Retention Behavior of Nitrogen-Containing Heterocyclic Compounds in Reversed-Phase and Hydrophilic HPLC–MS

Aromatic five- and six-membered nitrogen-containing heterocyclic compounds are biologically active substances widely used in biochemistry and medicine. In addition, such compounds are known as ecotoxicants formed during oxidative processes in industrial wastewaters. High-performance liquid chromatography–mass spectrometry has been widely used to determine nitrogen-containing heterocycles in various complex mixtures; octadecyl silica is used as the main sorbent for chromatographic separation. However, with octadecyl silica, satisfactory separation cannot be achieved in all cases when highly polar isomeric molecules are present, and more selective chromatographic approaches are needed. One such approach is hydrophilic chromatography, which enables the separation of polar compounds. The aim of this work was to characterize and compare the retention of several five- and six-membered nitrogen-containing heterocyclic compounds in reversed-phase and hydrophilic liquid chromatography modes coupled with mass spectrometric detection. Additionally, spectrophotometric measurements were carried out at a wavelength of 210 nm. Octadecyl- and bare silica adsorbents were used to achieve the aim of the work. High-resolution mass spectrometric detection in the electrospray ionization mode was performed. Nineteen nitrogen-containing heterocyclic compounds were studied. Formic acid and diethylamine were chosen as acid–base additives to aqueous acetonitrile mobile phases. Ammonium formate was used as an additive in the hydrophilic chromatography mode. In the reversed-phase chromatography mode, it was shown that the injection of a 0.1% formic acid aqueous solution improved the ionization and signal intensity of the mass spectrometer, but not always allow achieving sufficient isomer separation in elution from the octadecyl silica surface. The injection of a 0.01% diethylamine solution does not result in a pronounced improvement in the separation compared to the mobile phase without modifiers. The use of bare silica in the hydrophilic chromatography mode allows the separation of isomeric five-membered heterocycles by obtaining narrow symmetric peaks but does not lead to the selective separation of isomeric diazines. Thus, the retention of electrospray-ionized nineteen nitrogen-containing heterocyclic compounds in reversed-phase HPLC and HILIC modes was investigated in this work. It was found that hydrophilic chromatography on bare silica allows the satisfactory separation of isomeric five-membered heterocyclic compounds of different classes. For the separation of six-membered heterocycles, satisfactory separation is achieved by elution from the surface of octadecyl silica using an aqueous acetonitrile mobile phase without modifiers.