QUANTITATION OF URINARY 7-METHYLADENINE BY GAS-CHROMATOGRAPHY MASS-SPECTROMETRY USING ISOTOPICALLY LABELED INTERNAL STANDARDS

We have developed a procedure for isolating and quantifying 7-methylad enine from rat urine following the administration to the rat of methyl ating agents, such as dimethylnitrosamine. Urinary 7-methyladenine and its trideutero isomer, added as an internal standard, were precipitat ed with silver nitrate, the precipitate was extracted with HCl, and th e extract was further purified by C-18-Sep-Pak chromatography. The rec overed 7-methyladenine was then derivatized with pentafluorobenzyl bro mide at alkaline pH for analysis by gas chromatography-mass spectromet ry, indicating a bis(pentafluorobenzyl) conjugate, m/z 509. The mass s pectrum of this derivative shows a major fragmentation ion at mit 328 (and 331 for the trideutero derivative) resulting from the loss of one pentafluorobenzyl group. Levels of urinary 7-methyladenine above 150 pg could be detected from the ratio of the gas chromatography peak are as for these ions, using selective-ion monitoring. The method was sele ctive for the 7-methyl isomer. The procedures developed for the synthe ses of deuterated and tritiated 7-methyladenine, which were required f or these studies, are also described. (C) 1994 Academic Press, Inc.