Analysis of pyrene metabolites in marine snails by liquid chromatography using fluorescence and mass spectrometry detection

As part of a study of the metabolism of aromatic compounds in marine gastropods, a sensitive and selective method was developed to detect, identify and quantify pyrene (PY) and four of its metabolites in tissues: 1-hydroxypyrene (PYOH), pyrene sulfate (PYOS), pyrene glucuronide (PYOG) and pyrenediol disulfate (PYDS). Liquid chromatography (LC) with fluorescence detection was first used to detect the PY derivatives in the visceral mass of whelks exposed to PYOH. The identification of metabolites was accomplished through a combination of retention time and spectral matching with standards, enzymatic hydrolysis, solid phase extraction and LC coupled with electrospray ionization mass spectrometry. In addition to four known PY derivatives, two novel metabolites were identified as pyrenediol glucuronide sulfate and a second isomer of PYDS. The methanol extraction of metabolites from tissue gave excellent mean recoveries, ranging from 67 to 97%, for the available standards PY, PYOH, PYOS and PYOG spiked in both the muscle and visceral mass of Buccinum spp. The mean recoveries of a surrogate standard, 2-hydroxyfluorene, spiked in all tissue samples were 100% and 95% for visceral and muscle tissue samples, respectively. The method limits of detection for these compounds were all below 0.2 ng/g of wet tissue, low enough to detect metabolites in reference animals. Results from the application of this method to the quantitative analysis of biotransformation products in the visceral mass of the whelk Neptunia lyrata exposed to PYOH contaminated food are also presented. This method will be useful to apply to the analysis of PY metabolites in soft tissues of other animals.