A new method for the HPLC determination of gamma-hydroxybutyric acid (GHB) following derivatization with a coumarin analogue and fluorescence detection: Application in the analysis of biological fluids

Abstract

A new method of the determination of gamma-hydroxybutyric acid (GHB) in human biological fluids cerebrospinal fluid (CSF) and saliva after off-line derivatization is described. The proposed method was based on the reaction of 4-bromomethyl-7-methoxy coumarin (Br-MMC) with GHB in the presence of dibenzo-18-crown-6-ether (acting as reaction catalyst) to produce a fluorescent derivative. The formed derivative was monitored fluorimetrically at λ ext. = 330 nm and λ em. = 390 nm. The effect of derivatization parameters such as the concentration of Br-MMC, reaction time and the temperature was investigated in order to achieve the maximum method's sensitivity. The separation was achieved by use of a C 18 analytical column (Kromasil ® 250 mm × 4 mm i.d., 5 μm) while the injected sample volume was set to 25 μL. A binary gradient elution program of methanol versus phosphate buffer (40 mM, pH 3) was selected for the quantitative analysis of GHB. The method showed satisfactory linearity ( R 2 = 0.9979) in a linear range from 2.4 × 10 −6 to 7.2 × 10 −5 M. Ultrafiltration method was employed for the pre-treatment of the cerebrospinal fluid (CSF) prior to the analysis of GHB. The limit of detection (LOD) of the method was 3 × 10 −7 M in saliva and 2 × 10 −7 M in CSF samples, respectively, while the limit of quantitation (LOQ) was 1 × 10 −6 M for both specimens. The proposed protocol offers sensitivity comparing with the existing HPLC analytical methods or the CE indirect UV methods and can function as an attractive alternative to be used in clinical and toxicological analysis.