Levofloxacin structural formula Levofloxacin assay in rabbit plasma: an UPLC method optimization and validation 23060 A. Sitovs (Rigas Stradins University) D. Kustovs (Rigas Stradins University) M. Giorgi (University of Pisa) L. Kovalcuka (Latvia University of Agriculture) L. Voiko (Latvia University of Agriculture) S. Purviņa (Rīga Stradiņš University, Faculty of Pharmacy, Department of Pharmacology) D. Bandere (Rīga Stradiņš University, Faculty of Pharmacy) INTRODUCTION AND THE AIM OF THE STUDY RESULTS Levofloxacin (LVX) is a highly effective synthetic antibacterial agent (third-generation fluoroquinolone) with great potential in veterinary field. The aims of the present study are to optimize the liquid chromatography (UPLC) bioanalytical assay method for LVX detection in rabbit plasma and to validate it according to the EMA Guideline on bioanalytical method validation. The method is rapid, selective and has good peak separation (LVX peak retention time 1.2 min, IS time 1.7 min) with no peaks due to matrix observed. LLOQ is 0.01 µg/ml. The calibration curve is linear in range from 0.01 to 5 µg/ml (R2 = 0.9978). The recovery of LVX was close to 100%. The method is precise (intra-day and inter-day CV values not exceed 15%) and accurate (bias % not exceed 15%). IS Levofloxacin structural formula LVX MATERIALS AND METHODS Levofloxacin and enrofloxacin (internal standard, IS) pure compounds’ solutions in methanol were used as stocks. Pooled drug-free rabbit plasma was used for method validation. The 6-point calibration curve was prepared at LVX concentrations of 0.01, 0.05, 0.1, 0.5, 1 and 5 µg/ml. 100 µl of 10 µg/ml IS methanol solution was added to each sample. LVX and the IS were extracted from plasma via liquid-liquid extraction using 4 ml of 5:1 chloroform:isopropanol (v/v) mixture (with consequent organic phase evaporation under nitrogen stream) and reconstituted in 200 µl of mobile phase. All standards prepared in triplicate. The mobile phase consisted of 83% aqueous phase (0.02M KH2PO4 and 0.012M tetraethylammonium bromide, pH=2.5) and 17% acetonitrile (v/v). Flow rate was set to 0.3 ml/min. Chromatographic separation was performed using Waters Acquity H-class system on Waters Acquity UPLC BEH C18 Column (75 x 2.1 mm, 1.7µm particle size) and fluorimetric detector (excitation and emission wavelengths 295 and 490 nm, respectively). Injection volume 1 µl, run time 5 minutes. The selectivity, accuracy, precision was established. Example of chromatogram representing 1 µg/ml LVX standard extracted from plasma CONCLUSION This UPLC analytical method is considered reliable and is already used for the quantification of LVX in rabbit plasma samples in pharmacokinetic studies.