8/2/2019 Kratom Poster

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Chemical Characterization of Kratom and Associated AlkaloidsTaylor 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

Introduction

Conclusion

References

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.

Methods

Acknowledgements

We would like to thank Arcadia University and the Center for

Forensic Science Research and Education for support and funding.

Results

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 Kratomillegal. 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 theiodoplatinate 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.

 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. Kratom’s 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 State’s 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.

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.

GC/MS

Detected mitragynine in all

samples

Detected o-desmethyltramadol

in both 80x ExtractsPercent Recovery

5 µg- 66%

10 µg-75%

15 µg-50%

Color Tests1. 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 the10 cm line

4. Observe under UV light

5. Spray with developing

reagent iodoplatinate and

observe

Microscopic Examination

1. Place sample on slide

2. Observe characteristics

Liquid-Liquid Extraction

1.Weigh out 0.01g of sample

2. Add 500 μL of water

3. Add 8 μg of yohimbine internal

standard and vortex

4. Add 250 μL 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 andremove organic layer

8. Evaporate to dryness

9. Reconstitute with 100 μL of 

methanol

10. Analyze on the GC/MS

Babu, Kavita M., Christopher R. McCurdy, and Edward W. Boyer. "Opioid

Receptors and Legal Highs: Salvia Divinorumand Kratom." Clinical

Toxicology46 (2008): 146-52.

Bell, Suzanne. Forensic Chemistry. Upper Saddle River, NJ: Pearson Prentice

Hall, 2006.

Dayer, P., J. Desmeules, and L. Collart. "Pharmacology of Tramadol."08 July

2011.

"DEAResources, Microgram, March 2006." Welcome to the United States

 Department of Justice. Web. 24 June 2011.

<http://www.justice.gov/dea/programs/forensicsci/microgram/mg0306/ 

mg0306.html>.

"ErowidKratom (Mitragyna Speciosa) Vault." Erowid . Web. 24 June 2011.

<http://www.erowid.org/plants/kratom/kratom.shtml>.

Houghton, Peter J., AishahLatiff, and Ikram M. Said. "Alkaloids from

MitragynaSpeciosa."Phytochemistry30 (1991): 347-50.

"HowStuffWorks"Foren sic Drug Testing"" HowStuffWorks"Science" Web. 24

June 2011. <http://science.howstuffworks.com/forensic-lab-

technique2.htm>.

Kronstrand, Robert, Markus Roman, GunillaTheland er, and Anders Eriksson.

"Unintentional Fatal Intoxications with Mitragynineand O-

Desmethyltramadrol from the Herbal Blend Krypton." Journal of 

 Analytical Toxicology (2011): 242-47. Print.

Lee, C. R., D. Tavish, and E. M. Sorkin. "Tramadol. APreliminary Review of 

Its Pharmacodynamic and Pharmacokinetic Properties, and Therapeutic

Potential in Acute and Chronic Pain States." 08 July2011.

Maurer, Hans H. "Chemistry, Pharmacology, and Metabolismof Emerging

Drugs of Abuse." The Drug Monit 32 (2010): 544-49.

Paar, W. D., H. J. Dengler, J. Gerloff, and S. Poche. "Polymorphic CYP2D6

Mediates O-demethylation of the OpioidAnalgesic Tramadol." 23 July

1997. 8 July2011.

Philipp, AnikaA. "Monitoring of Kratom and Krypton Intake in Urine Using

GC-MS in Clinical and Forensic Toxicology." Anal Bioanal

Chem (2011): 127-35

Saferstein, Richard. Forensic Science Handbook . 2nd ed. Vol. 2. Upper Saddle

River: Pearson Prentice Hall, 2005.

Sunwanlert, Sangun. "AStudy of KratomEaters in Thailand." (1975): 21-27.

T., Arndt. "KratomAlkalo ids and O-desmethyltramadol in Urine of a "Krypton"

Herbal Mixture Consume." Forensic SciInt. (2011): 47-52.

"Thin Layer Chromatography." CU Boulder Organic Chemistry Undergraduate

Courses. 24 June 2011.

<http://orgchem.colorado.edu/hndbksupport/TLC/TLC.html>.

"Tramadol." Pub Med Health. National Center for Biotechnology Information,

U.S. National Libraryof Medicine, 1 Feb. 2011.. 11 July201 1.

<http://www.ncbi.nlm.nih.gov/pubmedhealth/PMH0000960/>.

Valle, Marta, MaríaJ. Garrido, Juan M. Pavón, Rosario Calvo, and IñakiF.

Trocóniz. "Pharmacokinetic-Pharmacodynamic Modeling of the

Antinociceptive Effects of Main Active Metabolites of Tramadol, ( )-O-

Desmethyltramadol and (−)-O-Desmethyltramadol, in Rats." Journal of 

Pharmacology and Experimental Therapeutics . 08 July2011.

Duquenois-Levine Test

1. Add sample

2. Add 0.5 mL of Duquenois reagent

and 0.5 mL of hydrochloric acid

3. Heat4. Add 1 mL of chloroform

5. Observe any color changes

SampleReagent and

HClHeating Chloroform

Marijuana

Powdered

Samples

Indo Kratom

Bali Kratom

Negative Control

Table 2. Duquenois- Levine color test: color reaction

that was observed at each step of the process

Figure 2. Microscopic

examination: hair like

fibers observed

Sample R  f StandardDeviation

% Differencefrom

Mitragynine

Mitragynine 0.83 0.03

Pill 0.80 0.04 3.4

O. Super 0.84 0.02 1.4

O. Max 0.84 0.03 1.4

K2 Sex 0.84 0.02 1.8

Super 0.80 0.02 3.8

Max 0.82 0.03 0.6

Regular 0 .84 0.06 1.3

S.P. Powder 0.85 0.06 2.7

Resin 0.83 0.03 0.0

Tincture 0 .90 0.03 7.8

Bali 0.83 0.03 0.1

Indo 0.86 0.02 4.1

Table 1. TLC retention factors showing the

samples are consistent with mitragynine

Figure 1. Structure of mitragynine