A new method based on liquid chromatography/tandem mass spectrometry has been developed for the direct determination of specific urinary mercapturic acids 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 diastereoisomers were separated on a C18‐DB (7.5 cm, 3 µm) column using variable proportions of 20 mM aqueous ammonium formate buffer and methanol at a flow‐rate of 0.5 mL/min. The analytes were ionized by electrospray, in negative‐ion mode. Operating in selected‐reaction monitoring mode, linearity of the MS response versus analyte concentration was established over 4 orders of magnitude, the detection limits being 0.7–1.0 µg/L for all the mercapturates. Precision of the method determined at 50 µg/L (n = 12), expressed as relative 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) were respectively 2.0, 3.6 and 5.5%. The method was applied to the quantitative analysis of conjugated metabolites in urine samples from workers occupationally exposed to styrene. The diastereoisomers R,R‐M1 and S,R‐M2 accounted respectively for 50 and 40% of total mercapturates, whereas the proportion of R,R‐M2 was 7% and only minor amounts of S,R‐M1 were detectable. Styrene 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 characterized by the genetic polymorphisms of the drug‐metabolizing enzymes to be identified.
A new method for the analysis of styrene mercapturic acids by liquid chromatography/electrospray tandem mass spectrometry
BERGAMASCHI, Enrico;
2000-01-01
Abstract
A new method based on liquid chromatography/tandem mass spectrometry has been developed for the direct determination of specific urinary mercapturic acids 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 diastereoisomers were separated on a C18‐DB (7.5 cm, 3 µm) column using variable proportions of 20 mM aqueous ammonium formate buffer and methanol at a flow‐rate of 0.5 mL/min. The analytes were ionized by electrospray, in negative‐ion mode. Operating in selected‐reaction monitoring mode, linearity of the MS response versus analyte concentration was established over 4 orders of magnitude, the detection limits being 0.7–1.0 µg/L for all the mercapturates. Precision of the method determined at 50 µg/L (n = 12), expressed as relative 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) were respectively 2.0, 3.6 and 5.5%. The method was applied to the quantitative analysis of conjugated metabolites in urine samples from workers occupationally exposed to styrene. The diastereoisomers R,R‐M1 and S,R‐M2 accounted respectively for 50 and 40% of total mercapturates, whereas the proportion of R,R‐M2 was 7% and only minor amounts of S,R‐M1 were detectable. Styrene 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 characterized by the genetic polymorphisms of the drug‐metabolizing enzymes to be identified.
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