Ultrahigh-performance liquid chromatography-tandem mass spectrometry quantitation of o-cresol in hydrolyzed human urine to assess toluene exposure.

Abstract

Toluene is a volatile organic compound used in domestic and industrial applications. The main routes of workplace exposure to toluene are inhalation and dermal contact. As toluene exposure can cause severe nervous system damage, its quantification is crucial to prevent occupational illness. Toluene is metabolized mainly as hippuric acid, S-benzylmercapturic acid and epoxides. These are rapidly converted to o-/p-cresol, which is then excreted in the urine as conjugated glucuronides and sulfates. o-Cresol and its conjugates can be chemically hydrolyzed to form free o-cresol, which can then serve as a urinary biomarker of toluene exposure. Current analytical methods for quantifying o-cresol in hydrolyzed urine are, however, either weakened by interference, are not sensitive enough or require water-sensitive sample preparation. Development of a liquid chromatography-tandem mass spectrometry method for assessing exposure to toluene is thus required. Urine samples were acidified and heated to form free o-cresol and then derivatized with dansyl chloride and diluted. Extracts were separated by reverse-phase chromatography on a BEH phenyl column and then analyzed using a triple quadrupole instrument in selected reaction monitoring mode. The dansyl chloride derivatization step was optimized to produce the derivative within a reaction time of 3min. Hydrolysis efficiency in forming free o-cresol from conjugated metabolites was evaluated using o-cresol-β-d-glucuronidespiked human urine: complete hydrolysis occurred in 45 min. Dynamic range was 0.4 to 40 μM, and the method was useful for toluene monitoring in non-occupational (0.1μmol/mmol creatinine) as well as occupational (0.3μmol/mmol creatinine) exposure. The calculated limit of detection and limit of quantitation of the method were 0.06 and 0.21 μM, respectively. Intraday and interday precisions were 3.2% and 4.4%, respectively. Method accuracy was established as 99% using ClinChek® urine controls. An ultrahigh-performance liquid chromatography-tandem mass spectrometry method for analysis of o-cresol was developed for biological monitoring of toluene exposure in human urine. This is the method of choice used by occupational health and safety practitioners in the province of Québec, Canada.