Isolation of 2-hydroxycarboxylic acids with a boronate affinity gel

Boric acid has been known to make a complex with 2-hydroxycarboxylic acids. Based on this principle, we have developed a new method for isolation of vanillylmandelic acid, p-hydroxymandelic acid, vanillyllactic acid, and p-hydroxyphenyllactic acid in urine. The technique involves two steps: extraction of lipophilic compounds from urine with n-butanol and selective isolation of 2-hydroxy acids in the n-butanol phase on a phenylboronate gel column. 2-Hydroxy acids were eluted from the column with 1.5 ml of 2% acetyl chloride in methanol with the recoveries of ca. 70%. According to analysis of urinary extract by gas chromatography--mass spectrometry, the mean excretion rates of the above four compounds in 12 healthy subjects were 3.68, 1.93, 0.091, and 1.08 mg/day, respectively.