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 To study the effect of alcohol on endogenous GHB content in human blood. Methods LC-MS/MS inspection method was established in the blood for γ- Hydroxybutyric acid （GHB）, γ- butyrolactone （GBL） and 1,4-butanediol （1,4-BD）. 22 healthy subjects were selected as the research subjects to simulate the real cases of drinking alcohol. The levels of GHB in their blood were detected before and after drinking 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 had 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 can provide the 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 0.15～50 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
（µg/mL）基质
效应相对标准
偏差RSD提取
回收率相对标准
偏差RSDGHB 1 20.0 4.6 81.9 6.3 5 20.1 3.8 86.2 4.2 25 19.8 6.1 92.3 4.8 1,4-BD 1 18.2 4.6 81.6 6.4 5 18.4 3.9 87.4 4.5 25 19.0 2.3 89.3 6.7 GBL 1 19.2 6.2 83.7 4.4 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
（%）GHB 1 0.96 5.4 −4.0 1.02 4.8 −2.0 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 1,4-BD 1 0.95 4.3 −5.0 0.98 7.4 −2.0 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 GBL 1 1.07 8.8 7.0 0.98 9.8 −2.0 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 GHB量 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 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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