Development of a UPLC–MS/MS method for determining γ-hydroxybutyric acid (GHB) and GHB glucuronide concentrations in hair and application to forensic cases

We present a series of forensic cases measuring concentrations in hair of γ-hydroxybutyric acid (GHB) and its glucuronide. The compounds were extracted from hair by incubation for 1.5 h in a 25:25:50 (v/v/v) mixture of methanol/acetonitrile/2 mM ammonium formate (8 % acetonitrile, pH 5.3). The compounds were determined by ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS). The limits of quantitation (LOQ) were 0.32 and 0.48 ng/mg hair for GHB and its glucuronide, respectively, and linearity up to 50 ng/mg. Extraction recoveries were 62–92 % for the analytes, and the accuracy was 90–108 %. Precision values expressed as relative standard deviations (%) obtained from daily controls were 9.1–11.3 % for GHB and 12.2–17.9 % for GHB glucuronide. Endogenous levels were up to 1.0 ng/mg for GHB and 1.2 ng/mg for GHB glucuronide in control subjects. In two GHB abusers, GHB was highly elevated (461–591 ng/mg), whereas GHB glucuronide was only slightly elevated in one of the subjects (1.7–3.1 ng/mg) and below the LOQ in the other. Thus, GHB glucuronide is not incorporated in hair in any appreciable amounts in GHB abusers and does not contribute diagnostically with regard to detection of exogenous exposure. To our knowledge, this is the first report to present GHB glucuronide in human hair.