SUBNANOMOLAR QUANTIFICATION OF CAFFEINES IN-VITRO METABOLITES BY STABLE-ISOTOPE DILUTION GAS-CHROMATOGRAPHY MASS-SPECTROMETRY

A method for the quantification of subnanomolar levels of in vitro met abolites of caffeine by an isotope dilution gas chromatographic-mass s pectrometric (GC-MS) assay has been developed and applied. Trideuterom ethylated analogs of each primary metabolite were synthesized and adde d after incubations of caffeine with human liver microsomes high in cy tochrome P4501A2. KPLC separation of the metabolites prior to GC-MS qu antification allowed the isolation of theobromine and paraxanthine whi ch coeluted by GC and enabled quantification over a larger dynamic ran ge. Quantitative analysis was performed on the n-propylated derivative s by selected-ion monitoring of either the M+.ions for the dimethylxan thines or [M-C3H6](+).ions for 1,3,7-trimethyluric acid. For the least abundant metabolite (1,3,7-trimethyluric acid), the detection level o n column was 200 pg. Replicate analyses exhibited intra-and inter-day variability of 4.2 and 7.9%, respectively. This assay has been success fully used in the quantification of caffeine's primary metabolites in more than 180 incubations, at varying substrate concentrations and wit h multiple enzyme sources. (C) 1998 Elsevier Science B.V.