Simultaneous quantification of gatifloxacin, moxifloxacin, and besifloxacin concentrations in cornea and aqueous humor by LC-QTOF/MS after topical ocular dosing.

The fourth-generation fluoroquinolones are widely used as ophthalmic antimicrobials. This study aimed to validate a new analytical technique for simultaneous quantification of gatifloxacin, moxifloxacin, and besifloxacin concentrations in the cornea and aqueous humor by liquid chromatography (LC) coupled to quadrupole time-of-flight mass spectrometry (QTOF/MS) at 10min and 1h after instillation of topical ophthalmic antimicrobial suspensions. It was used twenty-two male dogs without ocular lesions verified by ophthalmic and histologic examinations. Methanol:water (4:1) was used for the extraction procedure for cornea and acetonitrile:water (4:1) was used for aqueous humor. The chromatographic separations were carried out on a C18 column with a linear gradient of water and methanol, both containing 0.1% formic acid. The total chromatographic run time was 4min. Mass spectrometry analyses were performed on a Xevo™ G2-S QTof tandem mass spectrometer, operated in a positive ion electrospray ionization (ESI+) mode. The retention times were approximately 1.42min for gatifloxacin, 1.87min for moxifloxacin, and 3.01min for besifloxacin. No interference peak was detected for the three tested antimicrobials in samples obtained from both cornea and aqueous humor, ensuring that the peak response was exclusive to the analyte of interest. The limit of detection for the three antimicrobials was 0.11μg/mL and the limit of quantification was 0.42μg/mL for both cornea and aqueous humor samples. At both time points post instillation of the three antimicrobials, moxifloxacin had the highest corneal concentration and besifloxacin demonstrated the highest concentration in the aqueous humor.