2023 FDA Science Forum
Analytical Method Validation of Metformin and Estrone-3-Sulfate for In Vitro Uptake Assays
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Contributing OfficeCenter for Devices and Radiological Health
Abstract
Metformin and estrone-3-sulfate are well-known probe substrates for renal (OCT2) and hepatic (OATP1B1/3, OAT3) transporters, respectively, and are utilized in experiments to predict clinical drug-drug interactions. This study was performed to develop and validate analytical methods using UV-HPLC and LC-MS/MS to quantify, with high accuracy and precision, metformin and estrone-3-sulfate, respectively. Both assays were optimized and validated for precision, accuracy, 37°C stability, freeze-thaw stability, benchtop stability, selectivity, and injection carry-over according to FDA’s 2018 bioanalytical method validation guidance. Linearity range of the analytical methods for metformin and estrone-3-sulfate were constructed according to the preliminary kinetic experiments in human embryonic kidney (HEK293) cells transfected with each uptake transporter. A UV-HPLC method for metformin was developed using a pentafluorophenyl C18 column. Metformin was detected with a high sensitivity at 280 nm. The lower and upper limit of quantitation were 800 nM and 11 µM, respectively. The results showed that the method was accurate (102.5 ± 3.5%; 103 ± 10%) and precise (<1%; <2%) for surrogate (10% acetonitrile in water, v/v) and cell-lysate matrices, respectively. A sensitive method for estrone-3-sulfate was developed using triple quadrupole LC-MS/MS with negative electrospray ionization operating in multiple reaction monitoring mode with an ion transition m/z 349.2 > 269.25 and 354.2 > 274.25 for estrone-3-sulfate and its internal standard, respectively. Linearity was established over a concentrate range of 3-150 nM using a BEH C18-Acquity UPLC column which provides excellent chromatographic separation with no interference from the cell-lysate matrix. The method was accurate (104.8 ± 1.7%; 106.5 ± 1%) and precise (<7%; <5%) for surrogate (same as above) and cell-lysate matrices, respectively. Surrogate matrix and cell-lysate matrix were diluted with an equal volume of methanol before injection. The analytical methods meet all predefined performance acceptance criteria for specificity, sensitivity, and all stability experiments. The validated methods will be utilized to optimize in vitro uptake assays in transfected cells and to screen different transporter inhibitors for drug interaction predictions.
