Triacylglycerol regioisomers in human milk resolved with an algorithmic novel electrospray ionization tandem mass spectrometry method

•A sensitive LC-MS/MS method for analysis of triacylglycerol regioisomers was developed.•An optimization algorithm for quantitative analysis is included.•Hundreds of regioisomers of Finnish and Chinese human milk TAGs were analyzed.•Regioisomers of TAGs with standard deviation <17% covered 65 mol% of the total TAGs.•The method is applicable for analysis of a wide range of fats and oils.

A highly sensitive mass spectrometric (MS) method was developed and validated to analyze ratios of regioisomeric triacylglycerols (TAGs) in fats and oils. UPLC resolution of lithiated TAGs followed by daughter scan MS/MS of positive ions revealed several indicative ions for quantitative analysis. Reference TAGs containing C14-C20 fatty acids (FAs) showed good linear response. Analysis of Finnish and Chinese pooled human milk samples revealed hundreds of regioisomeric TAGs. At least 64 mol% of the TAGs were quantified with relative standard deviation <17%. When present in the same TAG molecule together with C18 FAs, palmitic acid was typically in the sn-2 position. When together with FAs 10:0, 12:0, 14:0, 20:1 and 20:2, the sn-2 preference of 16:0 was less clear. Oleic acid occupied typically the sn-1/sn-3 positions but when together with FAs 20:1, 20:2, 18:2, 14:1, 12:0 or 10:0 the positioning of 18:1 did not follow these rules.