This study was aimed to develop a simple, sensitive RP-HPLC-UV method for the detection of fluconazole (FLZ) in rat plasma. The proposed method is selective, reproducible, reliable and highly sensitive. Chromatic separation of fluconazole and internal standard were achieved by carrying out on a octadecyl silane (ODS-3, www.glsciencesinc.com) Hypersil C18 column (250 mm × 4.6 mm × 5 µm) using water (pH 5.2, adjusted with orthophosphoric acid) and acetonitrile (80:20, v/v) as the mobile phase at a flow rate of 2.5 mL/min with detection at 260 nm. The drug sample was prepared by one-step liquid-liquid extraction (LLE) from rat plasma using sodium hydroxide and dichloromethane. Here methyl paraben (MP) (0.5 µg/mL) was used as internal standard (IS). The chromatographic mean retention times of drug and methyl paraben were less than 8 min and 14 minutes respectively. The calibration standards were prepared in the range of 0.1-20 µg/mL with an average coefficient of variation less than 0.05 %. The accuracy was within a range of 97.41- 99.33%. The lower limit of detection (LLOD) and quantification (LLOQ) was 0.0013 µg/mL and 0.013µg/mL respectively where no interferences detected in the chromatograms. The stability study of forced and non forced degradation was also performed with plasma, drug and internal standard to demonstrate the stability-indicating power of the method. This HPLC method was applied successfully to the pharmacokinetic study in rat after iv bolus injection and monitoring in receiving the fluconazole therapy.
Keywords: Fluconazole (FLZ), Methyl paraben (MP), Reverse phase- high performance liquid chromatography (RP- HPLC), Plasma pharmacokinetics, Validation