Determination of gemifloxacin on dried blood spots by hydrophilic interaction liquid chromatography with fluorescence detector: application to pharmacokinetics in rats

Abstract

A highly selective, sensitive and rapid hydrophilic liquid interaction chromatographic method was developed and validated for determination of gemifloxacin on dried blood spots. The chromatographic separation was achieved on a reversed-phase zwitterionic hydrophilic interaction liquid chromatographic ZIC®HILIC-C₁₈ (4.6 × 100 mm; 5 µm) column using acetonitrile-10 mM ammonium acetate (pH 3.5; 80:20, v/v) as a mobile phase in an isocratic elution mode at a flow rate 0.6 mL/min at 27 °C. An on-line fluorescence detector set at excitation and emission wavelengths of 269 and 393 nm, respectively was used for monitoring column eluents. Ciprofloxacin was used as an internal standard. The method was validated for accuracy, precision, linearity and selectivity by design of experiments following ICH guidelines. The assay exhibited a linear range of 25-5000 ng/mL for gemifloxacin on dried blood spots. The lower limit of detection was found to be 10 ng/mL. The intra- and inter-assay coefficients of variation did not exceed 7.4% deviation of the nominal concentration. The recovery of GFX from dried blood spots was >95.0% and its stability was excellent with no evidence of degradation during sample processing for at least 3 months storage in a freezer at -20 °C.