[Determination of ofloxacin in human plasma and studies of its pharmacokinetics using HPLC method]

Ofloxacin is a new broad-spectrum oral bactericidal antimicrobial agent. Its primary effect is the inhibition of bacterial DNA gyrase. This paper describes the development of a simple method for its determination using HPLC with UV detection. We used a Waters liquid chromatograph equipped with a Model 490 E multi-wavelength detector, a Model 510 pump and a U6K injector. The separation was performed on a Spherisorb C18 column (200 mm x 4.6 mm ID, 5 microns) with a mobile phase of methanol-0.01 mol/L phosphate buffer-0.5 mol/L tetrabutylammonium bromide (35:65:4, pH 2.50). The flow-rate was 1.0 ml/min and detection was at 294 nm. A specimen (0.2 ml) was spiked with the internal standard (norfloxacin) and deproteinized by adding 1.0 ml methanol. The precipitated mixture was shaken and then centrifuged at 3000 x g for 10 min, the supernatant was evaporated at 75 degrees C under a nitrogen stream. The residue was taken up with 0.4 ml of the mobile phase and 50 microliters aliquots were injected into the system. The minimal detectable concentration in plasma is 20 ng/ml. There is a linear relationship between the peak area ratio over the range of 0.5-4.0 micrograms/ml with r = 0.9999. The method has been applied to assay ofloxacin concentration in human plasma. The pharmacokinetic characteristics were studied.