柱前衍生-超高效液相色谱-串联质谱法测定人体尿样中的对羟基苯甲酸

Determination of p-hydroxybenzoic acid in human urine by precolumn derivatization-ultra performance liquid chromatography-tandem mass spectrometry

目的建立丹磺酰氯柱前衍生-超高效液相色谱-串联质谱法测定人体尿液中对羟基苯甲酸的方法。方法样品酶解后,丹磺酰氯衍生,乙腈提取。目标化合物采用Waters ACQUITY UPLC BEH C_(18)色谱柱(50 mm×2.1 mm,1.7μm)分离,以乙腈和0.1%甲酸水溶液为流动相梯度洗脱,在电喷雾正离子源和多反应监测模式下进行测定,同位素内标法定量。结果对羟基苯甲酸在5.0~1000μg/L浓度范围内线性关系良好,相关系数大于0.999,回收率为97.5%~102.0%,精密度为1.53%~5.80%(n=6),基质效应为100.7%~112.0%(n=6),检出限和定量限分别为0.5和1.5μg/L。应用本方法测定267份学生尿液样本,对羟基苯甲酸的检出率为100%,浓度范围为686~60842μg/L。结论该方法有效提高定性准确性,可用于人体尿液中对羟基苯甲酸的定量分析。

OBJECTIVE A method has been established for the determination of p-hydroxybenzoic acid(p-HBA)in human urine based on the derivatization of p-HBA with dansyl chloride coupled with ultra-performance liquid chromatography-tandem mass spectrometry.METHODS The samples were derivatized with dansyl chloride and extracted with acetonitrile after enzymatic hydrolyzation.The target compound was separated by Waters ACQUITY UPLC BEH C_(18)(50 mm×2.1 mm,1.7μm)column with the gradient elution of acetonitrile and 0.1%formic acid in water.The electrospray source was in the positive ion mode and monitored in the multiple reactions monitoring mode.Isotope internal standard dilution technique was used for the quantification analysis.RESULTS There was a good linear relationship of p-HBA in the range of 5.0-1000μg/L with a correlation greater than 0.999.The recovery ranged from 97.5%to 102.0%,with the relative standard deviations(RSDs)of 1.53%-5.80%.Matrix effect was between 100.7%and 112.0%.The limit of detection(LOD)and the limit of quantitation(LOQ)were 0.5 and 1.5μg/L,respectively.The detection rate of p-hydroxybenzoic acid in 267 pupils’urine was 100%,with the concentration ranged from 686-60842μg/L.CONCLUSION The established method improved qualitative accuracy effectively.It can be used for the quantitative analysis of p-HBA in human urine.