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 δ13C, δ15N, 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 δ13C and δ15N 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 δ13C (-19.91‰), δ15N (14.87‰), and Pr (0.49 μg 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.

Department of Analytical Chemistry Faculty of Chemical Technology University of Pardubice Studentska 573 HB D Pardubice CZ 532 10 Czech Republic

Department of Chemistry Life Sciences and Environmental Sustainability University of Parma 43124 Parma Italy

Department of Food and Drug University of Parma 43126 Parma Italy

Department of Veterinary Medicine and Animal Sciences Milan University 26900 Lodi Italy

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