自动固相萃取-超高效液相色谱-串联质谱法测定人尿中15种防晒剂和防腐剂

Determination of 15 UV absorbers and preservatives in human urine by automated solid-phase extraction-ultra performance liquid chromatography-tandem mass spectrometry

本文建立了同时快速测定人尿液中15种防晒剂和防腐剂的高通量自动固相萃取-超高效液相色谱-串联质谱测定方法(automatic SPE-UPLC-MS/MS).人尿液经β-葡萄糖醛酸酶过夜酶解后,以Waters HLB 96-well Plate(30 mg,30μm)为基础,使用全自动固相萃取装置进行前处理,经25%乙腈溶液淋洗,甲醇-乙腈溶液(V:V=1:1)洗脱,氮吹至近干,20%乙腈溶液复溶后,采用Waters Acquity BEH C18色谱柱(100 mm×2.1 mm,1.7μm)、二元梯度洗脱系统(流动相A为水,流动相B为乙腈)进行色谱分离.质谱方法采用多反应监测(MRM)模式、电喷雾电离负离子模式和稳定同位素内标法进行数据采集和定量分析.15种分析物在相应线性范围内的线性相关系数(r)均大于0.999,3个加标水平的加标回收率为89.1%-110%,日内和日间精密度分别为0.9%-13%和3.1%-14%,样本稳定性、提取液稳定性、上样周期稳定性和反复冻融稳定性的相对标准偏差均在10%之内.将本方法应用于300名志愿者随机尿液的测定,结果显示该人群尿液的主要检出成分为4种防腐剂,分别是羟苯甲酯(MP)、羟苯乙酯(EP)、羟苯丙酯(PP)和对氯间二甲苯酚(PCMX),检出率分别为100%、92%、82%和64%,中位浓度分别为11.5、0.53、0.65、2.09 ng·mL^(-1).综上,本方法可应用于人尿液中15种防晒剂和防腐剂的快速提取、净化和定量分析,具有操作简便、灵敏度高、效率高、人员友好和绿色环保等优势,适用于大批量样品的检测应用.

A high-throughput automated solid-phase extraction-ultra performance liquid chromatography-tandem mass spectrometry(Automatic SPE-UPLC-MS/MS)method was developed for the simultaneous and rapid determination of 15 UV absorbers and preservatives in human urine.After overnight digestion byβ-glucuronidase,human urine was extracted with a Waters HLB 96-well Plate(30 mg,30μm)using a fully automated solid-phase extraction device.The sample was washed with 25%acetonitrile solution and eluted with methanol:acetonitrile(V:V=1:1)solution.The eluate was evaporated to dryness under a gentle stream of nitrogen and reconstituted with 20%acetonitrile solution.The chromatographic separation was performed on a Waters Acquity BEH C18 column(100 mm×2.1 mm,1.7μm)with a binary gradient elution system(water as mobile phase A and acetonitrile as mobile phase B).The mass spectrometry was operated in multiple reaction monitoring(MRM)mode and negative electrospray ionization mode.Stable isotope internal standard was adopted for quantification.The linear correlation coefficients(r)of 15 target analytes were all greater than 0.999 in the corresponding linear ranges.The recovery rates at three spiked levels ranged from 89.1%to 110%.The intra-day and inter-day precisions were 0.9%-13%and 3.1%-14%,respectively.The deviations of sample stability,extract stability,auto-sampler stability,and freeze-thaw stability were found to be within 10%.This method was applied to 300 urine samples collected from volunteers,and found methyl paraben(MP),ethyl paraben(EP),propyl paraben(PP),and chloroxylenol(PCMX)had the highest detection rates(100%,92%,82%,and 64%,respectively).The median concentrations were 11.5,0.53,0.65,and 2.09 ng·mL-1 for MP,EP,PP,and PCMX,respectively.In conclusion,this method can be applied to the rapid extraction,purification,and quantitative of 15 UV absorbers and preservatives in human urine.The method had the advantages of easy operation,high sensitivity,high efficiency,personnel-friendly,and eco-friendly.It is suitable for the application of detecting samples on a large scale.