Validated Liquid Chromatography-Tandem Mass Spectroscopy Method for the Simultaneous Quantification of Four Antimycotic Agents in Human Serum

A liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous quantification of voriconazole, fluconazole, posaconazole, itraconazole, and hydroxyitraconazole in human serum. A simple protein precipitation was used as sample pretreatment with ketoconazole as the internal standard. Chromatographic separation was performed on a Waters Alliance 2795 liquid chromatography system using a XBridge RP18 column. The mass spectrometer from Micromass was equipped with an electrospray ionization probe operating in the positive mode using multiple reaction monitoring. The method was linear over the range of 0.01 to 5.00 mg/L for itraconazole, 0.01 to 5.00 mg/L for OH-itraconazole, 0.02 to 10.00 mg/L for voriconazole, 0.06 to 30.00 mg/L for fluconazol, and 0.02 to 10.00 mg/L for posaconazole. The between-and within-run accuracy ranged from 92% to 110%. The between-and within-run imprecision of the method was less than 11%. The reported method provided the necessary linearity, precision, and accuracy to allow the determination of four antimycotic agents for therapeutic drug monitoring and pharmacokinetic-pharmacodynamic studies.