高效液相亲水作用色谱-串联质谱法同时测定人尿液中烟碱及其9种代谢物

Simultaneous Determination of Nicotine and Its Nine Metabolites in Human Urine Using Hydrophilic Interaction High Performance Liquid Chromatography-Tandem Mass Spectrometry

建立了同时测定人尿液中烟碱及其9种代谢物的高效液相亲水作用色谱-串联质谱分析方法。尿液样品经乙腈和甲醇混合溶剂(体积比3∶1)稀释20倍,过0.22μm滤膜后直接进样。采用Atlantis HILIC Silica柱(3.0 mm×100 mm,3.0μm),以10 mmol/L甲酸铵水溶液(用甲酸调至pH 3.0)-乙腈为流动相进行梯度分离,电喷雾正离子电离模式和多反应监测扫描模式下测定,以同位素内标法定量。结果表明,烟碱及其9种代谢物在各自的质量浓度范围内线性关系良好,相关系数(r)均大于0.997,检出限(LOD)和定量下限(LOQ)分别为0.03~0.24 ng/mL和0.10~0.80 ng/mL;在低、中和高3个加标水平下的加标回收率为81.9%~110%,日间和日内相对标准偏差(RSD)为0.50%~6.7%。该方法操作简单、灵敏度高、稳定性好,能够满足大批量人尿液中烟碱及其9种代谢物的检测需求。

Determination of nicotine metabolites in human urine provides an ideal tool for the assessment of the tobacco use-related nicotine exposure,provided that the considered metabolites comprise a large share of the amount exposed.Nicotine and its nine metabolites mentioned in this research comprised of more than 90%of the total nicotine metabolites.A hydrophilic interaction high performance liquid chromatography-tandem mass spectrometry was developed for the simultaneous determination of nicotine and its nine metabolites in human urine.The urine sample was diluted 20 times with a mixed solvent of acetonitrile and methanol(3∶1,by volume),and subsequently filtered through a 0.22μm membrane for direct analysis.Then these metabolites were separated on an Atlantis HILIC Silica column(3.0 mm×100 mm,3.0μm)by gradient elution,with 10 mmol/L ammonium formate aqueous solution(adjusted to pH 3.0 with formic acid)-acetonitrile as mobile phases,then detected by tandem mass spectrometry with electrospray ionization in positive ion mode under multiple reaction monitoring(MRM),and finally quantified by isotope-labelled internal standards.Results showed that the calibration curves for nicotine and its nine metabolites were linear in the certain concentration range with correlation coefficients(r)larger than 0.997.The limits of detection(LOD)and quantitation(LOQ)were in the ranges of 0.03-0.24 ng/mL and 0.10-0.80 ng/mL,respectively.The recoveries at low,medium and high spiked levels in real samples ranged from 81.9% to 110%,with intra-day and inter-day relative standard deviations of 0.50%-6.7%.The developed method could meet the requirements for high-throughput detection of nicotine and its nine metabolites in human urine with simplicity,high sensitivity and stability.