WO2023179228A1 - 一种推算饮酒时间的方法 - Google Patents

一种推算饮酒时间的方法 Download PDF

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WO2023179228A1
WO2023179228A1 PCT/CN2023/075150 CN2023075150W WO2023179228A1 WO 2023179228 A1 WO2023179228 A1 WO 2023179228A1 CN 2023075150 W CN2023075150 W CN 2023075150W WO 2023179228 A1 WO2023179228 A1 WO 2023179228A1
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etg
ets
drinking
alcohol
mobile phase
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贠克明
王乐乐
张伟
王瑞龙
光永立
张大明
张潮
胡萌
尉志文
张文芳
郭中元
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山西医科大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/98—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving alcohol, e.g. ethanol in breath
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
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    • G—PHYSICS
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    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
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    • G01N2030/067—Preparation by reaction, e.g. derivatising the sample
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/86—Signal analysis
    • G01N30/8603—Signal analysis with integration or differentiation
    • G01N2030/862—Other mathematical operations for data preprocessing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
    • G01N2030/8822—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving blood

Definitions

  • the invention relates to the technical fields of analytical chemistry and forensic identification, and in particular to a method for estimating drinking time.
  • Alcohol as a psychoactive substance with dependence properties, has been widely abused around the world. Due to the chronic excessive drinking of alcohol among the general population, the number of alcohol-related accidents has been increasing, such as violence caused by alcohol abuse, traffic accidents, etc. Therefore, the technical identification of alcohol is still one of the most commonly encountered tasks in the identification work of public prosecutors and judicial officers in our country.
  • the metabolic process of alcohol in the body is mainly completed through the oxidation reaction (90-92%) of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH).
  • ADH alcohol dehydrogenase
  • ADH aldehyde dehydrogenase
  • a small amount of alcohol ( ⁇ 1%) undergoes non-oxidative metabolism under the action of different enzymes and directly combines with other substances to generate non-oxidative metabolites.
  • ethyl glucuronide Etthyl-bD-6-glucuronide, EtG
  • UDP-glucuronyltransferase UDP-glucuronyltransferase
  • alcohol and glucuronic acid combined.
  • EtG and EtS are the two main non-oxidation products of alcohol metabolism. Although the content is small, the detection window is long. Even if alcohol has been completely metabolized, the presence of EtG and EtS can be detected in a variety of body fluids or tissues. Therefore, EtG and EtS are expected to be sensitive and specific biomarkers for identifying alcohol intake.
  • Drinking time is an important clue in the analysis process of alcohol-related cases and is of great significance in assessing the nature of the case. Therefore, the estimation of drinking time is also one of the more common contents in the identification of alcohol-related cases.
  • ratio calculations eliminate dose effects.
  • research on inferring medication time based on the time-varying pattern of the concentration ratio of the original substance and metabolite, or metabolite and metabolite has been emerging.
  • some studies have shown that alcohol is metabolized rapidly in the living body, and it is difficult to detect the presence of alcohol 8 hours after drinking, and the alcohol concentration after death may change due to postmortem redistribution and postmortem production.
  • the accuracy of alcohol concentration testing will only be recognized within 24 hours of death and when the temperature is below 20°C. Therefore, the application of the concentration ratio of alcohol proto-metabolites has great limitations, and a more reliable method is needed to infer drinking time.
  • EtG and EtS as non-oxidative metabolites of alcohol, not only have higher concentrations and longer detection windows, so that even if the alcohol itself cannot be detected after alcohol ingestion, the detection of EtG and EtS can still be used to make a preliminary judgment on the case.
  • relevant literature has proven that EtG and EtS are not produced after death and are relatively stable under low temperature conditions. Therefore, the concentration ratio of EtG to EtS can be considered to estimate the time of last drinking.
  • the purpose of the present invention is to provide a method for estimating drinking time, using the change pattern of the concentration ratio between alcohol non-oxidative metabolites with time to infer the drinking time, so as to avoid the inevitable external interference in the traditional method. question.
