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De Wolf K.,Ghent University | Balcaen L.,Ghent University | Van De Walle E.,Ghent University | Cuyckens F.,Johnson and Johnson Pharmaceutical RandD | Vanhaecke F.,Ghent University
Journal of Analytical Atomic Spectrometry | Year: 2010

In this study, a novel method was developed for tracing down reactive drug metabolites, the formation of which in the human body constitutes an important health risk as a result of their capability to bind to body proteins and DNA. Clozapine was used as a model because this drug forms both reactive and stable metabolites. Glutathione, which forms complexes with reactive metabolites, was added in order to trap reactive species of clozapine, formed by degradation in an electrochemical cell, thereby mimicking the real drug metabolism process. The method developed is based on the use of reversed-phase HPLC as a chromatographic separation technique and inductively coupled plasma-mass spectrometry (ICP-MS) for monitoring of Cl and S. Via Cl-monitoring, all metabolites of the Cl-containing clozapine can be detected, whereas S-monitoring allows for the detection of the S-containing molecule glutathione and its conjugates with reactive metabolites. The spectral overlap of the signals of 32S+ and 34S+ with those of 16O16O+ and 16O18O +, respectively, was tackled in 2 ways. On one hand, a quadrupole-based ICP-MS instrument, equipped with a dynamic reaction cell, was used. O2 was used as a reaction gas to convert the S+ ions to a sufficient extent into the corresponding SO+ species. This did not yield optimal results, due to pronounced ArC+ signals at mass 48 and 50 upon introduction of methanol into the ICP. On the other hand, a sector-field ICP-MS instrument operated at medium mass resolution permitted interference-free monitoring of the S+-signals. A new type of skimmer cone - termed X-skimmer - was evaluated and its use resulted in a 4-fold increase in the sensitivity in a methanolic environment, providing a limit of detection of 1 μg L-1 for S. The chromatograms obtained via HPLC-SF-ICP-MS permitted differentiation between reactive and stable metabolites. As a result, the method developed looks very promising for the detection of glutathione conjugates and shows potential for their quantification in early stages of drug development when a radiolabeled compound is not yet available. © 2010 The Royal Society of Chemistry. Source

Dearfield K.L.,U.S. Department of Agriculture | Thybaud V.,Sanofi S.A. | Cimino M.C.,U.S. Environmental Protection Agency | Custer L.,Bristol Myers Squibb | And 16 more authors.
Environmental and Molecular Mutagenesis | Year: 2011

Appropriate follow-up actions and decisions are needed when evaluating and interpreting clear positive results obtained in the in vitro assays used in the initial genotoxicity screening battery (i.e., the battery of tests generally required by regulatory authorities) to assist in overall risk-based decision making concerning the potential effects of human exposure to the agent under test. Over the past few years, the International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute (HESI) Project Committee on the Relevance and Follow-up of Positive Results in In Vitro Genetic Toxicity (IVGT) Testing developed a decision process flow chart to be applied in case of clear positive results in vitro. It provides for a variety of different possibilities and allows flexibility in choosing follow-up action(s), depending on the results obtained in the initial battery of assays and available information. The intent of the Review Subgroup was not to provide a prescriptive testing strategy, but rather to reinforce the concept of weighing the totality of the evidence. The Review Subgroup of the IVGT committee highlighted the importance of properly analyzing the existing data, and considering potential confounding factors (e.g., possible interactions with the test systems, presence of impurities, irrelevant metabolism), and chemical modes of action when analyzing and interpreting positive results in the in vitro genotoxicity assays and determining appropriate follow-up testing. The Review Subgroup also examined the characteristics, strengths, and limitations of each of the existing in vitro and in vivo genotoxicity assays to determine their usefulness in any follow-up testing. © 2010 Wiley-Liss, Inc. Source

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