Improved Detection of Kratom Alkaloids in Forensic Toxicology

Kratom is a botanical drug with psychoactive properties that produces both stimulant and opiate effects depending on the dosage. Its major psychoactive components are mitragynine and 7-hydroxymitragynine. The drug may not be included as part of routine toxicological screening and as such, its use may be underreported. This research seeks to improve the analysis of kratom alkaloids in toxicological specimens, and increase overall understanding related to their properties and drug metabolism. Five Mitragyna alkaloids including speciociliatine (SC), mitragynine (MG), paynantheine (PY), speciogynine (SG), and 7-hydroxymitragynine (7-MG-OH) were analyzed in blood and urine using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q/TOF-MS). Each method was validated in accordance with published guidelines for forensic use. LC-Q/TOF-MS was used throughout the study due to its selectivity and sensitivity. Recombinant human cytochrome P450 isoenzymes (rCYPs) were used to investigate the biotransformational pathways involved during drug metabolism. Phase I metabolism was attributed to four rCYPS (CYP3A4, CYP2D6, CYP2C19, and CYP2C18) producing a total of four metabolites (9-O-demethylmitragynine, 16-carboxymitragynine, 9-O-demethyl-16-carboxymitragynine, and 7-hydroxymitragynine). The pH and temperature dependent stability of MG, SC, SG, PY, and 7-MG-OH were also investigated. All five alkaloids were acid labile, with 7-MG-OH being the most unstable. However, SC, SG, and PY were more stable than MG and 7-MG-OH. In addition, two degradation products of mitragynine were identified. Chemical and enzymatic hydrolysis of conjugated metabolites of mitragynine were investigated using postmortem urine specimens. Acid and base hydrolysis, in addition to nine enzyme systems (β-glucuronidase: Escherichia coli, Patella vulgata, Helix pomatia; sulfatase: abalone entrails, Aerobacter aerogenes. Patella vulgata, Helix pomatia; recombinant systems; BGTurbo™, BGS™, ASPC™, IMCSzyme) were evaluated. Ultimately, hydrolysis did not improve the analysis of 9-O-demethylmitragynine and 7-hydroxymitragynine, but did improve the detection of 16-carboxymitragynine. Postmortem blood (n=40), urine (n=16), and tissue (n=20) specimens from 20 fatalities were also evaluated. Speciociliatine and speciogynine were shown to be biomarkers of kratom use, with concentrations higher than mitragynine in the majority of specimens. In addition, the majority of blood and urine specimens contained several metabolites of mitragynine, with 9-O-demethylmitragynine and 7-hydroxymitragynine being the most prominent.