  • a method for calculating drinking time includes the following steps:
  • x represents the average concentration ratio C EtG /C EtS
  • y represents the sampling time
  • the blood alcohol concentration after drinking is 0.22-0.66 mg/mL.
  • the amount of alcohol consumed by drinking alcohol is 0.72g/kg.
  • sampling times of the blood samples used in making the quadratic regression equation were 0h, 0.5h, 2h, 3h, 5h, 8h, 12h, 24h, 36h, 48h, and 120h respectively.
  • the separation conditions of liquid chromatography include the following parameters:
  • the chromatographic column adopts Inertsil ODS-3 column, 2.1mm ⁇ 100mm, 3 ⁇ m; the column temperature is 35°C;
  • the mobile phase A of the elution system is water-0.1% formic acid solution; the mobile phase B is acetonitrile-0.1% formic acid solution; the flow rate is 0.2mL/min; the gradient elution procedure is as follows:
  • the volume ratio of mobile phase A to mobile phase B is 95:5;
  • the volume ratio of mobile phase A to mobile phase B is 10:90;
  • the volume ratio of mobile phase A to mobile phase B is 10:90;
  • the volume ratio of mobile phase A to mobile phase B is 95:5.
  • the ratio of reagents here is based on volume ratio.
  • the detection conditions of mass spectrometry include the following parameters:
  • Electrospray ionization in negative mode was used; the ion spray voltage was -4000V and the temperature was 500°C.
  • the concentration of internal standard EtG-D 5 is 1 ⁇ g/mL; the concentration of internal standard EtS-D 5 is 1 ⁇ g/mL.
  • the method of the present invention mainly uses the change pattern of the average concentration ratio between alcohol non-oxidative metabolites with time to infer the drinking time, so as to avoid the inevitable external interference problem in the traditional method.
  • a good correlation model was obtained for the relationship between the average concentration ratio of ethyl glucuronate and ethyl sulfate and the time of alcohol use.
  • the average concentration ratio of EtG and EtS was substituted into the equation through the back-extraction method to calculate the theoretical value of drinking time.
  • the inference error was calculated using the formula "(theoretical value - measured value)/actual drinking time", and it was found that the error was basically less than 10%.
  • the present invention aims to establish a method for estimating the length of time after drinking by studying the pharmacokinetics of EtG and EtS in the blood after drinking, and also provides the pharmacokinetics of EtG and EtS in the Chinese population after oral administration of appropriate amounts. learning parameters.
  • the maximum concentration, maximum concentration, and elimination half-life of ethyl glucuronate in the blood are 4.12 ⁇ 1.07h, 0.31 ⁇ 0.11mg/L, and 2.56 ⁇ 0.89h respectively; the maximum concentration, maximum concentration, and elimination half-life of ethyl sulfate are 3.02 ⁇ 0.70 respectively. h, 0.17 ⁇ 0.04mg/L, 2.04 ⁇ 0.76h.
  • Figure 1 is a graph of the average concentration-time curves of alcohol, EtG, and EtS in blood.
  • Figure 2 is the LC-MS chromatogram (500ng/mL) of EtG and EtS and the internal standards EtG-D 5 and EtS-D 5 .
  • Figure 3 is an LC-MS chromatogram of a blank blood sample.
  • Figure 4 is an LC-MS chromatogram (500ng/mL) of blank blood samples added with EtG and EtS as well as internal standards EtG-D 5 and EtS-D 5 .
  • the drinking time mentioned in the present invention refers to the time from the start of drinking when sampling and testing.
  • the alcohol content in blood samples was determined using the headspace gas chromatography internal standard method, with tert-butyl alcohol as the internal standard. Add 1 mL of blood and 1 mL of tert-butyl alcohol (IS, 87 mg/mL) into the headspace bottle, dilute with 3 mL of ultrapure water, seal and mix, and then analyze by headspace gas chromatography.
  • the content of metabolites EtG and EtS in blood samples was determined by liquid chromatography tandem mass spectrometry (LC-MS/MS), with EtG-D 5 and EtS-D 5 as internal standards. Take 100 ⁇ L each of the internal standards EtG-D 5 (IS, 1 ⁇ g/mL) and EtS-D 5 (IS, 1 ⁇ g/mL) and mix them evenly to obtain a mixed internal standard; take 100 ⁇ L of blood and add 100 ⁇ L of the mixed internal standard to improve the Identification and quantification of metabolites (EtG and EtS). Then add 800 ⁇ L of 80% acetonitrile-methanol solution and precipitate at 0°C for 10 min.
  • LC-MS/MS liquid chromatography tandem mass spectrometry
  • Chromatographic separation was performed through the LC-20A system.
  • the chromatographic conditions are as follows:
  • Chromatographic column Inertsil ODS-3 column (2.1mm ⁇ 100mm, 3 ⁇ m; Shimadzu, Japan); The column temperature is 35°C.
