21
Fri, Sep
0 New Articles

Identification of “legal-high” Plant Species in Forensic Labs

Forensics

by Kelly Elkins, Towson University

Despite recent efforts to legalize marijuana, the plant remains a Schedule I listed substance under the United State Drug Enforcement Agency (DEA) Controlled Substances Act (CSA). Indeed, the plant remains the most common plant-based drug submitted into evidence in forensic labs in the United States; however, other, less common plant-based drugs are increasingly showing up in forensic labs, as adolescents and young adults increasingly seek out “legal high” drugs.1 In a May 2016 case, three Massachusetts students brought Hawaiian baby woodrose, Heavenly blue morning glory, and sleepy grass seeds to their school. Two of the students consumed the seeds, while the third spit them out. In another case, several students consumed seeds at a junior-prom high school dance in a nearby town, sickening many and sending one to the hospital. Boston television news station WCVB5 and the Daily Mail reported upon the use of these seeds as emerging “party drugs.”2,3

In a case reported in 2010, one male reportedly died after consuming Hawaiian baby woodrose seeds with a friend.4 The death occurred when one of the users became agitated and jumped out of a window.4 The surviving male ingested 6 seeds and could not recall how many the other consumed.4 Other cases have involved 3-8 seeds5 It was also reported that the men in this case had been smoking marijuana in addition to consuming the seeds.4 Concurrent use of morning glory seeds and marijuana has also been reported.5

Many commonplace plants and their seeds may be abused for their stimulant, hallucinogenic, analgesic, euphoric, and sedative effects. Since they are legal, people often falsely believe that these substances are safe to consume. As they may be advertised and sold as organic and natural products or in pill or capsule form or as extracts, these substances often appear to be beneficial to one’s health. In addition, doctors and pharmacists often may not be familiar with their active ingredients or potential effects, or be ill prepared to advise on any appropriate use. Users may risk serious adverse reactions with other medications, herbals and vitamins they are taking. In a 2011 U.K. study, the authors reported that the top three abused plants were Salvia divinorum, Kratom, and Hawaiian baby woodrose.6

Hawaiian baby woodrose is known by biologists as Argyreia nervosa. Ipomoea purpurea is morning glory.1,5,7 The seeds for both plants are legal and easy to obtain as they are sold over the internet and at hardware, nursery and garden stores. These seeds are commonly used to grow beautiful ornamental plants, and but can be soaked, crushed, or used as a source from which to extract a substance with powerful narcotic effects when ingested.5 Other reports have shown the seeds being used to make teas. Documented doses of morning glory seeds include anywhere from 150 to 700 seeds and 170-700 seeds. 4,5 A common dose of Hawaiian baby woodrose seeds is 5 to 10 seeds.4

Morning glory and Hawaiian baby woodrose plants produce d-lysergic acid amide, LSA, a drug that is similar in structure to the more famous lysergic acid diethylamide (LSD).5 A dose of 2 to 5 mg of LSA will produce hallucinations in users.4 LSA is a Drug Enforcement Agency Controlled Substances Act Schedule III substance in the United States1,4

LSA is classified as a Class A drug under the Misuse of Drugs Act in the United Kingdom.4 LSA users and doctors have documented that it causes flushing, dilated pupils, nausea, loss of a sense of time, memory loss, anxiety, hallucination, tachycardia, hypertension, and suicide.4 Reported fatalities due to drug use often reflect the concurrent use or consumption of one or more other drug such as alcohol or marijuana.4,5

