Highly-Sensitive Detection of Multiple Porcine-Specific Peptides in Processed Foods by LC/MS/MS Method

Significance of the topic

The unauthorized addition of pork to processed foods poses economic, safety and ethical challenges, especially for consumers observing halal or kosher dietary laws. DNA-based assays often fail after heat treatment, so a peptide-based approach offers greater reliability for confirming permissible food status.

Objectives and overview of the study

This study consolidated seven heat-stable porcine-specific peptide markers into a single LC–MS/MS MRM method to detect and semi-quantify trace amounts of pork in processed food matrices, ensuring sensitive and selective halal authentication.

Methodology and instrumentation

Sample preparation comprised four steps:

• Protein extraction from meat or processed food using urea/thiourea buffer.
• Reduction with dithiothreitol, alkylation with iodoacetamide, and tryptic digestion.
• SPE cleanup of digest using methanol and formic acid conditioning.
• UHPLC–MRM analysis targeting seven porcine peptides.

Used instrumentation

Shimadzu LCMS-8060 triple-quadrupole mass spectrometer coupled to Nexera X2 UHPLC. Phenomenex Aeris Peptide XB-C18 column (150×2.1 mm, 1.7 µm) with a water/acetonitrile gradient (0.1 % formic acid).

Main results and discussion

Specificity tests confirmed no cross-reactivity with beef and distinguished one homologous peptide in chicken by unique fragment ions. All seven markers remained detectable after heating at 200 °C for 30 min. Screening of commercial items correctly identified pork in non-halal products and no markers in certified halal foods. Spiking experiments in mutton curry demonstrated detection limits down to 0.1 % (wt) for key peptides YDII, LVVI and SALA. Calibration curves for four peptides showed linearity (R2 ≥ 0.995) across 0.1–5 % spiking with inter-day RSD ≤16 %.

Benefits and practical applications

• High sensitivity and selectivity for processed and cooked foods.
• Reliable detection at levels as low as 0.1 % pork in complex matrices.
• Supports halal and kosher certification, food safety and quality control.

Future trends and potential applications

Further developments may include:

• Expanding peptide panels for multi-species food authentication.
• Automating and streamlining sample preparation workflows.
• Incorporating high-resolution MS and advanced data analysis tools.
• Validating the method across diverse processed food matrices.

Conclusion

The presented LC–MS/MS MRM assay offers a robust, heat-stable peptide-based solution for detecting trace porcine material in processed foods, enhancing the accuracy of halal and kosher compliance testing.

References

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