Determination of endogenous GHB in ante-mortem whole blood, urine, and oral fluid by LC-MS/MS: The effect of different additives and storage conditions on the stability of GHB in blood.

Two challenges in detecting γ-hydroxybutyric acid (GHB) intake are its endogenous presence and in vitro production after sampling. This study developed an LC-MS/MS method for selective GHB determination in human antemortem blood, urine, and oral fluid at endogenous concentrations. Furthermore, the stability of GHB in blood samples and its endogenous concentrations in samples taken under controlled circumstances were investigated. Samples were extracted in methanol/acetonitrile and processed by anion exchange solid-phase extraction. GHB was separated from structural isomers using a reversed-phase LC column with anion properties. The validated limit of quantification was 0.005 µg/mL in blood and 0.010 µg/mL in urine and oral fluid, at which the relative reproducibility standard deviation and bias were <15 %. The mean extraction recovery was ≥90 %. The average GHB concentration increased by 1.2 µg/mL in fluoride/citrate- preserved blood after 28 days of storage at 4°C; however, in fluoride/oxalate (FX)-preserved blood, the mean concentration increased by only 0.055 µg/mL. No change was observed at -20°C. In 105 randomly selected samples of FX-preserved blood collected for forensic antemortem toxicological analysis, all concentrations were <0.066 µg/mL, even after long-term storage at -20°C. In blood, urine, and oral fluid samples from a clinical study of GHB intake, endogenous baseline levels from 30 participants ranged from 0.0069-0.050, 0.024-0.38, and 0.034-0.93 µg/mL, respectively. These results demonstrate that the current cut-off level of 5 µg/mL for discriminating between endogenous and exogenous GHB in antemortem blood could be considerably lower for FX-preserved blood stored at -20°C.