Analysis of quaternary amine compounds by hydrophilic interaction chromatography/mass spectrometry (HILIC/MS)

Hydrophilic interaction chromatography (HILIC) was explored for the separation of selected quaternary amine compounds of biological and environmental importance, including acetylcholine, choline, betaine, chlormequat, and mepiquat. The HILIC method was successful in separating the model quaternary amine compounds without the need for ion-pairing reagents. The present study indicates that the amide phase provides much stronger retention for the quaternary amine compounds than the aminopropyl phase, and also exhibits different selectivity toward the model compounds. Separation conditions including acetonitrile content, column temperature, buffer salt type, and concentration were found to have significant impact on the separation of the model compounds. In addition, the HILIC separation was coupled to a single quadruple mass spectrometer in this study, and an extremely low limit of detection of approximately 0.4 fmol for choline was achieved using selective ion monitoring. The liquid chromatography/mass spectrometry sensitivity of the hydrophilic interaction chromatography/mas spectrometry method was 75 times higher than that of the reversed-phase liquid chromatography/mass spectrometry method reported in the literature.