  • Mobile phase mobile phase A (ultrapure water-0.1% formic acid) and mobile phase B (acetonitrile-0.1% formic acid); gradient elution (see Table 1); flow rate is 0.2mL/min; total elution time is 14.0min; The injection volume is 5 ⁇ L.
  • Detection of target compounds was performed by tandem mass spectrometer (TRAP4000, Sciex, AB). The specific conditions are as follows:
  • Ion source electrospray ion source (ESI).
  • the ion spray voltage is -4000V and the temperature is 500°C.
  • the curtain gas, atomizer (Gas1) and heated auxiliary gas (Gas2) are 40psi, 50psi and 35psi respectively.
  • the observed value represents the theoretical observation time, that is, the estimated time of the last drinking session
  • the actual value represents the actual time, that is, the actual sampling time since the latest drinking session.
  • DAS3.0 software was used to calculate pharmacokinetic parameters according to the non-compartmental model. All data were summarized using descriptive statistics. Some key data are provided, including the arithmetic mean and standard deviation of target concentration and detection time points, pharmacokinetic parameters and other results. All statistical analyzes were performed using IBM Software (SPSS Inc., Chicago, IL, USA) version 13.0 was performed.
  • the limits of detection (LOD) and limits of quantification (LOQ) of EtG and EtS in blood samples were 0.02 ⁇ g/mL and 0.05 ⁇ g/mL, respectively.
  • LOD limits of detection
  • LOQ limits of quantification
  • the present invention is mainly based on pharmacokinetic research and uses the changing pattern of the average concentration ratio between alcohol non-oxidative metabolites with time to infer the drinking time.
  • a regression equation was established based on the average concentration ratio of EtG and EtS in the blood and the drinking time.
  • the relationship between the average concentration ratio of EtG and EtS and the time of alcohol use has a good correlation.
  • the average concentration ratio of EtG and EtS is substituted into the equation to calculate the theoretical value of drinking time through the back deduction method.
  • "(theoretical value-actual measurement Value)/actual drinking time” formula was used to calculate the inference error, and it was found that the error was basically less than 10%.
  • the LOD and LOQ of alcohol non-oxidative metabolites (EtG and EtS) in blood samples are 0.02 ⁇ g/mL and 0.05 ⁇ g/mL respectively, which shows that the method of the present invention can effectively quantify lower concentrations of EtG and EtS in blood.
  • the BAC of 0.72g/kg alcohol dose is 0.22-0.66mg/mL, which is close to the existing blood alcohol concentration (BAC) standard for qualitative drunk driving (more than 0.2mg/mL), indicating that this The method of the embodiment of the invention is suitable for monitoring most drunk driving cases in China.
  • the present invention calculates the pharmacokinetic parameters of alcohol, EtG, and EtS in the blood based on the non-compartmental model and shows that alcohol reaches the peak concentration C max (441.65 ⁇ 113.86 mg/L (0.44 ⁇ 0.11 mg/mL) at 2.02 ⁇ 0.54 h. )), compared with previous studies, the absorption phase of the alcohol obtained in the embodiment of the present invention is longer, that is, the absorption is slower. Furthermore, we found that blood alcohol could be detected in participants' blood within 3-8 hours, and the mean elimination half-life of alcohol was 1.24 ⁇ 1.09h (0.30-4.23h).
  • the present invention calculates the pharmacokinetic parameters of alcohol, EtG, and EtS in the blood based on the non-compartmental model, which shows that the metabolites EtG and EtS have a longer detection window, and confirms that the metabolic rate of EtG is higher than that of EtG. Alcohol is slow, and the elimination half-life of EtG in the blood is 2.56 ⁇ 0.89h.
  • EtS is another non-oxidative metabolite of alcohol metabolism with a concentration-time profile similar to that of EtG.
  • the peak concentration C max of EtS was 0.17 ⁇ g/mL (range 0.08 ⁇ g/mL ⁇ 0.28 ⁇ g/mL), and the peak time T max was 3.02 h.
  • the detection window and peak concentration C max of EtS are significantly lower than that of EtG.
  • EtS is relatively stable and insensitive to bacteria. Therefore, EtS can provide supplementary data for identifying alcohol intake.
  • the present study has established an idea and method for inferring drinking time using the average concentration ratio of EtG/EtS. After further verification, it is expected to provide a useful analysis and monitoring for the identification of drunk driving in my country and the inference of related drinking time. tool.
  • the sensitive LC-MS/MS methods developed and validated in the present embodiments can be applied to drunk driving and other forensic cases involving alcohol, while the long detection windows of EtG and EtS support their use as useful markers for detecting alcohol consumption.

