DNA Barcoding and Chemical Profile Using UHPLC, GC-MS and LC-MS/QTOF of Mitragyna speciosa Variation and Allied Species for Quality Control of Kratom Materials

Kratom (Mitragyna speciosa Korth.) has gained increasing global attention due to its traditional use, psychoactive properties, and emerging therapeutic potential; however, concerns regarding adulteration, substitution, and inconsistent quality of commercial products necessitate robust authentication strategies. This study aimed to integrate DNA barcoding and comprehensive chemical profiling to authenticate kratom variants and discriminate them from closely allied Mitragyna species for quality control and forensic applications. Nine DNA barcoding regions were analyzed, alongside chemical characterization using UHPLC, GC–MS, and LC–MS/QTOF. Among the tested loci, the internal transcribed spacer (ITS) and ITS2 regions exhibited the highest interspecific variation and effectively distinguished kratom from allied species. UHPLC and GC–MS analyses confirmed that mitragynine was exclusively detected in kratom variants, with Kan Khiao exhibiting the highest content (94.33 ± 0.14 mg/g) when quantified against the mitragynine standard using UHPLC analysis. LC–MS/QTOF profiling revealed an alkaloid-rich chemotype in kratom dominated by mitragynine and 7-hydroxymitragynine, whereas M. diversifolia, M. hirsuta, and M. rotundifolia showed distinct profiles enriched in phenolic acids and flavonoid glycosides. Multivariate analyses further identified procyanidin B1, datiscetin-3-O-rutinoside, mitragynine, and 7-hydroxymitragynine as key discriminatory markers. Overall, the combined molecular and chemical workflow provides a robust framework for kratom authentication, supporting regulatory monitoring, quality assurance, and forensic identification of kratom materials.