Development and Validation of a High-Precision LC-MS/MS Method for the Quantification of Azithromycin in Human Plasma Using Imipramine as an Internal Standard: Analytical Performance and Application to Bioequivalence Studies
Keywords:
LC-MS/MS , azithromycin, bioequivalence, internal standardAbstract
This study presents the development and validation of a high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of azithromycin in human plasma, utilizing imipramine as an internal standard. The method was rigorously validated according to the ICH M10 guidelines, ensuring its specificity, precision, accuracy, and robustness for bioequivalence studies. The calibration curve was constructed over a concentration range from 5 ppb to 640 ppb, with a lower limit of quantification (LLOQ) of 5 ppb, and demonstrated excellent linearity (R² > 0.99). Precision (within-run and between-run) and accuracy were evaluated across low, medium, and high concentration levels, with coefficients of variation (CV%) consistently below 6%. The matrix effect was quantified, revealing minimal interference from plasma components, and the method displayed excellent stability under various conditions, including short-term, freeze-thaw, and long-term storage. The results of carry-over, specificity, and sensitivity tests affirmed the reliability of the method. Furthermore, the proposed method was successfully applied to a bioequivalence study of azithromycin, demonstrating reproducible pharmacokinetic profiles and supporting its suitability for clinical and regulatory applications. This analytical approach provides a highly sensitive, reproducible, and reliable tool for the pharmacokinetic analysis of azithromycin in plasma, facilitating bioequivalence assessments in drug development and clinical settings.
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