Kenneth Tupper, from the Department of Educational Studies, University of British Columbia, has sent an e-mail Sep. 08th explaining the following:

“In December 2005, the Journal of Analytical Toxicology (JAT) published a case report by Sklerov et al. of a fatality of a 25-year-old American man who, it seemed, had drunk something like ayahuasca (see below). After reading this case report, a few members of the Multidisciplinary Association for Psychedelic Studies — many of whom were cited in it — decided that a response was needed. It was their opinion that the case report unfairly maligned ayahuasca, as the reported facts were inconsistent with the representation of danger implied by the authors.

The details of the objection are outlined in the letter to the editor of JAT (see below), but in brief, the young man who died could not have been drinking only a traditional plant-based ayahuasca preparation. The opinion of the experts who signed this letter was that he may have started with a herbal preparation, but at some point he must also have ingested a massive amount of synthetic 5-MeO-DMT (100 mg. at minimum was one estimate). The original case report was crafted in such a way that it did not communicate the true nature of the risks (or lack thereof) of traditional ayahuasca preparations, which have little or no 5-MeO-DMT in them. Given the controversial legal status of ayahuasca in many countries, and the number of court cases involving the tea in recent years, the authors of the letter felt that they could not let the Sklerov, et al. case report stand unchallenged.”

FULL LETTER TO THE EDITORS OF THE JOURNAL OF ANALYTICAL TOXICOLOGY IN 2006

Reference: Callaway, J.C., Grob, C.S., McKenna, D.J., Nichols, D.E., Shulgin, A. & Tupper, K.W. (2006). A demand for clarity regarding a case report on the ingestion of 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT) in an ayahuasca preparation. Journal of Analytical Toxicology. 30(6), 406-7.

Referenced to the original: Sklerov, J., Levine, B., Moore, K.A., King, T. & Fowler, D. (2005). A fatal intoxication following the ingestion of 5-methoxy-N,N-dimethyltryptamine in an ayahuasca preparation. Journal of Analytical Toxicology. 29(8), 838-841.

To the editor

The case report “A Fatal Intoxication Following the Ingestion of 5-Methoxy-N,N-Dimethyltryptamine in an Ayahuasca Preparation” by Sklerov et al. is misleading as to the nature and toxicity of ayahuasca.

The authors of the article state that: “the decedent ingested a preparation from a South American tree bark ‘ooasca’ (sic) and approximately 4 h later ingested tryptamines” (p. 839). However, elsewhere in the article they assert that this is a case of “administration of an ayahuasca-like preparation containing 5-MeO-DMT” (p. 838, italics added) and further, that “this is the only reported case of death following ingestion of hallucinogenic tryptamines contained in an ayahuasca preparation” (p. 841, italics added).

The article’s title also purports that this is a case of “5-Methoxy-N,N-Dimethyltryptamine in an Ayahuasca Preparation” (italics added). It is highly improbable that the levels of 5-MeO-DMT found in the decedent’s heart blood came from (or was “in”) a plant-based preparation similar to the ayahuasca medicine/sacrament used for centuries by indigenous healers of the Amazon regions of South America. Because there is no known plant or animal source that would provide such a large amount of 5-MeO-DMT, we believe that the decedent must have ingested synthetic material. “Ayahuasca” refers to decoctions made from Banisteriopsis caapi and usually other admixture plants. One of these, Diplopterys cabrerana, does contain trace amounts of 5-MeO-DMT. The most common ayahuasca admixture, however, is Psychotria viridis, which like B. caapi, does not contain 5-MeO-DMT. It is not clear from the evidence presented which admixtures might have been in the herbal preparation, but the disproportionately high level of 5-MeO-DMT reported in the decedent’s heart blood (1.88 mg/L) suggests that synthetic 5-MeO-DMT was taken subsequent to a more traditional herbal preparation. This point is not insignificant, as 5-MeO-DMT is far more potent than its analogue DMT, and a confusion between the two could have been the cause of an unintentional overdose of the former, particularly in combination with potentiating beta-carbolines. Most salient, however, is that there is no evidence that the 5-MeO-DMT ingested by the decedent was “in” (i.e. a constituent of) anything resembling a traditional ayahuasca brew or that his death from “hallucinogenic amine intoxication” can in any way be attributed to ayahuasca per se, as a superficial or uninformed reading of the article insinuates.

A number of important details, to which the authors may have been privy but are not reported on in the article, could help to set the record straight on this matter: What exactly was the herbal “ooasca” preparation made from? What were the “tryptamines” taken four hours after the “ooasca” herbal preparation? Is there any estimation of the quantity of tryptamine taken four hours after the “ooasca” preparation? (The ratio of 5-MeO-DMT to the other psychoactive alkaloids found in the decedent’s heart blood is grossly disproportional). Answers to these questions would provide clarity about the nature of this particular case and the generalizations that may be extended from it.

This clarification is important, as readers may otherwise make unwarranted inferences about the toxicity and harmfulness of ayahuasca. Anthropological (and growing contemporary scientific) evidence suggests the contrary: ayahuasca has long been revered as a traditional therapeutic agent over a large geographical region and throughout a wide variety of cultures, and it arguably deserves more attention from modern medical researchers and clinical practitioners. Furthermore, scientific information and analysis played a crucial role in a recent United States Supreme Court case involving the legality of ceremonial ayahuasca use under the Religious Freedom Restoration Act. Misleading aspects of the case report by Sklerov, et al. could influence future legal decisions on this matter in the United States and in other countries, should they be considered on their superficial merits. Moreover, scientific and intellectual integrity demand clarity on this complex issue.