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Abstract

一种推算饮酒时间的方法，包括以下步骤：在开始饮酒的0～120h内抽取多个血液样本，然后检测对应血液样本中酒精、EtG和EtS的浓度，并以此获得EtG和EtS的平均浓度比值C EtG/C EtS；以平均浓度比值C EtG/C EtS为横坐标，取样时间为纵坐标得到二次回归方程；取待检血液样本，测定其C EtG/C EtS，根据二次回归方程中饮酒时间与平均浓度比值C EtG/C EtS之间的关系，计算饮酒时间。利用酒精非氧化代谢物之间的浓度比值随时间的变化规律推断饮酒时间，规避传统方法中不可避免的外部干扰问题。

Description

一种推算饮酒时间的方法 技术领域
本发明涉及分析化学及司法鉴定技术领域，具体涉及一种推算饮酒时间的方法。
背景技术
酒作为一种具有依赖属性的精神活性物质，一直在世界范围内被广泛滥用。由于普遍人群长期过量饮酒，造成饮酒相关事故的数量一直在不断增加，例如因酗酒造成的暴力行为、交通事故等。因此，对于酒精的技术鉴定依然是我国公检法鉴定工作中最常遇到的任务之一。
酒精在体内的代谢过程主要通过乙醇脱氢酶(ADH)和乙醛脱氢酶(ALDH)的氧化反应(90-92％)完成。此外，还有少量的酒精(<1％)会在不同酶的作用下进行非氧化代谢，并与其它物质直接结合，生成非氧化代谢产物。例如，乙基葡萄糖醛酸苷(乙基-b-D-6-葡糖醛酸苷，Ethyl glucuronide,EtG)是在UDP-葡萄糖醛酸转移酶(UDP-glucuronyltransferase)的催化下，酒精与葡萄糖醛酸结合而成。另外，在磺酸基转移酶(sulfotransferase)的作用下，酒精还可以与其它硫酸酯进行结合反应，从而生成乙基硫酸酯(Ethyl sulphate,EtS)。EtG和EtS作为酒精代谢的两个主要的非氧化产物，虽然含量很少，但是检测窗口长，即便是酒精已被完全代谢，也可以在多种体液或者组织中检测到EtG和EtS的存在。因此，EtG和EtS有望成为鉴定酒精摄入的敏感性和特异性的生物标志物。
饮酒时间是酒精相关案件分析过程中的重要线索，对评估案件性质具有重大意义。因此，饮酒时间推算也是酒精相关案件鉴定工作中比较常见的内容之一。
通常情况下，比值的计算可以消除剂量的影响。近年来，通过原体与代谢物，或代谢物与代谢物浓度比值随时间变化的规律来进行服药时间推断的研究不断出现。但是，有研究表明，酒精在活体内代谢迅速，饮酒8h后就较难检测出酒精的存在，并且在死后的酒精浓度可能会因死后的再分布和死后的产生而发生变化，其只有在死后24小时内，且温度低于20℃时，才会认可酒精浓度检测的准确性。因此，酒精原体与代谢物的浓度比的应用有很大的局限性，需要一种更可靠的方法来进行饮酒时间的推断。EtG和EtS，作为酒精的非氧化代谢产物，不仅具有更高浓度和更长的检测窗口，使得酒精摄入后即使检测不到酒精本身，也可以通过EtG和EtS的检测来对案件进行初步判断，而且相关文献已经证明，EtG和EtS不会在死后产生，且低温条件下相对稳定。因此，可以考虑EtG与EtS的浓度比来估计最后一次饮酒的时间。
发明内容
为解决上述技术问题，本发明的目的在于提供一种推算饮酒时间的方法，利用酒精非氧化代谢物之间的浓度比值随时间的变化规律推断饮酒时间，以规避传统方法中不可避免的外部干扰问题。
为实现上述目的，本发明的技术方案如下。
一种推算饮酒时间的方法，包括以下步骤：
在开始饮酒的0～120h内抽取多个血液样本，然后检测对应血液样本中EtG和EtS的浓度，并以此获得EtG和EtS的平均浓度比值CEtG/CEtS；
以平均浓度比值CEtG/CEtS为横坐标，取样时间为纵坐标拟合得到二次回归方程：y＝1.646x2-0.9599x+0.0878，R2＝0.9904；
式中，x表示平均浓度比值CEtG/CEtS，y表示取样时间；
取待检血液样本，测定其CEtG/CEtS，根据二次回归方程得到饮酒时间与平均浓度比值CEtG/CEtS之间的关系，并计算饮酒时间。
进一步，饮酒后血液酒精浓度在0.22-0.66mg/mL。
更进一步，饮酒所摄入的酒精量为0.72g/kg。
进一步，制作二次回归方程时所用血液样本的取样时间分别为0h、0.5h、2h、3h、5h、8h、12h、24h、36h、48h、120h。
进一步，对应血液样本中EtG和EtS的浓度的检测方法如下：
S1、样品预处理
将血液样本转移至加有内标EtG-D5和EtS-D5的离心管中，加入80％(v/v)乙腈-甲醇溶液，在0℃下沉淀并离心，转移上清液后干燥，用5％(v/v)乙腈-水溶液复溶，再次离心，取上清液得到待测样品；
S2、对S1的待测样品用液相色谱-串联质谱法测定血液样本中EtG和EtS的浓度。
更进一步，S2中，液相色谱的分离条件包括以下参数：
色谱柱采用Inertsil ODS-3柱，2.1mm×100mm，3μm；柱温为35℃；
洗脱系统的流动相A为水-0.1％甲酸溶液；流动相B为乙腈-0.1％甲酸溶液；流速为0.2mL/min；梯度洗脱程序如下：
0～2min，流动相A与流动相B的体积比为95：5；
2～6min，流动相A与流动相B的体积比为10：90；
6～8min，流动相A与流动相B的体积比为10：90；
8.5～14min，流动相A与流动相B的体积比为95：5。
此处试剂的配比采用体积比。
更进一步，S2中，质谱的检测条件包括以下参数：
采用负向模式的电喷雾电离；离子喷雾电压为-4000V，温度为500℃。
更进一步，S1中，内标EtG-D5的浓度为1μg/mL；内标EtS-D5的浓度 为1μg/mL。
本发明的有益效果：
