Objectives: To find a method to distinguish exogenous gamma-hydroxybutyrate (GHB) from endogenous GHB by establishing ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) based on exosome for quantitative detection of GHB in the rat blood.
Methods: Adult male SD rats were divided into 1 h, 5 h, 10 h administration group and control group. After 1 h, 5 h and 10 h of single precursor of GHB gamma-butyrolactone (GBL) intraperitoneal injection in administration groups, 5 mL blood was collected from the abdominal aorta. Meanwhile, the control group was given a same dose of normal saline, and 5 mL blood was collected at 1 h. Among the 5 mL blood, 0.5 mL was directly detected by HPLC-MS after pretreatment, and exosomes were extracted from the remaining blood by differential centrifugation and detected.
Results: The concentration of GHB in the control group was (87.36±33.48) ng/mL, and the concentration with administration at 1 h, 5 h and 10 h was (110 400.00±1 766.35) ng/mL, (1 479.00±687.01) ng/mL and (133.60±12.17) ng/mL, respectively. The results of exosome detection showed that no peak GHB signal was detected in the control group and the 10 h administration group, and the concentrations of GHB at 1 h and 5 h administration groups were (91.47±33.44) ng/mL and (49.43±7.05) ng/mL, respectively.
Conclusions: GHB was detected in blood exosome by UPLC-MS, which indicated that exogenous GHB could be detected in plasma exosomes, while endogenous GHB could not be detected, suggesting that this method may be used as a basis to determine whether there is exogenous drug intake.
目的: 建立基于外泌体的超高效液相色谱-质谱法（ultra-high performance liquid chromatography-mass spectrometry，UPLC-MS）定量检测大鼠血液中的γ-羟基丁酸（gamma-hydroxybutyrate，GHB），寻找区分内源性GHB和外源性GHB的方法。方法: 将成年雄性SD大鼠分为给药1 h、5 h、10 h组与对照组，给药组单次腹腔注射GHB前体物质γ-丁内酯（gamma-butyrolactone，GBL）后，分别于注射后1 h、5 h、10 h腹主动脉采血5 mL，对照组给予等量生理盐水后1 h采血5 mL。其中0.5 mL血液经前处理后直接进行UPLC-MS检测，剩余血液经差速超速离心提取外泌体后进行检测。结果: 对照组大鼠血液中GHB质量浓度为（87.36±33.48） ng/mL，给药1 h、5 h和10 h组大鼠血液中GHB质量浓度分别为（110 400.00±1 766.35）、（1 479.00±687.01）和（133.60±12.17） ng/mL。外泌体检测结果显示，对照组及给药10 h组均未检测到GHB峰值信号，给药1 h和5 h组GHB质量浓度分别为（91.47±33.44）和（49.43±7.05） ng/mL。结论: 通过提取大鼠血液外泌体后经UPLC-MS法检测GHB，证实外源性GHB可在血液外泌体中检出，而内源性GHB则不能检出，提示该方法可能作为判断是否有外源性药物摄入的依据。.
Keywords: blood; exosome; forensic pathology; gamma-butyrolactone (GBL); gamma-hydroxybutyrate (GHB); rats; ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS).