A new method for the analysis of styrene mercapturic acids by liquid chromatography/electrospray tandem mass spectrometry

A new method based on liquid chromatography/tandem mass spectrometry has be en developed for the direct determination of specific urinary mercapturic a cids arising from the conjugation of (R)-and (S)enantiomers of styrene 7,8- oxide with glutathione (GSH), i.e. (R,R)- and (S,R)-N-acetyl-S-(1-phenyl-2- hydroxyethyl)cysteine (R,R-M1 and S,R-M1) and (R,R)- and (S,R)-N-acetyl-S-( 2-phenyl-2-hydroxyethyl)- cysteine (R,R-M2 and S,R-M2), The four diastereoi somers were separated on a C18-DB (7.5 cm, 3 mum) column using variable pro portions of 20 mM aqueous ammonium formate buffer and methanol at a flow-ra te of 0.5 mL/min. The analytes were ionized by electrospray, in negative-io n mode. Operating in selected-reaction monitoring mode, linearity of the MS response versus analyte concentration was established over 4 orders of mag nitude, the detection limits being 0.7-1.0 mug/L for all the mercapturates. Precision of the method determined at 50 mug/L (n = 12), expressed as rela tive standard deviation, was respectively 3.1, 4.8 and 6.9% within the run, intra-day and inter-day. The corresponding figures at 1.0 mg/L (n = 12) we re respectively 2.0, 3.6 and 5.5%. The method was applied to the quantitati ve analysis of conjugated metabolites in urine samples from workers occupat ionally exposed to styrene, The diastereoisomers R,R-M1 and S,R-M2 accounte d respectively for 50 and 40% of total mercapturates, whereas the proportio n of R,R-M2 was 7% and only minor amounts of S,R-M1 were detectable. Styren e mercapturates represented a minor fraction of total styrene metabolites, less than 1% on average. The ratio mercapturates/main metabolites (mandelic + phenylglyoxylic acid) showed a bimodal distribution, the medians of the two subgroups being 0.2 and 1%, respectively, Such subgroups are probably c haracterized by the genetic polymorphisms of the drug-metabolizing enzymes to be identified. Copyright (C) 2000 John Wiley & Sons, Ltd.