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 (HPLC) 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 developed. In contrast to earlier published methods, the present method allows detection of all possible isomers of DMHA in a single analysis. The DMHAs were extracted from urine with dichloromethane. After evaporation, the residue was dissolved in mobile phase and analysed by a stepwise 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% acetonitrile in water containing 0.3% acetic acid) was used for completion of the analysis at approximately 90 min. The DMHA isomers were chromatographed on a reversed phase Radial-Pak C18 column (4 microns; 100 mm x 5 mm inner diameter). The detection limit for the six isomers was 1.5 micrograms/ml (range 0.5-3.4, 100 microliters injection volume). The precision of the method was 4.2% relative standard deviation (range 3.8-4.4; 100 micrograms/ml). Standard curves of the DMHAs were linear over the interval 10-500 micrograms/ml in human urine. Individual DMHAs or the sum of DMHA isomers may be used as biological indicators of occupational exposure to TMBs.