Development, Validation, and Application of a Quantitative LC-MS/MS Method for Major Urinary Naphthalene Metabolites

Naphthalene is a volatile, lipid soluble compound and an important component of various fossil fuels, cigarette smoke, and jet fuel (1-3% by weight). Studies in rodents have shown that the compound produces dose-dependent cytotoxicity in the respiratory tract but targets are highly selective for airway epithelial cells in mice and nasal olfactory cells in both mice and rats; human susceptibility is unknown. The cytotoxicity of naphthalene is dependent on cytochrome P450 dependent metabolic activation and clearance is primarily dependent on the elimination of phase II metabolites in urine. Gas chromatography-mass spectrometry (GC/MS) methods are available for monitoring the levels of conjugates derived from 1-naphthol in urine but these ignore the likely substantial contributions of thioether-derived conjugates that represent primary detoxification products generated from naphthalene epoxide. The current studies present a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for quantitative measurement of not only the glucuronide and sulfate conjugates derived from 1-naphthol but also the mercapturic acids and N-acetyl glutathione derivative generated from naphthalene epoxide. Standard curves were linear over 2 log orders. On column limits of detection were metabolite-dependent and varied from 0.91 to 3.4 ng and limits of quantitation from 1.8 to 6.4 ng. The accuracy of measurement of spiked urine standards varied from -13.1 to + 5.2% of target and intra-day and inter-day variability averaged 7.2 (+/- 4.5) and 6.8 (+/- 5.0) %, respectively. Matrix effects were negligible. Application of the method to urine collected from mice exposed to naphthalene for 4 hours daily for 7 days at the OSHA short term exposure limit (15 ppm) showed that glutathione-derived metabolites accounted for 60-70% of the total measured metabolites and that sulfate and glucuronide conjugates were eliminated in approximately equal amounts. A 30% increase in metabolites was observed in day 6-7 urine compared to day 0-1, much of which was accounted for by glutathione derived metabolites. The methods presented here provide robust, relatively fast and direct measurements of several of the major metabolites of naphthalene including those derived from glutathione conjugation of naphthalene epoxide.