Simultaneous determination of styrene, toluene, and xylene metabolites in urine by gas chromatography/mass spectrometry

Exposure to styrene, toluene, and xylene (STX) frequently occurs in various industrial settings leading to several adverse health effects. Therefore, the biological monitoring by determination of urinary mandelic acid (MA) and phenylglyoxylic acid (PGA), hippuric acid (HA), and 2-, 3-, and 4-methylhippuric acids (2-, 3-, and 4-MHAs), the metabolites of STX, is required or at least recommended in case of workers exposed by these agents. Considering the fact that co-exposure to STX frequently occurs, methods that have been described for the separate analysis of these compounds in urine samples cannot be used effectively for monitoring. Therefore, a reliable gas chromatographic/mass spectrometric (GC/MS) method was developed for the simultaneous identification and quantification of these metabolites. Following solid phase extraction of the urine samples, the extracts were silylated and analyzed by GC/MS using a HP-5MS capillary column. The method was evaluated for linearity, limits of detection and quantification, and specificity, as well as for precision, extraction efficiency, and stability at three different concentrations prepared in urine. The assay was linear up to 0.16 mg/ml for MA, and 0.32 mg/ml for PGA, HA, and 2-, 3- and 4-MHAs. The limits of detection and quantification of STX metabolites varied between 0.001 and 0.02 mg/ml, and from 0.01 to 0.04 mg/ml, respectively. The within-day and between-day precision, determined at low, medium, and high concentrations, ranged from 2 to 12% and 2 to 19%, respectively. The extraction efficiency was 70-80%. No degradation of the metabolites occurred in the urine samples under the possible working conditions. The method was applied for the analysis of the urine samples from exposed workers. The cost- and time-effectiveness, the technical advantages and validity parameters of this GC/MS analysis make it suitable for biological monitoring of mixed exposure to styrene, toluene and xylene.