In addition to Hawaiian baby woodrose, morning glory, and sleepy grass, several other herbal plants have been reported for drug use.1,7,8 These include Salvia divinorum, nutmeg, Jimson weed, poppy, Mahuang, Betel nut, Yohimbe, Kava, cloves, and wormwood (used to produce Absinthe).1,7 Salvia divinorum is a perennial herb from the mint family with large green leaves and white flowers that is endogenous to Mexico and produces the hallucinogen Salvinorin A. It has been used by the Mazatec Indians for medicinal and ritual purposes.9 The U.S. DEA lists it as a “drug of concern” but it is not currently controlled under the Controlled Substances Act.9 Several U.S. states, European countries, Japan and Australia, however, have controlled Salvia divinorum and / or Salvinorum A. The rest of the plants are legal and not under consideration for control at this time. Nutmeg, Myristica fragrans, is a tropical tree endogenous to the Spice Islands of Indonesia and produces the hallucinogens myristicin and elemicin.1 Jimson weed grows naturally in tropical regions and produces atropine, an acetylcholinergic antagonist, and the hallucinogens scopolamine and hyoscyamine.1,7 Ma-huang, Ephedra sinica, is a shrub that produces ephedra alkaloid stimulants.1 Betel nut, Areca catechu, is the source of arecoline, a stimulant that reportedly gives a buzz like that felt after drinking six cups of coffee.1 Yohimbine is found in the bark of the Yohimbe tree (Pausinystalia yohimbe) and has stimulant and hallucinogenic effects.1,7 Kava, Piper methysticum, produces the Kava lactones and is used as an anxiolytic.5 The cloves tree, Syzygium aromaticum, grows in Indonesia and produces eugenol and nicotine which act as analgenic and stimulant drugs, respectively. 1 Poppy (Papaver somniferum) plants are natural sources of morphine and codeine narcotic drugs.1,7 Most of these natural narcotics are extracted from poppies grown in Asia, Latin America, and the Middle East in countries such as Afghanistan.1,7 Finally, Absinthe contains thujone, a drug with euphoric and hallucinogenic effects, from the wormwood plant (Artemisia absinthium) that is used to make it.1

Other plants may be sold containing synthetic cannabinoid designer drug compounds that have been sprayed on or soaked into the material. 6,10 These are widely distributed on the internet and sold as “Spice” or “K2” with labels including “incense,” “plant food,” “bath salts,” and “not for human consumption.” 6,10 A U.K. group performed an evaluation of UK-based websites and “legal high” products.6 These include Galium aparine, a plant that contains less caffeine than the coffee plant beans, as well as mint, liquorice, tea, raspberry, clover, and marigold plants. Sometimes, they are also applied to psychotropic plants including marijuana, Salvia divinorum, or Kratom.6,9,10

Forensic chemists are tasked with the identification of the drug evidence submitted to the crime lab.Forensic chemists are trained to identify marijuana using color tests, microscopic evaluation of their trichomes and cystolithic hairs, and using gas-chromatography or thin layer chromatography to separate out and identify the main hallucinogenic component, Δ9- tetrahydrocannabinol, or THC. However, in cases where the plant material has been smoked or ground, it may be difficult or impossible to identify morphologically, even using a microscope. In some cases the concentration of THC is too low to detect. In these cases, the marijuana plant can be identified using DNA analysis.11-16

Species identification has been shown to be important for solving cases; however, forensic labs typically do not employ forensic botanists, specialized staff who can differentiate the flowers, leaves and seeds of plants by their morphology. For example, the season or month of the year can be determined using pollen grains or leaf fragments to understand the timeline of a case. Species identification can be used to verify alibis. Components of trace mixtures on drug paraphernalia can be determined by species identification.11-16

New forensic DNA tests identify and differentiate selected species of plants used as drugs. One method identifies Salvia divinorum.17 Another method is able to differentiate four “legal high” plants – including the morning glory consumed by the students in the recent cases. The assay also detects Hawaiian woodrose, Jimson weed, and marijuana. The assay employs the polymerase chain reaction (PCR) that probes and copies selected segments of an organism’s DNA and then uses a post-PCR melt method to analyze the DNA copies that were produced. To develop the test, the team targeted unique regions of the plants’ genomes and tested the focused PCR reagents against two dozen species. The process was continued iteratively until PCR amplification was strong and the reagent proportions were optimized for them to work in concert.18 While multiplexing PCR primers to analyze tens of sites simultaneously is routine in human DNA typing, this study was the first to demonstrate simultaneously determining the identities of four “legal high” plants in a multiplex using post-PCR melt curve peak temperatures.18 The assay development and testing was published in the Journal of Forensic Sciences.18 An assay to detect Kratom (Mitragyna speciosa), a plant in the coffee family used for its opiate-like pain relief, is under development.19