1、本发明的方法主要是利用酒精非氧化代谢物之间的平均浓度比值随时间的变化规律来推断饮酒时间，以规避传统方法中不可避免的外部干扰问题。
2、本发明依据血液中EtG和EtS平均浓度比值与饮酒时间建立回归方程，得到在0-8h窗口期内的回归方程为y＝1.646x2-0.9599x+0.0878，R2＝0.9904；说明血液中葡萄糖醛酸乙酯与硫酸乙酯的平均浓度比与酒精使用时间的关系得到良好的相关模型，通过反推的方法将EtG和EtS的平均浓度比代入该方程中计算饮酒时间理论值，同时采用“(理论值-实测值)/实际饮酒时间”公式计算其推断误差，发现误差基本小于10％。
3、本发明旨在通过饮酒后血液中EtG和EtS的药代动力学研究，建立一种估算饮酒后时间长度的方法，而且还能提供适量口服后在中国人群中EtG和EtS的药代动力学参数。血液中葡萄糖醛酸乙酯最大浓度、最大浓度、消除半衰期分别为4.12±1.07h、0.31±0.11mg/L和2.56±0.89h；硫酸乙酯最大浓度、最大浓度、消除半衰期分别为3.02±0.70h、0.17±0.04mg/L、2.04±0.76h。
附图说明
图1是血液中酒精、EtG、EtS的平均浓度-时间曲线图。
图2是EtG与EtS以及内标EtG-D5和EtS-D5的LC-MS色谱图(500ng/mL)。
图3是空白血液样品的LC-MS色谱图。
图4是空白血液样品内添加EtG与EtS以及内标EtG-D5和EtS-D5的LC-MS色谱图(500ng/mL)。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
本发明所述的饮酒时间是指取样检测时距离开始饮酒的时间。
下述各实施例中所述实验方法如无特殊说明，均为常规方法；所述试剂和材料，如无特殊说明，均可在市场上购买得到。
实施例1
1、材料和方法
1.1、化学品和试剂
酒精(10mg/mL)                  美国Accustandard；
叔丁醇(AR，≥99.0％)           阿拉丁，上海；
EtG(100mg/mL)                  美国Cerilliant；
内标EtG-D5(IS；1μg/mL)        美国Cerilliant；
内标EtS-D5(IS；1μg/mL)        美国Cerilliant；
EtS-Na(98％)                   日本TSI；
甲醇(HPLC级)                   美国Merke；
乙腈(HPLC级)                   美国Merke；
甲酸(LC/MS级)                  中国百灵威；
超纯水                         美国Milli-Q超纯水系统
1.2、参与者和实验方法
在山西医科大学医学伦理委员会的批准下(2018LL349)，团队共招募了26名成年人参与该研究，其中包括14名男性和12名女性。所有参与者均无躯体或精神疾病史、无饮酒史或用药史，中位年龄24.5岁(范围是22 岁到27岁)，平均体重指数20.9kg/m2(范围是16.8kg/m2到34.6kg/m2)。
研究开始前，参与者签署知情同意书。为了安全起见，所有参与者饮酒后在校医院观察至少24小时，且在饮酒期间和饮酒后3天均对其进行了相应的医学评估。
禁食12小时后，参与者在30分钟内按照0.72g/kg(与参与者的体重成比例)的剂量标准伴食饮酒(汾酒，含有40％的酒精)，并于饮酒前(0h)和饮酒后0.5h、1.5h、2h、3h、5h、8h、12h、24h、36h、48h、120h，分别从肘正中静脉留置导管中抽取5mL血液作为待检测的血液样品。所有样品均在-20℃下保存至分析结束。
1.3、样品制备
1.3.1、含内标叔丁醇的待测样品的制备
血液样品中酒精含量的检测采用顶空气相色谱内标法测定，以叔丁醇为内标。将1mL血液和1mL叔丁醇(IS，87mg/mL)加入顶空瓶中，用3mL超纯水稀释后，密封混合，然后用顶空气相色谱分析。
1.3.2、含内标EtG-D5和EtS-D5的待测样品的制备
血液样品中代谢物EtG和EtS含量的检测采用液相色谱串联质谱(LC-MS/MS)法测定，以EtG-D5和EtS-D5为内标。分别取内标EtG-D5(IS，1μg/mL)和EtS-D5(IS，1μg/mL)各100μL，混合均匀，得到混合内标；取血液100μL，加入混合内标100μL，以提高对代谢物(EtG和EtS)的鉴定和定量。然后加入800μL 80％乙腈-甲醇溶液，在0℃下沉淀10min。之后以13000rpm离心5min，取出上清液，用35℃的氮气吹干。然后用400μL 5％乙腈-水溶液重新溶解，以13000rpm再次离心5min。从上清液中取3μL，注入液相色谱串联质谱(LC-MS/MS)中进行分析，见图2-4。
1.4、质谱分析
通过LC-20A系统，进行色谱分离。色谱条件如下：
色谱柱：采用Inertsil ODS-3柱(2.1mm×100mm，3μm；日本岛津)； 柱温35℃。
流动相：流动相A(超纯水-0.1％甲酸)和流动相B(乙腈-0.1％甲酸)；梯度洗脱(见表1)；流速为0.2mL/min；总洗脱时间14.0min；进样量为5μL。
表1梯度洗脱条件
通过串联质谱仪(TRAP4000，Sciex，AB)进行目标物的检测。具体条件如下：
离子源：电喷雾离子源(ESI)。离子喷雾电压为-4000V，温度为500℃。气帘气、雾化器(Gas1)和加热辅助气(Gas2)分别为40psi、50psi和35psi。
扫描方式：负离子+多反应监测(MRM)。
各分析物其它特定MRM参数见表2。
表2各分析物的特征离子对和质谱数据

