Determination of urinary mercapturic acids of styrene in man by high-performance liquid chromatography with...

A method for the determination of urinary N-acetyl-S-(1-phenyl-2-hydroxyethyl)-L-cysteine (M1) and N-acetyl-S-(2-phenyl-2-hydroxyethyl)-L-cysteine (M2) in man was developed. Clean-up of urine samples was obtained by a chromatographic technique, using a short reversed-phase precolumn; purified samples were then deacetylated with porcine acylase I for 16 h at 37 degrees C and deproteinized by centrifugal ultrafiltration. Derivatization was performed with o-phthaldialdehyde and 2-mercaptoethanol and the fluorescent derivatives were separated on a reversed-phase analytical column with a gradient mobile phase consisting of 50 mM acetate buffer (pH 6.5) and methanol. The retention times of the diastereoisomers of M1 (M1-"S" and M1-"R") were 52.8 and 73.7 min, respectively: M2 diastereoisomers eluted as a single peak at 70.5 min. The fluorescence detector was set at 330 nm (excitation) and 440 nm (emission). The detection limit (at a signal-to-noise ratio of three) was about 7 micrograms/1. The method was applied to 25 urine samples from workers exposed to styrene. A relationship was found between urinary mandelic and phenylglyoxylic acids and mercapturic acids specific for styrene. Urine samples from ten non-exposed subjects showed no detectable amounts of analytes.