A new derivatization reagent for LC-MS/MS screening of potential genotoxic alkylation compounds.

A screening method for trace analysis of potentially genotoxic alkylating compounds has been developed using butyl 1-(pyridin-4-yl) piperidine 4-carboxylate (BPPC) as a new, selective pre-column derivatization reagent for their subsequent analysis by hydrophilic interaction liquid chromatography (HILIC) hyphenated with tandem mass spectrometry (LC-MS/MS). The new derivatization reagent is a modification of 4-dimethylaminopyridine (4-DMAP) previously used for the determination of potentially genotoxic compounds. By using the new reagent the screening potential was enhanced without compromising reactivity. Derivatization at a high pH value was carried out and the reaction time at 60°C was 24h to anticipate for alkyl chlorides showing to be less reactive. The new reagent was designed to obtain reagent related fragmentation of the whole reagent as well as a side group of the reagent. Collision energies for detection of alkylating components derivatized using the new reagent are shown to be significantly more universal than with 4-DMAP. Neutral loss scanning on the fragmentation related to the build in side group remedies shortcomings in the screening for alkyl halides observed when using 4-DMAP. The new approach allows for screening of alkyl halides and alkyl sulfonates at trace levels down to 1 mg kg(-1) and target analysis at about a factor of 10 lower without a significant effect of the active pharmaceutical ingredient (API) matrix. The synthesis of the reagent, investigation of reactivity, the specificity of the fragmentation of derivatives and screening conditions in MS/MS analysis are described.

A new approach for generic screening and quantitation of potential genotoxic alkylation compounds by pre-column derivatization and LC-MS/MS analysis.

A generic LC-MS/MS method was developed for the analysis of potentially genotoxic alkyl halides. A broad selection of alkyl halides were derivatized using 4-dimethylaminopyridine in acetonitrile. The reaction conditions for derivatization, i.e., solvent, reaction time, temperature and concentration of alkyl halide, active pharmaceutical ingredient (API), and reagent, were optimized for sensitivity and robustness. The interference of the matrix and the API and the presence of water on the derivatization reaction were investigated for a model drug product (paracetamol/caffeine tablets). Hydrophilic interaction liquid chromatography was used to allow a quantitative determination of the derivatives by tandem mass spectrometry. The derivatization reaction was shown to be selective for alkyl halides, although some reactivity was also observed for an aromatic sulfonate, which is also genotoxic. Even though differences in reaction efficiencies have been observed, the enhanced sensitivity obtained by the derivatization allows the majority of the alkyl halides to be detected by MS/MS at relevant levels for genotoxic impurity evaluation, i.e., 10 mg kg(-1). Another key advantage is that for the majority of derivatives, reagent-related fragments are produced, which allows low-level screening for alkyl halides. Highly specific MS detection can be performed using neutral loss and precursor ion scan experiments. The applicability of a generic screening method will make the genotox evaluation less dependent on the quality of assessments based on predictions only, and it will provide essential information during the development of new chemical entities. In addition to screening, target analysis in the low milligrams per kilogram range can be performed. A similar response of the derivatized compounds was obtained in the range of 1-100 mg kg(-1) with a reproducibility better than 10%, which is sufficient for the determination of alkyl halides in APIs and drug products.