Determination of Mitragynine and 7‐Hydroxymitragynine in Raw Kratom Plant Material by a Multisegment Injection Capillary Zone Electrophoresis‐Tandem Mass Spectrometry

A novel validated analytical approach implementing multisegment injection strategy into the capillary zone electrophoresis-tandem mass spectrometry hyphenation for determination of two psychoactive alkaloids, mitragynine (MIT), and 7-hydoxymitraginine (7-OH-MIT), in kratom raw samples was introduced. A thorough optimization of the multisegment injection strategy and extraction procedure of the alkaloids from raw powdered plant material was realized. The separation of the two secondary plant metabolites was performed in the background electrolyte composed of 100 mM formic acid. The developed and optimized analytical approach showed favorable operation and validation parameters, including linearity (r² > 0.99), selectivity, precision (relative standard deviation in the range of 3.3%-9.4%), accuracy (relative error in the range of 92.2%-108.4%), recovery (85.1%-99.7%), or robustness (95.2%-99.7%). Furthermore, the method demonstrates high sensitivity, with the limit of detection at the concentration level of 1 and 2 ng/mL for MIT and 7-hydroxymitragynine, respectively. Finally, the method was successfully applied to determine the content of the psychoactive alkaloids in six raw kratom samples marketed as collector's items. The content of MIT and 7-OH-MIT in the kratom samples (original powdered raw material) varied from 23.15 to 35.51 and 0.43 to 0.72 µg/mg, respectively. Moreover, the developed innovative approach was assessed to evaluate sample preparation sustainability, alignment with green analytical chemistry principles, and practicality. The proposed method with enhanced sample throughput represents a very effective and promising green alternative to traditional chromatographic techniques and has high potential for implementation in pharmaceutical and toxicological practice.