注：a表示定量离子对。
1.5、估计最后一次饮酒的时间
采用血液样本中EtG与EtS平均浓度比值CEtG/CEtS与取样时间的关系来估算最后一次饮酒时间；用下式计算观察时间与实际时间之间的误差：
误差＝(|观察值-实际值|/实际值)*100％。
式中，观察值表示理论观察时间，也即估算的最后一次饮酒的时间；实际值表示实际时间，也即距离最近一次饮酒的实际采样时间。
1.6、统计量
采用DAS3.0软件按非房室模型计算药代动力学参数。所有数据均采用描述性统计的方法进行汇总。提供了一些关键数据，包括目标物浓度和检测时间点、药代动力学参数等结果的算术平均值和标准差。所有的统计分析均采用IBM软件(SPSS Inc.，芝加哥，IL，USA)的13.0版本进行。
2、结果
2.1、方法验证
血液样本中EtG和EtS的检测限(LOD)和定量限(LOQ)分别为0.02μg/mL和0.05μg/mL。我们用100μL的5％乙腈水溶液溶解前处理中所得的残渣，以定量LOQ以下的浓度。
表3血液中EtG和EtS的线性范围及检测限
表4血液中EtG、EtS的精密度、回收率和基质效应

如表3-4所示，所有分析物，包括酒精、EtG、EtS、EtG-d5和EtS-D5分离良好，未观察到内源性峰被分析物洗脱，方法得到了完全验证。
2.2、估计最后一次饮酒的时间
表5人血液中酒精及其代谢物的平均浓度(x±S(min-max)，n＝26)

