Chemical Characterization of Kratom and Associated Alkaloids

Taylor Curtis, Byron Dees, Theresa Scott1, Jill Yeakel1,2, Warren Korn2, Barry Logan1,2 1MSFS Program, Arcadia University, Glenside, PA 2The Center for Forensic Science Research and Education, Willow Grove, PA

Abstract
Originally, Thai agricultural workers chewed the leaves of the Kratom tree for its stimulating effects at low doses. It has been found that Kratom has depressant effects at high doses causing the Drug Enforcement Administration (DEA) to list it as a drug of concern. Consequently, it is important to characterize Kratom in the event that legislation is passed to make Kratom illegal. Chemically characterizing Kratom will assist in differentiating it from other drugs of similar appearance and structure. The effects of Kratom are produced by its most prevalent alkaloid, mitragynine. This analyte was the main component investigated to develop standard forensic chemical tests to characterize Kratom. For the samples thought to be Kratom, color tests, thin layer chromatography (TLC), and gas chromatography/mass spectrometry (GC/MS) were used to test for the presence of mitragynine. Microscopic examination was used to analyze the physical appearance of Kratom in leaf form. Methanolic extractions were utilized to remove the analytes from the original sample matrix in order to analyze the samples using color tests and TLC. Positive results from the color tests produced colors that are similar to the original color of the extract. Therefore, color tests are not ideal presumptive tests for Kratom. For TLC, it was determined that a chloroform:methanol (9:1) solvent system created the best separation and the iodoplatinate spray allowed for superior visibility. This method succeeded in producing results consistent with mitragynine. Liquid-liquid extraction (LLE) followed by GC/MS analysis confirmed the presense of mitragynine in all samples. This process of TLC followed by GC/MS allows Kratom to be successfully characterized through standard forensic chemical tests. GC/MS Detected mitragynine in all samples Detected o-desmethyltramadol in both 80x Extracts Percent Recovery 5 g- 66% 10 g- 75% 15 g- 50%

Results
Table 1. TLC retention factors showing the samples are consistent with mitragynine
Sample Mitragynine Pill O. Super O. Max K2 Sex Super Max Regular S.P Powder . Resin Tincture Bali Rf 0.83 0.80 0.84 0.84 0.84 0.80 0.82 0.84 0.85 0.83 0.90 0.83 0.86 Standard Deviation 0.03 0.04 0.02 0.03 0.02 0.02 0.03 0.06 0.06 0.03 0.03 0.03 0.02 3.4 1.4 1.4 1.8 3.8 0.6 1.3 2.7 0.0 7.8 0.1 4.1 % Difference from Mitragynine

Methods
Microscopic Examination 1. Place sample on slide 2. Observe characteristics
Duquenois-Levine Test 1. Add sample 2. Add 0.5 mL of Duquenois reagent and 0.5 mL of hydrochloric acid 3. Heat 4. Add 1 mL of chloroform 5. Observe any color changes

Color Tests 1. Add 1-2 drops of reagent 2. Add sample 3. Observe any color changes
Thin Layer Chromatography 1. Mark with a line 10 cm from loading area 2. Prepare the chamber with 100mL of 9:1 chloroform:methanol solvent system, let equilibrate 3. Place the plate into the chamber and remove once the solvent has reached the 10 cm line 4. Observe under UV light 5. Spray with developing reagent iodoplatinate and observe

Figure 1. Structure of mitragynine

Indo

Table 2. Duquenois- Levine color test: color reaction that was observed at each step of the process
Sample Reagent and HCl Heating Chloroform

Liquid-Liquid Extraction 1.Weigh out 0.01g of sample 2. Add 500 L of water 3. Add 8 g of yohimbine internal standard and vortex 4. Add 250 L of pH 11 1M tris buffer and vortex 5. Add 1.5 mL of ethyl acetate 6. Rotate for 20 minutes 7. Centrifuge for 10 minutes and remove organic layer 8. Evaporate to dryness 9. Reconstitute with 100 L of methanol 10. Analyze on the GC/MS

Introduction
Mitragyna speciosa, common name Kratom, is a tree found mainly in Asian countries. It can grow up to fifty feet tall and fifteen feet around and can be purchased in leaf, powder, and liquid extract forms. Kratom is considered an herbal substance and many of its effects are due to its most prevalent alkaloid, mitragynine. Kratoms first primary users were Thai agricultural workers who chewed the leaves to endure the harsh work conditions. At low doses, Kratom has analgesic and stimulating effects but in higher dosages, depressant effects are experienced. Due to these effects, Kratom is banned in Thailand, Malayasia, and Myanmar where it originated. Kratom is also banned in Australia where it is a schedule 9 drug meaning it has no accepted medical use and can only be used for research purposes. Schedule 9 in Australia is similar to the United States schedule 1. The DEA has listed Kratom as a drug of concern however it remains legal in the USA. Due to the prevalence of mitragynine, it will be the main analyte used in helping characterize Kratom.

