Measurement of phenolic environmental estrogens in human urine samples by HPLC–MS/MS and primary discussion the possible linkage with uterine leiomyoma

Abstract

A method was established for the determination of three phenolic environmental estrogens, namely bisphenol A (BPA), nonylphenol (NP) and octylphenol (OP), in urine from women of uterine leiomyoma group (n=49) and control group (n=29), by using solid-phase extraction (SPE) coupled with liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Urine samples were spiked with 2,4,6-tribromophenyl-terminated tetrabromobisphenol-A carbonate oligomer (TBBPA) and nonylphenol D8 (NP-D8) as internal standard (I.S.) and de-conjugated by adding β-glucuronidase and sulfatase before the SPE. The extraction recoveries of BPA, NP and OP were more than 73.3%; the standard curve was linear over the validated concentrations in the range of 1.0-100.0ng/mL and the limits of detection (LOD) of BPA, NP and OP were 0.32ng/mL, 0.18ng/mL and 0.15ng/mL, respectively. Moreover, by analysing quality control urine samples in 5 days, the results showed that the method was precise and accurate, for the intra- and inter-day CV% within 15.2%. Except that OP was not found (<LOQ) in any of the control urine samples, the three phenolic environmental estrogens were detected in all urine samples. For the uterine leiomyoma women, the mean concentrations of BPA, NP and OP were 13.9±12.7ng/mL, 2.77±2.22ng/mL and 4.09±5.51ng/mL (mean±SD), respectively. For the control group, the mean concentrations of BPA and NP were 8.50±12.2ng/mL and 3.84±3.90ng/mL (mean±SD), respectively. The Wilcoxon rank sum test was employed for the comparison of BPA and NP between and control in 2 subgroups defined by the number of gravidity (≤3 and >3). NP levels were significantly higher in uterine leiomyoma patients than control group in low gravidity subgroup. Though BPA levels in experimental and control groups were not significantly different, the mean levels and concentration distribution were different. The study suggested that there is certain relationship between exposure concentrations of phenolic environmental estrogens and uterine leiomyoma disease.