Simultaneous UHPLC-MS/MS method of estradiol metabolites to support the evaluation of Phase-2 metabolic activity of induced pluripotent stem cell derived hepatocytes

The goal of this study was to develop and validate a high-throughput UHPLC-MS/MS method for simultaneous quantitation of three estradiol metabolites namely estradiol 3-β-D-glucuronide (E3G), estradiol 17-β-D-glucuronide (E17G) and estradiol 3-sulfate (E3S) in cell culture medium to support the characterization of metabolic function of induced pluripotent stem cell (iPSC) derived hepatocytes. To achieve this goal, a simple protein precipitation method was used for sample cleanup. All the metabolites were separated chromatographically using a C-18 column where 10 mM ammonium acetate and acetonitrile was used in gradient flow for 4 min. The analytes were quantitated by triple quadrupole mass spectrometer with the use of isotopically labeled internal standard (IS). This method was validated as per the U.S Food and Drug Administration's Bioanalytical Method Validation, Guidance for Industry. Linearity for E3G and E17G was in the range of 2–1500 ng/mL whereas for E3S it was 0.3–500 ng/mL. Inter-day and intra-day accuracy and precision of this method were in the acceptable limits. In addition, multiple stability tests (freeze thaw, autosampler, water bath (37 °C), bench top and long term) were performed for all the metabolites in cell culture medium. All the metabolites were stable up to 3 freeze thaw cycles at −20 °C and − 80 °C, 48 h in autosampler, 24 h at 37 °C, 48 h at room temperature and 173 days at −20 °C. Extraction recoveries for the metabolites were reproducible and were in the range of 94–108%. This method was used to quantitate estradiol metabolites generated by iPSC hepatocytes in-vitro studies. •Method for characterization of iPSC hepatocyte metabolic activity•Simultaneous determination of three estradiol metabolites•Simple protein precipitation method used for extraction•High through-put analysis with shorter runtime, low injection volume and good sensitivity•Stable isotope internal standard was used to compensate losses during extraction and to reduce matrix effect.