尿中苯和甲苯代谢物苯巯基尿酸和苄基巯基尿酸的固相萃取-液相色谱-串联质谱同时测定法

Simultaneous determination of metabolites of benzene and toluene in human urine S-phenylmercapturic acid and S-benzylmercapturic acid by liquid chromatography tandem mass spectrometry

目的建立同时测定尿中苯和甲苯的代谢产物——苯巯基尿酸（s—phenylmercapturicacid，S-PMA）和苄基巯基尿酸（S乩enzylmercapturicacid，S-BMA）的固相萃取-液相色谱-串联质谱（LC～MS—MS）法。方法尿样中结合态的苯巯基尿酸和苄基巯基尿酸在酸性环境（pH2．5）下水解20min成游离态的S-PMA和S—BMA，以C。固相萃取小柱富集、净化，以甲醇洗脱，采用WatersX—TerraC50色谱柱，流动相为乙腈-0．1％甲酸溶液梯度淋洗分离后进入质谱测定；在喷雾电压4．5kV，毛细管温度300oC下，以d2-苯巯基尿酸为内标，采用SRM模式负离子扫描方式测定，内标法定量。结果在0．05—50．0ng／ml的范围内，该方法所得S-PMA和S-BMA的回归方程均具有良好的线性，r值分别为0．9955和0．9993，检出限分别为0．03、0．004ng／ml，平均回收率分别为86．4％～99．6％和80．3％-108．0％，RSD分别为10．7％和11．3％。结论该方法具有分离效果好、稳定性强、操作简便、快捷、回收率高等特点，可用于苯和甲苯低暴露人群尿中两者代谢产物S—PMA和S—BMA的检测。

Objective To develop a method for simultaneous determination of metabolites of benzene and toluene in human urine, i.e. S-phenylmercapturic acid（S-PMA） and S-benzylmercapturic acid（S-BMA） by solid-phase extraction-liquid chromatography tandem mass（LC-MS-MS）. Methods The urine sample was added with internal standard （dE-S-phenylmercapturic acid） and was hydrolyzed by formic acid （pH2.5） first, the hydrolyzing time was 20 min, then was cleaned up and concentrated on Cs SPE column,then was determined with LC-MS-MS. The targeted compounds were analyzed by LC-MS-MS under negative ion mode using Waters X-Terra C8 column as the analyzing column. The mobile phase was CH3CN and HCOOH＂ H20 （0.1%） gradient elution. The spray voltage was 4.5 kV. The capillary temperature was 300 ~C. Results The linear ranges were 0.05-50.0 ng/ml. The related coefficients were 0.995 5 and 0.999 3. The limits of detection were 0.03 and 0.004 ng/ml. The rats of recovery were 86.4%-99.6% and 86.4%-108.0%. The relative standard deviations were 10.7% and 11.3%（n=6）. Conclusion The method is simple, fast and applicable to simultaneous determination of metabolites of benzene and toluene in human urine.