In vitro production of gamma-hydroxybutyrate in antemortem blood under various storage conditions

gamma-Hydroxybutyrate (GHB) is an endogenous compound and central nervous system depressant drug used recreationally for its intoxicating effects. In a medico-legal context, the interpretation of blood GHB concentrations can be complicated by its endogenous nature and potential for formation during storage. In Canada, the per se limit for GHB in blood is 5 mg/L. Endogenous GHB concentrations in blood are typically well below 5 mg/L; however, there is a paucity of literature regarding the potential production of GHB in antemortem blood during storage. Changes in GHB concentrations were evaluated over the course of 306 days in preserved and unpreserved antemortem blood stored at 4°C and 21°C. Results were compared to 22 impaired driving cases in Ontario between 2019 and 2022 where GHB was detected in antemortem blood by toxicological analysis at the Centre of Forensic Sciences. Preservative was effective at minimizing GHB production (< 2.5 mg/L) regardless of storage temperature, whereas significant in vitro production of GHB occurred in unpreserved antemortem blood. GHB production occurred rapidly in unpreserved blood stored at 21°C; a significant increase was detected after 5 days. The rate of GHB production in unpreserved blood stored at 4°C occurred more gradually but increased significantly by day 30 and reached a maximum concentration of 10 mg/L at 114 days. In unpreserved blood, GHB concentrations were significantly lower at 4°C compared to 21°C for the first 44 days; however, refrigeration had no significant effect then onward. Blood concentrations of GHB detected in the majority of impaired driving cases were markedly higher than the maximum concentration of 10 mg/L detected in the study; however, in 4 of the 22 cases, concentrations were below 10 mg/L. The results demonstrate that concentrations of GHB less than 10 mg/L in blood collected for the purposes of a drug-impaired driving investigation should be carefully interpreted.