Standardisation and validation of an HPLC method for determining serum posaconazole levels in Colombia

Background Colombia currently does not have a specialised service for measuring antifungal levels in serum, which is of prime importance for the proper treatment and correct management of invasive fungal infections. Aims To standardise and validate a simple, sensitive, and specific protocol, based on high performance liquid chromatography, complying with the parameters recommended by the Food and Drug Administration, to detect, identify, and quantify serum concentrations of posaconazole. Methods A high performance liquid chromatography Agilent series-1 200 equipment was used with ultraviolet diode array detector and analytical column-Eclipse XDB-C18. Posaconazole-SCH56592 (batch IRQ-PAZ-10-X-103) was used as the primary control and itraconazole (batch ZR051211PUC921) was used as an internal control. The validation was performed taking into account all criteria recommended by the Food and Drug Administration (selectivity, calibration curves, recovery, accuracy, precision, sensitivity, reproducibility, and stability of the sample). Results The most suitable chromatographic conditions were the following: column temperature 25 °C, ultraviolet detection at 261 nm, 50 μl injection volume, flow volume 0.8 ml/min, 10 min running time, mobile phase of acetonitrile:water (70:30), and final retention times of 3.4 and 7.2 min for posaconazole and itraconazole, respectively, with a wide and reliable quantification range (0.125 μg/ml to 16 μg/ml). Using these parameters, the method was selective, R² in the calibration curves was ≥ 0.99, and the percentage recovery was 98.7%, with a coefficient of variation less than 10%. The relative error for accuracy and the coefficient of variation for precision were less than 15%, all meeting the acceptance criteria recommended by the Food and Drug Administration. Conclusions The selectivity and chromatographic purity of the obtained signal, as well as the standardised limits of detection and quantification, make this method an excellent tool for therapeutic monitoring of patients treated with posaconazole.