DETERMINATION OF DIMETHYLHIPPURIC ACID ISOMERS IN URINE BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

In order to analyse metabolites in urine after trimethylbenzene (TMB) exposure a method based on high-performance liquid chromatography (HPL C) for determination of the six dimethylhippuric acids (2,3-DMHA, 2,6- DMHA, 2,5-DMHA, 2,4-DMHA, 3,4-DMHA and 3,5-DMHA) in urine has been dev eloped. In contrast to earlier published methods, the present method a llows detection of all possible isomers of DMHA in a single analysis. The DMHAs were extracted from urine with dichloromethane. After evapor ation, the residue was dissolved in mobile phase and analysed by a ste pwise gradient HPLC system with ultraviolet (UV) detection at 225 nm. Mobile phase A (1.25% acetonitrile and 0.3% acetic acid in water) was used up to a retention time of 59.5 min and mobile phase B (5% acetoni trile in water containing 0.3% acetic acid) was used for completion of the analysis at approximately 90 min. The DMHA isomers were chromatog raphed on a reversed phase Radial-Pak C-18 column (4 mu m; 100 mm x 5 mm inner diameter). The detection limit for the six isomers was 1.5 mu g/ml (range 0.5-3.4, 100 mu l injection volume). The precision of the method was 4.2% relative standard deviation (range 3.8-4.4; 100 mu g/ ml). Standard curves of the DMHAs were linear over the interval 10-500 mu g/ml in human urine. Individual DMHAs or the sum of DMHA isomers m ay be used as biological indicators of occupational exposure to TMBs.