Chemical identification methods, such as gas chromatography-mass spectroscopy, liquid chromatography, and infrared spectroscopy, can be used for identification if the active drug from the plants has previously been identified and is a member of the labs’ identification databases. As an advantage to the other methods, the new plant DNA detection assays can be used by forensic scientists with no specialized training in botany to identify the plants using instrumentation widely available in their labs.17-18

In addition to “legal high” plant species identification, a recent study focused on the identification of psychedelic mushrooms in forensic cases.20 Another PCR-based species identification assay was developed to differentiate psychedelic “magic” mushrooms and marijuana in a single test tube.21

Having assays available is integral to law enforcement’s efforts identify these plants and trace material to understand the circumstances of cases and control misuse. If a drug or plant is not restricted, there is no criminal penalty. However, local, state, and federal crime labs, medical examiners’ office, and public health offices have a mission to keep their inhabitants safe. Laws concerning controlled substances continue to evolve. For example, the Smoking Opium Exclusion Act of 1909 banned the importation, possession and use of “smoking opium.” The 2005 Combat Methamphetamine Epidemic Act limited the sale of cold medicines containing pseudoephedrine (a starting material for making methamphetamine) and required photo identification for purchase. Stores are required to keep logs for two years. While federally the U.S. government still has marijuana classified as a CSA Schedule I substance (banned and no acceptable medical use), twenty nine states and the District of Columbia have passed legislation allowing the use of marijuana for medical use and seven states have legalized recreational use.

The DEA considered controlling kratom last year as a result of its effects on users but the plant remains legal in the United States even as many countries around the world – including Thailand, Malaysia, and Burma where it is endogenous – have banned it. In the United States, Kratom is sold as a homeopathic or herbal remedy in shops across the country. For now, the local home improvement store in Somerset, Massachusetts nearby the schools whose students ingested the seeds last year has been asked to stop selling the Hawaiian baby woodrose, Heavenly blue morning glory, and sleepy grass plant seeds2,3 to restrict access but a tailored approach is needed as many species are toxic at some level including many flowering plants.

Acknowledgements

Towson University Masters in Forensic Science graduate students Ashley Cowan, Anjelica Perez and Alicia Quinn are acknowledged for their contributions to developing the assays discussed herein.

References 1. Ghosh A, Ghosh

T. Herbal Drugs of Abuse. Sys Rev Pharm 2010 Jul-Dec;1(2):141-5.

2. http://www.wcvb.com/article/home-improvement-storeasked- to-pull-seeds-with-lsd-like-effects-offshelves/ 8234558

3. http://www.dailymail.co.uk/news/article-3575339/Are- FLOWER-SEEDS-new-party-drug-Police-warnteenagers- hospitalized-eating-garden-products-psychotichigh. html

4. Klinke HB, Mueller IB, Steffenrud S, Dahl-Sørensen R. Two cases of lysergamide intoxication by ingestion of seeds from Hawaiian Baby Woodrose. Forensic Sci Int 2010;197(1–3):e1–5.

5. Juszczak GR, Swiergiel AH. Recreational use of D-lysergamide from the seeds of Argyreia nervosa, Ipomoea tricolor, Ipomoea violacea, and Ipomoea purpurea in Poland. J Psychoactive Drugs 2013;45(1):79–93.

6. Schmidt MM, Sharma A, Schifano F, Feinmann C. “Legal highs” on the net-Evaluation of UK-based Websites, products and product information. Forensic Sci Int 2011 Apr;206(1-3):92-7.