注：“-”表示未检测到；BAC表示血液酒精浓度；所有值被保留在小数
点后两位。在摄入后24h、36h、48h和120h均未检测到酒精、EtG和EtS。 采集血液样本的时间选择0～120h，是基于有文献报道称酒精非氧化代谢物检测窗口比酒精原体长的事实；但在本发明实施例的检测过程中发现，各个目标物在24h后即检测不到。
根据表5所示的血液样本中EtG和EtS的平均浓度，我们计算了EtG与EtS的平均浓度比值CEtG/CEtS，并分析了单次口服后的比值与最后使用时间的关系。结果发现，以平均浓度比值CEtG/CEtS为横坐标，取样时间为纵坐标拟合得到二次回归方程：y＝1.646x2-0.9599x+0.0878，R2＝0.9904；式中，x表示平均浓度比值CEtG/CEtS，y表示取样时间。
表6从二次函数推导出的时间与实际最后一次饮酒时间之间的误差

CI:Confidence Interval(95％)。
如表6所示，使用回归方程(y＝1.646x2-0.9599x+0.0878，x表示比率，y表示时间，R2＝0.9904)，将EtG和EtS的浓度比代入回归方程，计算其饮酒时间的观察值，通过误差计算公式(误差＝(|观察值-实际值|/实际值)*100％)得到观察值与实际值在8h内的误差基本小于10％。
2.3、药代动力学分析
人血液中各时间点的酒精及其代谢物的平均浓度见表5，酒精及其代谢物的检测限数据见表7。
表7血液中酒精及其代谢物的检测限数据(x±S(min-max)，n＝26)

结果表明，参与者饮酒0.72g酒精/kg后，1.5h后血液中酒精的平均浓度达到0.41±0.11mg/mL，然后逐渐下降，检测窗口时间(最大观察值)为3-8h。
代谢物EtG(0.29±0.12μg/mL)和EtS(0.16±0.04μg/mL)分别在5h和3h达到峰值。
此外，如图1所示，在研究过程中，我们发现EtG的浓度始终高于EtS的浓度。
基于非房室模型，参与者饮酒0.72g酒精/kg后，计算血液中酒精以及代谢物EtG、EtS的药代动力学参数，获得药代动力学模型，结果如表8所示。
表8人血液中酒精及其代谢物的药代动力学参数(x±S，min-max，n＝26)

注：AUC(0-t)表示曲线下面积；t1/2z表示半衰期；Tmax表示达峰时
间；Cmax表示达峰浓度；Vz/F表示表观分布容积；Clz/F表示清除率。
结果发现，酒精在2.02±0.54h时达到峰值浓度(441.65±113.86mg/L (0.44±0.11mg/mL))。代谢物在4.12±1.07h和3.02±0.70h时达到峰值浓度(0.31±0.11mg/L和0.17±0.04mg/L)。酒精和EtG、EtS的t1/2z分别为1±1.09h和2.56±0.89h、2.04±0.76h。酒精的Clz/F为0.49±0.33L/h；然而，由于酒精代谢物(EtG和EtS)的入体剂量未知，无法准确计算二者的Vz/F和CLz/F。
3、讨论
本发明主要是基于药代动力学研究，利用酒精非氧化代谢物之间的平均浓度比值随时间的变化规律来推断饮酒时间。具体是依据血液中EtG和EtS的平均浓度比值与饮酒时间建立回归方程，得到在0-8h窗口期内的回归方程为y＝1.646x2-0.9599x+0.0878，R2＝0.9904；说明血液中EtG和EtS的平均浓度比与酒精使用时间的关系具有良好的相关性，通过反推的方法将EtG和EtS的平均浓度比代入该方程中计算饮酒时间理论值，同时采用“(理论值-实测值)/实际饮酒时间”公式计算其推断误差，发现误差基本小于10％。
血液样本中酒精非氧化代谢物(EtG和EtS)的LOD和LOQ分别为0.02μg/mL和0.05μg/mL，这说明本发明的方法能够有效定量血液中较低浓度的EtG和EtS。本发明实施例中0.72g/kg酒精剂量的BAC为0.22-0.66mg/mL，这与现有的定性酒后驾驶的血液酒精浓度(BAC)标准(大于0.2mg/mL)相接近，说明本发明实施例的方法适用于中国大多数酒后驾驶案例的监测。
本发明基于非房室模型计算血液中酒精和EtG、EtS的药代动力学参数表明，酒精在2.02±0.54h时达到了峰值浓度Cmax(441.65±113.86mg/L(0.44±0.11mg/mL))，与以往的研究相比，本发明实施例得到的酒精的吸收相更长，也即吸收更慢。此外，我们发现参与者的血液中可以在3-8小时内检测到血液中的酒精，并且酒精的平均消除半衰期为1.24±1.09h(0.30-4.23h)。
本发明基于非房室模型计算血液中酒精和EtG、EtS的药代动力学参数表明，代谢物EtG和EtS具有较长的检测窗口，且证实了EtG的代谢速度比 酒精慢，EtG在血液中的消除半衰期为2.56±0.89h。EtS是酒精代谢的另一个非氧化代谢物，其浓度-时间曲线与EtG相似。在我们的研究中，在0.72g/kg剂量下，EtS的达峰浓度Cmax为0.17μg/mL(范围0.08μg/mL～0.28μg/mL)，达峰时间Tmax为3.02h。此外，我们的研究还发现，EtS的检测窗口和达峰浓度Cmax均显著低于EtG，但是，EtS比较稳定，且对细菌不敏感，因此，EtS可以为识别酒精摄入提供补充数据。
综上所述，本发明研究建立了一种利用EtG/EtS平均浓度比进行饮酒时间推断的思路和方法，经进一步验证后，有望为我国酒驾鉴定及相关饮酒时间的推断提供一个有用的分析监测工具。此外，我们还研究了EtG和EtS在中国人群血液中的药代动力学，并获得了二者药代动力学参数。本发明实施例中开发和验证的敏感LC-MS/MS方法可应用于酒后驾驶和其他涉及酒精的法医案件，而EtG和EtS的长检测窗口支持将它们作为检测酒精消费的有用标记物。
以上仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。

