Simultaneous determination of mandelic acid enantiomers and phenylglyoxylic acid in urine by high-performance liquid chromatography with precolumn derivatization

A reversed-phase HPLC method for the simultaneous quantitative determination of mandelic acid enantiomers (MA) and phenylglyoxylic acid (PGA) in urine is described. MA and PGA were extracted with ethyl acetate from urine at acidic pH and derivatized with S-(−)-1-(1-naphthyl) ethylamine. A ZORBAX SB-C18 column (250 mm × 4.6 mm i.d., 5 μm, Agilent, USA) was used with a mobile phase composed of methanol–10 mmol/L phosphate buffer [pH 2.5 (65:35, v/v)] at a flow-rate of 0.8 ml/min. Detection was set at UV wavelength of 254 nm. The mean absolute recoveries were 94.2%, 91.9%, 92.5% and 86.3% for S-MA, R-MA, PGA and salicylic acid (I.S.), respectively. The intra- and inter-day precisions determined at three different concentrations ranged from 2.8% to 4.8%, 0.7% to 7.7% and 1.3% to 6.8%, respectively. The lower limits of detection for MA enantiomers and PGA in urine were 1 μg/ml and the lower limits of quantification were 5 μg/ml (R.S.D. < 10%, n = 5). The method has been applied to determine the urinary excretion of MA enantiomers and PGA from Sprague–Dawley rats after orally administered with styrene.