Quantification of kratom alkaloids mitragynine, 7-hydroxy mitragynine, mitragynine pseudoindoxyl, and dihydro-7-hydroxy mitragynine (MGM-15) in forensic casework and medicolegal death investigations

Mitragynine (MG) is the primary psychoactive component of the kratom plant (Mitragyna speciosa). 7-Hydroxy mitragynine (7OH-MG) is found naturally in the plant but in small amounts (<2%). MG is metabolized to 7OH-MG and further to mitragynine pseudoindoxyl (MP), with the latter documented via in vitro enzymatic activity. Lack of scheduling has caused unregulated markets to proliferate with gas stations, smoke shops, and online retailers selling semi-synthetic formulations of these alkaloids under the umbrella term "kratom," though distinct from botanical kratom. Products containing 7OH-MG, MP, and synthetically derived dihydro-7-hydroxy mitragynine (DH-7OH-MG, aka MGM-15) exhibit greater potencies and activities at the µ-opioid receptor compared to MG and are now highly implicated in intoxications and deaths. These alkaloids present complex analytical and interpretive challenges due to their isomeric forms, incompatibility with certain extraction and instrumental techniques promoting analyte interconversion, instability in blood, and lack of published concentration ranges. A quantitative high-resolution mass spectrometry method for MG, 7OH-MG, MP, and DH-7OH-MG, which prevents interconversion and separates isomers, is presented herein along with stability data and concentrations from more than 200 authentic cases. MG, MP, and DH-7OH-MG were relatively stable in blood, whereas 7OH-MG decreased quickly and by 89% over 112 days with concurrent increase in MP. In a case subset (n = 139), mean ± SD concentrations in blood were 146 ± 477 ng/mL for 7OH-MG (median: 58 ng/mL, range: 0-5,000 ng/mL) and 194 ± 239 ng/mL for MP (median: 120 ng/mL, range: 4.8-1,900 ng/mL). In a second subset (n = 17), the mean ± SD DH-7OH-MG concentration was 324 ± 669 ng/mL (median: 170 ng/mL, range: 50-2,900 ng/mL). Testing for kratom alkaloids is currently extremely limited and/or subject to potential inaccuracy due to interconversion, leaving adverse events and fatalities under-investigated. Continued vigilance is needed as the kratom alkaloid product landscape rapidly evolves.