Abstract
A rapid and sensitive reverse phase high performance liquid chromatography method was developed for assay of fluconazole in olive oil based nano-emulsions. Separation of fluconazole from excipients present in nanoemulsion formulation was achieved on high efficiency core shell, micro-bore reverse phase C18 column (50 x 2.1 mm, 2.6 mu) maintained at a temperature of 30 degrees C by column oven. Mobile phase containing 75 volumes of 10 mM potassium dihydrogen phosphate pH 3 adjusted with orthophosphoric acid and 25 volumes of methanol was introduced at a flow rate of 0.3 ml/minute. After a very short run time of 4-5 minutes, isocratic elution followed detection at 210 nm by UV-visible detector. A sharp and symmetric peak was obtained at the retention time of 2.9 minutes. The optimized method was validated for linearity, accuracy, precision, and robustness. The linear regression data for the calibration plot are indicative of a good linear relationship between peak area and concentration (r(2)= 0.996) over a wide concentration range (1 mu g/ml to 50 mu g/ml). Limit of detection and limit of quantification values were determined to be 0.10 and 0.5 mu g/ml, respectively. The overall accuracy of the method was 98.2% with RSD of 0.71% indicating the acceptable accuracy of the method. Precision of the method was evaluated at two levels i. e intraday precision (repeatability) and inter-day precision (intermediate precision) with overall RSD of all determinations less than 1% indicating the acceptable precision of the method. The developed and validated method was successfully applied for the quantification of fluconazole in olive oil based nano-emulsions.