Qualitative and quantitative analysis of pork in beef food with LC-MS/MS

Importance of the topic

Global food fraud, particularly the substitution or mixing of meat species, poses significant economic, ethical, and health challenges. Reliable identification and quantification of unauthorized meat components—such as pork in beef products—are vital for consumer protection, regulatory compliance, and preventing religious or allergenic risks. Traditional immunoassays (ELISA) and DNA-based methods (PCR) can yield false positives/negatives or suffer from DNA degradation, making proteomic approaches by LC-MS/MS a compelling alternative.

Objectives and study overview

This work aims to develop a targeted LC-MS/MS multiple reaction monitoring (MRM) method for the qualitative and quantitative determination of pork adulteration in beef. Key goals include the selection of species-specific peptides, optimization of collision energies, and establishment of calibration curves over a wide adulteration range (5–95%). The approach was validated for accuracy, linearity, and sensitivity in mixed meat matrices.

Instrumentation

• Liquid Chromatograph: Shimadzu LC-30A
• Mass Spectrometer: Shimadzu LCMS-8050 with ESI+ interface
• Sample Disruption: Ultra-Turrax T-25 homogenizer
• SPE Cleanup: Waters HLB cartridges

Methods and materials

• Sample preparation: Beef samples were spiked with pork at ratios of 5%, 10%, 20%, 40%, 60%, and 80%. Two grams of minced tissue were extracted in 7 M urea/2 M thiourea/50 mM Tris-HCl (pH 8) under high-speed homogenization.
• Protein processing: Extracts underwent reduction with DTT, alkylation with IAA, and tryptic digestion followed by HLB SPE cleanup.
• Chromatography: Separation on a Shim-pack GISS column (2.0 mm×150 mm, 2.1 µm) at 40 °C using a water/ACN gradient with 0.1% formic acid, flow 0.3 mL/min, injection 5 µL.
• Mass spectrometry: MRM acquisition (dwell time 10–20 ms), interface 300 °C, DL 250 °C, heating block 400 °C, nebulizing gas 3 L/min, drying/heating gas 10 L/min.

Results and discussion

Characteristic peptides for pork (SALAHAVQSSR, TLAFLFAER, YDIINLR, LVVITAGAR) and beef (TLALLFSGPASGEAEGGPK, EASGPINFTVFLNMFGEK, HPSDFGADAQAAMSK, ALEDQLSELK, LVIITAGARF) were evaluated. Collision energies were optimized in Skyline software, and transitions m/z 534.30→853.45 (pork) and 901.45→1290.60 (beef) were selected for quantitation. MRM chromatograms demonstrated clear separation of target peptides in mixed samples. Calibration curves constructed over 5–80% pork and 20–95% beef exhibited excellent linearity (r = 0.9960 and 0.9982) with accuracy ranges of 87.4–110.2% (pork) and 95.2–104.9% (beef).

Benefits and practical applications

Compared to immunoassays and PCR, the LC-MS/MS MRM approach provides higher specificity, reduced false results, and direct quantification of peptide markers. The method is adaptable to routine food authenticity testing, quality control in meat processing, and regulatory surveillance of adulteration.

Future trends and possibilities

Advances may include expanding the peptide library to cover more species, integration with high-resolution MS for untargeted screening, automation of sample preparation, and development of multiplexed assays for comprehensive meat profiling. Combined proteomic and metabolomic workflows could further strengthen food authentication capabilities.

Conclusion

A robust LC-MS/MS MRM method was established for detecting and quantifying pork adulteration in beef products. Optimized peptide markers and collision energies yielded highly linear and accurate calibration across relevant adulteration levels. This proteomic strategy addresses limitations of ELISA and PCR, offering a reliable tool for meat authenticity testing.