Direct determination of free phenylacetic acid in human plasma and urine by column-switching high performance liquid chromatography with fluorescence detection.

A simple and highly sensitive column-switching high performance liquid chromatographic method with fluorescence detection for the determination of free phenylacetic acid (PAA) in human plasma and urine is described. The method is based on the direct derivatization of plasma and urine PAA with 6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone-3-propionylcarboxylic acid hydrazide (DMEQ-hydrazide). The derivatization reaction proceeds in aqueous solution in the presence of pyridine and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide at 37 degrees C. The resulting DMEQ derivative of PAA is separated from endogenous interfering substances by a column-switching chromatographic system consisting of a precolumn (YMC-Pack C4) for sample clean-up and an analytical column (L-Column ODS) for the complete separation of the derivative. The derivative is detected fluorimetrically at 445 nm with excitation at 367 nm. The detection limits (signal to noise ratio = 3) for PAA is 10 fmol in a 10 microL injection volume. The recoveries from plasma and urine are 75 and 96%, respectively. The present method is highly sensitive and simple compared to conventional liquid-liquid extraction procedures. The sensitivity allows the direct determination of free PAA in an extremely small amount (5 microL) of plasma and urine.