Development and validation of a HPLC–QTOF-MS method for the determination of GHB-β-O-glucuronide and GHB-4-sulfate in plasma and urine

Gamma-hydroxybutyric acid (GHB) is used as a so-called knock-out drug in drug-facilitated sexual assault. Due to GHB’s short detection window in blood and urine, crime verification remains problematic in many cases. Phase II metabolites can enhance the detectability and prolong the detection window of some lower-molecular-mass molecules, as exemplified by ethanol. GHB-β-O-glucuronide (GHB-Gluc) was recently introduced as one phase II metabolite, and GHB-4-sulfate (GHB-Sulf) was also postulated to represent a urinary metabolite of exogenous GHB. To unambiguously verify the presence of GHB-Sulf in urine, the reference standard material of GHB-Sulf was synthesized, together with its deuterated analogue, GHB-Sulf-d ₆. This reference standard material, together with GHB-Gluc and GHB-Gluc-d ₄, was employed to develop and validate a quantitative method for determining both target analytes in plasma and urine samples. Compounds were separated by high-performance liquid chromatography on a Hypercarb column and detected by electrospray ionization time-of-flight-mass spectrometry. Calibration curves were linear over the entire calibration range up to 20 mg/L, and quantification limits of <0.01 mg/L were achieved. Accuracy and interday and intraday precision fulfilled all forensic guidelines criteria. In plasma, only GHB-Gluc was detected, while both metabolites were clearly identified in urine, even in real human specimens. Stability was investigated under different temperature conditions for up to 4 weeks, and both phase II metabolites were found to be stable under refrigerated conditions. This method proved to be suitable for this purpose, and allowed for the first time the analysis and quantification of two different GHB metabolites from plasma and urine samples.