Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 72 No. 4 pp. 747ñ755, 2015 ISSN 0001-6837 Polish Pharmaceutical Society NATURAL DRUGS IDENTIFICATION CHALLENGES IN EXAMINATION OF COMMERCIAL PLANT MATERIAL OF PSYCHOTRIA VIRIDIS ANNA P. KOWALCZUK1*, ANNA £OZAK1, ROBERT BACHLI—SKI3, ANNA DUSZY—SKA3, JOANNA SAKOWSKA1 and JORDAN K. ZJAWIONY2 1National Institute of Medicines, Che≥mska 30/34, 00-725 Warszawa, Poland 2Department of Pharmacognosy and Research Institute of Pharmaceutical Sciences, School of Pharmacy, The University of Mississippi, MS 38677, USA 3Central Forensic Laboratory of the Police, Aleje Ujazdowskie 7, 00-583 Warszawa, Poland Abstract: Psychotria viridis (chacruna) is a hallucinogenic plant with psychoactive properties associated with the presence of N,N-dimethyltryptamine (DMT). This species is primarily known as an ingredient of the bev- erage Ayahuasca, but dry leaves are also smoked by recreational users. The plant is controlled in Poland and France and its proper identification poses many challenges due to the fact that genus Psychotria is relatively large and there are other species that are easily confused with chacruna. The aim of the present work was to develop an effective authentication procedure for the dried and shredded leaves of P. viridis, to be used in com- parison of chemical and botanical characteristics of its commercial products. Dried leaves of P. viridis origi- nating from Brazil, Peru and Hawaii were purchased from Internet providers. For DMT identification, thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) methods have been elaborated, validated and applied. In order to clarify the existing differences among samples, chemometric methods have been used. Botanical features and the gas chromatography tandem mass spectrometry (GC-MS) chromatograms have been analyzed using hierarchical cluster analysis (HCA). Our studies revealed significant variety among plant material marketed as P. viridis. Grouping of samples based on their micromorphology features and GC- MS results did not correspond well with the presence of DMT. Based on our results an indisputable identifica- tion of dried specimens as P. viridis is very problematic. It is necessary to postulate changes in legislation regarding regulation of P. viridis and replace it with DMT as controlled substance. Keywords: Psychotria viridis, identification, chromatography, micromorphology, cluster analysis Psychotria viridis Ruiz & Pavon from the indole-3-acetic acid which is not able to cross the Rubiaceae family is known as a popular hallucino- blood-brain barrier and produce hallucinogenic genic plant derived from the Amazon lowlands (1). effects (4). In the Amazon region, leaves of P. viridis Leaves of P. viridis known also as chacruna contain are used in combination with stems of Banisteriopsis N,N-dimethyltryptamine (DMT), an indole alkaloid caapi in the form of a drink called Ayahuasca (5). responsible for the psychoactive properties of the The alkaloids included in B. caapi block MAO and plant. The action of DMT resembles to some extent thus allow for the hallucinogenic effect after drink- LSD, however, the caused sympathomimetic effects ing Ayahuasca beverage. The dry leaves of P. viridis are more intense and onset and duration of effects are also smoked by recreational users and can be are much shorter (2). As LSD, DMT belongs to the used for extraction of DMT (6). group of serotonergic hallucinogens activating sev- P. viridis is controlled in France and Poland (7, eral 5-HT receptors. Although the mechanism of 8). In the other countries not the plant, but its main action of DMT is not yet fully understood, its hallu- active compound DMT is illegal. According to US cinogenic effects are attributed to its agonist activi- law live plants and seeds can be legally bought and ty to 5-HT2A and 5-HT2C receptors (3). sold (9). DMT is not active orally due to oxidative Identification of plant material is difficult due to deamination by monoamine oxidase A (MAO) to the fact that the genus Psychotria comprises nearly * Corresponding author: e-mail: [email protected]; phone 22 841 06 23 (383) 747 748 ANNA P. KOWALCZUK et al. 2000 species (10, 11), moreover, some other genera in matograms obtained with GC-MS method for South American flora are being confused with methanol, ethyl acetate, dichloromethane, chloro- chacruna. The problem with proper plant identification form and hexane extracts have been analyzed with results also from the divergent information about the hierarchical cluster analysis (HCA). contents of DMT. The list of Psychotria spp. contain- ing DMT varies depending on the literature source. EXPERIMENTAL There are reports that some Psychotria species suppos- edly containing DMT are actually lacking it (12-14). Plant materials Nevertheless, P. viridis is always recognized as The examined products were purchased from being the one that possesses hallucinogenic proper- the Internet through the http://www.maya-ethno- ties associated with DMT presence. botanicals.com/ website. The shredded leaves of P. The aim of the present work was to develop