Development and Validation of a Quantitative LC-MS/MS Analytical Method for the Simultaneous Detection of Isotretinoin and Nifedipine in Plasma: A Comprehensive Study on Calibration, Specificity, and Performance
Keywords:
Isotretinoin, Nifedipine, LC-MS/MS, Plasma, Analytical Method Validation, Calibration CurveAbstract
This study presents a highly sensitive and validated LC-MS/MS method for the simultaneous quantification of isotretinoin (IST) and nifedipine (NIF) in plasma. A systematic approach was employed, including preparation of stock solutions and calibration standards for both drugs. Analytical validation was performed following the International Council for Harmonisation (ICH M10) guidelines, with special emphasis on specificity, linearity, precision, and accuracy. Calibration curves were constructed using standard isotretinoin concentrations ranging from 2.5 ppb to 640 ppb, with NIF used as an internal standard (IS) to correct for potential matrix effects. The specificity of the method was confirmed by testing blank plasma samples and ensuring no interference at the retention times of IST and IS. The developed method demonstrated excellent linearity (R² > 0.99) across the concentration range, with precision values meeting the acceptance criteria for both intra- and inter-day variability. The method’s robustness was validated by analyzing plasma samples from a set of volunteer participants, with accurate recovery rates observed for both analytes. The optimized LC-MS/MS conditions included the use of a quaternary solvent system, Agilent Zorbax SB-C18 column, and positive electrospray ionization (ESI), with data acquisition facilitated by Mass Lynx software. This method provides a reliable, high-throughput analytical tool for monitoring isotretinoin and nifedipine concentrations in plasma, supporting both clinical and pharmaceutical research applications.
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