Avoiding False Identification of 7-Hydroxymitragynine in Kratom Products Using a Multicriteria LC-MS Confirmation

Kratom (Mitragyna speciosa) products have become widely available in diverse commercial formulations, raising increasing analytical and regulatory interest due to the presence of potent alkaloids such as mitragynine (MG), 7-hydroxymitragynine (7-OH), and mitragynine pseudoindoxyl (MGP). The accurate identification of these compounds is analytically challenging because kratom matrices contain numerous structurally related alkaloids and isomeric species that produce similar mass spectral features. In this study, an ultrahigh-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) workflow was developed to characterize kratom alkaloids in authentic leaf materials and 38 commercial kratom products representing multiple formulations. Chromatographic separation combined with accurate mass measurement and diagnostic MS/MS fragmentation enabled reliable differentiation of MG, 7-OH, MGP, and related isomers. Multiple overlapping chromatographic peaks were observed at m/z 415.2227, demonstrating that reliance solely on accurate mass or commonly monitored transitions such as m/z 415 → 190 can lead to false-positive identification of 7-OH in complex botanical matrices. Application of the multicriteria confirmation strategy revealed substantial variability in alkaloid composition across commercial products, including the presence of 7-OH and MGP in several tablet and capsule formulations. Untargeted metabolomic profiling further distinguished kratom plant extracts, including the authentic leaf materials, from processed products enriched in specific alkaloids. These results highlight the importance of integrating chromatographic resolution with orthogonal MS/MS criteria for reliable identification of kratom alkaloids and provide an analytical framework for the characterization of complex botanical products.