Discrimination of cold-slaughtered versus freshly slaughtered chicken meat using a heat-stable hemoglobin-derived...

Cold-slaughtered (CS) meat obtained from poultry that die prior to slaughter, poses serious ethical, safety, and regulatory concerns, as its consumption and marketing are prohibited under food laws in several countries. In this first-of-its-kind study, an integrated targeted proteomic method to distinguish cold-slaughtered (CS, dead-on-arrival chicken) from freshly slaughtered (FS) chicken meat was developed. The MALDI-TOF/MS analysis of SDS-PAGE protein bands (∼15 kDa) identified hemoglobin-derived peptide IAGHAEEYGAETLER (m/z 1645.81) as reliable marker. To enhance analytical specificity, a Multiple Reaction Monitoring (MRM) MS method was developed using synthesized peptide. A distinct precursor ion (m/z 823.1; RT = 4.20 min) with four MRM transitions were detected and validated across field samples (n = 50). The developed method exhibited excellent sensitivity with an LOD of 0.121 μg/mL and a LOQ of 0.5 μg/mL for the synthetic peptide. Receiver Operating Characteristic (ROC) analysis established discriminative cut-off values for both raw and cooked meat, with performance positivity (PP) scores ≥0.315 indicating CS chicken, achieving 100 % sensitivity and specificity. This pioneering study provides conclusive evidence that protein extraction followed by LC-MS-based MRM targeting hemoglobin-derived peptide IAGHAEEYGAETLER constitutes a robust, heat-stable biomarker-based approach for authenticating CS meat.