Black Pepper Authenticity Analysis Workflow with LC-QTOF and Novel Software Solution

Significance of the Topic

Black pepper is one of the most widely traded spices globally, often subject to geographic origin fraud due to opaque supply chains and economic incentives. Reliable analytical methods to verify provenance are essential for protecting consumers, maintaining regulatory compliance, and ensuring fair trade practices.

Objectives and Study Overview

This study aimed to develop and demonstrate a streamlined workflow for untargeted authenticity analysis of black pepper using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF) combined with novel software (MassHunter Explorer 2.0). The focus was on differentiating samples from five countries of origin and simplifying data processing for routine use.

Methodology

Ground black pepper (20 mg) was extracted with methanol/water (4:1) using vortexing, shaking, sonication, centrifugation, and filtration. Quality control samples were injected regularly to monitor system stability.

Used Instrumentation

• Agilent 1290 Infinity III LC system with Zorbax Eclipse Plus C18 column (2.1 × 150 mm, 1.8 µm)
• Revident LC-Q-TOF with iterative AutoMS/MS acquisition
• Mobile phases: water and methanol each with 4.5 mM ammonium formate, 0.5 mM ammonium fluoride, 0.1% formic acid
• Gradient elution over 24 min, column at 45 °C, flow rate 0.4 mL/min
• Mass spectrometry: MS at 3 spectra/s, AutoMS/MS at 3 spectra/s, gas temperatures 325 °C and 375 °C, capillary voltage 2500 V

Main Results and Discussion

Over 25 000 molecular features were detected; filtering by abundance, variability, and frequency reduced this to 6000 compound groups. One-way ANOVA and fold-change analysis highlighted 239 discriminating features. Principal component analysis revealed clear separation of samples by origin, although Vietnam and Cambodia samples clustered closely. MassHunter Explorer 2.0 guided users through six steps (setup, find and align, normalization, filtering, statistics, identification) using an integrated interface.

Identification combined accurate mass, isotope pattern matching, and MS/MS spectral comparison against curated libraries. Direct export to external tools (SIRIUS CSI:FingerID, NIST MS Search) enabled annotation of features not present in internal libraries, for example revealing a piperlonguminine derivative.

Benefits and Practical Applications

• Simplified workflow reduces need for specialized data-processing expertise
• Rapid identification of authenticity markers for quality control
• Capability to integrate third-party spectral libraries enhances annotation depth
• Robust detection of geographic origin differences supports regulatory and commercial requirements

Future Trends and Opportunities

Advances may include integration of machine-learning algorithms for feature selection, expanded spectral databases for comprehensive identification, and automation of reporting for real-time authenticity monitoring. Cloud-based platforms could facilitate collaborative validation across laboratories and industries.

Conclusion

The combination of LC-QTOF analysis with MassHunter Explorer 2.0 delivers a powerful, user-friendly workflow for black pepper authenticity testing. The approach effectively distinguishes geographic origins, streamlines data processing, and enhances identification through integrated and third-party library searches.

References

• Mialon N, et al. Untargeted metabolomic approaches in food authenticity: A review that showcases biomarkers. Food Chemistry. 2022;398:133856.