Kousoulos Constantinos, Tsatsou Georgia, Apostolou Constantinos, Dotsikas Yannis, Loukas Yannis L
Laboratory of Pharmaceutical Analysis and Bioequivalence Services (GLP Compliant), Department of Pharmaceutical Chemistry, School of Pharmacy, University of Athens, Panepistimioupoli Zografou, 157 71, Athens, Greece.
Anal Bioanal Chem. 2006 Jan;384(1):199-207. doi: 10.1007/s00216-005-0159-6. Epub 2005 Dec 7.
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.
建立了一种半自动液相色谱 - 串联质谱（LC/MS/MS）方法，用于同时定量测定人血浆中的抗真菌药物伊曲康唑（ITZ）及其活性代谢物羟基伊曲康唑（OH-ITZ）。血浆样品在2.2 mL 96孔深孔板中进行液 - 液萃取（LLE）。使用乙腈（ACN）和甲基叔丁基醚（MTBE）的混合物作为有机溶剂从血浆中萃取ITZ、OH-ITZ和内标（IS）R51012。由于其组成，这种特定混合物对测定性能有显著影响。所有液体转移步骤，包括校准标准品和质量控制样品的制备以及内标的添加，均使用机器人液体处理工作站自动进行，以实现平行样品处理。涡旋、离心和冷冻后，蒸发上清液中的有机溶剂。将分析物和内标溶解在少量重构溶液中，取一份等分试样通过反相LC/MS/MS联用进行分析，采用正离子电喷雾电离和TurboIonSpray接口，使用多反应监测（MRM）。该方法对ITZ和OH-ITZ均显示出灵敏性和特异性，在2 - 500 ng mL⁻¹的ITZ浓度范围和4 - 1000 ng mL⁻¹的OH-ITZ浓度范围内具有出色的线性，非常准确，并且具有很好的日间和日内精密度。所提出的高通量方法用于口服两片100 mg伊曲康唑片剂后的生物等效性研究，并使该研究能够在四天内完成。
