A Semiquantitative Method for the Determination of Reactive Metabolite Conjugate Levels in Vitro Utilizing Liquid Chromatography-Tandem Mass Spectrometry and Novel Quaternary Ammonium Glutathione Anal

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• 作者：John R. Soglia ; Leonard G. Contillo ; Amit S. Kalgutkar ; Sabrina Zhao ; Cornelis E. C. A. Hop ; James G. Boyd ; and Mark J. Cole
• 刊名：Chemical Research in Toxicology
• 出版年：2006
• 出版时间：March 2006
• 年：2006
• 卷：19
• 期：3
• 页码：480 - 490
• 全文大小：301K
• 年卷期：v.19,no.3(March 2006)
• ISSN：1520-5010

文摘

An in vitro semiquantitative reactive metabolite detection assay is described that incorporates NADPH-supplemented human liver microsomes, a novel quaternary ammonium glutathione analogue conjugatingagent (QA-GSH), and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for detection.The assay was developed to have high sample capacity and the potential for high sample throughput.MS/MS detection is selective and sensitive for the QA-GSH conjugating agent and semiquantitation ofQA-GSH-reactive metabolite conjugates is performed using QA-GSH standards added to samples priorto analysis [i.e., internal standards (ISs)]. The reactive metabolite trapping capability of the free thiolgroup in QA-GSH was assessed using model drugs acetaminophen, clozapine, and flutamide, which arebioactivated to afford reactive metabolites. MS signal responses of equimolar amounts of QA-GSHstandards were compared to assess the feasibility of using a QA-GSH IS approach to semiquantify reactivemetabolite levels in vitro. The full scan Q1 MS response for each standard was within 3.3-fold of oneanother even though the "parent" moiety structure of each QA-GSH conjugate standard differedsignificantly. Standard curve analysis using selected reaction monitoring for each QA-GSH standardgave slope values that differed by only 1.5-fold. The QA-GSH IS semiquantitation method was tested bydetermining the level of QA-GS-acetaminophen conjugate formation at three different concentrations ofacetaminophen and comparing the results to those from linear regression of authentic standards. Thecalculated levels of conjugate formed compared closely with those calculated from linear regression dataof authentic standard curves. These results show that the QA-GSH semiquantitation assay describedherein is a viable method for semiquantitatively assessing the bioactivation potential in vitro and is well-suited for use in early drug discovery high throughput screening paradigms.