Ion cluster technique in conjunction with gas chromatography-mass spectrometry (GC-MS) was used for the identification and quantitation of major metabolites of cocaine (1a) in rat and humans. In a typical experiment, a female rat weighing 250 gm was intraperitoneally administered a 20-mg/kg mixture of 1a, NCD3-cocaine (1b), OCD3-cocaine (1c), and 4T2-cocaine (1d). The urine was collected, extracted with organic solvents, and separated into several fractions using TLC and HPLC techniques. Tritium radioactivity in a metabolically stable position in 1d was useful in the separation of metabolites, while the deuterium labeled 1(b + c), creating an artificial isotopic cluster, provided specific identification of metabolites by mass spectrometric interpretation. Norcocaine (2), benzoylnorecgonine (3), N-hydroxynorcocaine (4), methylecgonidine (5), benzoylecgonine (11), ecgonine methyl ester (9), hydroxycocaine (7), hydroxymethoxycocaine (10), and dimethoxyhydroxycocaine (6) were found to be the major metabolites of 1a in the rat urine as well as in plasma. The whole brain analysis showed significant amounts of unmetabolized 1a and 2, and minor concentrations of 9, 5, 7, and 10, and traces of 6. Some of these metabolites have been reported earlier by us as well as other investigators and are unequivocally confirmed in this work. Unmetabolized 1a, its pharmacologically active metabolite 2, and other major metabolites were quantitated in the rat brain, plasma, and urine using stable isotope-labeled analogues as internal standards and selected ion monitoring (SIM) mass spectrometry. The pharmacokinetic profiles of 1a and 2 indicate half-lives of less than 20 min in the brain and plasma. These data are in good agreement with widely reported short-lived behavioral effects of cocaine.(ABSTRACT TRUNCATED AT 250 WORDS)