Abstract
A method was developed for the simultaneous determination of 10 phenolic acids, including homovanillic acid, vanilmandelic acid, and 5-hydroxyindole-3-acetic acid, in urine by gas chromatography with hydrogen flame ionization detection. It consists of extraction of the free acids in urine into ether followed by isobutyloxycarbonylation of the phenolic hydroxyl groups with isobutyl chloroformate in aqueous alkaline medium and methylation of carboxylic acid groups with diazomethane. The derivatives thus obtained have good chromatographic properties and are stable under normal laboratory conditions. The calibration graphs of each phenolic acid for quantitation were constructed with the use of trans-m-cinnamic acid as an internal standard. Separation of the derivatives of phenolic acids from other urinary constituents was achieved by using two columns of 1.5% OV-17 and 0.65% Poly-A-101A. The peaks of phenolic acid derivatives derived from urine were identified by means of gas chromatography-mass spectrometry. The results of recovery experiments with urine samples fortified with a known amount of each phenolic acid were satisfactory, the average recovery ranging from 89.5 to 105.0%.
