QUANTIFICATION OF LEVOFLOXACIN IN HUMAN PLASMA BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY AND THE IMPACT OF THE ANTICOAGULANTS TYPE

Levofloxacin has low concentration in plasma, thus it requires sensitive and selective analysis method. Many kinds of anticoagulant are often used to obtain plasma as analytical matrix from whole blood. Citrate, heparin, and ethylenediaminetetraacetic acid (EDTA) are anticoagulant commonly used in analyzing drug in human plasma. This study was focused on analyzing levofloxacin in human plasma with three types of anticoagulants. The analysis was performed using High Performance Liquid Chromatography (HPLC) – photodiode array with column C18 SunfireTM (250 x 4.6 mm), 5 mm; temperature of 45°C, mobile phase consisting of 0.5% triethylamine pH 3.0 and acetonitrile (88:12 v/v); flow rate of 1.25 mL/minute, and ciprofloxacin HCl was used as internal standard. The method was linear at concentration range of 50.0 – 10.000.0 ng/mL with r>0.9994. Accuracy and precision for citrate, heparin, and EDTA plasma fulfilled the acceptance criteria of both intra-batch and inter-batch. There was no significant difference for stability and recovery of levofloxacin in citrate, heparin, and EDTA plasma (p>0.05; ANOVA), but it showed significant difference for peak area ratio (p<0.05), between citrate-EDTA plasma and heparin-EDTA plasma for low concentration and between citrate-heparin plasma and citrate-EDTA plasma for mid and high concentration. There was interference on retention time of less than 8 minutes in blank EDTA plasma, while on citrate and heparin plasma there was no interference. The method can be applied for pilot bioequivalence study using heparin as anticoagulant with six healthy subjects which were administered two 500 mg levofloxacin formulations.