To identify a novel optimized strategy for preventing fraudulent substitutions of squid species and origins, forty European squids (Loligo vulgaris) and forty flying squids (Todarodes sagittatus) from the Mediterranean Sea and Atlantic Ocean were analyzed for 613C, 615N, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Yb, and Lu using isotope ratio mass spectrometry and inductively coupled plasma-mass spectrometry. While 613C and 615N variations were mainly species-related, they alone could not reliably distinguish samples. To address this issue, decision rules were developed using Classification and Regression Tree analysis. Threshold values for 613C (-19.91%o), 615N (14.87%o), and Pr (0.49 mu g kg- 1) enabled successful discrimination among Mediterranean European squids, Atlantic European squids, Mediterranean flying squids, and Atlantic flying squids, achieving over 90% accuracy, 81% precision, 80% sensitivity, and 93% specificity. This method holds promise for enhancing traceability and safety in the seafood industry, ensuring product integrity and consumer trust.
