Partial three-dimensional chromatography of pulse triacylglycerols with triple parallel mass spectrometry

Authors: Byrdwell, W Craig; KOTAPATI, HARI - University Of Maryland

Submitted to: Separations
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/22/2023
Publication Date: 12/5/2023
Citation: Byrdwell, W.C., Kotapati, H.K. 2023. Partial three-dimensional chromatography of pulse triacylglycerols with triple parallel mass spectrometry. Separations. 10: 594. https://doi.org/10.3390/separations10120594.
DOI: https://doi.org/10.3390/separations10120594

Interpretive Summary: Fats and oils can be extremely complex, with hundreds of individual fats, called triacylglycerols, TAGs. A thorough and detailed analysis of the TAGs in fats and oils sometimes requires a better separation than can be accomplished using normal separation methods. For such samples, a multi-dimensional separation using multiple instruments joined together into a single analysis can provide the extra separation necessary to identify the many molecular species. To address this difficulty, we developed a three-dimensional (3D) separation system that partially separates molecules in the first dimension, and then further pulls them apart using two different second dimensions that employ different mechanisms to separate molecules based on different characteristics. We demonstrated the 3D separation by analyzing the oils extracted from legume seeds, called pulses, so we could compare the new results to previous results for these same samples. Using this 3D method we were able to determine the individual components in the oils of ten pulses: baby lima beans, black beans, black-eyed peas, butter beans, cranberry beans, garbanzo beans, green split peas, lentils, navy beans, and pinto beans. The compositions of pulses clustered into three groups having similar compositions.

Technical Abstract: We analyzed ten pulses (dried seeds of legumes), baby lima beans, black beans, black-eyed peas, butter beans, cranberry beans, garbanzo beans, green split peas, lentils, navy beans, and pinto beans, using three-dimensional liquid chromatography (3D-LC) with parallel second dimensions, LC × (LC + LC). We have combined non-aqueous reversed-phase (NARP) chromatography as the first dimension separation, 1D, with argentation UHPLC for separation based on degree of and location of unsaturation in the first second dimension, 2D(1), and multi-cycle NARP-UHPLC in the second second dimension, 2D(2). Pulses contained 1.9% to 2.7% lipids, except garbanzo beans, which contained 6.2% lipids. High-resolution, accurate-mass (HRAM) orbitrap mass spectrometry (MS) was used to perform lipidomic analysis of the 2D(1) and percent relative quantification, showing that the most abundant average triacylglycerol (TAG) molecular species across all pulses were PLL at 10.67% and PLLn at 10.45%. Common beans (Phaseolus vulgaris) clustered together by principal component analysis (PCA), showing the highest levels of linolenic acid, C18:3, in mo-lecular species such as PLnLn, LLnLn, and OLLn, with palmitic (P), C16:0, linoleic (L), 18:2, and oleic (O), 18:1, FAs. Calibration curves made from interweaved sets of regioisomer standards allowed absolute quantification of 1,2- and 1,3-regioisomers for a subset of TAGs.