an viridis originated from Brasil (samples 1 and 2), effective authentication procedure for the dried and Hawai (sample 3) and Peru (samples 4-7) according shredded leaves of P. viridis. We based our method to the providerís declaration. on DMT detection and microscopic confirmation of identity of the plant material. In the search for diag- Reagents and materials nostic features of P. viridis we used micromorpho- Chloral hydrate was purchased from Carl Roth logical examination of live plant material. To con- GmbH (Germany), Sudan III from Merck firm the presence of DMT in the samples, the low (Germany), isopropanol pure p.a. from Chempur cost chromatographic methods commonly used in (Poland) and glycerol pure p.a. from Standard police laboratories were developed: thin layer chro- (Poland). Sudan III reagent was prepared according matography as a first screening tool and high per- to United States Pharmacopeia (USP). formance liquid chromatography method when the Methanol, chloroform, ethyl acetate, dichloro- highest sensitivity is required. methane and hexane pure p.a. were purchased from The known methods of DMT identification in POCH (Poland), triethylamine pure p.a. from Sigma P. viridis and in Ayahuasca involve use of gas chro- Aldrich (Belgium), anisaldehyde from Sigma matography tandem mass spectrometry (GC-MS) Aldrich (Germany), 99.9% acetic acid from POCH (15, 16), capillary electrophoresis (CE) (17), liquid (Poland), and 95% sulfuric acid pure p.a. from chromatography tandem mass spectrometry (LC- Chempur (Poland). Anisaldehyde solution was pre- MS) (18, 19) and nuclear magnetic resonance spec- pared according to European Pharmacopoeia. troscopy (NMR) (20). Currently popular, barcoding HPLC reagents such as acetonitrile (MeCN) methods, can be a good solution for the species iden- HPLC grade S were purchased from Rathburn tification of powdered plant material. However, they Chemicals Ltd. (UK), sodium hexanesulfonate pure are still in the process of development and charac- p.a. from Sigma Aldrich (Switzerland), and 85% teristic DNA sequences have to be found. This is orthophosphoric acid from Merck (Germany). challenging in case of P. viridis as the genus com- Deionized water was obtained with deionizating filter prises a large number of species. system Water PRO PS from Labconco Corp. (Kansas At the same time, there is a lack of fast and City, USA). Membrane filters Chromafil Pet-45/25 simple methods like thin layer chromatography from Macherey-Nagel GmbH & Co.KG (Germany). (TLC) or high performance liquid chromatography N,N-Dimethyltryptamine (DMT) standard with (HPLC), for identification of DMT in P. viridis. One the purity 99.4% was purchased from Lipomed AG of the difficulties in HPLC analysis of DMT in plant (Switzerland) lot 00013890-9019. samples may be attributed to relatively strong basic character of the compound. Utilizing basic proper- Apparatus ties of DMT the ion pair chromatography method Light microscope Biolar C Ti hal with digital has been developed, as well as fast and cost effective video optikam Pro5 from PZO (Poland) and Optica method of thin layer chromatography. Both methods Vision software; automatic TLC Sampler from had been validated and successfully applied in the Camag (Switzerland); Video-scanner Desaga with a analysis of all seven chacruna samples. software for video documentation ProVidoc Our studies revealed significant variety among (Germany); HPLC Ultima 300 System Dionex, with plant material marketed as P. viridis. In order to clar- UV-VIS detector, 2 pumps, autosampler and Chrome- ify the existing differences among samples, pharma- leon Datasystem; GC-MS Agilent GC7890A+ cognostic examination and chemometric methods 5975C VLMSD instrument from Agilent Techno- have been used. Botanical features and the chro- logies (USA) were used. Identification challenges in examination of commercial plant... 749 For the statistical analysis ìStatistica 10î soft- HPLC method ware from StatSoft (USA) was applied. HPLC analysis was performed on Luna C18(2) 250 ◊ 4.6 mm, 5 µm column from Phenomenex Methods (USA). The mobile phases were: A: 10 mM solution of Sample and standard preparation for the chro- sodium hexanesulfonate monohydrate adjusted to pH 2 matographic analysis with orthophosporic acid, B: acetonitrile. The analysis Powdered dried plant material (0.5 g) was was performed in gradient: A : B 80 : 20 (2 min), 65 : extracted with 5.0 mL of methanol in an ultrasound 35 (20 min), 20 : 80 (26 min), 80 : 20 (30 min). The bath for 30 min and filtered through membrane fil- detection wavelength was λ 254 nm, injection volume ters. The standard solution was prepared as a 0.5 10 µL, flow rate 1 mL/min, time of analysis 30 min. mg/mL ethanol solution of DMT. The solution for DMT standard solution, methanolic extracts from the the method validation in specificity parameter was Psychotria viridis samples mixture of equal parts of also prepared by mixing 1 mL of extract from the extract solution from Hawaiian Psychotria and stan- Hawaii sample and 1 mL of the standard solution of dard solution (mix) and blank were injected.