Separation of triacylglycerols containing allenic and acetylenic fatty acids by enantiomeric liquid chromatography-mass spectrometry

Abstract

Triacylglycerols (TAGs) containing less common fatty acids (FAs) were isolated from the seeds of three plants (Santalum album, Crepis foetida, and Leucas aspera). These FAs had allenic (laballenic acid, Lb) and acetylenic (crepenynic, C; ximenynic acids, Xi) bonds. TAGs were analyzed on reversed-phase and chiral columns. High-resolution tandem mass spectrometry identified TAGs by positive electrospray ionization (ESI+). Twenty-two molecular species of TAGs isolated from the seed oil of Santalum album were separated by RP-HPLC and chiral HPLC methods and identified by positive electrospray ionization tandem MS detection (ESI+-MS). Two major enantiomers, i.e., sn-OOLb and sn-LLLb (O represents oleic acid; and L represents linoleic acid), were synthesized from the appropriate phosphatidylcholines. This allowed the identification of enantiomers after separation by chiral chromatography by tandem mass spectrometry. Similarly, TAGs from the seeds of Crepis foetida, and Leucas aspera were analyzed by reversed-phase chromatography and identified by mass spectrometry. Four enantiomers (sn-OOC, sn-LLC, sn-OOXi, and sn-LLXi) were synthesized. A total of six and three enantiomers of TAGs containing crepenynic and ximenynic acids, respectively, were identified by chiral column analysis. The retention times of TAGs containing allenic and acetylenic bonds were always greater on the reversed-phase column than TAGs with the same number of carbon atoms and the same unsaturation (e.g., LLL versus LLLb). From the chiral column, the regioisomers and enantiomers were eluted in the order of symmetric-asymmetric-asymmetric (i.e., sn-OCO, sn-COO, and sn-OOC). Through tandem mass spectrometry, we were able to identify and distinguish regioisomer [DAG]+-type ions, i.e., [MNH4NH3RCOOH]+, that can be considered diagnostic. Unfortunately, enantiomers and TAGs with the same numbers of carbon atoms and the same unsaturation levels have identical mass spectra, such as LLL and LLLb.