predict elemental composition for all the characteristic fragment ions of the metabolite generated by in-source CID. The structure of the metabolite and its fragment ions can be proposed based on the elemental composition, the metabolite spectra, and its fragments. The known structure of the parent drug with its corresponding CID mass spectrum can facilitate elucidation of metabolite structures. Thus, confirmation of a proposed metabolite is accomplished by the agreement between its proposed structure, the accurate mass, and elemental composition of each ion in its mass spectrum. Metabolic Profile. For the metabolite profile study, only one fixed nozzle voltage was applied (no nozzle switching was involved for this experiment). The total ion chromatogram and extracted ion chromatograms for the microsomal samples described in this work are shown in Figure 2. These data indicated the presence of five metabolites (M1-M5). These data parallel those reported previously.12 The mass shift for each metabolite was 16 Da relative to protonated glyburide, suggesting that several monooxygenated metabolites had been formed. Nicotinamide adenine dinucleotide phosphate (NADPH) was present during the incubation. Oxidation products were therefore expected.12 The chromatographic peaks for the metabolites were readily observed in the total ion
Table 1. Summary of Measurements of the Exact Mass Difference between Each Metabolite (M1-M5) and Parent Drug, for Triplicate Injections of the 10 µM Human Incubation Sample injection no. metab
1
2
3
M1 M2 M3 M4 M5
15.9948 15.9997 15.9945 15.9950 15.9998
15.9943 15.9922 15.9945 15.9962 15.9945
15.9974 15.9944 15.9962 15.9932 15.9932
average CV (%)a 15.9955 15.9954 15.9951 15.9948 15.9958
0.01 0.02 0.01 0.01 0.02
REb 3.8 × 10-5 3.3 × 10-5 1.0 × 10-5 -6.3 × 10-6 5.8 × 10-5
a CV, coefficient of variation. b RE, relative error. The theoretical mass of oxygen is 15.9949 Da.
chromatogram due to the relatively good sensitivity of the TOF system and the relatively high levels of the metabolites in the sample. The previous investigation of in vitro glyburide metabolites in human microsomes using an ion trap mass spectrometer reported a total of seven monooxygened metabolites.12 The two metabolites missing here are isomers of cyclohexyl hydroxylation products. Four cyclohexyl hydroxylation isomers were observed in this study, and six were observed in the previous study.12 Mass spectrometry often cannot distinguish between these isomers. Therefore, it is helpful to separate isomers chromatographically or they may not be distinguishable. The chromatography conditions are different between this study and the previous one, and the present one has less chromatographic resolution, which may lead to not detecting the other metabolites. The other possible reasons include that these two missing metabolites may not have been formed in this study or their concentration may be below the detection limits. Elemental Composition of Metabolites. The exact mass differences between each of the metabolites and the parent drug were determined based on triplicate analyses of the 10 µM human incubation samples and the results are summarized in Table 1. The average exact mass difference for each of the five metabolites corresponded to a unique elemental composition, oxygen, whose theoretical exact mass is 15.9949 Da. Consequently, the elemental composition for each of the protonated metabolites is C23H29ClN3O6S, based upon the known elemental composition of C23H29ClN3O5S for the protonated parent drug. The determined elemental composition of metabolites may be used as an upper limit for calculation of fragment ion elemental compositions. The relative mass errors in Table 1 are within 58 ppm. However, the possible elemental compositions dramatically decrease with the decrease of masses of ions. For mass as low as 16 Da, 58 ppm of mass error still leads to unique elemental composition, oxygen. Glyburide. The CID mass spectrum for the parent drug, glyburide, was characterized to aid in the interpretation of its metabolite mass spectra. Figure 3 shows the backgroundsubtracted in-source CID mass spectrum of the parent drug, glyburide, obtained from injection of a 10 µM human incubation sample. The background ion counts from mobile phase and reference standard solution have been subtracted and so they are not observed in Figure 3. The signal intensities of the mass peaks of reference ions are approximately 300 and 500 counts and those of investigated ions are from 200 to 550 counts. Thus, the signal intensities of mass peaks of reference ions and investigated ions
Figure 3. LC/MS in-source CID mass spectrum and accurate mass determination of the parent drug, glyburide, determined from the in vitro incubation samples. See Figure 2 for incubation and experimental conditions.
