Abstract
Background:
Detecting porcine gelatin in gelatin powders and food products containing gelatin is crucial for ensuring compliance with dietary restrictions, ethical considerations, and regulatory standards.

Aim:
This study presents a non-targeted proteomic approach utilizing liquid chromatography coupled with high-resolution Orbitrap mass spectrometry (LC-Orbitrap HRMS) for the identification and quantification of porcine gelatin in gelatin powder and food products such as gummy candies and marshmallows.

Methods:
Peptide sequences unique to bovine and porcine gelatin were identified through comparative analysis of mass spectrometry data, enabling reliable differentiation from gelatin sourced from other animals. Both extraction buffers, including Tris-HCl buffer pH 8.0 and ammonium bicarbonate pH 8.0 were effective for protein and peptide extraction in gelatin. A number of peptide markers of porcine gelatin were obtained in both extraction buffers. In addition, this study revealed that the reduction and alkylation process is unnecessary, thereby simplifying the extraction steps.

Results:
This method was successfully tested with gelatin used in complex food matrices such as gummy candies and marshmallows. Porcine gelatin was detected in 3 samples of gummy candies and 3 samples of marshmallows.

Conclusion:
Therefore, future research such as the targeted approach and standardization is strongly required to obtain a valid, reliable, and reproducible method for the analysis of porcine gelatin in food and pharmaceutical product applications for halal authentication purposes.

Key words: Porcine gelatin; Bovine gelatin; Non-targeted proteomics; LC-HRMS; Halal authentication.