Romański Michał, Teżyk Artur, Zaba Czesław, Główka Franciszek K
Department of Physical Pharmacy and Pharmacokinetics, Poznan University of Medical Sciences, 6 Święcickiego Street, 60-781 Poznań, Poland.
Department of Forensic Medicine, Poznan University of Medical Sciences, 6 Święcickiego Street, 60-781 Poznań, Poland.
Talanta. 2014 Sep;127:123-32. doi: 10.1016/j.talanta.2014.03.067. Epub 2014 Apr 4.
For the first time a high performance liquid chromatography method with tandem mass spectrometry detection (HPLC-MS/MS) was developed for simultaneous determination of a pro-drug treosulfan (TREO) and its active monoepoxide (S,S-EBDM) in biological matrices. Small volumes of rat plasma (50 μL) and the brain homogenate supernatant (100 μL), equivalent to 0.02 g of brain tissue, were required for the analysis. Protein-free TREO, S,S-EBDM and acetaminophen, internal standard (IS), were isolated from the samples by ultrafiltration. Complete resolution of the analytes and the IS was accomplished on Zorbax Eclipse column using an isocratic elution with a mobile phase composed of ammonium formate - formic acid buffer pH 4.0 and acetonitrile. Detection was performed on a triple-quadrupole MS via multiple-reaction-monitoring following electrospray ionization. The developed method was fully validated according to the current guidelines of the European Medicines Agency. Calibration curves were linear in ranges: TREO 0.2-5720 μM and S,S-EBDM 0.9-175 μM for plasma, and TREO 0.2-29 μM and S,S-EBDM 0.4-44 μM for the brain homogenate supernatant. The limits of quantitation of TREO and S,S-EBDM in the studied matrices were much lower in comparison to the previously used bioanalytical methods. The HPLC-MS/MS method was adequately precise (coefficient of variation≤12.2%), accurate (relative error≤8.6%), and provided no carry-over, acceptable matrix effect as well as dilution integrity. The analytes were stable in acidified plasma and the brain homogenate supernatant samples for 4 h at room temperature, for 4 months at-80°C as well as within two cycles of freezing and thawing, and demonstrated 18-24h autosampler stability. The validated method enabled determination of low concentrations of TREO and S,S-EBDM in incurred brain samples of the rats treated with TREO, which constitutes a novel bioanalytical application.
首次开发了一种采用串联质谱检测的高效液相色谱法（HPLC-MS/MS），用于同时测定生物基质中的前体药物苏消安（TREO）及其活性单环氧化物（S,S-EBDM）。分析所需的大鼠血浆体积小（50μL），脑匀浆上清液体积小（100μL），相当于0.02g脑组织。通过超滤从样品中分离出无蛋白的TREO、S,S-EBDM和对乙酰氨基酚（内标，IS）。在Zorbax Eclipse柱上采用等度洗脱，流动相由pH 4.0的甲酸铵-甲酸缓冲液和乙腈组成，实现了分析物和内标的完全分离。通过电喷雾电离后采用多反应监测在三重四极杆质谱仪上进行检测。根据欧洲药品管理局的现行指南对所开发的方法进行了全面验证。校准曲线在以下范围内呈线性：血浆中TREO为0.2 - 5720μM，S,S-EBDM为0.9 - 175μM；脑匀浆上清液中TREO为0.2 - 29μM，S,S-EBDM为0.4 - 44μM。与先前使用的生物分析方法相比，所研究基质中TREO和S,S-EBDM的定量限要低得多。HPLC-MS/MS方法具有足够的精密度（变异系数≤12.2%）、准确度（相对误差≤8.6%），且无残留、基质效应可接受以及稀释完整性良好。分析物在酸化血浆和脑匀浆上清液样品中于室温下4小时、-80°C下4个月以及冻融两个循环内均稳定，并在自动进样器中稳定18 - 24小时。经过验证的方法能够测定用TREO处理的大鼠脑样品中低浓度的TREO和S,S-EBDM，这构成了一种新的生物分析应用。
