Screening of potentially genotoxic impurities in pharmaceuticals by LC-MS and CE-MS

Abstract

Every day we are at risk for exposure to toxic components present in the environment and in food. Also medicines may contain traces of potentially genotoxic impurities (PGI), resulting from residues of process impurities or degradation.The presence of well-defined functional groups in impurities is indicative for potential genotoxicity. Reaction of these components with DNA can result in genetic mutations with the risk of the development of cancer. Current guidelines require control of PGI in medicines down to a level of 1.5 μg per day, however these fully rely on a theoretical assessment on the presence of these components. If components were missed in the assessment, these are not covered in this control strategy. Analytical methods capable of screening unknown PGI at levels down to 1 mg.kg-1 are lacking. Taking use of the reactivity of the PGI an analytical screening strategy was developed for alkylating compounds to minimize subjectivity of the control strategy. In this thesis an analytical strategy for semi-targeted screening of potentially genotoxic alkylating compounds in pharmaceuticals at trace levels has been described. The approach is based on a selective derivatization of the alkylating compounds, i.e. alkyl halides and sulfonate esters. The characteristics of the derivatives are utilized for preconcentration, separation and detection. Derivatization was performed using 4-dimethylaminopyridine (DMAP) and a new reagent, butyl 1-(pyridinyl-4yl) piperidine 4-carboxylate (BPPC). Derivatization results in natively charged derivatives with a high sensitivity in MS detection. Generally a very similar retention behavior and response in detection is obtained for a wide range of PGIs allowing generic application of the methods. Moreover, a common marker is found in MS/MS detection. This does not only result in sensitive target analysis, i.e. down to 0.05 mg.kg-1, but also allows for selective semi-targeted screening of unknown PGI at levels down to 1 mg.kg-1. The application of the new reagent BPPC, designed to obtain fragmentation related to the loss of a side group in the reagent in addition to the loss of the complete reagent, resulted in an even more comprehensive and specific approach relative to the commercially available DMAP. Detection of PGIs can be hampered when co-elution with the excess of reagent or API takes place. In such case, the response of the derivatives may significantly decrease as a result of ionization suppression. As separation of the derivatives from the API is an important prerequisite, two highly selective orthogonal separation methods, using Hydrophilic interaction liquid chromatography (HILIC) and capillary electrophoresis (CE) were developed. Making optimally use of the characteristics of the derivatives, applying preconcentration by electrokinetic injection and effective separation using highly concentrated non-volatile background electrolytes, resulted in successful application of CE-MS for PGI analysis. The potential demonstrated for alkylation compounds may be extended to other classes of PGIs, and analysis of PGI in food and environmental samples.