The retention features of nitrogen-containing heterocyclic compounds in reversed-phase and hydrophilic HPLC-MS modes
Abstract
Aromatic five- and six-membered nitrogen-containing heterocyclic compounds are biologically active substances and are widely used in biochemistry and medicine. In addition, such compounds are known as ecotoxicants formed during oxidation processes in industrial wastewater. High-performance liquid chromatography-mass spectrometry is widely used to determine nitrogen-containing heterocycles in various complex mixtures. Octadecyl silica gel is the main sorbent for chromatographic separation. However, octadecyl silica gel does not always enable satisfactory separation in the presence of highly polar isomeric molecules, and more selective chromatographic approaches are required. One of these is hydrophilic chromatography, which facilitates the separation of polar compounds.
The aim of the study was to comparatively characterize the retention of a number of five- and six-membered nitrogen-containing heterocyclic compounds during reversed-phase and hydrophilic liquid chromatography with mass spectrometric detection. In addition, spectrophotometric detection at a wavelength of 210 nm was carried out. In the study, we used sorbents based on octadecyl silica gel and unmodified silica gel. High-resolution mass spectrometry was used in the electrospray ionization mode. We studied 19 nitrogen-containing heterocyclic compounds. We used aqueous acetonitrile mobile phases with added aqueous solutions of formic acid and diethylamine as acid-base modifiers. An ammonium formate solution was used as a modifier during hydrophilic chromatography. Reversed-phase chromatography showed that the introduction of 0.1% aqueous formic acid solution increased the ionization and the mass spectrometer signal intensity, but did not always result in a satisfactory separation of isomers upon elution from the octadecyl silica gel surface. The introduction of 0.01% diethylamine solution provided no significant improvement in the separation compared to the mobile phase without modifiers. The use of unmodified silica gel in hydrophilic chromatography mode allowed us to separate isomeric five-membered heterocycles, which was demonstrated by narrow symmetric peaks, but did not result in selective separation of isomeric diazines.
Thus, we studied the retention of 19 nitrogen-containing heterocyclic compounds using reversed-phase and hydrophilic liquid chromatography with electrospray ionisation. It was determined that hydrophilic chromatography on unmodified silica gel could be used for satisfactory separation of isomeric five-membered heterocyclic compounds of different classes. For six-membered heterocycles, satisfactory separation was achieved by elution from the surface of octadecyl silica gel using aqueous acetonitrile mobile phase without modifiers.
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References
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