Marijuana Powdered Samples Indo Kratom Bali Kratom Negative Control

Acknowledgements
Figure 2. Microscopic examination: hair like fibers observed

We would like to thank Arcadia University and the Center for Forensic Science Research and Education for support and funding.

References

Conclusion
To differentiate Kratom from marijuana in the lab, microscopic examination and Duquenois-Levine color tests are adequate. Performing a methanolic extraction of samples followed by thin layer chromatography with a solvent system of chloroform:methanol (9:1) and a reagent spray of iodoplatinate, shows that the samples contain an analyte consistent with mitragynine. A LLE using pH 11 1M Tris buffer followed by GC/MS analysis confirms the presence of mitragynine and thus Kratom. Mitragynine has been detected in the Super Premium Powder, Regular, Max, Super, K2 sex, Orisha Max, Orisha Super, Pill, two 80x extracts purchased two months apart, Indo Kratom, Bali Kratom, Resin, and Tincture samples. O-desmethyltramadol, metabolite of the synthetic opioid tramadol, has been detected in the two 80x extracts.

Objective
Characterize Kratom and its associated alkaloids using standard forensic chemical tests including microscopic examination, color tests, thin layer chromatography, and gas chromatography/mass spectrometry.

Babu, Kavita M., Christopher R. McCurdy, and Edward W. Boyer. "Opioid Maurer, Hans H. "Chemistry, Pharmacology, and Metabolism of Emerging Receptors and Legal Highs: Salvia Divinorum and Kratom." Clinical Drugs of Abuse." The Drug Monit32 (2010): 544-49. Toxicology 46 (2008): 146-52. Paar, W. D., H. J. Dengler, J. Gerloff, and S. Poche. "Polymorphic CYP2D6 Bell, Suzanne. Forensic Chemistry. Upper Saddle River, NJ: Pearson Prentice Mediates O-demethylation of the Opioid Analgesic Tramadol." 23 July Hall, 2006. 1997. 8 July 2011. Dayer, P., J. Desmeules, and L. Collart. "Pharmacology of Tramadol."08 July Philipp, Anika A. "Monitoring of Kratom and Krypton Intake in Urine Using 2011. GC-MS in Clinical and Forensic Toxicology." Anal Bioanal "DEA Resources, Microgram, March 2006." Welcome to the United States Chem (2011): 127-35 Department of Justice. Web. 24 June 2011. Saferstein, Richard. Forensic Science Handbook. 2nd ed. Vol. 2. Upper Saddle <http://www.justice.gov/dea/programs/forensicsci/microgram/mg0306/ River: Pearson Prentice Hall, 2005. mg0306.html>. Sunwanlert, Sangun. "A Study of Kratom Eaters in Thailand." (1975): 21-27. "Erowid Kratom (Mitragyna Speciosa) Vault." Erowid. Web. 24 June 2011. T., Arndt. "Kratom Alkaloids and O-desmethyltramadol in Urine of a "Krypton" <http://www.erowid.org/plants/kratom/kratom.shtml>. Herbal Mixture Consume." Forensic Sci Int. (2011): 47-52. "Thin Layer Chromatography." CU Boulder Organic Chemistry Undergraduate Houghton, Peter J., Aishah Latiff, and Ikram M. Said. "Alkaloids from Courses. 24 June 2011. Mitragyna Speciosa." Phytochemistry30 (1991): 347-50. <http://orgchem.colorado.edu/hndbksupport/TLC/TLC.html>. "HowStuffWorks "Forensic Drug Testing"" HowStuffWorks "Science" Web. 24 "Tramadol." Pub Med Health. National Center for Biotechnology Information, June 2011. <http://science.howstuffworks.com/forensic-labU.S. National Library of Medicine, 1 Feb. 2011.. 11 July 2011. technique2.htm>. <http://www.ncbi.nlm.nih.gov/pubmedhealth/PMH0000960/>. Kronstrand, Robert, Markus Roman, Gunilla Thelander, and Anders Eriksson. Valle, Marta, Mara J. Garrido, Juan M. Pavn, Rosario Calvo, and Iaki F. "Unintentional Fatal Intoxications with Mitragynine and OTrocniz. "Pharmacokinetic-Pharmacodynamic Modeling of the Desmethyltramadrol from the Herbal Blend Krypton." Journal of Antinociceptive Effects of Main Active Metabolites of Tramadol, ( )-OAnalytical Toxicology (2011): 242-47. Print. Desmethyltramadol and ()-O-Desmethyltramadol, in Rats." Journal of Lee, C. R., D. Tavish, and E. M. Sorkin. "Tramadol. A Preliminary Review of Pharmacology and Experimental Therapeutics. 08 July 2011. Its Pharmacodynamic and Pharmacokinetic Properties, and Therapeutic Potential in Acute and Chronic Pain States." 08 July 2011.