Separation of highly hydrophilic nicotinamide metabolites using a COSMOSIL PBr column

Highly hydrophilic compounds such as nicotinamide metabolites are very difficult to separate via high-performance liquid chromatography (HPLC) using octadecyl (C(18)) columns. In general, for the separation of hydrophilic compounds, hydrophilic interaction liquid chromatography (HILIC) columns are used instead of reversed phase chromatography using C(18) columns. However, HILIC columns generally obey complex separation mechanisms because ionic interactions are involved in the retention process, which hinders the optimization of the separation conditions. Additionally, the resulting peak shapes are disturbed when large amounts of aqueous samples are injected. This study demonstrates that COSMOSIL PBr columns, in which both hydrophobic and dispersive interactions occur, show high retention for various hydrophilic compounds under similar separation conditions as those used with C(18) columns. Specifically, using a COSMOSIL PBr column, 11 nicotinamide metabolites could be separated under simpler conditions than those used previously with C(18) columns, affording better peak shape for each compound. The applicability of the method was evaluated using a tomato sample, from which the nicotinamide metabolites were successfully separated. The results show that the COSMOSIL PBr column is a good alternative to the C(18) column for a good separation of all the peaks, including impurity peaks.