Anal. Chem. 1995, 67,2931-2936
Integrated Application of Capillary HPLC/ Continuous-Flow Liquid Secondary ion Mass Spectrometry to Discovery Stage Metabolism Studies Chun LI,* Nathalie Chauret, Yves Duchanne, Laird A. Trimble, Deborah A. Nicoll.OrWtth, and James A. Yergey Medicinal Chemistry Department, Merck Frost Centre for Therapeutic Research, P.0. Box 1005, Pointe-Claire-Dolval, Quebec, Canada H9R 4P8
The application of capillary HPLC/continuous-flowliquid secondary ion mass spectrometry (CF-LSIMS) as part of an integrated approach for characterizingdiscovery stage in vitro metabolites, using a spedic inhibitor for 5-lipoxygenase as a model compound, was investigated. CFLSIMS demonstrated excellent sensitivity in detecting the metabolites in both the positive and the negative ion modes, with a good full-scan mass spectrum obtained when 5 pmol of metabolitewas injected onto the capillary column. Strong pseudomolecular ions and key fragment ions were observed in the primary spectra of the parent drug and its three oxidative in vitro metabolites, allowing the site of metabolism to be pinpointed to particular substructures. This technique demonstrated versatility and offered a very rapid screening procedure for metabolite identification. Continuous-flow fast atom bombardment interfaces were first developed by Ito et al.’ as “frit-FAB” and Caprioli et alS2as continuous-flow FAB. They were designed to eliminate some of the major disadvantages of using direct-probe FAB or liquid secondary ion mass spectrometry (LSIMS), while retaining the essential advantages of this bombardment ionization process. Direct-probe FAB or LSI mass spectra can often be complicated by the high level of chemical noise due to the high concentration of matrix on the probe tip, with the result that the structurally informative fragment ions may be obscured by the matrix ions. Continuous-flow FAB or LSIMS entails the use of a sample introduction probe that provides a continuous flow of liquid containing small amounts of matrix to the probe tip. The chemical noise is significantly reduced, and therefore, the signal-to-noise ratio is greatly enhan~ed.~ The combination of liquid chromatography (LC) and CF-FAB or CF-LSIMS provides on-line coupling for HPU: separation, and the soft ionization allows direct analysis of polar and labile (1) Ito, Y.; Takeuchi,T.; Ishii, D.; Goto, M.; Mizuno, T. /. Chromatogr. 1985, 358,201. (2) Capnoli, R M.; Fan, T.; Cottrell, J. S. Anal. Chem. 1986,58,2949. (3) Caprioli, R. M.; Fan, T.Biochem. Biophp. Res. Commun. 1986,141, 1058. (4) Caprioli, R M.; Moore, W. T.; Fan, T. Rapid Commun. Mass Spectrom. 1987, 1, 15. (5) Ashcroft, A E.; Chapman, J. R.; Cottrell, J. S . /. Chromutogr. 1987,394, 15. (6) Boulenguer, P.; Leroy, Y.; Alonso, J. M.; Montreuil, J.; Kcart, G.; Colbert, C.; Duquet, D.; Dewaele, C.; Foumet, B. Anal. Biochem. 1988,168, 164. 0003-2700/95/0367-2931$9.00/0 0 1995 American Chemical Society
