Standardization and validation of a high-efficiency liquid chromatography with a diode-array detector (HPLC-DAD) for voriconazole blood level determination

Abstract

Introduction. A specialized service for antifungal blood level determination is not available in Colombia. This service is essential for the proper follow-up of antifungal therapies.Objective. To standardize and validate a simple, sensitive, and specific protocol based on high-performance liquid chromatography with a diode array detector for voriconazole blood level quantification.Materials and methods. We used an Agilent HPLC™ series-1200 equipment with a UVdiode array detector with an analytical column Eclipse XDB-C18 and pre-column Eclipse-XDB-C18 (Agilent). We used voriconazole as the primary control and posaconazole as an internal control. We performed the validation following the Food and Drug Administration (FDA) recommendations.Results. The best chromatographic conditions were: Column temperature of 25°C, UV variable wavelength detection at 256 nm for voriconazole and 261 nm for posaconazole (internal standard); 50 μl of injection volume, 0,8 ml/min volume flow, 10 minutes of run time, and mobile phase of acetonitrile:water (60:40). Finally, retention times were 3.13 for voriconazole and 5.16 minutes for posaconazole. Quantification range varied from 0.125 μg/ml to 16 μg/ml.Conclusion. The selectivity and chromatographic purity of the obtained signal, the detection limits, and the standardized quantification make this method an excellent tool for the therapeutic monitoring of patients treated with voriconazole.