7. Marnell T, editor. Drug Identification Bible. 4th Edition. Colorado: Denver, 1999.

8. Shulgin AT, Drugs of abuse in the future. Clin Toxicol. 1975 8(4):405-56.

9.https://www.deadiversion.usdoj.gov/drug_chem_info/sal via_d.pdf

10. Ogata J, Uchiyama N, Kikura-Hanajiri R, Goda Y. DNA sequence analyses of blended herbal products including synthetic cannabinoids as designer drugs. Forensic Sci Int 2013 Apr;227(1-3):33-41.

11. Coyle HM. Nonhuman DNA Typing: Theory and Casework Applications. CRC Press, 2007.

12. Lee C-L, Miller Coyle H, Lee HC. Genetic analysis of individual seeds by amplified fragment length polymorphism method. Croat Med J 2007 Aug;48(4):563-565.

13. Ferri G, Alù M, Corradini B, Beduschi G. Forensic botany: species identification of botanical trace evidence using a multigene barcoding approach. Int J Legal Med. 2009 Sep;123(5):395-401.

14. Allgeier L, Hemenway J, Shirley N, LaNier T, Miller Coyle H. Field testing of collection cards for Cannabis sativa samples with a single hexanucleotide DNA marker. J. Forensic Sci 2011 Sept;56(5):1245-1249.

15. Shirley N, Allgeier L, Lanier T, Miller Coyle H. Analysis of the NMI01 marker for a population database of cannabis seeds. J Forensic Sci 2013 Jan;58 Suppl 1:S176-82.

16. Curtis K, Coyle HM. Development of a Quantitative Real-Time Polymerase Chain Reaction (RT-PCR) Assay for Plant Species. J Forensic Res Crime Stud 2014 Mar;1:1-5.

17. Murphy TM, Bola G. DNA identification of Salvia divinorum samples. Forensic Sci Int 2013 Jan;7(1):189-193.

18. Elkins KM, Perez ACU, Quinn AA. Simultaneous identification of four “legal high” plant species in a multiplex PCR high resolution melt assay. J Forensic Sci 2017, 62:593-601.

19. Cowan AC, Elkins KM, unpublished results.

20. Kowalczyk M, Sekuła A, Mleczko P, Olszowy Z, Kujawa A, Zubek S, Kupiec T. Practical aspects of genetic identification of hallucinogenic and other poisonous mushrooms for clinical and forensic purposes. Croat Med J. 2015 Feb; 56(1):32–40.

21. Cowan AF, Elkins KM. Detection and identification of Psilocybe cubensis DNA using a real-time polymerase chain reaction high resolution melt (PCR-HRM) assay. J Forensic Sci 2017, DOI: 10.1111/1556-4029.13714.

By Kelly Elkins, Towson University

Kelly Elkins, PhD, is an Assistant Professor of Chemistry in the Chemistry Department and Master of Science in Forensic Science Professional Science Master’s program at Towson University (TU) in Towson, Maryland; TU offers undergraduate and graduate forensic science programs accredited by the Forensic Science Education Programs Accreditation Commission (FEPAC). Prior to her current appointment, she was Director of Forensic Science and Assistant Professor of Chemistry at Metropolitan State College (University) of Denver in Denver, Colorado where she managed the FEPAC-accredited undergraduate forensic science program, and internships and undergraduate research program in Criminalistics. She is the author of Forensic DNA Biology: A Laboratory Manual published by Elsevier in 2013, four peer-reviewed book chapters, and 25 peer-reviewed papers in journals including the Journal of Forensic Sciences, Drug Testing and Analysis, and Medicine, Science and the Law. Her upcoming Forensic Chemistry textbook is expected to be published in 2018. She is a member of the American Chemical Society and the American Academy of Forensic Sciences where she has been recommended for promotion to Fellow at the 2018 national meeting. She has been interviewed by NBC 9News Denver, FOX 31 News Denver, ABC 7 News Denver, The Denver Post, and Forensic Magazine as a forensic expert. Her research was recently highlighted by GenomeWeb.com.

Sign up via our free email subscription service to receive notifications when new information is available.