Quantification of ciprofloxacin in plasma and nasal mucosa matrices by high-performance liquid chromatography with fluorescence detection

J Sousa1,2, G Alves2,3, A Fortuna1,2, A Falcão1,2. 1University of Coimbra, Faculty of Pharmacy - 3000-548 Coimbra, Portugal, 2University of Coimbra, CNC - Center for Neurosciences and Cell Biology - 3004-517 Coimbra, Portugal, 3University of Beira Interior, CICS-UBI – Health Sciences Research Center - 6200-506 Covilhã, Portugal

Introduction: Fluoroquinolones are synthetic antibiotics that have gained a considerable interest over the last decades due to their broad spectrum of activity and improved pharmacokinetic properties. The high prevalence of upper respiratory tract infections has motivated preliminary studies of the use of topical nasal antibiotics for the treatment of acute and chronic rhinosinusitis (1). The intranasal administration is a promising route that enables direct drug delivery in biophase, improving therapeutic efficacy and minimizing systemic toxicity. Ciprofloxacin (CIP) is one of the most commonly prescribed fluoroquinolone agents and due to its excellent activity against H. influenza and M. catarrhalis it can be used in the treatment of rhinosinusitis.

This work aims at developing and validating high-performance liquid chromatographic methods with fluorescence detection (HPLC-FD) for the quantification of CIP in human plasma and in nasal mucosa.

Methods: Blank human plasma samples (500 µL) were spiked with known amounts of CIP and levofloxacin used as internal standard (IS). Sample preparation consisted in a single step deproteinization with 20% trichloroacetic acid. The supernatant was directly injected into the HPLC-FD system. Blank nasal tissue samples (approximately 20 mg) of CD-1 mice, as surrogate to blank human nasal matrix, were spiked with CIP and lomefloxacin (IS), homogenised and extracted with acetonitrile using an Ultra-Turrax disperser. After centrifugation, the supernatant was evaporated, reconstituted and injected into the HPLC-FD system. The chromatographic analysis was performed on a LiChroCART Purospher® Star (C18, 3µm, 55mm x 4mm) column with 0.1% aqueous formic acid (pH 3.0, triethylamine)-methanol (82:18, v/v) as mobile phase, pumped at 1.2 mL/min. The excitation/emission wavelengths were 278/450 nm for human plasma samples and 278/445 nm for nasal samples. The human plasma assay was fully validated according to the Food and Drug Administration guideline. Owing to the limited supply of blank human nasal samples and the invasive nature of the collection procedures, a partial validation in mice nasal mucosa was performed.

Results: In both assays, CIP and IS were separated within 8 min and no endogenous interferences were detected at their retention times. Calibration curves were linear (r2≥0.996) over the range of 0.02-5.0 µg/mL in plasma and 0.8-200 ng in nasal tissue. The overall intra and inter-day precision (% CV) did not exceed 7.07% for both matrices while the intra and inter-day accuracy (% bias) ranged from 1.10 to 9.16% in human plasma and 1.79 to 13.93% in nasal mucosa. Human plasma sample dilution (1/5) for concentrations above 5.0 µg/mL demonstrated to be precise and accurate. The overall mean relative recovery from human plasma was between 91.2 and 98.5% and the mean absolute recovery from nasal tissue ranged from 33.19 and 43.76%.

Conclusion: Selective, accurate and reproducible HPLC-FD methods were developed and validated for the quantification of CIP in plasma and nasal mucosa matrices. Hence, it is a useful tool to support pharmacokinetic studies of CIP in both matrices.

References: 1 - Sahin-Yilmaz A, et al. Otolaryngol Head Neck Surg. 138:3 (2008) 340-346.

Acknowledgements: FCT (SFRH/BD/69378/2010) and Portuguese Blood Institute, Portugal.