All on one high-performance thin-layer chromatography plate: solvent-free nanomole-scaled on-surface synthesis, workup and online high-resolution mass spectrometry for elucidation of two new degradation products in an ifosfamide formulation

Abstract

On-surface reactions were introduced as a new strategy for rapid structure elucidation. This strategy is illustrated by the miniaturized synthesis-guided identification of two new degradation products (impurities) occurring in a pharmaceutical formulation of the anti-cancer drug ifosfamide, especially in the presence of urea. Synthesis on the silica gel surface bypassed the need for solvents, as the large nm-porous surface favoured a fast conversion of the reaction partners. For the on-surface synthesis of the impurities, the respective reagents were accurately and automatedly applied in the nanomole scale on a high-performance thin-layer chromatography (HPTLC) silica gel plate. After a fast reaction, the workup of the reaction mixture was performed by development of the HPTLC plate followed by online high-resolution mass spectrometry. As proof of concept and for benchmarking, a reaction mixture obtained from conventional preparative synthesis in a round-bottom flask was analysed in parallel as well as different formulations. The use of adsorbents as inert layer turned out to be highly efficient for a rapid generation and confirmation of impurities, as the synthesis on the HPTLC layer revealed them within 10 min. Image evaluation was simply performed by videodensitometry. The advantageous combination of all steps on one HPTLC plate and its resulting efficiency made surface synthesis on chromatographic phases an optimal tool for signal highlighting in mass spectrometry, and thus for the assignment of impurities in drugs. The chemistry process scale was miniaturized down to the μg-level per synthesis (in total 30-60 μg chemicals/reaction), setting a new state-of-the-art standard. All material savings clearly contribute to green chemistry, and this strategy substantially reduces the consumption of chemicals and greatly enhances the analytical efficiency, when adapted by scientists for the quality control of any other chemical product. The combination of synthesis, workup and detection in a miniaturized process, contributes to optimized workflows in a lean laboratory.