Yousra elmazahy*, Ahmed Farag, Lobna shalaby,Mohamed Nagy,
A fast and selective Reversed-Phase (RP) liquid chromatography-Diode Array (DAD) method was developed and validated to determine Voriconazole in human plasma. Thanks to the high throughput and sensitivity of UltraHigh-Performance Liquid Chromatographic (UHPLC) Chromatography, there were only 7.5 minutes of run time, and no buffers were used. 200 μl serum and 200 μl internal standard fluconazole were added. After simple protein precipitation with 400 μl methanol, 10 μl of the clear supernatant separated on Agilent Zorbax Eclipse Plus® C18 Column (50*2.1 mm, 1.8 μm). The column temperature was maintained at 40°C. The flow rate was 0.4 ml/min using a gradient elution of methanol and water. The retention time of Fluconazole and Voriconazole were 4.2 and 6.2 min, respectively. Detection was performed using a diode array detector set at a wavelength of 261 nm. The method was optimized and validated per FDA Guidance for Industry, Bioanalytical method validation. System suitability, in terms of theoretical plates, resolution, tailing and injection precision in terms of retention time and area, was performed to ensure the suitability and effectiveness of the chromatographic system as per ICH guidelines. The developed method provided outstanding recovery, accuracy, precision, selectivity, stability and reproducibility results. The calibration curve was linear over a range of 0.2-12 μg/ml with a correlation coefficient 0.9997c, adequately covering the therapeutic range for appropriate drug monitoring. The limit of detection and quantification were 0.2 μg/ml and 0.5 μg/ml, respectively. The intra-day accuracy and precision CVs were 91.2%-120% and 0.8%-6.8%, respectively, and the inter-day accuracy and precision were 92.6%-115% and 2.5% to 7.5%, respectively. Absolute recovery was 100% ± 15% for Voriconazole.