Sørensen Lambert K, Faldborg Kathrine B, Andersen Charlotte U, Hasselstrøm Jørgen B
Section for Forensic Chemistry, Department of Forensic Medicine, Aarhus University, Palle Juul-Jensens Boulevard 99, Aarhus N 8200, Denmark.
Section for Forensic Chemistry, Department of Forensic Medicine, Aarhus University, Palle Juul-Jensens Boulevard 99, Aarhus N 8200, Denmark.
Forensic Sci Int. 2024 Dec;365:112286. doi: 10.1016/j.forsciint.2024.112286. Epub 2024 Nov 1.
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.
检测γ-羟基丁酸（GHB）摄入存在两个挑战，即其内源性存在以及采样后的体外生成。本研究开发了一种液相色谱-串联质谱（LC-MS/MS）方法，用于测定人死后血液、尿液和口腔液中内源性浓度的GHB。此外，还研究了GHB在血样中的稳定性及其在受控情况下采集的样本中的内源性浓度。样本用甲醇/乙腈萃取，并通过阴离子交换固相萃取进行处理。使用具有阴离子特性的反相液相色谱柱将GHB与结构异构体分离。验证后的定量限在血液中为0.005μg/mL，在尿液和口腔液中为0.010μg/mL，此时相对重现性标准偏差和偏差<15%。平均萃取回收率≥90%。在4°C储存28天后，氟化物/柠檬酸盐保存的血液中GHB平均浓度增加了1.2μg/mL；然而，在氟化物/草酸盐（FX）保存的血液中，平均浓度仅增加了0.055μg/mL。在-20°C时未观察到变化。在为法医生前毒理学分析采集的105份随机选择的FX保存血液样本中，即使在-20°C长期储存后，所有浓度均<0.066μg/mL。在一项GHB摄入的临床研究的血液、尿液和口腔液样本中，30名参与者的内源性基线水平分别为0.0069 - 0.050、0.024 - 0.38和0.034 - 0.93μg/mL。这些结果表明，对于在-20°C储存的FX保存血液，目前用于区分生前血液中内源性和外源性GHB的5μg/mL的截断水平可能会低得多。