Figure 4. Proposed fragmentation scheme for glyburide.
are comparable. The mass spectrum is the same as that obtained from an injection of a glyburide analytical standard under the same LC/MS conditions. Signals were observed for the protonated molecule at m/z 494.1510 and its abundant fragment ions at m/z 369.0671 and 169.0050. The measured accurate masses and the corresponding mass measurement errors in parts per million (ppm are also indicated in Figure 3). The isotopic ratio for each of the above ion clusters supports the presence of a chlorine atom in their elemental compositions. When the upper limit of possible elements of all the fragments is set at C23H29ClN3O5S, pursuant to the known composition of protonated glyburide, and the lower limit is set at one chlorine atom, the elemental composition calculation generates a unique elemental composition for m/z 169.0050 and 369.0671 ions, respectively. These compositions would be C8H6ClO2 for m/z 169.0050 and C16H18ClN2O4S for m/z 369.0671. The proposed sequential fragmentation mechanism leading to the formation of m/z 369.0671 and 169.0050 are shown in Figure 4 along with the theoretical masses. The mass errors of the individual isotopic ions for glyburide, whose intensities are greater than 5% relative to the isotopic “A” ion, were also examined and are presented in Table 2. The mass errors were less than 6 ppm in all cases. The accurate isotopic A + 1, A + 2, and A + 3 masses may be used as constraints for elemental composition determination, and an example will be demonstrated (vide infra). A comparison of measured and theoretical isotopic distribution of the protonated glyburide is shown in Table 3. The isotopic distribution can also be used to eliminate the number of possible elemental compositions, which will be referenced in subsequent discussions. Analytical Chemistry, Vol. 72, No. 14, July 15, 2000
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Table 2. Accurate Isotopic Mass Errors between Experimental and Theoretical Values for Protonated Glyburide and Its Fragments, Obtained from LC/MS Analysis of the 10 µM Human Incubation Sample (from the LC/MS Run of Figure 4) isotopic mass error (ppm) isotopic mass
m/z 169.0051a C8H6ClO2
m/z 369.0670a C16H18ClN2O4S
m/z 494.1511a C23H29ClN3O5S
-0.2 -1.0 -3.3
0.2 -0.9 -3.3 -5.2
-0.2 -5.5 -5.3 -2.3
A A+1 A+2 A+3 a
Theoretical value.
Table 3. Comparison of Isotopic Ratios between Experimental and Theoretical Values for Protonated Glyburide, m/z 494, Obtained from LC/MS Analysis of the 10 µM Human Incubation Sample (from the LC/MS run of Figure 4) relative intensity (%)
a
isotopic mass
experimentala
theoretical
A A+1 A+2 A+3
100 28.7 40.6 11.3
100 28.0 41.2 10.8
The experimental relative intensity is based on the mass peak area.
Table 4. Elimination of Two of Three Possible Elemental Compositions To Obtain the Elemental Composition for m/z 169.0050 Based on Isotopic Mass Error isotopic mass error (ppm) exptl exact mass (amu)
C8H6ClO2
C2H7N4OPCl
CH6N6SCl
169.0050 170.0081 171.0011
0.6 2.4 7
5 18 5.8
4 18 8.8
The fragments of lower mass usually generate fewer possible elemental compositions and often the unique elemental compositions can be assigned, as shown below. The experimental m/z 169.0050 in Figure 3 generates 16 possible elemental compositions when the mass tolerance is set at 9 ppm and the elements C, H, O, N, P, S, K, Na, and Cl are considered without limiting the number of each element. The experimental isotopic ratio for A/(A + 2) is ∼3, which reduces the possible elemental compositions to: C8H6ClO2, C2H7N4OPCl, and CH6N6SCl. Table 4 shows the mass errors between determined individual isotopic masses and theoretical isotopic mass. Obviously C8H6ClO2 is the only composition whose isotopic masses (A, A + 1, and A + 2) agree within 9 ppm of experimental values. In addition, the experimental relative abundance between A/(A + 1) supports this conclusion. In Figure 3 two relatively abundant ions at m/z 532.1077 and 570.0627, in addition to the protonated molecule of glyburide at m/z 494.1510, were observed. The isotopic pattern of the ion at m/z 532.1077 is similar to that of protonated glyburide except that the ratio between the isotopic ion A + 2 and A is ∼7% higher than that of protonated glyburide. In addition, these two ions are 3346 Analytical Chemistry, Vol. 72, No. 14, July 15, 2000
Figure 5. LC/MS in-source CID mass spectrum and accurate mass determination of the cyclohexyl hydroxylation metabolite(s) (M1) of glyburide formed in vitro by human liver microsomes. See Figure 2 for incubation and experimental conditions.
