Quantification of the Conjugated Forms of Dichlorobisphenol A (3,3′-Cl₂BPA) in Rat and Human Plasma Using HPLC-MS/MS

Abstract

Background: 

Bisphenol A (BPA) is a ubiquitous contaminant that has endocrine-disrupting effects. Chlorinated derivatives of BPA are formed during chlorination of drinking water and have higher endocrine-disrupting activity. Dichlorobisphenol A (Cl₂BPA) is the most abundant chlorinated BPA derivative found in several human biological matrices. Recent in vitro experiments have shown that Cl₂BPA is metabolized in sulpho- and glucuro-conjugated compounds. To date, no assay has been developed to quantify the sulfo- and glucuro-conjugates of 3,3′-Cl₂BPA (3,3′-Cl₂BPA-S and 3,3′-Cl₂BPA-G, respectively).

Methods: 

A high-performance liquid chromatography-tandem mass spectrometry assay for the determination of 3,3′-Cl₂BPA conjugated forms in plasma samples was developed and validated according to the European Medicines Agency guidelines. Quantification was performed in the multiple reaction monitoring mode for all target analytes using a SCIEX 6500 + tandem mass spectrometer with an electrospray source operating in the negative ionization mode. Chromatographic separation was achieved using a C18 column maintained at 40°C and a binary mobile phase delivered in the gradient mode at a flow rate of 0.35 mL/min. Sample was prepared via simple precipitation using acetonitrile. The assay was validated and applied to rat and human plasma samples.

Results: 

Linearity was demonstrated over the range of 0.006–25 ng/mL for 3,3′-Cl₂BPA-G and 0.391–100 ng/mL for 3,3′-Cl₂BPA-S. Intraday and interday bias values were in the 95%–109% range, and the imprecision <9%. Internal standard corrected matrix effects were also investigated. This method enabled quantification of the conjugated forms of 3,3′-Cl₂BPA in plasma samples.

Conclusions: 

This is the first report on the development and validation of an analytical method for the quantification of 3,3′-Cl₂BPA-G and 3,3′-Cl₂BPA-S in the plasma matrix. This study is also the first report on the in vivo occurrence of these metabolites.