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Validation of an efficient LC-microdialysis method for gemifloxacin quantitation in lung, kidney and liver of rats

https://doi.org/10.1016/j.jchromb.2013.01.006Get rights and content

Abstract

A liquid chromatography method has been established for the reliable determination of unbound gemifloxacin concentrations in kidney, lung and liver microdialysates of rats. Microdialysis probes were inserted into tissues of rats, and then dialysates were collected at regular time intervals after intravenous administration of gemifloxacin (40 mg kg−1). A pilot study was performed to assess gemifloxacin penetration in lung, kidney and liver of rats. Gemifloxacin was separated on a C18 column eluted using triethylamine solution (0.5%, v/v), adjusted to pH 3.0 ± 0.1 with 85% phosphoric acid, methanol and acetonitrile (71:15:14, v/v/v) as mobile phase at a flow rate of 1.1 mL min−1. The fluorescence detector was set at excitation and emission wavelengths of 344 nm and 399 nm, respectively. The limit of quantitation was found to be 50 ng mL−1. Linearity was found to be over a concentration range of 50–2000 ng mL−1. The intra-assay and inter-assay precision and accuracy values were determined from the analysis of six quality control samples. The results obtained at three concentration levels showed R.S.D. values lower than 6.06% and 4.10% for repeatability and intermediate precision, respectively. The accuracy (R.E.%) ranged from 90.0 to 106.5%. The chromatographic run time of each sample was performed in 9 min. Drug stability in microdialysates was shown at room temperature for 8 h, after three freeze–thaw cycles, in freezer at −80 °C for 14 days, and in the autosampler after processing for 8 h. The relative recoveries determined by extraction efficiency (EE) and retrodialysis (RD) in vitro employing a flow rate of 1.5 μL min−1 were 29.24 ± 3.67% and 23.67 ± 3.31%, respectively. In vivo recoveries determined by RD in Wistar rats’ kidney, lung and liver were 27.69 ± 2.09%, 23.12 ± 3.79% and 17.38 ± 0.68%, respectively. The method was successfully applied to investigate tissue penetration of unbound gemifloxacin into the kidney, lung and liver of rats.

Highlights

► An efficient method for quantitation of unbound gemifloxacin concentrations is reported. ► Gemifloxacin was successfully determined in kidney, lung and liver of rats. ► Gemifloxacin unbound tissue concentrations were kidney > liver > lung.

Introduction

Gemifloxacin is a newer generation fluoroquinolone with suitable pharmacokinetic and pharmacodynamic properties to treat pulmonary diseases caused by Streptococcus pneumoniae, as well as atypical pathogens and Gram-negative respiratory ones [1], [2]. Many considerations derived from saliva, urine, tissue biopsies and indirect modeling of tissue concentrations from plasma curves act as surrogates for true target site concentrations. The in vivo assessment of drug distribution by microdialysis and target site pharmacokinetics has become to better obtain more realistic information from drugs [3]. Microdialysis can be used to acquire concentration variations of protein-free molecules located in interstitial or extracellular spaces and relies on the passive diffusion of analyte across a dialysis membrane [4].

Fluoroquinolones are well distributed in tissues, as seen in several articles published in the literature. Different techniques to determine gemifloxacin tissue distribution were employed, e.g. blister technique [5], tissue homogenate [6], [7], radiochemistry [8] and human-microdialysis [9]. Joukhadar et al. [9] investigated the free gemifloxacin concentrations in the interstitial space fluid of skeletal muscle and subcutaneous adipose tissue in human but no one has investigated the free gemifloxacin in the biophase of interest (lungs) yet. Tissue penetration of quinolones is variable among this class of antimicrobial drugs. For example, the penetration of ciprofloxacin was found to be 0.89 and 1.23 for subcutaneous adipose tissue and skeletal muscle, respectively, considering tissue and plasma levels [10]. Levofloxacin ratios between 0.85 in skeletal muscle and 1.1 in subcutaneous adipose tissue were also observed [11], [12]. So, it is important to determine the free levels in different tissues to better know the antimicrobial penetration. Membrane transport process such as an active influx and/or efflux also may contribute to tissue distribution of antimicrobials. The efflux pump (P-glycoprotein) can corroborate with different levels of antimicrobials into tissues and the investigation of free levels can be explained considering this observation.

In the present study, we employed microdialysis to investigate the potential of gemifloxacin to penetrate lung, kidney and liver tissues in healthy rats. To achieve this goal, an efficient HPLC method is described here. To our knowledge, there have been no reports of liquid chromatography method to determine free gemifloxacin in lung, liver and kidney of rats sampled by microdialysis.

Section snippets

Chemicals and reagents

Gemifloxacin mesylate (purity > 99.5%) was a donation from Aché Pharmaceutical Laboratory (São Paulo, Brazil). LC grade methanol, acetonitrile and triethylamine were purchased from Merck (Darmstadt, Germany). Other chemicals used in the experiment were of analytical reagent grade and purchased from commercial sources. Urethane was purchased from Sigma (St. Louis, USA). Distilled water was prepared using a Milli-Q water purification system from Millipore. Ringer's solution consisted of 149 mM NaCl,

Method validation

Representative chromatograms of blank Ringer's solution (a), rat dialysate spiked with gemifloxacin 500 ng mL−1 (b) and 0.5 h dialysate sample (c) from kidney (i), lung (ii) and (iii) liver of rat are presented in Fig. 1. The results indicated that there were no significant endogenous interferents at the retention time of gemifloxacin peak (around 6.6 min), showing the specificity of the method.

The linearity of the standard curves was checked in six different runs after calculating individual

Conclusion

An efficient, stable, precise, accurate and selective method with fluorescence detection for quantitation of unbound gemifloxacin concentrations was validated. The in vitro and in vivo performance of the microdialysis technique was established for the study of gemifloxacin. The method was successfully applied to investigate this anti-infective drug into the kidney, lung and liver of rats.

Acknowledgments

The authors would like to thank FAPERGS (Brazil) and University of Caxias do Sul for financial support. The authors also acknowledge Laboratory Aché (Brazil) for the donation of gemifloxacin.

References (15)

  • B. Roy et al.

    J. Pharm. Biomed. Anal.

    (2010)
  • B. Roy et al.

    J. Pharm. Biomed. Anal.

    (2010)
  • B.K. Yoo et al.

    Ann. Pharmacother.

    (2004)
  • R.C. Owewns et al.

    Diagn. Microbiol. Infect. Dis.

    (2005)
  • B.H. Westerink et al.

    Handbook of Microdialysis: Methods, Applications and Perspectives

    (2007)
  • T. Tsai

    Application of Microdialysis in Pharmaceutical Science

    (2011)
  • T. Gee et al.

    J. Antimicrob. Chemother.

    (2001)
There are more references available in the full text version of this article.

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