Carbon isotopic ratio analysis by gas chromatography/ combustion/isotope ratio mass spectrometry for the detection of gamma-hydroxybutyric acid (GHB) administration to humans Christophe Saudan 1 * , Marc Augsburger 2 , Patrice Mangin 1,2 and Martial Saugy 1 1 Laboratoire Suisse d’Analyse du Dopage, Institut Universitaire de Me ´decine Le ´gale, Centre Hospitalier Universitaire Vaudois et Universite ´ de Lausanne, Chemin des Croisettes 22, 1066 Epalinges, Switzerland 2 Laboratoire de Toxicologie et de Chimie Forensiques, Institut Universitaire de Me ´decine Le ´gale, Centre Hospitalier Universitaire Vaudois et Universite ´ de Lausanne, Rue du Bugnon 21, 1005 Lausanne, Switzerland Received 30 July 2007; Revised 26 September 2007; Accepted 3 October 2007 Since GHB (gamma-hydroxybutyric acid) is naturally produced in the human body, clinical and forensic toxicologists must be able to discriminate between endogenous levels and a concentration resulting from exposure. To suggest an alternative to the use of interpretative concentration cut-offs, the detection of exogenous GHB in urine specimens was investigated by means of gas chromatog- raphy/combustion/isotope ratio mass spectrometry (GC/C/IRMS). GHB was isolated from urinary matrix by successive purification on Oasis MCX and Bond Elute SAX solid-phase extraction (SPE) cartridges prior to high-performance liquid chromatography (HPLC) fractioning using an Atlantis dC18 column eluted with a mixture of formic acid and methanol. Subsequent intramolecular esterification of GHB leading to the formation of g -butyrolactone (GBL) was carried out to avoid introduction of additional carbon atoms for carbon isotopic ratio analysis. A precision of 0.3% was determined using this IRMS method for samples at GHB concentrations of 10 mg/L. The 13 C/ 12 C ratios of GHB in samples of subjects exposed to the drug ranged from S32.1 to S42.1%, whereas the results obtained for samples containing GHB of endogenous origin at concentration levels less than 10 mg/L were in the range S23.5 to S27.0%. Therefore, these preliminary results show that a possible discrimination between endogenous and exogenous GHB can be made using carbon isotopic ratio analyses. Copyright # 2007 John Wiley & Sons, Ltd. GHB (gamma-hydroxybutyric acid) is a metabolite of g -aminobutyric acid (GABA), the primary inhibitory neuro- transmitter in the central nervous system. 1 Since the 1960s GHB has been used medically for the induction of anesthesia, treatment of narcolepsy, and for alcohol and opiate with- drawal. In addition to its therapeutic effects, this drug has also gained popularity amongst ravers or club attendees for its intoxicating effects such as euphoria, reduced inhibition, sedation and muscle relaxation. Moreover, its ease of administration in spiked drinks and the potential amnesia have resulted in the use of GHB as a sexual assault related drug. In both clinical and forensic investigations on biological tissues, it is usually required to determine if any GHB detec- ted is due to endogenous production or exogenous ingestion. There have been increasing data suggesting that endogenous GHB concentrations are less than 10 mg/L in ante-mortem urine samples, except for specimens of individuals suffering from 4-hydroxybutyric aciduria. 2,3 The presence of GHB in post-mortem fluid has also been evaluated, although the available data have been subject to controversy. 4,5 Very recently, interpretative cut-offs based on GHB concentrations measured in cases unrelated to GHB ingestion have been proposed for both post-mortem blood and urine. 6 It has been estimated that any GHB detected is endogenous for urine specimens with a concentration less than 20 mg/L. The rapid rise in the amount of GHB in post-mortem fluid is attributed to the reduction in Krebs cycle activity 1 as well as to a possible role of microorganisms. 7 We propose here a method for determination of absolute carbon isotopic ratio 13 C/ 12 C(d 13 C values) of GHB in urine samples using an extensive clean-up procedure followed by conversion of GHB into GBL (gamma-butyrolactone) prior to analysis by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). To demonstrate the potential of this method to distinguish between endogenous and exogenous sources, we investigated the carbon isotope RAPID COMMUNICATIONS IN MASS SPECTROMETRY Rapid Commun. Mass Spectrom. 2007; 21: 3956–3962 Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/rcm.3298 *Correspondence to: C. Saudan, Laboratoire Suisse d’Analyse du Dopage, Institut Universitaire de Me ´decine Le ´gale, Chemin des Croisettes 22, 1066 Epalinges, Switzerland. E-mail: christophe.saudan@chuv.ch Copyright # 2007 John Wiley & Sons, Ltd.