Determination of free and total phenylacetic and p- and m-hydroxyphenylacetic acids in human urine by liquid chromatography with fluorimetric detection

• Published in: Journal of chromatography. Biomedical applications Vol. 414; pp. 275 - 284
• Main Authors: Yamaguchi, Masatoshi, Matsunaga, Reiko, Fukuda, Kazuya, Nakamura, Masaru
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 1987
• ISSN: 0378-4347
• DOI: 10.1016/0378-4347(87)80053-1

A highly sensitive and rapid liquid chromatographic method for the determination of free and total phenylacetic and p- and m-hydroxyphenylacetic acids in human urine is described. After extraction of urine with diethyl ether, these acids and phenylpropionic acid (internal standard) are converted into the corresponding fluorescent derivatives by treatment with 3-bromomethyl-6,7-dimethoxy-1-methyl-2(1 H)-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 C 18) with aqueous 65% (v/v) methanol and detected fluorimetrically. The detection limits for phenylacetic and p- and m-hydroxyphenylacetic acids are 5, 30 and 100 fmol, respectively, at a signal-to-noise ratio of 5 in a 20-μl injection volume. This sensitivity permits precise determination of the free and total acids in 20 μl of normal human urine.