A semi-automated liquid chromatography-tandem mass spectrometry (LC/MS/MS) method was developed for the simultaneous quantification of the antifungal drug itraconazole (ITZ) and its coactive metabolite hydroxyitraconazole (OH-ITZ) in human plasma. The plasma samples underwent liquid-liquid extraction (LLE) in 2.2 mL 96 deepwell plates. ITZ, OH-ITZ and the internal standard (IS) R51012 were extracted from plasma, using a mixture of acetonitrile (ACN) and methyl t-butyl ether (MTBE) as the organic solvent. This specific mixture, due to its composition, had a significant impact on the performance of the assay. All liquid transfer steps, including preparation of calibration standards and quality control samples as well as the addition of the IS, were performed automatically using robotic liquid handling workstations for parallel sample processing. After vortexing, centrifugation and freezing, the supernatant organic solvent was evaporated. The analytes and IS were dissolved in a small volume of a reconstitution solution, an aliquot of which was analyzed by combined reversed phase LC/MS/MS, with positive ion electrospray ionization and a TurboIonSpray interface, using multiple reactions monitoring (MRM). The method was shown to be sensitive and specific to both ITZ and OH-ITZ, it revealed excellent linearity for the range of concentrations 2-500 ng mL(-1) for ITZ and 4-1000 ng mL(-1) for OH-ITZ, it was very accurate and it gave very good inter- and intra-day precisions. The proposed high-throughput method was employed in a bioequivalence study after per os administration of two 100 mg tablets of ITZ, and it allowed this study to be completed in under four days.

A fully automated high-throughput liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed for terbinafine quantification in human plasma. The plasma samples were treated by liquid-liquid extraction (LLE) in 2.2 mL 96-deepwell plates. Terbinafine and the internal standard (IS) N-methyl-1-naphthalenemethylamine were extracted from human plasma by LLE, using a mixture of methyl t-butyl ether (MTBE)-hexane (70:30, v/v) as the organic solvent. All liquid transfer steps, including preparation of calibration standards and quality control samples, as well as the addition of the IS, were performed automatically by using robotic liquid handling workstations. After vortexing, centrifugation and freezing, the supernatant organic solvent was evaporated and reconstituted in a small volume of a reconstitution solution. Sample analysis was performed by reversed-phase LC-MS/MS, with positive ion electrospray ionization, using multiple reaction monitoring (MRM). The method had a very short sample preparation time and a chromatographic run time of 2.2 min. It was proved to have excellent sensitivity, specificity, accuracy as well as inter- and intraday precision for the quantification of terbinafine in human plasma. The calibration curve was linear for the range of concentrations 5.0-2000.0 ng/mL. The proposed method was applied to the rapid and reliable determination of terbinafine in a bioequivalence study after per os administration of 250 mg tablet formulations of terbinafine.

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A semi-automated liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for the determination of glimepiride in human plasma. The plasma samples were treated by liquid-liquid extraction (LLE) in 1.2 mL 96-well format micro-tubes. Glimepiride and the internal standard (IS) glibenclamide were extracted from human plasma by LLE, using a mixture of ethyl acetate/diethyl ether 50:50 (v/v) as the organic solvent. After vortexing, centrifugation and freezing, the supernatant organic solvent was evaporated. The analyte and IS were dissolved in a small volume of a reconstitution solution, an aliquot of which was analyzed by reversed-phase LC/MS/MS with positive ion electrospray ionization, using multiple reaction monitoring. The method proved to be sensitive and specific for both drugs, and statistical evaluation revealed excellent linearity for the range of concentrations 2.0-500.0 ng/mL with very good accuracy and inter- and intra-day precisions. The proposed method enabled the rapid and reliable determination of glimepiride in pharmacokinetic or bioequivalence studies after per os administration of a 3 or 4 mg tablet of glimepiride.

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