Differentiation of White and Red Galangal Rhizomes by Pseudo-Targeted LC-MS/MS Metabolomics

The rhizomes of white galangal (Alpinia galanga) and red galangal (Alpinia purpurata) are valued as herbal medicines and pesticides. However, these two plants may exhibit differing bioactivities and side effects due to variations in their metabolite compositions. Differentiating the rhizomes of the two plants in dried, chopped, or powdered form can be challenging, raising concerns about misidentification and fraud. This study aimed to distinguish between the two rhizomes using a pseudo-targeted metabolomics approach integrated with multivariate analysis and MS/MS spectra-based molecular networking. Metabolites were extracted via ultrasonic treatment in methanol, followed by untargeted LC-MS Q-Orbitrap and pseudo-targeted LC-MS triple quadrupole analyses. Principal component analysis (PCA) effectively separated the two rhizomes and highlighted metabolite features contributing to this separation. Hierarchical cluster analysis (HCA) clustered samples by plant type and location, and also showed differences in metabolite distribution patterns. Molecular networking, interpreted in conjunction with PCA loading scores, revealed distinct molecular cluster patterns. Finally, the study confirmed that differences in metabolite composition and molecular patterns enable accurate differentiation. This approach offers a reliable method for authenticating rhizomes, ensuring herbal quality control, and preventing counterfeiting.