Aims: Because of its short detection window, uncovering the intake of gammahydroxybutyric
acid (GHB) still constitutes a problem. Aim of the experiments was to
develop and evaluate new tools for a possible extension of the detection window after the
intake of GHB. Methods: Blood, plasma and urine samples (each n=49) of volunteers and of
patients (n=3, patient 1 and 2 chronical intake, patient 3 single intake) therapeutically taking
up to 4.5 g GHB (Xyrem®) per night were collected at different points in time after the intake
up to 72 h. Additionally, hair samples of the patients were taken. Concentration profiles of
GHB (high-performance liquid chromatography-tandem mass spectrometry), GHB-β-
glucuronide and GHB-4-sulfate (high-performance liquid chromatography-quadrupole timeof-
flight-mass spectrometry) in plasma and urine were recorded over time. Hair samples were
analyzed by a validated LC-MS/MS method for GHB and GHB-β-glucuronide. Alterations in
gene expression of ALDH5A1, AKR7A2, EREG and PEA15 in blood, genes of interest which
code for enzymes involved in GHB metabolism, was investigated via quantitative PCR using
an empirically derived normalization strategy. Furthermore, possible discrimination of
endogenous from exogenous GHB in urine via isotope ratio MS was tested. Results and
discussion: The parent compound could be quantified above the usual cut-offs for 4-6 hours
both in blood and urine. No discrimination endogenous/exogenous by neither using the phase
II metabolites of GHB nor using the expression of the genes of interest was possible. In the
hair samples of patients GHB and its glucuronide could not be determined in concentrations
higher than the control group. A discrimination endogenous/exogenous GHB was only
possible using isotope ratio mass spectrometry, however carbon isotope ratios in urine did not
differ longer than GHB was detectable above the cut-off limit. Conclusion: Therefore, these
methods do not seem to be able to extend the detection window of exogenous GHB.