LC-Ion Trap-MS Method for the Determination of Fluconazole in Plasma for Bioequivalence Studies of Pharmaceutical Formulations Using Semi-Automated Sample Handling

Abstract

An LC–ion trap-MS method for the determination of fluconazole in human plasma has been developed to be applied for bioequivalence studies. The human plasma samples were extracted with methyl-tert-butyl ether. The extract was evaporated to dryness, reconstituted with mobile phase and injected into the LC–MS apparatus. Fluconazole d₄ was used as the internal standard (IS). A C₁₈ analytical column was used with a mobile phase consisting of 40% formic acid 0.1% and 60% acetonitrile. A robotic liquid handler was used to increase the method’s throughput. Fluconazole was detected by monitoring the mass transition of m/z 307.1 to m/z 238.0 (m/z 311.1 to m/z 242.0 for the IS). The method was validated according to the European Medicines Agency (EMEA) regulations and was found to be accurate, precise, and selective throughout the calibration range (0.05–10.0 µg/mL). Compared to the published methods for the determination of fluconazole in its usual therapeutic levels, the method provides the shortest sample runtime. For the first time, it was demonstrated that ion trap mass spectrometry can be used for the determination of fluconazole in plasma.

Keywords:

• bioequivalence studies
• fluconazole
• ion trap
• liquid chromatography
• plasma
• robotic liquid handler