Authentication of ground black pepper. An AP-MALDI-MS approach

Food fraud in the spice sector is an increasingly critical issue, with ground black pepper (Piper nigrum L.), susceptible to adulteration from both exogenous contaminants and endogenous by-products. In this study we applied Atmospheric Pressure Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (AP-MALDI-MS), an ambient mass spectrometric (AMS) technique, to assess the authenticity of ground black pepper samples using a non-targeted approach. A total of 58 samples, including authentic, adulterated, and pure adulterants, were analyzed. The AP-MALDI source was coupled to a linear ion trap mass spectrometer, enabling MSⁿ experiments for tentative metabolite identification. Spectral fingerprints revealed diagnostic ions including piperine, dimeric alkaloids, plant-specific flavonoid glycosides, and triacylglycerols. The analytical performance of the method was evaluated using multivariate statistics (principal component analysis, PCA, and partial least squared discriminant analysis, PLS-DA) and two machine learning models were built: random forest (RF) and least absolute shrinkage and selection operator (LASSO). Random forest achieved the highest performance with 91.8% area under the curve (AUC) in cross-validation, 81.71% on the withheld test set and 85.0% accuracy on the test set. These results support AP-MALDI-MS as a robust, high-throughput approach for ground black pepper authentication, and demonstrate its applicability for screening spice authenticity with minimal sample preparation.