Quantitation of Urinary 7-Methyladenine by Gas Chromatography-Mass Spectrometry Using Isotopically Labeled Internal Standards

Abstract

We have developed a procedure for isolating and quantifying 7-methyladenine from rat urine following the administration to the rat of methylating agents, such as dimethylnitrosamine. Urinary 7-methyladenine and its trideutero isomer, added as an internal standard, were precipitated with silver nitrate, the precipitate was extracted with HCl, and the extract was further purified by C18-Sep-Pak chromatography. The recovered 7-methyladenine was then derivatized with pentafluorobenzyl bromide at alkaline pH for analysis by gas chromatography-mass spectrometry, indicating a bis(pentafluorobenzyl) conjugate, m/z 509. The mass spectrum of this derivative shows a major fragmentation ion at m/z 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 areas for these ions, using selective-ion monitoring. The method was selective for the 7-methyl isomer. The procedures developed for the syntheses of deuterated and tritiated 7-methyladenine, which were required for these studies, are also described.