Untargeted Lipidomics Profiling for Halal Authentication of Meatball Products from Mixed Meat Sources

Food adulteration with non-halal components in processed meat products presents significant challenges for halal certification. While conventional DNA- and protein-based methods are compromised by thermal degradation, this study developed an untargeted lipidomics approach using UHPLC-HRMS to authenticate halal meatballs containing pure beef, goat, pork (90% meat and 10% tapioca/salt), and their 1:1 binary mixtures (n=3 per group) processed at 100°C for 30 minutes. Lipid profiling in ESI+/ESI- modes identified glycerophospholipids as the dominant lipid class (72-85% of total lipids), with 800-1,475 lipid ions detected per sample. Chemometric analysis revealed clear separation between halal and non-halal samples, with PCA showing 78.4% variance in ESI+ and PLS-DA achieving excellent classification (R²=0.994, Q²=0.952, 98.7% accuracy). Five lipid markers (LPE(16:0p)+H, LPE(16:1e)+H, LPE(18:1p)+H, PC(10:0p/23:0)+H, PC(8:0/24:1)+H) were identified for single-meat differentiation, while twelve additional markers (including PE(13:0/21:6)+H and PS(6:0/19:0)) distinguished mixed samples, enabling detection of 5% pork adulteration (AUC=0.987). The method demonstrated superior thermal stability versus DNA-based techniques (92.4% lipid vs. 31.2% DNA recovery post-processing, p<0.01), effectively overcoming limitations of conventional authentication methods. These findings establish lipidomics as a robust solution for the halal verification of thermally processed meat products, with identified markers providing specific targets for the regulatory monitoring of pork adulteration in complex meat matrices.