A sensitive, selective, accurate and precise ultra-high performance liquid chromatography-Tandem mass spectrometry (MS/MS) method was developed and validated for the simultaneous determination of drugs used as eye drops in cataract surgery in aqueous humor. Cataract surgery requires a powerful mydriatic eye drops combination such as cyclopentolate hydrochloride and phenylephrine hydrochloride to dilate the pupil and facilitate eye lens replacement and also requires strong fluoroquinolone antibiotic such as lomefloxacin hydrochloride. The method was performed with positive ion electrospray ionization and the analytes were quantified and monitored on a triple quadrupole mass spectrometer using multiple reaction monitoring scanning mode. Liquid-liquid extraction was used for the purification and preconcentration of analytes from rabbit aqueous humor matrix. Chromatographic elution was performed using an Phenomenex Luna® C18 (150 mm × 2.1 mm, 1.6 μm) column and moxifloxacin hydrochloride as internal standard with a mobile phase consisting of methanol:water:formic acid (70:29:1, by volume) at flow rate of 0.2 mL/min. Satisfactory results regarding linearity, recovery, stability, accuracy and precision of the analytes were obtained. Full validation of the procedure was performed according to the US Food and Drug Administration guidance for industry: bioanalytical method validation and European Medicines Agency (EMA) guideline on bioanalytical method validation.
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