| Objective:Alcohol as “road killer” is a worldwide social problem. About50%to60%of traffic accidents are related to drunk driving which is obviouslythe primary cause of death in all accidents. Currently, the BAC (BloodAlcohol Content) test is mainly used to identify the drunk driving. Due to thefast metabolism, ethanol cannot be detected in body after6to8hoursbecause of the long time interval between the occurrence of accident and thetime of BAC test, especially in hit-and-run cases. Therefore, it is insufficientto identify the drunk driving only by the BAC test.Ets is a directnon-oxidative metabolite of ethanol,and the clearance pathway is catalyzedby the sulfortransferase.the Ets can be detected in serum about8hours and inurine about3-5days after using alcohol.So Ets can be the importantbiomarker that can be used to identiy drunk driving in forensic medicine.This experiment established detection method of ethyl sulfate in the bloodand urine by High Performance Liquid Chromatography tandem massspectrometry (HPLC–MS/MS). Volunteer’s Ets in the blood and urine wasdetermined by the method of HPLC–MS/MS. So we can understand the kinetics of ethyl sulfate in people and then use for judging ethanolconcentration in medicolegal identification.Methods:1. Urine samples were spiked with the internal standard solution andmethanol. After agitation，centrifugation, the supernatant was evaporatedto dryness under nitrogen at40oC, then resolved by methanol, aftercentrifugation take supernatant sample analysis.2. EtS in serum and urine were detected by HPLC-MS/MS.chromatographic condition: chromatographic column: BOSTON-C18（100mm×2.1mm ID，dp3μm）; mobile phase: acetonitrile (A)-water(B)（contain0.1%methane acid）、 gradient elute； flow rate：0.2mL·min-1；column temperature：40oC；injection volume：5μL；Mass spectrometry conditions： ion source is ESI source， theidentification and quantification were carried out by negative electrosprayionization and selected ion monitoring mode(SIM）， Using internalstandard method for quantitative.3. Seven volunteers had been abstained from alcohol during the weekpreceding the study，Ethanol (mixed with water) was consumed over a30min period at a fixed dose of0.60±0.07g/kg body weight，Blood sampleswere then collected every30min for the first4h, every1h between4and8hafter start of drinking，Urine collections were made at every voiding, which corresponded to approximately once every2h for the first8h. then wehave12blood samples and10urine samples form every volunteer. theconcentrations of Ets in serum and urine was detected by HPLC-MS/MS, theconcentrations of ethanol in serum and urine was detected by Gaschromatography with flame ionization detector, the concentrations of Cr inurine was determined by Automatic Bionchemistry Analyzer(ModularDDPP).Results:1.Ets in urine have good linear relationship range from10.96ng/mL to10960ng/mL.The regression equations of Ets in urine is Y=0.0008X+0.3329（r=0.989）,the detection limitation of standard Ets in urine is0.1ng/mL.The recoveries were between89.6%and117.4%, with the relative standarddeviations between2.8%and10.2%; Ets in serum have good linearrelationship range from5.48ng/mL to10960ng/mL.The regressionequations of Ets in serum is Y=0.001X+0.251（r=0.993）,the detectionlimitation of standard Ets in serum is0.1ng/mL. The recoveries werebetween80.4%and114.0%, with the relative standard deviations between1.4%and8.72%.2. When volunteers consumed0.60±0.07g ethanol/kg body mass，The resulting mean peak ethanol concentrations in serum was66.5±12.8mg/100ml，the mean peak EtS concentrations in serum was79.9±12.3ng/ml， the time of peak serum ethanol concentrations was0.86±0.24h，the time ofpeak serum EtS concentrations was3.36h±0.56h， the average longestdetection time of ethanol in serum was5.71±0.95h， EtS in serum can bedetected in all of final blood sample（8h）；The individual ethanol-to-EtSratios in serum (ethanol in mg/100ml, EtG in ng/ml) were>1for the first3hours（range2-3.5h）after drinking, decreasing to﹤1during the rest of thetime period studied. The resulting mean peak ethanol concentrations in urinewas193.1±26.2mg/100ml，the mean peak EtS concentrations in urine was6882.8±1095.0ng/ml，the time of peak urine ethanol concentrations was4h，the time of peak urine EtS concentrations was4.57±0.98h，the averagelongest detection time of ethanol in urine was13.9±0.6h，the averagelongest detection time of EtS in urine was37.7±5.5h. For serum, the areaunder curve of ethanol correlated with the peak concentration of ethanol(correlation coefficient：0.723; significance level p<0.05), the area undercurve of ethanol correlated with the peak concentration of EtS (correlationcoefficient：0.79; significance level p<0.05),the area under curve of EtScorrelated with the peak concentration of ethanol (correlationcoefficient:0.714; significance level p<0.05), the area under curve of EtScorrelated with the peak concentration of EtS (correlation coefficient:0.756;significance level p<0.05); For blood, the peak concentration of ethanolcorrelated with the peak concentration of EtS (correlation coefficient：0.684; significance level p<0.005). Conclusion:1. This experiment established detection method of ethyl sulfate in theblood and urine by HPLC–MS/MS. This method is proved to be simple,precision, fast, sensitive, convenient. It can be suitable for the determinationof EtS in human serum and urine. Then can use to judge ethanolconcentration in medicolegal identification.2. The research studied the kinetics of ethanol and EtS in both bloodand urine over the entire time course of absorption, distribution andelimination. The results indicated that EtS analysis in blood could be avaluable supplement to ethanol measurement, to estimate the time of alcoholconsumption. For this purpose, two subsequent increasing EtS values and ahigh ethanol-to-EtS ratio in blood supports information of recent ingestion.Thus EtS becomes the important biomarker that can be used for judgingethanol concentration in medicolegal, then provide evidence for forensicscience. |
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