Determination of endogenous/exogenous γ-hydroxybutyric acid (GHB) in urine

Abstract

γ-Hydroxybutyric acid (GHB) is an endogenous metabolite and a precursor of the neurotransmitter γ-aminobutyric acid. In some European countries GHB is also used as an anaesthetic agent and in the treatment of alcohol withdrawal. The purpose of the present study was to determine GHB concentrations in urine to enable discrimination between endogenous GHB and exogenous GHB derived from therapeutic administration (Alcover®, Laboratorio farmaceutico C.T.Srl, Sanremo). 34 urine samples from 17 alcoholics under treatment for alcohol withdrawal with GHB (group 1) and 12 urine samples from healthy volunteers without GHB intake (group 2) were collected anonymously and analysed by HPLC/MS/MS. The urine concentrations of GHB were determined from two standard curves with the following concentrations: 0.25, 0.50, 1.00, 2.50,5.00 and 10.00 μg/ml, and 5.00, 10.00, 25.00, 50.00 and 100.00 μg/ml. Acceptable linear regression was obtained for both calibration curves: R2 = 0.9950 and R2 = 0.9994, respectively. Intraday precision and accuracy were calculated as coefficient of variation (%) and bias (n = 10) on three positive control specimens at three different concentrations (0.50, 2.50 and 7.50 μg/ml). The limit of quantification was determined to be 0.50 μg/ml. The median and average concentrations of GHB in group 2 (no GHB intake) were 3.55 and 3.49 μg/ml, respectively, while 52.90% of group 1 (subjects treated with GHB) had urinary GHB concentrations less than the currently used cutoff of 10.00 μg/ml. In the absence of a rapid immunoassay for the detection of GHB in urine, LC/MS/MS analysis was found to be specific and reasonably fast for clinical and forensic toxicology applications. Of the subjects being treated with GHB, 52.90% were found to have a urinary GHB concentration less than 10.00 μg/ml, with GHB concentrations ranging from 2.95 to 8.50 μg/ml. Thus the widely accepted 10.00 μg/ml cut-off could be too high so that there is the risk of false-negative samples.