Meat authentication and adulteration testing by HPLC combined with high-resolution, accurate-mass (HRAM) mass spectrometry

Significance of the Topic

Globalization of food supply chains has led to a rise in economically motivated meat adulteration, posing health risks and undermining consumer trust. Traditional PCR and immunoassays often lack the sensitivity, specificity, and comprehensive molecular information needed for reliable species identification in complex meat matrices. High-resolution mass spectrometry coupled with liquid chromatography offers an advanced proteomic approach for routine meat authenticity testing, enabling precise detection of species-specific peptide biomarkers at trace levels.

Objectives and Study Overview

• Perform in silico bioinformatic characterization of key muscular proteins (myoglobin, myosin, hemoglobin) from common mammalian species.
• Identify and validate species-specific proteotypic tryptic peptides for targeted mass spectrometry.
• Develop and demonstrate a robust HPLC-HRAM MS assay using PRM and DIA modes to detect pork adulteration down to low percentage levels.

Methodology and Instrumentation

• Sample Preparation: Meat samples (2 g) were homogenized, sonicated, centrifuged, proteins precipitated with acetone, redissolved, reduced (DTT), alkylated (IAA), and trypsin-digested for 24 h.
• Chromatography: Thermo Scientific UltiMate 3000 UHPLC with Biobasic C8 microbore column (100 × 1 mm, 5 μm); binary gradient of 0.1% formic acid in acetonitrile and water; 75 μL/min flow; 2 μL injection.
• Mass Spectrometry: Thermo Q Exactive hybrid quadrupole-Orbitrap; positive ESI; full-scan at 140,000 resolution (m/z 500–1500); PRM at 17,500 resolution; DIA at 70,000 survey and 17,500 MS2 resolution; optimized AGC targets and collision energies.
• Bioinformatics: Sequence alignment (QIAGEN CLC Viewer), in silico digestion and fragment prediction (mMass), database searches (Proteome Discoverer, Mascot) to define proteotypic peptides and unique fragment ions.

Main Results and Discussion

The targeted bottom-up approach identified a conserved myoglobin tryptic peptide (residues 120–134) with species-specific mass shifts among beef, horse, pork, and lamb. PRM monitoring of y13 and y14 fragment ions yielded mass accuracies within ±1.5 ppm and provided clear chromatographic separation with no interfering signals. Pork adulteration in beef, lamb, and chicken was detected at the 1% (w/w) level; PRM extended detection down to 0.2% (w/w). DIA acquisitions offered broader data coverage for retrospective mining, though sensitivity below 1% was reduced. Additional proteotypic peptides from myosin-1 (619–638), myosin-2 (619–639), and β-hemoglobin (40–58) were identified for confirmatory multiplex assays.

Benefits and Practical Applications

• High specificity and sensitivity enable reliable detection of low-level meat adulteration in routine QC and regulatory laboratories.
• Targeted HRAM MS workflows can be validated with isotopically labeled internal standards for quantitative compliance testing.
• Method adaptability allows expansion to multiple species and integration into food safety monitoring programs.

Future Trends and Potential Applications

Expansion of data-independent acquisition and advanced bioinformatic pipelines will facilitate comprehensive proteomic surveillance of food products. Development of multiplexed assays covering a broader range of species and implementation of automated sample preparation platforms will enhance throughput. Integration of certified reference materials and standardized workflows promises to support regulatory harmonization and global food authenticity initiatives.

Conclusion

A targeted HPLC-HRAM MS strategy based on proteotypic peptide biomarkers provides a robust, sensitive, and specific solution for meat authentication and adulteration testing. The approach achieves detection limits below 1% (w/w) for pork in meat matrices and can be extended to multiple species through additional peptide markers. Routine adoption of this workflow will strengthen food safety and combat fraudulent practices.

References

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