Reversed-phase liquid chromatography-mass spectrometry of complex mixtures of natural triacylglycerols with chloride-attachment negative chemical ionization

Abstract

Short chain triacylglycerols of butteroil and long chain triacylglycerols of menhaden oil were resolved by conventional reversed-phase high-performance liquid chromatography (HPLC) using a linear gradient of 10–90% propionitrile in acetonitrile as the mobile phase. The triacylglycerol species were identified by positive chemical ionization mass spectrometry, which provided [MH-RCOOH] + and [MH] + ions and by negative chemical ionization mass spectrometry with chloride attachment, which yielded exclusively the pseudomolecular ions [M + Cl] −. The negative ions were produced by the inclusion of 1% methylene chloride in the HPLC mobile phase, which did not affect the triacylglycerol elution profile. The restriction of ionization to the chloride-attachment pseudomolecular ions increased about 100-fold the sensitivity of detection of all molecular species and facilitated their quantitation by mass spectrometry. By combining the results of positive and negative chemical ionization mass spectrometry of the eluted peaks it was observed that the complex short chain triacylglycerols showed extensive resolution within isologous saturated carbon number while the polyunsaturated fish oil triacylglycerols showed readily detectable resolution within isologous carbon and double bond number☆. These observations permit the identification and quantitation of molecular species of triacylglycerols in such complex mixtures as butterfat and fish oil, which have thus far proved difficult or impossible.