An LC–MS/MS method for the determination of ofloxacin in 20 μl human plasma

A sensitive and selective liquid chromatography–tandem mass spectrometry method was developed and validated for the determination of ofloxacin in 20 μl human plasma over the concentration range of 0.078–20 μg/ml. Sample preparation was achieved by protein precipitation with acetonitrile and methanol containing the internal standard (Gatifloxacin). Chromatographic separation was achieved on a Luna 5 μm PFP (110 A, 50 × 2 mm) column with acetonitrile and water containing 0.1% formic acid (50:50, v/v) as the mobile phase, at a flow rate of 400 μl/ml. The within-day and between-day precision determinations for ofloxacin, expressed as the percentage coefficient of variation, were lower than 7% at all test concentrations. Recovery of ofloxacin was greater than 70% and reproducible at the low, medium and high end of the dynamic range. No significant matrix effects were observed for the analyte or internal standard. The assay was successfully used to examine the pharmacokinetics of ofloxacin as part of a study to characterize the pharmacokinetics of a number of anti-tuberculosis drugs utilized in the treatment of multi-drug resistant tuberculosis (MDR-TB).