very stable and it is difficult to fragment them by in-source CID. The exact mass difference between the ion at m/z 532.1077 and the mass of the parent compound ([m/z 494 - H]) is 38.9640 Da, which is consistent with the mass of a potassium ion. The value of 38.9640 differs from the theoretical value of potassium ion by only 0.0008 mass unit (21 ppm). This suggests the ion at m/z 532.1077 contains a potassium ion adduct. The relative abundance ratio also fits well, since the theoretical abundance ratio of (A + 2)/A for potassium is 7.2%, which provides additional support for the potassium adduct. LC/MS/MS using a PE-Sciex API-3000 was performed and the product ion at m/z 39 was observed (data not shown), which further supports this conclusion. The same strategy may be applied to the ion at m/z 570.0627. The isotopic pattern of the ion at m/z 570.0627 is similar to the protonated glyburide except that the relative abundance ratio of the isotopic ion (A + 2)/A is 14.6% higher than that of protonated glyburide. If it is assumed two protons in the protonated molecule were replaced with two potassium ions, then the resulting exact mass of the potassium ion is 38.9631. The difference between the theoretical value and the determined value is only 0.8 ppm. Investigations13,14 have shown that alkali metal adducts including [M + K]+ and [M + 2K - H]+, formed by the strong interaction between a central metal ion and several coordination atoms of M, can be very stable in the ion source and do not undergo insource CID. All the evidence obtained, including resistance to insource CID dissociation, isotopic pattern, and the exact masses, suggests that the well-known hypothesis for potassium adducts holds true in this study. However, further investigation is needed to fully characterize the behavior of alkali metal adducts with glyburide. A common source of potassium is the glass containers, which are used as reservoirs for mobile phase, so there is a continuous supply of potassium from mobile phase. For the present study, the high levels of potassium salts from the sample may have contaminated the system, which also contribute to the formation of potassium adducts. Metabolites M1-M4. The same in-source CID mass spectral characteristics were observed for metabolites M1-M4 as the parent glyburide. Therefore, the mass spectral behavior of the parent drug may be used as a guide to aid interpretation of the metabolite spectra. Figure 5 shows a representative backgroundsubtracted, in-source CID mass spectrum (M1) obtained from LC/ (13) Kiehl, D. E.; Julian, R. K.; Kennington, A. S. Rapid Commun. Mass Spectrum. 1998, 12, 903-910. (14) Volmer, D. A.; Lock, C. M., Rapid Commun. Mass Spectrum. 1998, 12, 157162.
Figure 6. Proposed fragmentation scheme for cyclohexyl hydroxylation metabolites (M1-M4).
Figure 8. Proposed fragmentation scheme for ethylhydroxy metabolite (M5).
Figure 7. LC/MS in-source CID mass spectrum and accurate mass determination of the ethylhydroxy metabolite (M5) of glyburide formed in vitro by human liver microsomes. See Figure 2 for incubation and experimental conditions.
MS analysis of 10 µM of the incubation sample. The measured accurate masses are also indicated in Figure 5. The isotopic pattern for each of the observed ions indicated that there was a chlorine atom in their elemental compositions. When the upper limit of possible elements for all the fragments was set at C23H29ClN3O6S, which was expected from the protonated metabolite, with the number of chlorine atom confined too, the elemental composition calculation generates only one elemental composition for fragment ions m/z 169.0055 and 369.0671. These compositions would be C8H6ClO2 for m/z 169.0055 and C16H18ClN2O4S for m/z 369.0671. The same characteristic fragment ions at m/z 369.0671 and 169.0055 in the CID spectrum of the parent drug suggests that oxygenation occurs on the cyclohexyl ring and that metabolites M1-M4 are isomers (the CID spectra of M1-M4 were essentially the same). The proposed fragmentation pathway is shown in Figure 6. The mass errors between the experimental and theoretical values are also shown in Figure 5. Adduct ions at m/z 548.0998 and 586.0522 were observed for the metabolites. These correspond with ions at m/z 532.1077 and 570.0627 for glyburide. Using the same reasoning as described above, these two ions are proposed as very stable potassium adducts of metabolites M1-M4. Metabolite M5. Metabolite M5 reveals a different in-source CID behavior relative to the other metabolites. Figure 7 shows a representative background-subtracted, in-source CID mass spectrum obtained from injection and LC/MS analysis of the 10 µM incubation sample. The measured accurate masses are also indicated in Figure 7. The isotopic pattern for each of the ions indicates there is a chlorine atom in their elemental compositions.
