| Objective:1. A method based on ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed and optimized to investigate four phthalate monoesters, bisphenol A and nonylphenol in the urine of women in Nanjing.2. To explore the characteristics of the target analytes exposure among women of childbearing age in Nanjing.3. To evaluate a possible correlationship between phthalate, bisphenol A and nonylphenol exposure and unexplained recurrent spontaneous abortion.Methods:1. β-glucuronidase/sulfatase solution and solid-phase extraction were used to extract mono-n-butyl phthalate (MBP), mono-isobutyl phthalate (MiBP), mono-benzyl phthalate (MBzP), mono-2-ethylhexyl phthalate (MEHP), bisphenol A (BPA) and 4-n-nonylphenol (4-n-NP) in human urine. Samples were analyzed by using UPLC-MS/MS after pretreatment and then a quantitative method was established for each analyte.2.186 urine samples were collected from women of childbearing age in Nanjing residence at Zhong Da Hospital whose ages ranged from 20-40 years during June 2014 to March 2016. Informed consent was obtained from all participants in the study prior to collecting any data or samples. Participants were women whose gestational age was over 28 weeks (n=96) or women who had had at least one child and undergone therapeutic abortion without desire of conception. Both of them had never experienced spontaneous abortion. Both domestic and international studies were introduced to make a comparison and carry out the characteristic analysis.3. Case-control study was introduced to conduct the research. The case group (n=45) comprised women with unexplained recurrent spontaneous abortion while the control group (n=96) were preganant women whose gestational age was over 28 weeks and had never experienced spontaneous abortion. Wilcoxon rank sum test was employed to compare the differences in concentrations of MBP, MiBP, MBzP, MEHP, BPA and 4-n-NP levels between the control and case group then explore the relationship between phthalate, bisphenol A and nonylphenol exposure and unexplained recurrent spontaneous abortion.Results:1. The calibration curves showed good linearity within the range 1-100 ng/mL for MBP, MiBP, MBzP, MEHP, BPA and 4-n-NP. The limit of detection (LOD) for MBP, MiBP, MBzP and MEHP was 0.1 μg/L, while the LOD for BPA and 4-n-NP was 0.3 μg/L. The recovery ranged from 88.8%-115.4%. Both the intra-day and inter-day precision were less than 10%.2. The median concentrations of MBP, MiBP, MBzP, MEHP, BPA and 4-n-NP among 186 women of childbearing age were 3.14,1.70,0.44,4.20,0.78 and 1.47 ng/mL, respectively. While the detection rate for each analyte was 94.6%,91.9%, 82.8%,89.8%,58.6% and 68.3%, respectively.3. For the control group, the median concentrations of MBP, MiBP, MBzP, MEHP, BPA and 4-n-NP were 2.01,1.57,0.38,3.82,1.40 and 1.01 ng/mL. The median levels of the same analytes were 6.68,3.89,0.40,7.78,4.27 and 0.17 ng/mL, respectively, in the case group. There were significant differences (p<0.05) in MBP, MiBP, MEHP and BPA levels between case and control group.Conclusion:A UPLC-MS/MS method was developed and optimized to analyze BPA, NP and four phthalate monoesters in human urine. The method validated in this study has proven to be sensitive, accurate and precise, and can be applied to the evaluation of human exposure to phthalates, BPA and 4-n-NP. Women of childbearing age in Nanjing were widely exposed to Di-n-butyl phthalate (DBP), Di-isobutyl phthalate (DiBP), Benzyl butyl phthalate (BBzP) and Di-(2-ethylhexyl) phthalate (DEHP). There may be potential correlationship between DBP, DiBP, DEHP or BPA exposure and unexplained recurrent spontaneous abortion. |
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