A two-stage reversed-phase ultra high performance liquid chromatography (UHPLC) separation coupled with Orbitrap-MS was developed for the separation of triacylglycerols (TAGs) in milk, animal fats and vegetable oils. The method constitutes a significant improvement compared with existing methods, achieving separation of positional isomers and identifying a great number of TAGs with equivalent carbon numbers (ECNs) ranging between 26 and 56. This is achieved in a third of the time of the method most highly cited in the literature.
Unlike previously developed methods, the UHPLC-MS method is appropriate for the analysis of samples of a wide range of biological sources. A total of 90 samples analysed with the method include 37 fats from meat products, 37 oils and fats from plant material, 6 milk fat extracts, 2 infant formula milk fat extracts and 6 forensic samples suspected to contain residues of infant formula milk. Statistical analysis of the results resulted in groups of samples with the same or similar origins. Separate groups of olive oil, sunflower oil, sesame seed oil, soya, pork and cow milk samples were produced, along with a group including both beef and lamb samples and a group including different types of poultry samples (chicken, duck and goose). Milk and infant formula milk were categorised in two different groups, although the brand of infant milk formula could not be positively determined.
TAGs important for the discrimination between species were identified. Most abundant peaks for each type of sample were identified and were usually the most important variables between different types of samples. In olive oil those peaks were identified as OOO and OOP*, in rapeseed oil OOO and OOL*. In both sunflower and sesame seed oil the two most abundant TAGs were LLL and LLO, with LLL being the highest one in sunflower and LLO in sesame seed. The most abundant TAGs in beef and lamb were OOP*, POP* and OOS, while in pork they were OPO*, LPO and SPO*. For all three types of poultry analysed OOP* and OOO were amongst the highest abundance TAGs. Milk fat samples had significantly more TAG components than oils and animal fats, but all cow milk samples had higher abundances for PPC4, PMC4 and OPP. The TAGs with the highest abundance for goat milk were PPC4, PPCa and OPO. Positional isomers of TAGs are another indicator for the identity of samples, especially in the case of animal fats. Beef and lamb fat samples have very similar TAG distributions, but beef fat contains only the SOS* isomer while lamb fat contains both SOS* and SSO* and pork and poultry contain only SSO*.
This work shows that a single UHPLC method can be applied for the analysis and identification of TAGs in oils and fats of plant and animal origin and the variations of TAG distribution and relative abundances are sufficient for the identification of the sample’s origin in most cases.
