Simple quantitation of kratom components from urine by LC-MS/MS for forensic toxicology

Significance of the Topic

Kratom (Mitragyna speciosa) is an emerging psychoactive botanical with opioid-like properties, increasingly encountered in forensic and clinical toxicology. Reliable quantitation of its major alkaloids mitragynine and 7-hydroxymitragynine in human urine supports abuse monitoring and safety assessments.

Objectives and Study Overview

This study aims to develop a simple, accurate, and precise LC-MS/MS method for quantifying mitragynine and 7-hydroxymitragynine in human urine. The workflow integrates ammonia-supported SPE cleanup using HyperSep Retain PEP plates, rapid reversed-phase UHPLC separation, and tandem mass spectrometric detection.

Methodology and Instrumentation

Sample preparation employs polymeric SPE at high pH to stabilize 7-hydroxymitragynine, achieving selective cleanup from 100 µL urine. Chromatographic separation uses a Hypersil GOLD VANQUISH C18 1.9 µm column (2.1×100 mm) with a 6.5-minute gradient. Quantitation is performed on a Thermo Scientific TSQ Quantiva triple quadrupole MS in positive ion mode. Data acquisition and processing are managed by Chromeleon CDS.

• Vanquish Horizon UHPLC system
• Hypersil GOLD VANQUISH C18 1.9 µm column
• HyperSep Retain PEP 96-well plate
• TSQ Quantiva triple-stage quadrupole mass spectrometer
• Chromeleon CDS software

Main Results and Discussion

The method exhibits linear calibration over 15–1000 ng/mL (R2>0.996) with an LLOQ of 15 ng/mL. Intra-batch accuracy ranged within ±9.2% and precision (CV) below 9.3%. No carryover or matrix interferences were observed. Recoveries averaged 87.4% for mitragynine and 90.3% for 7-hydroxymitragynine. Matrix factor normalized by internal standards was 0.97–1.01, indicating minimal suppression or enhancement.

Benefits and Practical Applications

The described protocol combines rapid analysis with robust cleanup, enabling high-throughput screening of kratom alkaloids in toxicological and clinical laboratories. Small sample volume requirements and low LLOQ facilitate application in forensic casework and pharmacokinetic studies.

Future Trends and Opportunities

Future developments may extend this workflow to other kratom constituents, apply high-resolution MS for untargeted metabolite profiling, and integrate automated SPE for increased throughput. The approach can inform regulatory monitoring and clinical research on novel psychoactive substances.

Conclusion

The study delivers a streamlined LC-MS/MS assay for reliable quantitation of mitragynine and 7-hydroxymitragynine in urine. It demonstrates excellent linearity, accuracy, precision, and selectivity, supporting its utility in forensic toxicology and bioanalytical research.

References

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