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

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.