Lim Heng-Keang, Chen Jie, Sensenhauser Carlo, Cook Kevin, Subrahmanyam Vangala
Drug Metabolism and Pharmacokinetics, Global Preclinical Development, Johnson and Johnson Pharmaceutical Research Institute, 1000 Route 202 South, Raritan, NJ 08869, USA.
Rapid Commun Mass Spectrom. 2007;21(12):1821-32. doi: 10.1002/rcm.3024.
Performance evaluation of accurate mass measurement by the LTQ/Orbitrap, at a resolving power of 60,000 and in external calibration mode, indicated that the Orbitrap is capable of providing high mass accuracy of <2 ppm for over 24 h post-calibration. This, together with limited trade-off between sensitivity and resolving power plus a wide dynamic range for mass accuracy, suggested that the LTQ/Orbitrap is an ideal analytical tool for structural elucidation of metabolites. The application of the LTQ/Orbitrap to identification of human liver microsomal metabolites of carvedilol was evaluated, using parent mass list triggered data-dependent multiple-stage accurate mass analysis, at a resolving power of 60,000 in external calibration mode. A metabolite identification workflow was developed to utilize chemical formulas from high-resolution accurate mass measurements to confirm structures of product ions of a drug proposed by Mass Frontier, illustrated by identification of structures used to establish lineage of product ions of carvedilol, which later served as a template for identification of its metabolites. A total of 58 in vitro metabolites of carvedilol were detected using 5-ppm mass tolerance filters for theoretical m/z of protonated molecules of predicted metabolites in addition to product ions and neutral mass losses diagnostic of carvedilol. The chemical formulas with unsaturation numbers calculated from the accurate m/z of precursor and product ions can be used to assign, with a high degree of confidence, the structures of metabolites and the sites of metabolism. The mass accuracies obtained for all full scan MS and MSn spectra were <2 ppm. The majority of the metabolites identified agreed with those previously reported except for those that have not been reported before. For example, several glutathione conjugates of carvedilol were reported for the first time, which may explain the reported hepatotoxicity during clinical trials and recent clinical use.
在分辨率为60,000且处于外部校准模式下，对LTQ/Orbitrap精确质量测量的性能评估表明，Orbitrap能够在校准后超过24小时内提供小于2 ppm的高质量准确度。这一点，再加上灵敏度和分辨率之间有限的权衡以及质量准确度的宽动态范围，表明LTQ/Orbitrap是用于代谢物结构解析的理想分析工具。使用母体质列表触发的数据依赖多阶段精确质量分析，在分辨率为60,000且处于外部校准模式下，评估了LTQ/Orbitrap在鉴定卡维地洛人肝微粒体代谢物中的应用。开发了一种代谢物鉴定工作流程，利用高分辨率精确质量测量得到的化学式来确认由Mass Frontier提出的药物产物离子的结构，以卡维地洛产物离子谱系结构的鉴定为例进行说明，该结构随后用作其代谢物鉴定的模板。除了卡维地洛诊断性的产物离子和中性质量损失外，使用5 ppm质量容差过滤器对预测代谢物质子化分子的理论m/z进行检测，共检测到58种卡维地洛的体外代谢物。根据前体离子和产物离子的精确m/z计算出的不饱和度的化学式，可用于高度自信地确定代谢物的结构和代谢位点。所有全扫描MS和MSn光谱获得的质量准确度均小于2 ppm。除了以前未报道过的那些代谢物外，鉴定出的大多数代谢物与先前报道的一致。例如，首次报道了几种卡维地洛的谷胱甘肽缀合物，这可能解释了临床试验和近期临床使用中报道的肝毒性。