Claims (8)

  1. 一种推算饮酒时间的方法，其特征在于，包括以下步骤：
    在开始饮酒的0～120h内抽取多个血液样本，然后检测对应血液样本中EtG和EtS的浓度，并以此获得EtG和EtS的平均浓度比值CEtG/CEtS；
    以平均浓度比值CEtG/CEtS为横坐标，取样时间为纵坐标拟合得到二次回归方程：y＝1.646x2-0.9599x+0.0878，R2＝0.9904；
    式中，x表示平均浓度比值CEtG/CEtS，y表示取样时间；
    取待检血液样本，测定其CEtG/CEtS，根据二次回归方程得到饮酒时间与平均浓度比值CEtG/CEtS之间的关系，并计算饮酒时间。
  2. 根据权利要1所述的推算饮酒时间的方法，其特征在于，饮酒后血液酒精浓度在0.22-0.66mg/mL。
  3. 根据权利要2所述的推算饮酒时间的方法，其特征在于，饮酒所摄入的酒精量为0.72g/kg。
  4. 根据权利要求1所述的推算饮酒时间的方法，其特征在于，制作二次回归方程时所用血液样本的取样时间分别为0h、0.5h、2h、3h、5h、8h、12h、24h、36h、48h、120h。
  5. 根据权利要求1所述的推算饮酒时间的方法，其特征在于，对应血液样本中EtG和EtS的浓度的检测方法如下：
    S1、样品预处理
    将血液样本转移至加有内标EtG-D5和EtS-D5的离心管中，加入80％乙腈-甲醇溶液，在0℃下沉淀并离心，转移上清液后干燥，用5％乙腈-水溶液复溶，再次离心，取上清液得到待测样品；
    S2、对S1的待测样品用液相色谱-串联质谱法测定血液样本中EtG和 EtS的浓度。
  6. 根据权利要求5所述的推算饮酒时间的方法，其特征在于，S2中，液相色谱的分离条件包括以下参数：
    色谱柱采用Inertsil ODS-3柱，2.1mm×100mm，3μm；柱温为35℃；
    洗脱系统的流动相A为水-0.1％甲酸溶液；流动相B为乙腈-0.1％甲酸溶液；流速为0.2mL/min；梯度洗脱程序如下：
    0～2min，流动相A与流动相B的体积比为95：5；
    2～6min，流动相A与流动相B的体积比为10：90；
    6～8min，流动相A与流动相B的体积比为10：90；
    8.5～14min，流动相A与流动相B的体积比为95：5。
  7. 根据权利要求5所述的推算饮酒时间的方法，其特征在于，S2中，质谱的检测条件包括以下参数：
    采用负向模式的电喷雾电离；离子喷雾电压为-4000V，温度为500℃。
  8. 根据权利要求5所述的推算饮酒时间的方法，其特征在于，S1中，内标EtG-D5的浓度为1μg/mL；内标EtS-D5的浓度为1μg/mL。
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