A Preliminary Inquiry Into The Relevance Between The Exposure Of Bisphenol A And Uterine Leiomyoma

In this study,a method of solidphase extraction coupled with ultra performance liquid chromatography tandem mass spectrometry（SPE–UPLC–MS/MS）was established for the determination of bisphenol A（BPA）in human urine in order to investigate the possible linkage between the exposure concentration of BPA and uterine leiomyoma disease from epidemiological perspective.Fifty-one urine samples collected from patients with uterine leiomyoma were used as case group,forty-three urine samples from non-uterine leiomyoma and hormone independent gynecopathy women as normal group,sixty-six urine samples from cervicitis women（of which six were mild cases,thirty moderate and thirty severe）as negative control group.The urine samples were spiked with 2,4,6-tribromophenyl-terminated tetrabromobisphenol-A carbonate oligomer（TBBPA）as internal standard（I.S.）and was de-conjugated by addingβ-glucuronidase and sulfatase.After theenzymatic treatment,the samples were separated and passed through the pre-conditioned Supelclean C18 column solid phase extraction cartridges so as to be purified and concentrated.UPLC separation was performed on a Waters Acquity UPLC?BEH C18 column（100mm×2.1mm×1.7mm）,utilizing a gradient elution program of methanol and water（containing0.01mol·L^-11 ammonium acetate）as the mobile phase.Triple quadrupole mass spectrometry analyzer was used for the qualitative and quantitative analysis of UPLC-MS/MS system under negative electrospray ionization（ESI^-）in multiple reaction monitoring（MRM）mode.The results of validation study showed that the calibration curve was linear over the concentrations of BPA in the range of 1-100ng·mL^-11 with the correlation coefficient no less than 0.995（R²=0.9978）.The limit of detection（LOD）of BPA was 0.1ng·mL^-11 based on a signal‐to‐noise ratio（S/N）of 3 and the limit of quantitation（LOQ）was 0.3ng·mL^-11 based on a signal‐to‐noise ratio（S/N）of 10.The recovery and precision of the method were evaluated by analysing five replicates of human urine samples spiked with standards at 1ng·mL^-1、5ng·mL^-11 and 20ng·mL^-1.The average recoveries at three spiking levels range from 82.56%to 108.50%with the relative standard deviation（RSD）below of15.0%.The proposed method is simple,selective,accurate with high sensitivity and quick analytical rate which meets the requirements for the analysis of biological specimen.The proposed method was applied to determinate the concentration of BPA in the urine samples from normal group,negative control group and case group in order to evaluate possible linkage between the internal exposure concentration of BPA and uterine leiomyoma disease.For the normal group,BPA was detected in the urine of 42（97.7%）,the concentration distribution of BPA was 1.51ng·mL^-1(0.60ng·mL^-1,2.57ng·mL^-1);For the negative control group,BPA was detected in the urine of 61（92.4%）,the concentration distribution of BPA was 1.97ng·mL^-1(0.96ng·mL^-1,3.27ng·mL^-1);For the case group,BPA was detected in the urine of 50（98.0%）,the concentration distribution of BPA was 5.18ng·mL^-1(2.74ng·mL^-1,13.98ng·mL^-1).The Nonparametric tests was employed for the comparison of BPA among three groups.According to the results,levels of BPA in the urine from case group were significantly higher（P<0.05）than those from normal group and negative control group,but levels in the urine between normal group and negative control group as well as among mild,moderate and severe cervicitis groups did not show obvious difference（P﹥0.05）.There was not a significant correlation between the exposure level of BPA and the size as well as the number of fibroids by spearman correlation analysis（P﹥0.05）.The finding of the study suggested that BPA appeared to be prevalent in human urine and there is certain relationship between the exposure concentrations of BPA and uterine leiomyoma disease.This research provides a reliable method for analyzing the exposure level of bisphenol A in human body,and offers scientific evidence for the further study of the relavance between the development of estrogens dependent gynecologic oncology and bisphenol exposure.