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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-phenylmercapturic acid,S-PMA)和苄基巯基尿酸(S-benzylmercapturic acid,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(pH 2.5)下水解20 min成游离态的S-PMA和S-BMA,以C8固相萃取小柱富集、净化,以甲醇洗脱,采用Waters X-Terra誖C8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4.5 kV,毛细管温度300℃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0.05~50.0 ng/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0.995 5和0.999 3,检出限分别为0.03、0.004 ng/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SPMA和S-BMA的检测。
OBJECTIVE To establish a method for the simultaneous determination of benzene and toluene in urine by S-phenylmercapturic acid (S-PMA) and S-benzylmercapturic acid (S-BMA) - Tandem mass spectrometry (LC-MS-MS) method. Methods The bound Phenylmercaptopuric acid and benzylmercapto-uric acid in urine samples were hydrolyzed in acidic environment (pH 2.5) for 20 min to form free S-PMA and S-BMA. The samples were purified by C8 solid phase extraction Methanol eluting with a Waters X-Terra Paradox C8 column, the mobile phase was acetonitrile-0.1% formic acid solution gradient elution separation into the mass spectrometry determination; spray voltage 4.5 kV, capillary temperature 300 ℃, d2-phenylmercapturic acid As the internal standard, using negative ion mode SRM mode determination, internal standard method. Results The regression equations of S-PMA and S-BMA obtained in this method all showed good linearity with r values of 0.995 5 and 0.999 3 in the range of 0.05-50.0 ng / ml with detection limits of 0.03 and 0.004 ng / ml. The average recoveries were 86.4% -99.6% and 80.3% -108.0%, respectively, with RSDs of 10.7% and 11.3%, respectively. Conclusion The method has the advantages of good separation, strong stability, simple, fast and high recovery rate. It can be used to detect the metabolites SPMA and S-BMA in the urine of people with low benzene and toluene exposure.