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Zhang, Jun; Rodila, Ramona; Gage, Eric; Hautman, Mathew; Fan, Leimin; King, Linda L.; Wu, Huaiqin; El-Shourbagy, Tawakol A.
Analytica chimica acta, 02/2010, Letnik: 661, Številka: 2Journal Article
Simvastatin (SS) is an effective cholesterol-lowering medicine, and is hydrolyzed to simvastatin acid (SSA) after oral administration. Due to SS and SSA inter-conversion and its pH and temperature dependence, SS and SSA quantitation is analytically challenging. Here we report a high-throughput salting-out assisted liquid/liquid extraction (SALLE) method with acetonitrile and mass spectrometry compatible salts for simultaneous LC–MS/MS analysis of SS and SSA. The sample preparation of a 96-well plate using SALLE was completed within 20 min, and the SALLE extract was diluted and injected into an LC–MS/MS system with a cycle time of 2.0 min/sample. The seamless interface of SALLE and LC–MS eliminated drying down step and thus potential sample exposure to room or higher temperature. The stability of SS and SSA in various concentration ratios in plasma was evaluated at room and low (4 °C) temperature and the low temperature (4 °C) was found necessary to maintain sample integrity. The short sample preparation time along with controlled temperature (2–4 °C) and acidity (pH 4.5) throughout sample preparation minimized the conversion of SS → SSA to ≤0.10% and the conversion of SSA → SS to 0.00% The method was validated with a lower limit of quantitation (LLOQ) of 0.094 ng mL −1 for both SS and SSA and a sample volume of 100 μL. The method was used for a bioequivalence study with 4048 samples. Incurred sample reproducibility (ISR) analysis of 362 samples from the study exceeded ISR requirement with 99% re-analysis results within 100 ± 20% of the original analysis results.
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