Blanz Joachim, Délémonté Thierry, Pearson David, Luneau Alexandre, Ritzau Michael, Gertsch Werner, Ramstein Philippe, Dayer Jérôme, Desrayaud Sandrine, Braun Elisabeth, Aichholz Reiner
Analytical Sciences & Imaging, Novartis Institutes for Biomedical Research, CH-4002 Basel, Switzerland.
Analytical Sciences & Imaging, Novartis Institutes for Biomedical Research, CH-4002 Basel, Switzerland.
J Chromatogr B Analyt Technol Biomed Life Sci. 2015 May 1;989:1-10. doi: 10.1016/j.jchromb.2015.02.044. Epub 2015 Mar 10.
LC-MS based drug metabolism studies are effective in the optimization stage of drug discovery for rapid partial structure identification of metabolites. However, these studies usually do not provide unambiguous structural characterization of all metabolites, due to the limitations of MS-based structure identification. LC-MS-SPE-NMR is a technique that allows complete structure identification, but is difficult to apply to complex in vivo samples (such as bile collected during in vivo drug metabolism studies) due to the presence, at high concentrations, of interfering endogenous components, and potentially also dosage excipient components (e.g. polyethylene glycols). Here, we describe the isolation and structure characterization of seven metabolites of the drug development candidate 1-isopropyl-4-(4-isopropylphenyl)-6-(prop-2-yn-1-yloxy) quinazolin-2(1H)-one from a routine metabolism study in a bile-duct cannulated rat by LC-MS-SPE. The metabolites were isolated from bile and urine by repeated automatic trapping of the chromatographic peak of each metabolite on separate Oasis HLB SPE columns. The micropreparative HPLC/MS was performed on an XBridge BEH130 C18 HPLC column using aqueous formic acid/acetonitrile/methanol as mobile phase for the gradient elution. Mass spectrometric detection was performed on a LTQ XL linear ion trap mass spectrometer using electrospray ionization. Desorption of each metabolite was performed after the separation sequence. NMR spectra ((1)H, (13)C, 2D ROESY, HSQC and HMBC were measured on a Bruker AVANCE III spectrometer (600 MHz proton frequency) equipped with a 1.7 mm (1)H{(13)C,(15)N} Bruker Biospin's TCI MicroCryoProbe™.
基于液相色谱-质谱联用（LC-MS）的药物代谢研究在药物发现的优化阶段对于快速鉴定代谢物的部分结构非常有效。然而，由于基于质谱的结构鉴定存在局限性，这些研究通常无法对所有代谢物进行明确的结构表征。液相色谱-质谱联用-固相萃取-核磁共振（LC-MS-SPE-NMR）技术能够实现完整的结构鉴定，但由于体内复杂样品（如体内药物代谢研究中收集的胆汁）中存在高浓度的干扰内源性成分以及潜在的剂量辅料成分（如聚乙二醇），该技术难以应用于此类样品。在此，我们描述了在胆管插管大鼠的常规代谢研究中，通过LC-MS-SPE从药物研发候选物1-异丙基-4-（4-异丙基苯基）-6-（丙-2-炔-1-基氧基）喹唑啉-2（1H）-酮中分离并鉴定了七种代谢物的结构。通过在单独的Oasis HLB固相萃取柱上反复自动捕获每种代谢物的色谱峰，从胆汁和尿液中分离出代谢物。在XBridge BEH130 C18高效液相色谱柱上进行微制备型高效液相色谱/质谱分析，以甲酸水溶液/乙腈/甲醇作为流动相进行梯度洗脱。使用电喷雾电离在LTQ XL线性离子阱质谱仪上进行质谱检测。在分离序列结束后对每种代谢物进行解吸。在配备1.7 mm 1H{13C，15N}布鲁克生物自旋公司的TCI低温探头的布鲁克AVANCE III光谱仪（质子频率600 MHz）上测量核磁共振谱（1H、13C、二维旋转坐标系相关光谱（2D ROESY）、异核单量子相干谱（HSQC）和异核多键相关谱（HMBC））。
