Simultaneous High-Performance Liquid Chromatographic Analysis of Hippuric Acid and ortho-, meta-, and para-Methylhippuric Acids in Urine

A high-performance liquid chromatographic assay for the simultaneous analysis of hippuric and methylhippuric acids in urine is described. Compounds are first extracted from acidified urines after addition of o-methylbenzoylalanine (internal standard), using methyl-t-butyl ether. The organic phase is evaporated under nitrogen flow and the residue dissolved in the mobile phase which consisted of 91% potassium phosphate buffer (12.0mM, pH: 2.0), 4.5% methanol, and 4.5% tetrahydrofuran. The method described allows complete separation of meta- and para-methylhippuric acids in less than 20 min using a stainless steel column packed with octadecyl-dimethysilyl silica. The authors used this method to determine the urinary excretion of hippuric and methylhippuric acids in rabbits following exposure to a mixture of toluene and isomers of xylene. Data indicated that under the actual experimental conditions of exposure o-xylene is excreted to a lesser extent as a methylhippuric acid than the meta- and para-xylenes.