LC-MS/MS法测定饮酒者尿液中乙醇和5-羟色胺代谢物

Analysis of 5-hydroxytryptamine metabolites in urine by LC-MS/MS for alcohol consumer

目的建立尿液中5羟(基)-β吲哚乙醇葡萄糖苷酸(5-hydroxytryptophol glucuronide,GTOL)和5羟(基)吲哚-3-乙酸(5-hydroxyindole-3-aceticacid,5-HlAA)的高效液相色谱-串联质谱(LC-MS/MS)检测方法,为疑似饮酒案件的法医学鉴定提供实验依据。方法尿液经乙腈(1%甲酸)沉淀蛋白,经Captiva EMR-Lipid滤柱过滤,以0.1%甲酸水溶液~乙腈(90:10 V/V)等度洗脱,在多反应监测模式下检测GTOL和5-HIAA,并对38例案件尿液样本定性定量检测。结果 GTOL的线性范围是0.025μg/mL~2μg/mL,线性关系良好,R2> 0.999,检测限和定量下限分别为0.01μg/mL和0.02μg/mL。5-HIAA的线性范围是0.01μg/mL~50μg/mL,线性关系良好,R^(2)> 0.99,检测限和定量下限分别为0.02μg/mL和0.05μg/mL。GTOL和5-HIAA的批内和批间精密度均小于15%,准确度分别在90.52%~111.04%和96.81%~112.30%,稳定性变化率在-11.96%~3.71%。25例乙醇阳性尿液样本GTOL和5-HIAA比值均大于15(pmol/nmol),13例乙醇阴性尿液样本GTOL和5-HIAA比值均小于15(pmol/nmol)。结论本研究建立的尿液中GTOL和5-HIAA定性定量分析方法准确、灵敏,可快速、精确地得到GTOL和5-HIAA比值,为司法鉴定法工作中饮酒判定提供科学依据。

Objective To establish a liquid chromatography-mass spectrometry(LC-MS/MS) method for the determination of 5-hydroxytryptophol glucuronide(GTOL) and 5-hydroxyindole-3-aceticacid(5-HLAA) in urine, and to verify the feasibility of the method based on practical cases. Methods Urine was precipitated by acetonitrile(1% formic acid) and filtered by Captiva EMR-Lipid filter. The mobile phase was A, 0.1%(V/V) formic acid solution in water, and B, acetonitrile. A gradient elution was applied was 90:10. The urine samples from 38 cases were detected in multi-reaction monitoring mode. Results The linear range of GTOL was 0.025μg/mL~2μg/mL, and had a coefficient of determination R^(2)> 0.999. Limit of detection and the lower limit of quantitation were 0.01μg/mL and 0.02μg/mL, respectively. The linear range of 5-HIAA was 0.01μg/mL to 50μg/mL, and had a coefficient of determination R2> 0.99. Limit of detection and the lower limit of quantitation were 0.02μg/m L and 0.05μg/m L, respectively. The intra-batch and interbatch accuracies of GTOL and 5-HIAA were less than 15%, and the accuracies were 90.52%~111.04% and 96.81%~112.30%, respectively. The stability variation rates ranged from-11.96% to 3.71%. The ratios of GTOL and 5-HIAA in 25 ethanol-positive urine samples were greater than 15 pmol/nmol, and the ratio of GTOL and 5-HIAA in 13 ethanol-negative urine sample was less than 15 pmol/nmol. Conclusion The qualitative and quantitative analysis method of GTOL and 5-HIAA in urine samples established in this study is accurate and sensitive, and can obtain the ratio of GTOL and 5-HIAA quickly and accurately, which provides a scientific basis for the determination of alcohol consumption in the practice of forensic science.