Codetermination of antimicrobial agents in rabbit tear fluid using LC–MS/MS assay: Insights into ocular pharmacokinetic study

Managing ocular microbial infections typically requires pharmacotherapy using antibiotic eye drops, such as moxifloxacin hydrochloride (MFX), combined with an antifungal agent like amphotericin B (AB). We carried out and validated an LC–MS/MS assay to quantify these compounds in rabbit tear fluid in order to look into the pharmacokinetics of these two drugs. We employed a protein precipitation technique for the extraction of drugs under examination. A Waters Symmetry C₁₈ column was used to separate the analytes and internal standard. The composition of the mobile phase was like (A) 0.1% v/v formic acid in water and (B) methanol. The detection of MFX and AB was accomplished through the utilization of positive ion electrospray ionization under multiple reaction monitoring mode. The linearity curves for both analytes exhibited an acceptable trendline across a concentration range of 2.34–300 ng/mL for MFX and 7.81–1000 ng/mL for AB in surrogate rabbit tear fluid. The lower limit of quantitation for MFX was 2.34 ng/mL, while for AB, it was 7.81 ng/mL. The approach was strictly validated, encompassing tests of selectivity, linearity (with r² > 0.99), precision, accuracy, matrix effects, and stability. Consequently, we employed this method to evaluate the pharmacokinetics profiles of MFX and AB in rabbit tear fluid following single topical doses.