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A new, simple, specific, accurate and precise reversed phase-high performance liquid chromatography (RP-HPLC) method was developed for the determination of levofloxacin in rat plasma and saliva was developed. An HPLC system based on a Phenomenex Luna C18Column (250 × 4.6 mm) and a UV detector (λ = 296 nm) were used. A mixture of Acetonitrile: water (80:20 v/v) adjusted to pH 3.5 by orthophosphoric acid at a flow rate of 1.4 ml/min was used as mobile phase. The proteins were precipitated with methanol. The average recovery was 94.79 and 92.66%, respectively in plasma and saliva. The detection limit for levofloxacin in plasma and saliva was 1 µg/ml. The calibration curve was linear over the concentration range 1 to 16 mg/ml for plasma and saliva. The inter-day and intra-day assay coefficients of variation were found to be less than 5%. The present validated method was successfully used for pharmacokinetic studies of levofloxacin in plasma and saliva.       Key words: High-performance liquid chromatography (HPLC), levofloxacin, plasma, saliva, pharmacokinetics.

Development and validation of RP-HPLC assay for levofloxacin in rat plasma and saliva: Application to pharmacokinetic studies