Development and validation of a sensitive LC-MS/MS method for quantification of darifenacin in human plasma: application to pharmacokinetic and bioequivalence studies

Abstract

Background: Darifenacin is a selective muscarinic M3 receptor antagonist used in the management of overactive bladder syndrome. Accurate quantification of darifenacin in biological matrices is essential for pharmacokinetic studies, bioequivalence assessments, and therapeutic drug monitoring.

Objective: The present study aimed to develop and validate a rapid, sensitive, and robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of darifenacin in human plasma using liquid-liquid extraction.

Methods: Darifenacin and an appropriate internal standard were extracted from human plasma using liquid-liquid extraction technique. Chromatographic separation was achieved using a reverse-phase column with gradient elution. Detection was performed using electrospray ionization in positive ion mode with multiple reaction monitoring (MRM). The method was validated according to US FDA and ICH guidelines for bioanalytical method validation, encompassing selectivity, linearity, accuracy, precision, recovery, matrix effect, and stability parameters.

Results: The validated method demonstrated excellent linearity over the concentration range with correlation coefficient (r²) > 0.99. The lower limit of quantification (LLOQ) was adequate for pharmacokinetic studies. Intra-day and inter-day precision (% RSD) were within acceptable limits (< 15%). Accuracy ranged between 85-115% at HQC, MQC, LQC quality control levels and 80-120 % for LOQQC. Recovery was consistent and reproducible. The analyte remained stable under various storage and processing conditions including freeze-thaw cycles, bench-top stability, autosampler stability, and long-term storage.

Conclusion: A simple, sensitive, and validated LC-MS/MS method has been successfully developed for the quantification of darifenacin in human plasma. The method is suitable for application in pharmacokinetic and bioequivalence studies, meeting all regulatory requirements for bioanalytical method validation.