Validation of rapid and simple LC–MS/MS method for determination of voriconazole in rat plasma

A rapid, simple and sensitive LC–MS/MS analytical method was developed and validated for the determination of voriconazole (VRC) in rat plasma, using ketoconazole as internal standard (IS). Analysis was performed on a Shimadzu® HPLC system using a Shimadzu® C18 column and isocratic elution with acetonitrile–water–formic acid (60:40:0.05, v/v/v), at a flow of 1.0 mL/min (split ratio 1:5), and a mass spectrometer Micromass®, equiped with a double quadrupole and an electrospray ionization interface, operated in a positive mode. Plasma samples were deproteinized with methanol (1:2) and 30 μL of the supernatant was injected into the system. The retention times of VRC and IS were approximately 3.3 and 2.7 min, respectively. Calibration curves in spiked plasma were linear over the concentration range of 50–2500 ng/mL with determination coefficient >0.98. The lower limit of quantification was 50 ng/mL. The accuracy of the method was within 5%. Intra- and inter-day relative standard deviations were less or equal to 12.5 and 7.7%, respectively. The applicability of the LC–MS–MS method for pharmacokinetic studies was tested using plasma samples obtained after intravenous administration of VRC to male Wistar rats. The reported method provided the necessary sensitivity, linearity, precision, accuracy, and specificity to allow the determination of VRC in pre-clinical pharmacokinetic studies.