Development and Validation of a New Ultra-fast HPLC Method for Quantification of Levofloxacin in Rabbit Aqueous Humour: Application to a Pharmacokinetic Study

A specific, sensitive, reliable, low-cost, isocratic, ultra-fast HPLC-DAD for Levofloxacin quantification in rabbit aqueous humor was developed, validated and applied to a pharmacokinetic study, conducted on commercial formulation of Levofloxacin. Separations were obtained on a XB-C18 column (100A, 100 mm × 4.60 mm, 2.6 μm, Phenomenex) with an isocratic mobile phase consisting of 18% acetonitrile and 82% triethylamine 0.5% in water (pH adjusted to 2.5 with H3PO4) at a flow rate of 0.5 ml/min. Detection of levofloxacin was done at 292 nm, and the column temperature was 40°C. The total analysis run-time was 5 min per sample, and no interfering peaks from aqueous humor were detected. Method was found to be selective, linear (R2=0.99984), accurate (intra-day recovery, 98.24%-100.04%) and precise (RSD, ≤ 5.50%). Pharmacokinetic parameters in rabbit aqueous humors were calculated by PKSolver add in program and are in agreement with literature data. The outlined method can be efficiently applied for monitoring levofloxacin in aqueous humor in pharmacokinetics studies.