Determination of p-hydroxymandelic acid enantiomers in urine by high-performance liquid chromatography with electrochemical detection.

High-performance liquid chromatography (HPLC) with electrochemical detection using a chiral ligand-exchange column was developed for the enantioselective determination of p-hydroxymandelic acid (HMA), a metabolite of synephrine, with high sensitivity. A good linear relationship between current ratio and amount was noted for 0.5-500 pmol HMA, with a correlation coefficient of 0.999 for each HMA enantiomer. The relative standard deviation (R.S.D.) was 1.6% at 100 pmol d-HMA and 2.2% at 100 pmol l-HMA. The detection limit of each HMA enantiomer was 0.5 pmol (signal to noise ratio, S/N = 3). By this method, HMA in Citrus unshiu and in urine following the ingestion of C. unshiu was determined. Although no HMA was found in c. unshiu, d- and l-HMA were present in urine after the ingestion of C. unshiu. The time courses of HMA and conjugated synephrine enantiomers excreted in urine following the ingestion of C. unshiu for 24 h could be monitored. This method should prove applicable to the study of synephrine metabolism.