Comparative Mass Spectroscopy Profiling of Masala Spices: Unveiling Direct and Remote Antimicrobial Effects of Nonvolatile and Aroma Metabolites via Biochemometric Tools and Molecular Networking

ABSTRACT Herbal spices are widely used as food additives not only for their distinct aroma and flavor but rather for their health‐promoting properties. In this study, the chemical profiles of four major masala spices—chaat (CMP), pav‐bhaji (PMP), sambhar (SMP), and tandoori (TMP)—were analyzed using advanced multiplex‐mass spectrometry techniques (HS‐SPME–gas chromatography (GC)–MS and UPLC–MS/MS). A total of 45 aroma compounds were identified, with aldehydes emerging as the dominant constituents in PMP and CMP masalas at 74.7% and 56.1%, respectively, compared with 22.1% in SMP and 21.7% in TMP. Notably, cinnamaldehyde was the major compound, present at 70.1% in PMP, 48.5% in CMP, and 10.7% in SMP. Furthermore, 98 secondary metabolites were annotated, spanning nitrogenous compounds, gingerols, diarylheptanoids, phenolic acids, flavonoids, and saponins. Molecular networking highlighted five key clusters of bioactive compounds, including gingerols, capsaicin derivatives, piper alkaloids, and steroidal saponins. The antimicrobial properties of the masalas were evaluated using vapor phase (VP) and direct assays, revealing broad‐spectrum activity, and PMP exhibited the strongest antibacterial effect with the lowest VP minimum inhibitory concentration (VP‐MIC) values, attributed to cinnamaldehyde richness. The partial least squares (PLS) of identified metabolites against all tested microorganisms indicated that PMP was the most correlated to antimicrobial effect. These findings highlight the potential of masala spices not only as flavoring agents but also as food additives with antimicrobial properties. However, factors, such as batch variations, geographic origin, and processing methods, may affect their consistency and application. Future studies should correlate other biological effects, that is, antioxidant, using same model developed in this study for these species.