Division of Applied Regulatory Science, Office of Clinical Pharmacology, Office of Translational Sciences, Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, MD 20993, USA.
Division of Applied Regulatory Science, Office of Clinical Pharmacology, Office of Translational Sciences, Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, MD 20993, USA.
J Chromatogr B Analyt Technol Biomed Life Sci. 2019 Sep 15;1126-1127:121765. doi: 10.1016/j.jchromb.2019.121765. Epub 2019 Aug 13.
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.
本研究旨在开发并验证一种可同时定量检测三种雌二醇代谢物(即雌二醇 3-β-D-葡糖苷酸(E3G)、雌二醇 17-β-D-葡糖苷酸(E17G)和雌二醇 3-硫酸盐(E3S))的高通量 UHPLC-MS/MS 方法,以支持诱导多能干细胞(iPSC)衍生肝细胞代谢功能的表征。为了实现这一目标,采用简单的蛋白沉淀法进行样品净化。所有代谢物均采用 C-18 柱进行色谱分离,其中 10mM 乙酸铵和乙腈在 4min 内以梯度方式流动。采用三重四极杆质谱仪,利用同位素标记内标(IS)定量分析。该方法按照美国食品和药物管理局的生物分析方法验证、行业指南进行验证。E3G 和 E17G 的线性范围为 2-1500ng/mL,而 E3S 的线性范围为 0.3-500ng/mL。该方法的日内和日间准确度和精密度均在可接受范围内。此外,还对细胞培养介质中所有代谢物进行了多次稳定性测试(冻融、自动进样器、水浴(37°C)、台式和长期稳定性)。所有代谢物在 -20°C 和 -80°C 下可稳定 3 个冻融循环,在自动进样器中 48h,37°C 下 24h,室温下 48h,-20°C 下 173 天。代谢物的提取回收率具有重现性,范围为 94-108%。该方法用于定量检测 iPSC 肝细胞体外研究中生成的雌二醇代谢物。