Analysis of regioisomers of short-chain triacylglycerols by normal phase liquid chromatography–electrospray tandem mass spectrometry

Abstract

The regiospecific distribution of short-chain fatty acids in triacylglycerols (TAGs) influences their physico-chemical, nutritional, and organoleptic properties. This study demonstrates the applicability of normal phase HPLC with positive electrospray ionization mass spectrometry for the identification and structural analysis of regioisomers of short-chain triacylglycerols in nine interesterified mixtures of monoacid triacylglycerols. The finding that only one type of triacylglycerol adduct ion was formed without fragmentation or with minimal fragmentation was critical for liquid chromatography electrospray mass spectrometric quantification and identification of molecular species of TAGs. In the product ion electrospray tandem mass spectrometry, the 4–11 times lower abundance of the ion formed by cleavage of fatty acid from the secondary position relative to that formed by cleavage from the primary positions provided solid basis for differentiation between regioisomers of mono- and dibutyrates. Although the respective differences were lower, mono- and dicaproate regioisomers could be differentiated despite their incomplete separation. The tandem mass spectra of the short-chain triacylglycerols containing three different acyl groups revealed that the regioisomer having the shortest acyl chain in the secondary position, eluted in the first peak. The mixture of the other two regioisomers, where the shortest acyl chain was located in the primary positions, eluted in the second peak. In one instance only did the tandem mass spectra allow to differentiate between the reverse isomers eluting in the second peak. An effective chromatographic resolution of butyrate triacylglycerols and the linear relationship between the molar and area ratios of analyte and standard, enabled the mass spectrometric quantification of their regioisomers.