Development of a high-performance liquid chromatographic method for the simultaneous determination of pyrene-1,6- and 1,8-dione in animal and human urine

Abstract

A recent in vivo mass-balance study on the disposition of 14C-labelled pyrene in rats suggests that 1-hydroxypyrene (1-OHP) is not the major excreted metabolite. In that report, specific metabolites other than 1-OHP were not identified. The purpose of this study was to identify and quantify these unknown metabolites of pyrene in animal and human urine. Using a high-performance liquid chromatography (HPLC)-electrospray-mass spectrometry method, it was observed that dioxygenated pyrene metabolites (m/z 233) were present in significant amounts in urine samples of rats treated with pyrene. An HPLC method with fluorescence detection was then developed for the simultaneous determination of pyrene-1,6- and 1,8-dioxygenated metabolites, that is, the sum of hydroquinone, semiquinone, and quinone forms of these metabolites, after derivatization into 1,6-diacetoxypyrene (P16Da) and 1,8-diacetoxypyrene (P18Da). The mean limits of detection (± standard deviation) were 46 ± 22 nmol P16Da/L and 86 ± 32 nmol P18Da/L, as calculated from standard solution curves. The intraday coefficient of variation in rats was 5.5% for P16Da and 7.2% for P18Da; in humans, it was 6.8% for P16Da and 7.4% for P18Da (n = 36 in each case). The day-to-day coefficients of variation in rats were 12.5% for P16Da and 7.3% for P18Da; in humans, they were 10.1% for P16Da and 7.2% for P18Da (n = 12 in each case). The recovery rates of these metabolites ranged between 89 and 126% in rats and between 100 and 121% in humans (n = 36 in each case). Interestingly, rat data showed that P16Da molar amounts in 24-h urine samples (n = 4) exceeded those of 1-OHP by 64 to 121 times and P18Da amounts exceeded those of 1-OHP by 13 to 35 times. Similarly, P16Da molar concentrations in spot urine samples of human subjects (n = 4) exposed to pyrene exceeded those of 1-OHP by 4 to 12 times while P18Da concentrations were 0.4 to 2 times those of 1-OHP. Pyrene-1,6- and 1,8-dioxygenated metabolites are major metabolites of pyrene and, particularly in the case of the 1,6-isomers, potentially useful biomarkers of both environmental and occupational exposure to pyrene.