Determination of free and total phenylacetic acid in human and rat plasma by high-performance liquid chromatography with fluorescence detection.

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Abstract
A highly sensitive and simple high-performance liquid chromatographic method has been developed for the determination of free and total phenylacetic acid in human and rat plasma. After extraction with diethyl ether from plasma, phenylacetic acid and phenylpropionic acid (internal standard) are converted to the corresponding fluorescent derivatives by reaction with 3-bromomethyl-6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone in the presence of potassium hydrogen carbonate and 18-crown-6 in acetonitrile. The derivatives are separated on a reversed-phase column (Radial-Pak cartridge C18) with aqueous 65% (v/v) methanol and detected fluorimetrically. The detection limit for phenylacetic acid is 11 pmol/ml in plasma at a signal-to-noise ratio of 5. This sensitivity permits precise determination of free and total phenylacetic acid in 50 .mu.l of human and rat plasma. The method was applied to the determination of free and total phenylacetic acid in plasma from healthy volunteers, and control and "behavioral despair" rats.