Conversion of γ-hydroxybutyric acid to a fluorescent derivative: a method for screening

Abstract

Conversion of γ-hydroxybutyric acid (GHB) to a fluorescent derivative using 3-bromomethyl-6,7-dimethoxy-1-methyl-1,2-dihydroquinoxaline-2-one (Br-DMEQ), and its application to drug screening were studied. Br-DMEQ reacted with the carboxyl group of sodium GHB in the presence of a potassium salt and crown ether to produce a fluorescent derivative, which could be easily detected by thin-layer chromatography (TLC). An electrospray ionization mass spectrum of the fluorogenic product supported the expected structure. The Br-DMEQ-derivatized GHB gave an Rf value of 0.49 on TLC, which was easily distinguished from 19 other carboxylic acids; all of the latter had Rf values over 0.61. Various sodium carboxylates including sodium GHB reacted with Br-DMEQ in the presence of KCl, but the free forms of the carboxylic acids did not react under these conditions. When aqueous solution containing GHB was pretreated with sulfuric acid, GHB was converted to γ-butyrolactone, resulting in removal of its reactivity with Br-DMEQ. By such analysis following the derivatization, with and without the above pre-treatment with sulfuric acid, GHB added to human urine could be specifically detected, although the limit of detection was about 100μg/ml, which was 20 times higher than the endogenous blood GHB level. Therefore, the present method seems useful for screening GHB present in a solid state such as powder and tablets, and also in urine samples obtained in overdose cases.