Researches on the Effect of Alcohol on Endogenous GHB in Human Blood by LC-MS/MS Method
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摘要:
目的 运用LC-MS/MS法研究酒精对人体血液中内源性GHB含量的影响。 方法 建立血液中γ-羟基丁酸(GHB)、γ-丁内酯(GBL)和1,4-丁二醇(1,4-BD)的LC-MS/MS法,以22名健康受试者为研究对象,模拟真实案例饮酒,测定研究对象饮酒前、饮酒7 h后血液中内源性GHB的含量,比较饮酒前后血液中内源性GHB的含量。 结果 GHB在0.1~50 µg/mL浓度范围、GBL和1,4-BD在0.15~50 µg/mL浓度范围内,线性关系良好(R2 > 0.999),GHB、GBL和1,4-BD的检出限分别为0.015 µg/mL,0.025 µg/mL和0.01 µg/mL,定量限分别为0.1µg/mL,0.15 µg/mL和0.15 µg/mL。3种目标物的基质效应在18.2%~20.1%之间,回收率为81.6 %~92.3%,日内和日间精密度RSD均小于10%,准确度为-5.8%~7.0%。饮酒7 h后血液中内源性GHB浓度范围为0.71~3.81 µg/mL,相比饮酒前GHB含量升高,差异有统计学意义(P < 0.05)。 结论 饮酒7 h后人体血液中内源性GHB含量增高,但浓度低于4 µg/mL,为真实案例涉及的内外源性GHB的判定提供数据支持,丰富了GHB的法医毒物学数据。 Abstract:Objective Using LC-MS/MS method to study the effect of alcohol on endogenous GHB content in human blood . Methods To establish LC-MS/MS inspection method in the blood for γ- Hydroxybutyric acid (GHB), γ- butyrolactone (GBL) and 1,4-butanediol (1,4-BD). Taking 22 healthy subjects were selected as the research subjects and simulating real cases of drinking alcohol. Detecting the levels of GHB in their blood before and after drinking , and to compare the endogenous GHB levels in the blood before and after drinking. Results GHB in the concentration range of 0.1~50 μg/mL has a good linear relationship and there was a good linear relationship between GBL and 1,4-BD in the concentration range of 0.15~50 μg/mL (R2 > 0.999), The limits of detection of GHB, GBL and 1,4-BD were 0.015 μg/mL, 0.025 μg/mL and 0.01 μg/mL, respectively, and the limits of GHB, GBL and 1,4-BD quantification were 0.1 μg/mL, 0.15 μg/mL and 0.15 μg/mL. The matrix effects of the three compounds ranged from 18.2% to 20.1%, the recoveries were 81.6% to 92.3%, the intra-day and inter-day precision of RSD were less than 10%, and the accuracy was -5.8%-7.0%. The content of GHB in the blood after 7 hours of drinking were higher than that before drinking alcohol (P < 0.05), and the concentration range was 0.71-3.81 μg/mL. Conclusion The content of endogenous GHB in human blood has increased after 7 hours of drinking alcohol, but the concentration was lower than 4 μg/mL, which provided data support for the assessment of endogenous and exogenous GHB involved in real cases, enriching the forensic toxicology data of GHB . -
Key words:
- γ-hydroxybutyric acid /
- γ-Butyrolactone /
- 1,4-Butanediol /
- Alcohol /
- LC-MS/MS method
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表 1 HPLC梯度洗脱条件
Table 1. HPLC gradient elution conditions
