Ranasinghe Asoka, Ramanathan Ragu, Jemal Mohammed, D'Arienzo Celia J, Humphreys W Griffith, Olah Timothy V
Bristol-Myers Squibb, Research & Development, Bioanalytical & Discovery Analytical Sciences, Princeton, NJ 08543, USA.
Bioanalysis. 2012 Mar;4(5):511-28. doi: 10.4155/bio.12.13.
UHPLC coupled with orthogonal acceleration hybrid quadrupole-TOF (Q-TOF)-MS is an emerging technique offering new strategies for the efficient screening of new chemical entities and related molecules at the early discovery stage within the pharmaceutical industry. In the first part of this article, we examine the main instrumental parameters that are critical for the integration of UHPLC-Q-TOF technology to existing bioanalytical workflows, in order to provide simultaneous quantitative and qualitative bioanalysis of samples generated following in vivo studies.
MATERIAL & METHODS: Three modern Q-TOF mass spectrometers, including Bruker maXis™, Agilent 6540 and Sciex TripleTOF™ 5600, all interfaced with UHPLC systems, are evaluated in the second part of the article. The scope of this work is to demonstrate the potential of Q-TOF for the analysis of typical small molecules, therapeutic peptides (molecular weight <6000 Da), and enzymatically (i.e., trypsin, chymotrypsin and pepsin) cleaved peptides from larger proteins.
RESULTS & DISCUSSION: This work focuses mainly on full-scan TOF data obtained under ESI conditions, the major mode of TOF operation in discovery bioanalytical research, where the compounds are selected based on their pharmacokinetic/pharmacodynamic behaviors using animal models prior to selecting a few desirable candidates for further development. Finally, important emerging TOF technologies that could potentially benefit bioanalytical research in the semi-quantification of metabolites without synthesized standards are discussed. Particularly, the utility of captive spray ionization coupled with TripleTOF 5600 was evaluated for improving sensitivity and providing normalized MS response for drugs and their metabolites. The workflow proposed compromises neither the efficiency, nor the quality of pharmacokinetic data in support of early drug discovery programs.
超高效液相色谱（UHPLC）与正交加速混合四极杆-飞行时间（Q-TOF）质谱联用是一种新兴技术，为制药行业早期发现阶段高效筛选新化学实体及相关分子提供了新策略。在本文的第一部分，我们研究了对于将UHPLC-Q-TOF技术整合到现有生物分析工作流程至关重要的主要仪器参数，以便对体内研究后产生的样品进行同时定量和定性生物分析。
本文第二部分评估了三台与UHPLC系统联用的现代Q-TOF质谱仪，包括布鲁克maXis™、安捷伦6540和Sciex TripleTOF™ 5600。这项工作的范围是证明Q-TOF在分析典型小分子、治疗性肽（分子量<6000 Da）以及从较大蛋白质经酶解（即胰蛋白酶、糜蛋白酶和胃蛋白酶）得到的肽方面的潜力。
这项工作主要集中在电喷雾电离（ESI）条件下获得的全扫描TOF数据，这是发现型生物分析研究中TOF操作的主要模式，即在使用动物模型根据药代动力学/药效学行为选择化合物，然后再挑选一些理想的候选物进行进一步开发之前。最后，讨论了可能有益于生物分析研究中无需合成标准品进行代谢物半定量的重要新兴TOF技术。特别是，评估了与TripleTOF 5600联用的俘获喷雾电离在提高灵敏度以及为药物及其代谢物提供标准化质谱响应方面的效用。所提出的工作流程既不影响支持早期药物发现项目的药代动力学数据的效率，也不影响其质量。