The upper limit of possible elements of all the fragments is set as C23H29ClN3O5S, which is expected from protonated metabolites, and the lower limit is set to include one chlorine atom. Only one elemental composition was generated for m/z 169.0045 and 385.0624, respectively. The composition would be C8H6ClO2 for m/z 169.0045 and C16H18N2O5SCl for m/z 385.0624. Two elemental compositions were generated for the ion at m/z 367.0504: C16H16N2O4SCl and C19H12N2O4Cl. The theoretical (A + 2)/A isotopic ratios for C16H16N2O4SCl and C19H12N2O4Cl are 39 and 35, respectively. The experimental isotopic value observed for the ratio of (A + 2)/A in Figure 7 is 39. These results suggest the elemental composition of C16H16N2O4SCl for the ion at m/z 367.0504. The proposed fragmentation mechanism is shown in Figure 8. The mass error between the measured value and the theoretical value using a two-point internal calibration is also indicated in Figure 7. The fragment ion at m/z 385.0624 appears to result from elimination of cyclohexylisocyanate, while the fragment ion at m/z 367.0504 appears to result from elimination of both cyclohexylisocyanate and water from the protonated molecule. The elimination of cyclohexylisocyanate indicates that the cyclohexyl ring is not the site of oxidation. The elimination of H2O indicates that hydroxylation is not likely to have occurred on the aromatic ring. The presence of an ion at m/z 169.0045 further suggests that hydroxylation did not occur on the chlorinated aromatic ring since the m/z 169 ion would have been expected to shift to m/z 185. It has been previously proposed that hydroxylation occurs on the ethyl amide ethylene bridge,12 but the exact position cannot be confirmed using the technology described herein. The adduct ions at m/z 532.1077 and 570.0627 for glyburide were shifted to m/z 548.0998 and 586.0532 for the M5 metabolite. Using the same logic as detailed above, these ions are also proposed as very stable potassium adducts of metabolite M5. CONCLUSION A time-of-flight MS system, coupled with HPLC has proven to be a sensitive and reliable technique for the characterization of Analytical Chemistry, Vol. 72, No. 14, July 15, 2000
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glyburide metabolites derived from in vitro incubation samples. The automated nozzle potential-switching feature allows “on-thefly” in-source CID to be performed to obtain both protonated molecules and characteristic fragment ions, whose accurate masses can be determined using internal mass calibration. Observed mass errors are less than 9 ppm for both protonated molecules and fragment ions. Elemental compositions of metabolite fragment ions can be rapidly and uniquely assigned. On the basis of the elemental compositions and in-source CID spectra, structures of metabolites and their fragments may be proposed. The experimental isotopic abundance ratios also help to constrain the number of possible elemental compositions. The approach described herein is easily automated and may be used for rapid characterization of metabolites whose parent drug’s structure is known. The metabolism in this study is relatively simple, and small mass shifts such as 14, 16, and 32 Da between parent drug and metabolite can be used to obtain the elemental composition of metabolites using a low-resolution mass spectrometer. However, when relatively larger mass shifts are involved, the accurate mass
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measurement capability described can provide unambiguous identification. ACKNOWLEDGMENT We thank PE-Biosystems for the generous loan of a Mariner TOF instrument and Jones Chromatography for the loan of an Endurance autosampler. Financial support from Advanced BioAnalytical Services, Inc., and Merck Research Laboratories is gratefully acknowledged. We thank Randy Sozio and Peter Gorycki of SmithKline Beecham Pharmaceuticals for the preparation of in vitro samples. We also thank Dr. Tim Wachs for valuable discussions and Dr. Katja Heinig, who initiated this project. Special thanks go to Dr. Ed Takach of PE-Biosystems for valuable technical support and Ms. Laura Lauman of PE-Biosystems for her continued support of our research.
Received for review January 31, 2000. Accepted May 2, 2000. AC000089R
