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

Abstract

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 10 min and 1 h 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 4 min. 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 1.42 min for gatifloxacin, 1.87 min for moxifloxacin, and 3.01 min 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 0.11 μg/mL and the limit of quantification was 0.42 μg/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.

Introduction

Fluoroquinolones are antimicrobial drugs used worldwide for the treatment of infections in humans and animals. The pharmacological mechanism of action of fluoroquinolones involves binding to topoisomerase IV and gyrase in the presence of DNA, causing changes in the conformation of the formed protein. Quinolones are bacteriostatic agents because they prevent bacteria replication (Cheng et al., 2013, Aldred et al., 2014, Tse-Dinh, 2015). The fourth-generation quinolones are products of chemical modifications resulting in an extension of the antimicrobial spectrum of former generations to broad-spectrum activity covering gram-negative and gram-positive bacteria. These compounds are widely used for the prophylaxis and treatment of ocular infection (Mather et al., 2002, Scoper, 2008).

Fourth-generation fluoroquinolone ophthalmic solutions include gatifloxacin, moxifloxacin (McGee, Holt, Kastner, & Rice, 2005), and besifloxacin (Deschênes and Blondeau, 2015, Gu et al., 2016a). The ophthalmic suspension of besifloxacin, a more recent fluoroquinolone developed exclusively for ophthalmic use, contains a mucoadhesive polymer (Deschênes and Blondeau, 2015, Gu et al., 2016a, Chung et al., 2013) that may contribute towards its ability to maintain concentrations in both cornea and aqueous humor, perhaps by reducing elimination due to lacrimation.

For effective therapeutic action, these drugs must be present in certain concentrations at the target tissue. Intraocular penetration of the drug is hampered by defense mechanisms protecting the eyes – the two most important being the corneal barrier affecting drug permeability and lacrimal drainage of the drug (Samuelson, 2013, Nautscher et al., 2015). Drugs with high intraocular penetration and those that maintain adequate intraocular concentrations are considered the most effective topical ocular antimicrobials (Chung et al., 2013, Maggs, 2008).

Reported methods for measuring fluoroquinolones in ocular samples are high-performance liquid chromatography with fluorescence detection (FLD) (Basci et al., 1997, Davis et al., 2010, Matsuura et al., 2013) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Gu et al., 2016b, Proksch et al., 2009). Methods for measurement in plasma also include high-performance liquid chromatography with ultraviolet detection (HPLC-UV) (Cavazos-Rocha, Carmona-Alvarado, Vera-Cabrera, Waksman-de-Torres, & Salazar-Cavazos, 2014). To the best of our knowledge, liquid chromatography (LC) coupled to quadrupole time-of-flight mass spectrometry (QTOF/MS) has not been reported for determination of fluoroquinolones in ocular samples.

The aim of this study was to develop and validate an analytical method for the detection and quantification of gatifloxacin, moxifloxacin, and besifloxacin concentrations in cornea and aqueous humor by a LC-QTOF/MS employing QuanTOF technology. Another aim was to determine the concentrations of these antimicrobials in the cornea and aqueous humor of dogs, 10 min and 1 h after instillation of these topical ophthalmic suspensions.