The role of mass spectrometry, especially liquid chromatography/mass spectrometry (LC/MS), in qualitative drug metabolite identification has become evident and increasingly i m p ~ r t a n t . ~ - ~ ~ Drug metabolite identification in animals and human subjects is important for both basic science and drug discovery. It can have a strong impact on structure-activity relationship studies and the design of metabolically more stable drug candidates. The ability to characterize reactive intermediates using LC/MS8 is likely to affect the selection of drug candidates with respect to potential toxicological problems. There are several examples in the literature demonstrating the use of mass spectrometry for metabolite identification. Some involved isolation of the metabolites and characterization by off-line mass ~pectrometry;~ however, this process can be time consuming and may not be suitable for unstable metabolites. Thermospray LC/MS provided on-line analysis of metabolites, but the technique requires relatively large amounts (10 ng to 5 pg) of metabolites in order to get a full-scan mass spectrum.lOJ1 Recently, electrospray U / M S has also become a very popular technique for metabolite identification?J1JZ However, in most cases, only pseudomolecular ions were observed in the primary ESI mass spectra, with very little structural information obtained, necessitating further analysis by MS/MS for the characterization of unknown metabolites.*J2 CF-LSIMS has also been demonstrated to be useful for metabolite identification, providing characterization of glutathione conjugates using selected-ion monitoring at the low nanogram level13 and of metabolites of cycloates in radish leaves with detection limits of 15 ng.I4 This technique has been used effectively and routinely in our laboratory for metabolism studies.15 It provides good (7) Teffere, Y.; Baird, W. M.; Smith, D.M. Anal. Chem. 1991,63,453. (8)Davis, M.; Baillie, T. /. Mass Spectrom. 1995,30,57. (9) Vrbanac, J. J.; O’Leary, I. A; Baczynskyj, L. Biol. Mass Spectrom. 1992, 21,517. (10) Fujiwara, H.; Chott, R C.; Solsten, R T. Biol. Mass Spectrom. 1992,21, 431. (11) Iwabuch, H.; Kitazawa, E.; Kobayashi, N.; Watanabe. H.; Kanai M.; Nakamura, K Biol.Mass Spectrom. 1994,23, 540. (12) Jackson, P. J.; Brownsill, S . D.; Taylor, A R; Walther, B. J Mass Spectrom. 1995,30,446. (13) Moritz, T.; Schneider, G.; Jensen, E. Biol. Muss Spectrom. 1992,21, 554. (14) Onisko, B. C.; Barnes, J. P.; Stanb, R E.; Walker, F. H.; Kerlinger, N. Bid. Mass Spectrom. 1994,23, 626. (15) Nicoll-Griffith, D. A; Chauret, N.; Yergey, J. A; Trimble, L. A; Favreau, L; Zamboni, R Drug Metab. Dispos. 1993,21, 861. (16) Ducharme, Y.; Brideau, C.; Dube, D.;Chan, C. C.; Falgueyret, J. P.; Gillard, J. W.; Guay, J.; Hutchinson, J. H.; McFarlane, C. S.; Riendeau, D.; Scheigetz, J.; Girard, Y. /. Med. Chem. 1992,37,512.
Analytical Chemistry, Vol. 67, No. 17, September 1, 1995 2931
. .
O
-
OH
o
&'
L-702,539
0
Figure 1. Chemical structures of L-702,539 and L-702,618. L-702,618 is the open lactone form of L-702,539, designed to improve the absorption of L-702,539, which is the active drug form.
