Development and Validation of Quantification Method for Fluconazole in Human Serum Using Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry

Abstract

Fluconazole is one of the commonly prescribed antifungal with recommendation for therapeutic level monitoring to ensure its levels are within therapeutic range to reduce the risk of adverse effects, toxicity of this otherwise safe drug and to prevent occurence of resistant fungi. A sensitive and robust method was developed to quantitate the level of fluconazole in human serum using ultra-high performance liquid chromatography-tandem mass spectrometry. Using Agilent SB-C18 column (50 x 2.1mm, 1.8 µm particle size), fluconazole was eluted separately from the internal standard using mobile phase (A) 0.1% formic acid in water and (B) methanol. The flow rate was set at 0.4 mL/min in positive ionization mode. The method was linear from 156.25 ng/mL to 5,000 ng/mL (r2 = 0.999) with the recovery of 80-90%, and the accuracy and precision of 89-101% and 0-10%, respectively. Fluconazole was also proven stable under different test conditions with the accuracy of 87-114%. The developed method met the guidelines drawn by the European Medicines Agency, Food and Drug Administration and International Council of Harmonisation. The method used for purification of samples prior to injection was user-friendly and robust.