Mercapturic acids derived from toluene in rat urine samples: identification and measurement by gas chromatography-tandem mass spectrometry

Toluene is one of the most widely used CMR chemicals in industry. Worker exposure to this compound is regulated in France, but new, more sensitive methods are required to effectively monitor this exposure. A gas chromatography-tandem mass spectrometry (GC-MS/MS) method was developed and fully validated for the simultaneous determination of urinary toluene mercapturic acids derived from side chain and ring oxidation, i.e., benzylmercapturic acid and the three isomers o-, m- and p-toluylmercapturic acids, respectively. The method involves a simple and efficient two-step preparation procedure consisting of liquid-liquid extraction of the urinary acids followed by a microwave-assisted esterification of the isolated compounds using 2-propanol. The method meets all the required validation criteria: high selectivity, intra-day and inter-day precision ranges between 1.0 % and 12.4 %, with close to 100 % recovery. Linearity has been shown over the reduced concentration range 0.03-0.5 mg/L whereas a multiplicative model (ln-ln transformation) had to be used to describe the full range of concentrations 0.03-20 mg/L. The limits of detection for the four analytes, ranging from 2.8 to 5.5 mu g/L, made the method suitable for their identification and quantification in urine from rats inhaling toluene in the 2 to 200 ppm concentration range. All urine samples from exposed rats contained measurable amounts of all metabolites. This is the first time that o- and m-toluylmercapturic acids have been shown to occur. Our results confirm the hypothesis that toluene mercapturic acids derived from ring oxidation exist in three forms.