IMPROVED HIGH PERFORMANCE LIQUID CHROMATOGRAPHY DETERMINATION OF HIPPURIC ACID AND METHYLHIPPURIC ACID ISOMERS IN URINE

Abstract

A study was made on the effect of detection wavelength and separation mode of HPLC on determination of urinary hippuric acid (HA) and three isomers of methylhippuric acid (MHA). The interference of other constituents of urine in the determination was effectively decreased by detection at a short wavelength of 227.6nm. Meta and para MHAs were separated by the addition of β-cyclodextrine to the mobile phase. Four metabolites were successfully separated from other components of urine by the combination of ODS-silica packed-column and mobile phase (method F). The detection limits were found to be 50 and 5mg/l for HA and MHAs, respectively. MHAs could not be detected in the non-exposed subjects. Average levels (±SD) of HA in non-exposed males and females were 272.2 (±210.8) and 393.0 (±269.8)mg/l, respectively. The urinary levels of HA in females were significantly higher than those in males.