时间/min 流动相A(%) 流动相B(%) 0.00 90 10 0.5 90 10 2.5 10 90 3.0 10 90 3.1 90 10 6.0 90 10 表 2 GHB、GHB-d6、1,4-BD、GBL的d-MRM离子、碎裂电压及碰撞能
Table 2. The acquisition parameters ,fragmentation voltage and collision energy of d-MRM ,GHB,GHB-d6,1,4-BD,GBL
目标物 扫描模式 母离子(m/z) 子离子(m/z) 碎裂电压(v) 碰撞能(ev) GHB ESI- 103.0 103.0/85.1* 65 8 103.0/57.1 12 GHB-d6 ESI- 109.0 109.0/61.1* 60 8 109.0/90.0 16 1,4-BD ESI+ 91.1 91.1/73.0* 30 4 91.1/55.0 12 GBL ESI+ 87.0 87.0/45* 66 20 87.0/43 24 注:*为定量离子。 表 3 血液中GHB、1,4-BD、GBL的校准曲线、检出限和定量限
Table 3. Calibration curve ,detection limit and quantitation limit of GHB 1,4-BD GBL in blood
化合物 线性范围(µg/mL) 线性回归方程 R2 检出限(µg/mL) 定量限(µg/mL) GHB 0.1~50 У=22.288925x-0.955609 0.9993 0.015 0.1 1,4BD 0.15~50 У=151049.912950x+57464.367658 0.9992 0.01 0.15 GBL У=16316.378144x+105811.540171 0.9995 0.025 0.15 表 4 3种目标物的基质效应与提取回收率 (%)
Table 4. Matrix effects and extraction recovery rates of three target substances(%)
化合物
名称浓度 基质
效应相对标准
偏差RSD提取
回收率相对标准
偏差RSD1 20.0 4.6 81.9 6.3 GHB 5 20.1 3.8 86.2 4.2 25 19.8 6.1 92.3 4.8 1 18.2 4.6 81.6 6.4 1,4-BD 5 18.4 3.9 87.4 4.5 25 19.0 2.3 89.3 6.7 1 19.2 6.2 83.7 4.4 GBL 5 18.9 1.3 85.6 3.2 25 18.2 5.1 84.5 4.6 表 5 3种目标物的精密度及准确度
Table 5. Precision and accuracy of 3 target substances
目标物 添加浓度(µg/mL) 日内精密度 日间精密度 平均检测浓度
(µg/mL)相对标准偏差
RSD (%)偏倚值
(%)平均检测浓度
(µg/mL)相对标准偏差
RSD (%)偏倚值
(%)1 0.96 5.4 −4.0 1.02 4.8 −2.0 GHB 5 5.04 6.4 0.8 4.93 9.5 1.4 25 24.83 7.7 −0.68 24.92 3.8 −0.32 1 0.95 4.3 −5.0 0.98 7.4 −2.0 1,4-BD 5 4.71 2.2 −5.8 4.93 7.3 −1.4 25 24.87 2.6 −0.52 24.96 7.3 −0.16 1 1.07 8.8 7.0 0.98 9.8 −2.0 GBL 5 5.01 4.6 0.2 5.03 5.6 0.6 25 24.98 1.2 −0.08 25.02 1.4 −0.08 表 6 饮酒前和饮酒7 h后血液中GHB和酒精含量检测结果(µg/mL)
Table 6. GHB and alcohol content in blood before and 7 hours after drinking alcohol (µg/mL)
志愿者 性别 实验分组 饮酒量(mL) 饮酒前 饮酒7 h后 GHB
(µg/mL)GHB量
(µg/mL)1 女 低剂量组 1000 0.63 0.84 2 女 0.66 0.81 3 女 0.84 1.22 4 男 0.34 0.71 5 男 1.69 2.02 6 男 0.86 0.99 7 男 0.71 0.77 8 女 中剂量组 2000 0.28 1.26 9 女 0.76 1.58 10 女 0.49 1.34 11 女 1.29 1.71 12 男 1.52 3.81 13 男 1.14 3.14 14 男 1.50 1.87 15 男 0.96 2.86 16 女 高剂量组 4000 0.71 0.86 17 女 1.22 1.67 18 女 0.62 0.87 19 女 1.86 2.26 20 男 1.68 1.88 21 男 1.49 2.36 22 男 1.11 2.80 表 7 饮酒前和饮酒7 h后血液中内源性GHB含量的情况[($ \bar x \pm s $),µg/mL]
Table 7. The content of endogenous GHB levels in the blood before and 7 hours after drinking [($ \bar x \pm s $),µg/mL]
组别 n(例) 饮酒前 饮酒7 h后 饮酒前后差值 t P 实验组 22 1.02±0.47 1.71±0.87 0.69±0.40 −4.840 0.000* *P < 0.05 表 8 案例血液样品中GHB(μg/mL)、酒精(mg/100 mL)的含量
Table 8. Contents of GHB (μg/mL)and alcohol (mg/100 mL)in case blood samples
样品名称 目标物 检测结果 锁骨下静脉血 GHB 10 锁骨下静脉血 酒精 96 -
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