固相萃取-液相色谱-串联质谱测定人体尿液中有机磷酸酯及其二酯和羟基代谢物

Determination of organophosphate esters and their diester and hydroxylated metabolites in human urine by high performance liquid chromatography-tandem mass spectrometry combined with solid phase extraction

尿液中的有机磷酸酯二酯代谢物(di-OPEs)是现阶段识别和量化人体暴露于有机磷酸酯(OPEs)的首选生物标志物.目前鲜有研究同时对OPEs及其二酯代谢物、羟基代谢物(OH-OPEs)进行分析测定,且关注的目标化合物种类较少.本研究对尿液前处理过程中常用的净化浓缩方法进行了优化,建立了人体尿液样品中14种OPEs、7种di-OPEs和3种OH-OPEs的高效液相色谱-串联质谱分析方法(HPLC-MS/MS).取2 mL样品经β-葡萄糖醛酸酶/芳基硫酸酯酶酶解6 h后,加入2%甲酸/水调节pH,然后用STRATA-X-AW固相萃取柱进行净化,收集固相萃取过程中的2%甲酸/甲醇淋洗液和2%氨水/甲醇洗脱液氮吹浓缩后分别进行OPEs、OH-OPEs和di-OPEs的HPLC-MS/MS测定,OPEs和OHOPEs的质谱检测选用电喷雾正离子模式电离,di-OPEs选用负离子模式,在多重反应监测模式(MRM)下测定.尽管尿液样品中多数目标物质在检测时存在基质效应(均值24%-159%),但通过合适的同位素内标进行校正,可以抵消部分基质影响.在优化的条件下,24种目标物质在0.05-40 ng·mL^(−1)范围内线性关系良好(r>0.99),方法检出限(MDL)0.0008-0.32 ng·mL^(−1),加标回收率60%-131%,RSD为4%-22%.采用本方法对实际人体尿液样本进行分析,7种di-OPEs和3种OH-OPEs的总含量为0.07-7.04 ng·mL^(−1),中位含量为0.54 ng·mL^(−1),14种OPEs的总含量为<MDL-0.68 ng·mL^(−1),中位含量为0.05 ng·mL^(−1).两种具有直接工业生产应用的di-OPEs(磷酸二苯基酯DPHP和磷酸二丁酯DBP)检出率高于60%,其来源值得追溯.一种新型有机磷酸酯阻燃剂3-异丙基苯基二苯基磷酸酯(3IPPDPP)的检出率为66.7%,应该引起关注.

Organophosphate diester(di-OPE)metabolites in urine are commonly used as biomarkers to identify and quantify human exposure to organophosphate esters(OPEs).Few studies have determined OPEs and their diester and hydroxyl metabolites(OH-OPEs)at the same time,and there were fewer types of substances of concern.In this study,purification and concentration parameters in the pretreatment process were optimized,and a high-performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS)method was established for the detection of 14 OPEs,7 di-OPEs and 3 OH-OPEs in human urine samples.2 mL of the sample was digested byβ-glucuronidase/aryl sulfatase enzyme solution for 6 h.After adding 2%formic acid in water to adjust the pH value,the samples were cleaned up by a STRATA-X-AW solid phase extraction column,2%formic acid/methanol rinse and 2%ammonia/methanol eluent were collected and concentrated for the detection of OPEs,OH-OPEs and di-OPEs by HPLC-MS/MS.OPEs and OH-OPEs were ionized in electrospray ionization positive mode,di-OPEs were ionized in electrospray ionization negative mode,then the analytes were detected in the multiple reaction monitoring(MRM)mode.Although most of the target analytes in urine samples have matrix effects(average in range of 24%—159%),the matrix effects can be partially cancelled out by choosing suitable isotopic internal standards for correction.Under the optimized condition,the linear relationships of all the analytes were good in the range of 0.05—40 ng·mL^(−1)(r>0.99).The method detection limits(MDL)ranged from 0.0008 ng·mL^(−1)to 0.32 ng·mL^(−1).The average recoveries of 24 analytes in urine were in the range of 60%—131%and the RSDs were in the range of 4%—22%.The optimized method was applied to detect OPEs,di-OPEs and OH-OPEs in 15 human urine samples.The total concentrations of 7 di-OPEs and 3 OHOPEs ranged from 0.07 ng·mL^(−1)to 7.04 ng·mL^(−1),with a median concentration of 0.54 ng·mL^(−1),and the total concentrations of 14 OPEs ranged from<MDL to 0.68 ng·mL^(−1),with a median concentration of 0.05 ng·mL^(−1).Diphenyl phosphate(DPHP)and dibutyl phosphate(DBP),which have direct industrial production and applications,were detected at a rate higher than 60%,so their sources need to be further studied.3-isopropylphenyl diphenyl phosphate(3IPPDPP),a new organophosphate flame retardant,was detected at a rate of 66.7%,and it should be a cause for concern.