chromatographic resolution and excellent sensitivity. Most importantly, the technique provided a rapid screening and characterization of unknown metabolites: strong molecular ions and key fragment ions were often obtained in the primary mass spectra of both the drug and the metabolites, allowing the site of metabolism to be pinpointed on a particular substructure without the need for MS/MS experiments. This paper describes the application of capillary HPLC and continuous-flow liquid secondary ion mass spectrometry (HPLCI CF-LSIMS) as part of an integrated approach for characterizing discovery stage in vitro metabolites. The model compound used to exemplify the use of CF-LSIMS is L702,539 (Figure l), a tetrahydropyranylphenyl naphthalenic lignan lactone which is a potent and specific inhibitor of 5-lipoxygenase.16-20 A more complete discussion of the metabolic fate of L702,539, relating in vitro studies in a variety of species with metabolites observed in vivo, has already been published.2l EXPERIMENTAL SECTION
Materials. L702,539 and all the standards of the identified metabolites were synthesized in the Merck Frosst medicinal chemistry 1ab0ratories.l~Solid phase extraction cartridges were obtained from Varian (Harbor City, CA). All solvents used were obtained from commercial sources and were of HPLC grade. Microsome Incubations. Incubations were conducted with 50 pg of L702,539 and 1 mg of microsomal protein from rhesus monkey liver in the presence of an NADPH-generating system as previously described.22The h a l total drug concentration was 210 pM. Typically, the incubations were conducted for 1h at 37 "C and then quenched with an equal volume of acetonitrile, and the precipitated proteins were removed by centrifugation (Ep pendorf centrifuge model 5415C, 14 OOO rpm x 10 min). The supernatant was then injected directly onto the CF-LSIMSsystem. Analytical HPLC Analysis. Analytical HPLC analysis of the rhesus monkey microsome incubation was performed on a Waters HPLC system (Waters, Milford, MA), comprised of a 600MS pump, a model 715 autosampler, a 994 photodiode array detector controlled by Powerline software, and a Novapak C18 column (0.46 x 15 cm). A linear gradient from 45%A55%B to 90%Al0%B (17) Young, R N.; Girard, Y.; Gillard, J. W.; Trimble, L. A; Scheigtz, J.; Yergey, J. A; Ducharme, Y.; Nicoll-Griffith, D. A,; Hutchinson, J. H. US. Patent 5,227,399 July 13, 1993, Example 4. (18)Leukotrienes and Lipoxygenases: Chemical, Biological and Clinical Aspects, Rokach, J., Ed.; Elsevier: Amsterdam, 1989. (19) Borgeat, P.; Samuelsson, B. Proc. Natl. Acad. Sci. U.S.A. 1979,76, 2148. (20) Samuelsson, B. Science 1983,220, 568. (21) Chauret, N.; Li,C.; Ducharme, Y.; Trimble, L.A; Yergey, J. A; Ramachandran, C.; Nicoll-Griffith, D. A Drug Metab. Dispos. 1995,23, 65. (22) Organic Syntheses; Wiley: New York, Collect. 1943; Vol. 2, p 165.-
2932 Analytical Chemistty, Vol. 67, No. 77, September 7, 7995
over 40 min with a flow rate of 1mL/min was used for separation (eluant A, CH30H; eluant B, 20 mM NH40Ac adjusted to pH 5.0 with acetic acid). The detection wavelength was 245 nm. Isolation of Metabolites. To prepare enough of each isolated metabolite for NMR and further mass spectrometric characterization, incubations using rhesus monkey microsomes were scaled up appropriately. The isolation involved three steps. Fist, a crude solid-phase extraction (SPE) procedure was used to remove microsomal protein and concentrate the sample. The supernatant of the large-scale incubation mixture was diluted l@fold with distilled H20 so that it contained ~ 5 CH3CN. % The solution was applied to the 6 mL Varian C18 SPE cartridge which was preconditioned with 12 mL of CH30H followed by 12 mL of HzO. After sample application, the cartridge was washed with distilled H20, and the crude extract was eluted with 4 mL of methanol. Second,the crude extract was divided into four fractions, and each was diluted with 3 mL of HzO. Metabolites of interest were isolated by injecting each diluted fraction onto the preparative HPLC system, which consisted of a Waters 600MS pump, a Valco C6W injector fitted with a 10 mL loop, a preparative p-BondaPak C18 column (19 x 150 mm), and a Waters 990 diode array detector. A linear gradient of 60% A40% B to 90% AlO% B (A, CH3OH; B, 20 mM NH~OAC,pH 4.5) over 40 min with a flow of 10 mL/min was used for separation. Metabolite peaks were collected manually, and the same metabolite fractions collected from four separate HPLC isolations were combined. Third, the isolated materials were concentrated and desalted using a 1 mL Bond-Elute C18 SPE cartridge which was conditioned with 2 mL of CH30H followed by 2 mL of HzO. Following application of the isolated fractions (diluted to
