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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图 1 GHB、GHB-d6、1,4-BD和GBL 4种混标（1 µg/mL）的TIC色谱峰图
Figure 1. TIC chromatograms of GHB，GHB-d6，1,4-BD，and GBL mixed standards （1 µg/mL）
图 2 GHB、GHB-d6、1,4-BD、GBL 4种混标（1 µg/mL）的d-MRM色谱图
Figure 2. D-MRM chromatograms of GHB，GHB-d6，1,4-BD，and GBL mixed standards （1 µg/mL）
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 
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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。
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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
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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
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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
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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
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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
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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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