Analysis of Kratom’s Main Psychoactive Components: Mitragynine and 7-Hydroxymitragynine

Significance of the Topic

The increasing recreational use of Kratom and its active alkaloids, mitragynine and 7-hydroxymitragynine (7-HMG), raises concerns due to their interaction with opioid receptors and potential for abuse. Reliable detection and quantification of these compounds in biological matrices such as urine are essential for forensic toxicology, clinical monitoring, and regulatory decision-making.

Objectives and Study Overview

This study aimed to develop and validate a rapid, sensitive, and robust LC-MS/MS method using the scheduled MRM™ algorithm on the SCIEX QTRAP® 4500 system to quantify mitragynine and 7-HMG in human urine over a calibration range of 2–500 ng/mL.

Methodology and Instrumentation

Sample Preparation:

• Urine samples (100 µL) were treated with β-glucuronidase for hydrolysis, followed by a dilute-and-shoot workflow.
• Mitragynine-D3 and 7-HMG-D3 served as internal standards at 2 µg/mL.

Chromatography:

• ExionLC™ AC HPLC with a Phenomenex Biphenyl column (50 × 3 mm, 2.6 µm).
• Mobile phases: formic acid in water (MPA) and formic acid in methanol/acetonitrile (MPB).
• Gradient run time: 4.5 min, injection volume 10 µL.

Mass Spectrometry:

• SCIEX QTRAP® 4500 with Turbo V™ source and Curtain Gas™ interface.
• QJet® ion guide and curved LINAC® collision cell for enhanced sensitivity and fast scanning.
• Scheduled MRM™ algorithm to maximize dwell time and quantitative precision.

Results and Discussion

Chromatographic performance showed baseline resolution of mitragynine (2.23 min) and 7-HMG (1.96 min). Retention time reproducibility remained within minimal variability over 70 injections.

Calibration and Sensitivity:

• Linear response (R2 > 0.99) across three orders of magnitude (2–500 ng/mL).
• Lower limit of quantitation (LLOQ) at 2 ng/mL with signal-to-noise ratio > 10.

Precision and Accuracy:

• Evaluated at 10 and 200 ng/mL with ≤ 20% CV and ± 20% accuracy criteria met.
• No carryover detected after ULOQ injections; higher concentrations diluted into range.

Benefits and Practical Applications

This method provides a streamlined dilute-and-shoot protocol compatible with high-throughput forensic and clinical workflows. The combination of rapid LC separation and scheduled MRM on the QTRAP 4500 delivers high sensitivity, robust quantitation, and minimal chromatographic drift, making it well suited for routine urine screening and confirmation of Kratom use.

Future Trends and Opportunities

Emerging directions include:

• Extension to additional Kratom alkaloids and metabolites.
• Integration with high-resolution MS for non-targeted screening.
• Automation of sample preparation and data processing pipelines.
• Miniaturized and on-site testing platforms for rapid toxicology.

Conclusion

A validated LC-MS/MS method using the SCIEX ExionLC AC and QTRAP 4500 system enables reliable quantification of mitragynine and 7-HMG in human urine. The approach combines a simple sample preparation with high sensitivity, precision, and throughput, meeting the demands of forensic and clinical laboratories.

References

1. S. National Institute on Drug Abuse. DrugFacts: Kratom; 2018.
2. U.S. Drug Enforcement Administration. DEA Announces Intent to Schedule Kratom; August 30, 2016.