Development and validation of a liquid chromatography-tandem mass spectrometry method for the simultaneous determination of phthalates and bisphenol a in serum, urine and follicular fluid

Phthalates and bisphenol A interfere with the synthesis, secretion, transport, binding, metabolism, and excretion of endogenous hormones and, for this reason, are classified as endocrine disruptors. We are here presenting an analytical method for the simultaneous detection of six phthalates metabolites and bisphenol A in different biological fluids (urine, serum and follifular fluid) by liquid chromatography coupled to tandem mass spectrometry. The quantification was carried out in negative electrospray ionization mode using selected reaction monitoring as acquisition mode. Different extraction protocols, using either solid phase or liquid/liquid extraction, were comparatively evaluated to optimize the sample preparation procedure. Solid-phase extraction was chosen as it ensured the best recovery and overall sensitivity. The method was successfully validated: recovery varying in the range 71 ± 2%-107 ± 6% depending on the target analyte and the matrix considered, intra-assay and inter-assay precision  ≤ 12% for follicular fluid, ≤11% for serum and ≤ 10% for urine and accuracy ≤ 115% for follicular fluid, ≤113% for serum ≤ 115% for urine , linearity with R 2  > 0.99, with the exception of MEP (recovery 64 ± 8%, intra-assay precision  ≤ 20%, inter-assay precision ≤ 16% for follicular fluid). The actual applicability of the method developed and validated in this study was assessed by the analysis of real samples, including 10 specimens of follicular fluid, serum and urine samples, that showed the presence of phthalates metabolites and Bisphenol A, and confirming that the newly developed method can be applied in the routine clinical laboratory for the identification and quantitation of these endocrine-disrupting chemicals.