| Elcohol related accidents and cases has become a serious social problem in contemporar China which made Ethanol analysis the routine request in forensic practices, such as identification of drinving after drink, hit-and-run cases, and the source of ethanol in post mortal bodies.The bulk mount (95%-98%) of consumed ethanol is metabolized in the liver by alcohol dehydrogenase while the remaining (2%-5%) is excreted unchanged in exhaled air, urine and sweat; only a very small scale of ethanol (less than 0.1%) is conjugated with Uridine-5'-diphospho-glucuronoside and Sulphate, respectively and produces Ethyl Glucuronide (EtG) and Ethyl Sulphate (EtS). The two ethanol metabolites attract many researchers'interest for their non-oxidative, non-volatility profile and longer detectable time in urine and blood. EtG is the exclusive evidence in ethanol "negative" cases for drinving after drink and ethanol intake before death. These are several reports for EtG detection and metabolic kinetics, but there is no related report in China by far.In this paper, the Authors devoted to establish and evaluate analysis methods for EtG in blood and urine, and study the EtG metabolic kinetics in blood and urine of health volunteers. This work is demonstrated in six chapters, listed by time order.The first chapter summarized progress and application of EtG analysis in the published papers, and focusing on the pretreatment and detection method for EtG in serum and urine matrix; illustrated the meaning and the strategy of this study, which is study EtG metabolism based on Chinese population by developing reliable and convenient analysis method.Chapter 2 developed and evaluated a method analyzing EtG in blood with gas chromatograph coupled with tandem mass spectrum (GC-MS/MS). The limit of detection (LOD) is 0.05μg/mL, and the linearity range is 0.1μg/mL-10μg/mL (R2>0.999). The accuracy is 90%-110%, the intra-day RSD and the inter-day RSD are both less than 15%。This method was qualified to detecting EtG in blood sample by authentic sample analysis.Chapter 3 developed and evaluated a liquid chromatograph-linear ion trap-Orbitrap method to detect EtG in blood and urine matrix. The LOD are both 0.02μg/mL in blood and urine, and the linearity range are both 0.05-5.00μg/mL (R2> 0.998). The intra-day accuracy and the inter-day accuracy are both between 85%-110%, while the intra-day RSD and the inter-day RSD are both within 10%. What's more, the optimization of parameters for EtG detection is demonstrated in this chapter in details.Chapter 4 reported a liquid chromatograph coupled with tandem mass spectrum method for EtG detection in urine and blood. The LOD are both 0.05μg/mL, and the linearity range are both 0.1μg/mL-5μg/mL (R2>0.999). The accuracy are both 95%-105%, while intra-day RSD and inter-day RSD both within 15%. This chapter also reported the adjusting of chromatographic parameters for best analysis performance.Chapter 5 recorded the dose administrated drinking experiment. With the LC-MS/MS method established in Chapter 4, the author analyzed the blood and urine sample for EtG concentration, and with previous publish methods, the author analysis the ethanol concentration. Those data illustrated EtG metabolism and the mutual relationship between EtG and ethanol in blood and urine. EtG concentration in blood reach its peak at 4-6hrs after drinking and the detectable time is 8-21h,8-10hrs longer than ethanol; EtG concentration in blood reach its peak at 4-6hrs after drinking, detectable time is 20-85hrs,12-70hrs longer than ethanol. After statistic calculation with software (Stata 7.0), a lative rough equation estimating the time of drink from and ethanol concentration was established.Chapter 6 established a LTQ-Orbitrap method for detecting EtG in urine and blood, and the linearity range is 0.1μg/mL-μg/mL (R2>0.995)In this study, we established multiple analysis method for the detection of EtG in blood and urine with GC-MS/MS, LC-MS/MS and LTQ-Orbitrap which was a noval platform. EtG metabolism based on Chinese population was investigated and the relation between EtG and Ethonal in blood and urine was discussed. This work provides ethanol surveillance using EtG as bio-mark with solid technical support for identification of the hit-and-run suspects and the source of ethanol in post mortal bodies. |
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