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 spec- trometry coupled with high-performance liquid chroma- tography 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 2 0 0 μL/ min without postcolumn splitting. In-source collision-induced dis- sociation (CID) by automated nozzle potential switching was employed to obtain both abundant protonated mol- ecules and characteristic fragments whose accurate masses were measured simultaneously by internal mass calibra- tion, 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 composi- tions of all metabolite fragment ions were generated based upon the known compositional elements of the protonated molecule. The structures of metabolites and their frag- ment 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 ethylhy- droxy 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 triple- quadrupole or ion trap mass spectrometers, because of their capability for performing MS/ MS or MS n experiments. 1-3 API- time-of-flight mass spectrometry (API-TOF) has recently received renewed interest, because of advancements in the technology. This includes fast mass spectral acquisition speed with high full- scan 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 * Corresponding author: (e-mail) JDH4@ cornell.edu. (1) Bu, H. Z.; Poglod, M.; Micetich, R. G.; Khan, J. K. J. Mass Spectrom. 1999 , 34, 1185-1194. (2) Yu, X.; Cui, D. H.; Davis, M. R. J. Am Soc Mass Spectrom. 1999 , 10, 175- 183. (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. Anal. Chem. 2000, 72, 3342-3348 3342 Analytical Chemistry, Vol. 72, No. 14, July 15, 2000 10.1021/ac000089r CCC: $19.00 © 2000 American Chemical Society Published on Web 06/14/2000