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

doi:10.1016/j.jchromb.2013.08.032

Journal of Chromatography B

Volume 938, 1 November 2013, Pages 80-85

https://doi.org/10.1016/j.jchromb.2013.08.032 Get rights and content

Highlights

•
A SPE–HPLC–MS/MS method is established to detect BPA, NP and OP in human urine.
•
The exposure levels of BPA, NP and OP in uterine leiomyoma and control group urine sample are investigated.
•
There is linkage between the exposure concentration of BPA, NP and OP and uterine leiomyoma disease.

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 1.0–100.0 ng/mL and the limits of detection (LOD) of BPA, NP and OP were 0.32 ng/mL, 0.18 ng/mL and 0.15 ng/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 ± 12.7 ng/mL, 2.77 ± 2.22 ng/mL and 4.09 ± 5.51 ng/mL (mean ± SD), respectively. For the control group, the mean concentrations of BPA and NP were 8.50 ± 12.2 ng/mL and 3.84 ± 3.90 ng/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.

Section snippets

1 Introduction

Bisphenol A (BPA), nonylphenol (NP) and octylphenol (OP) are man-made alkyl phenols (APs) and are well known in the scientific research as environmental endocrine disruptors [1]. BPA, a primary raw material, is widely used in the industry as an important intermediate in the production of epoxy resins and polycarbonates plastic [2], [3]. NP and OP are extensively used in the production of elasticizers, technical grade abstergents and pesticide emulsifiers [4]. The exposure of the

Reagents and materials

BPA, NP and OP were purchased from Aldrich–Sigma (purity > 99.0%, St. Louis, MO, USA). NP-D8 and TBBPA (purity > 99.0%, HuaErBo Technology Co., Beijing, China), HPLC grade dichlormethane, methanol and ammonium acetate were purchased from TEDIA (Ohio, USA) and keMiO (Tianjing China). Analytical grade acetic acid and sodium acetate were obtained from Shanghai National Medicine Chemicals Corporation. β-Glucuronidase/sulfatase (β-glucuronidase 18 U/mg, arylsulfatase 16 U/mg) was purchased from

Calibration curves

The peak-area ratios of analytes/I.S. versus the nominal concentrations were plotted, and a least-square linear regression weighted by the reciprocal of the concentrations was applied to generate the calibration curves. The calibration curves prepared on three different days showed good linearity and acceptable data over a wide range of BPA, NP and OP concentrations of 1.0–100.0 ng/mL with R² ≥ 0.998.

The accuracy and precision were determined at each day. The BPA, NP and OP of mean accuracy were

Conclusions

A SPE procedure based on C18 cartridges coupled with HPLC–MS/MS was developed and validated for the determination of BPA, NP and OP in human urine samples. The analytical method established and validated in this study is simple, selective, sensitive, accurate and precise, which meets the requirements for the analysis of biological specimen. The proposed method was applied to investigate the exposure levels of BPA, NP and OP in uterine leiomyoma and control group urine sample. The results

Acknowledgements

This work received financial support from National Natural Science Foundation (Project No.: 81172721) of the People's Republic of China, Suzhou Social Development Projects (Project No.: SS201124), Nanjing Science and Technology Project (Project No.: 201201054), the National Natural Science pre-Research Project funds of the Southeast University (Project No.: 3290001102), Youth Projects of Jiangsu Provincial Health Department (Project No.:Q201305) and the SRTP Project of the Southeast University

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Citation Excerpt :
Finally, most previous studies were limited in their ability to control for confounding. Three of the five studies on this topic compared BPA concentrations across UL groups without controlling for any potential confounders (21–23). One of the other two studies was a cross-sectional analysis of the Endometriosis: Natural History, Diagnosis and Outcomes study, with adjustment for age, race/ethnicity, BMI, and smoking but not for socioeconomic status or parity (24).
To examine the association of urinary concentrations of phenols, parabens, and triclocarban with incidence and growth of uterine leiomyomata (UL; fibroids).
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Clinic visits at baseline and every 20 months for 60 months.
754 Black women aged 23–35 years residing in the Detroit, Michigan area (enrolled during 2010–2012).
None.
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Urinary biomarker concentrations were generally inversely associated with UL incidence, but the associations were weak and nonmonotonic. For example, hazard ratios comparing concentrations ≥90th with <50th percentile were 0.77 (95% CI: 0.46, 1.27) for bisphenol A, 0.72 (95% CI: 0.40, 1.28) for bisphenol S, and 0.76 (95% CI: 0.43, 1.33) for methylparaben. Biomarker concentrations were not strongly associated with UL growth.
In this study of reproductive-aged Black women, urinary phenols, parabens, and triclocarban biomarkers were neither strongly nor consistently associated with UL incidence and growth.
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754 mujeres negras de 23-35 años residiendo en Detroit, área de Michigan (incluidas durante 2010-2012).
Ninguna.
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¹: These two authors contribute to this paper equally.

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

Highlights

Abstract

Section snippets

1 Introduction

Reagents and materials

Calibration curves

Conclusions

Acknowledgements

Toxicol. Lett.

Reprod. Toxicol.

J. Chromatogr. B

Mol. cell. Endocrinol.

Chin. J. Ind. Med.

Toxicol. Appl. Pharmacol.

J. Chromatogr. B

J. Chromatogr. B

Environ. Health Perspect.