Determination of triacylglycerols in butterfat by normal-phase HPLC and electrospray-tandem mass spectrometry

Here, we report the identification and quantification of the molecular species of long-, medium-, and short-chain triacylglycerols (TAG) in butterfat (BF), including TAG with an odd number of acyl carbons (ACN) and TAG with unidentified molecular species. In the present study, in addition to auto-MS(2), a large number of methods, each recording MS(2) for 1-4 ions, were used for identification of TAG species. For the quantification of long-chain, odd ACN TAG, and TAG with unidentified molecular species, molar correction factors (MCF) were calculated from the uncorrected mol% (area mol%) of each ACN:DB (number of double bonds) class in randomized butterfat (RBF), and the respective mol% in the calculated random composition of RBF. The butyrate, caproate, and medium-chain (C(8), C(10)) TAG were quantified using regio- or acyl-chain-specific MCF calculated from their area mol% in RBF and mol% in the calculated random composition. These methods enabled us to identify CA. 450 TAG species in 184 quantified peaks of 88 ACN:DB classes. The proportions of saturated, monoene, diene, triene, tetraene, pentaene, and hexaene TAG were 40.0, 38.4, 16.2, 4.5, 0.6, 0.1, and 0.03 mol%, respectively. The proportions of TAG with not identified molecular species and odd ACN TAG were 11.8 and 5.7 mol%, respectively. The most abundant short-chain TAG species were butyroyldipalmitoylglycerol + butyroylmyristoylstearoylglycerol (5.25 mol%) and butyroylpalmitoyloleoylglycerol (4.08 mol%).