Development and Validation of an LC-MS/MS Method for Quantitative Determination of Alprazolam in Human Plasma: A Bioequivalence Study

This study details the development and validation of a highly sensitive LC-MS/MS method for the quantification of Alprazolam in human plasma. The analytical procedure adhered to guidelines provided by regulatory authorities, including EMEA and ICH M۱۰, ensuring comprehensive validation across parameters such as specificity, accuracy, precision, carry-over, linearity, matrix effect, and stability. The method employs a Quattro Micro quadrupole mass spectrometer with an electrospray ionization (ESI) source in positive mode, coupled with an Agilent Zorbax SB-C۱۸ column. Alprazolam and an internal standard, Lorazepam, were monitored using multiple reaction monitoring (MRM) with transitions of ۳۰۸.۹۰→۲۸۱.۰۰ and ۳۲۰.۸۰→۲۷۵.۰۰, respectively. Calibration curves were linear over a concentration range of ۰.۵–۱۶ ppb (R² > ۰.۹۹). The lower limit of quantification (LLOQ) was determined to be ۰.۵ ppb, meeting the acceptance criteria for signal-to-noise ratio (S/N > ۱۰). Validation results demonstrated intra- and inter-day precision with RSD ≤ ۶.۸% and accuracy deviations ≤ ۶.۱%. Matrix effects, evaluated using plasma samples from six individuals, showed mean deviation factors within acceptable limits (<۱۵%). Short-term, freeze-thaw, and long-term stability studies confirmed the robustness of the analyte under various conditions. Application of the validated method to a bioequivalence study involved plasma sample analysis from healthy volunteers after administration of test and reference formulations. Pharmacokinetic parameters such as Cmax, Tmax, and AUC were computed, confirming bioequivalence based on predefined regulatory criteria. This validated LC-MS/MS method offers high precision, reliability, and sensitivity, making it suitable for bioequivalence studies and therapeutic drug monitoring of Alprazolam.

کلیدواژه ها:

Alprazolam ، LC-MS/MS ، Bioequivalence ، Plasma quantification ، Analytical method validation ، Pharmacokinetics ، Matrix effects ، Stability analysis