Sincerely,
Jace C. Callaway, Ph.D.
Department of Pharmaceutical Chemistry, University of Kuopio, Finland

Charles S. Grob, M.D.
Professor of Psychiatry and Pediatrics, UCLA School of Medicine Director of the Division of Child Adolescent Psychiatry, Harbor-UCLA Medical Center

Dennis J. McKenna, Ph.D.
Centre for Spirituality & Healing, University Of Minnesota
Director of Ethnopharmacology, Heffter Research Institute

David E. Nichols, Ph.D.
Medicinal Chemistry and Molecular Pharmacology, Purdue University
Professor of Medicinal Chemistry and Molecular Pharmacology

Alexander Shulgin, Ph.D.
Pharmacologist, Chemist

Kenneth W. Tupper
Department of Educational Studies, University of British Columbia”

—–

ORIGINAL TEXT PUBLISHED AT THE JOURNAL OF ANALYTICAL TOXICOLOGY IN 2005

Reference: Sklerov, J., Levine, B., Moore, K.A., King, T. & Fowler, D. (2005). A fatal intoxication following the ingestion of 5-methoxy-N,N-dimethyltryptamine in an ayahuasca preparation. Journal of Analytical Toxicology. 29(8), 838-841.

“Abstract

A case of a 25-year-old white male who was found dead the morning after consuming herbal extracts containing β-carbolines and hallucinogenic tryptamines is presented. No anatomic cause of death was found at autopsy. Toxicologic analysis of the heart blood identified N,N-dimethyltryptamine (0.02 mg/l), 5-methoxy-N,N dimethyltryptamine (1.88 mg/l), tetrahydroharmine (0.38 mg/l), harmaline (0.07 mg/l), and harmine (0,17 mg/l). All substances were extracted by a single-step n-butyl chloride extraction following alkalinization with borate buffer. Detection and quantitation was performed using liquid chromatography-electrospray mass spectrometry. The medical examiner ruled that the cause of death was hallucinogenic amine intoxication, and the manner of death was undetermined.

Introduction

Ayahuasca, also commonly known as yagé, hoasca, and daime, refers to a decoction of the woody vines of Banisteriopsis caapi. The extract is prepared as a tea formulation, usually with additional plant material containing psychoactive drugs. Ayahuasca has a long history of use in various South American medical and sacramental practices, and its use has expanded into North America and Europe in recent years (1). The foundation of the ayahuasca beverage is the β-carboline alkaloids: harmine, harmaline, and tetrahydroharmine (THH) found in B. caapi. Harmine and harmaline are competitive, reversible inhibitors of monoamine oxidase type-A (MAO), whereas THH is believed to inhibit presynaptic serotonin uptake (2). Harmine, harmaline, and B. caapi extract have also demonstrated increased dopamine release in vitro (3). The harmala alkaloids themselves have mild sedative effects, possibly through interaction with the benzodiazepine receptor (4).

The ayahuasca brew is commonly prepared with the leaves of Psychotria viridis, which contains the U.S. Drug Enforcement Administration Schedule I hallucinogen N,N-dimethyltryptamine (DMT), although nicotine, cocaine, atropine, and other alkaloid combinations have been described (5). Hallucinogenic drugs are generally composed of two groups: phenethylamines and indoleamines. DMT is the prototypical drug of the tryptamine subgroup of the indoleamine hallucinogenic drug group. Other tryptamine derivatives that have been identified as hallucinogenic drugs include 5-methoxy-α-methyltryptamine, α-methyltryptamine, 5-methoxy-N,N-DMT (5-MeO-DMT), N,N-diethyltryptamine, N,N-dipropyltryptamine, and 5-methoxy-N,N-diisopropyltryptamine. All of these compounds have high affinity for the serotonin 5-HT2 receptors; hallucinogenic potency is well correlated with the relative affinity of these compounds for the 5-HT2 receptor (6).

DMT and its analogues are metabolized by MAO in the liver. When it is administered orally, first pass metabolism prevents significant absorption of DMT. However, when taken as part of an ayahuasca beverage, the presence of the harmala alkaloids significantly reduces the first-pass metabolism of DMT. As a result, more DMT is free to circulate to the central nervous system where it binds to serotonergic receptors and exerts its psychedelic effects (7). The onset of action of an ayahuasca preparation is between 35 and 40 min with effects felt for up to 3 or 4 h (8-10). Subjective effects have been reported to include nausea, dose-dependent visual and auditory perception changes, personal psychological introspection, increased alertness and stimulation, and changes in gauging the passing of time (7,9,11). Cardiovascular effects have included moderate increases in systolic/diastolic blood pressure and heart rate (7,11) with only diastolic increases significant over placebo (7).

We report a fatal case involving the recreational administration of an ayahuasca-like preparation containing 5-MeO-DMT. Quantitative measurements of 5-MeO-DMT, DMT, harmine, harmaline, and THH are reported for postmortem samples, 5-MeO-DMT has been reported to have greater potency than DMT when smoked (12). Presumably this translates to an equivalent potency difference when both are administered orally in combination with a monoamine oxidase inhibitor. This premise was advanced by the self-experimentation reported by Ott (8), which showed oral activity of 5-MeO-DMT at lower doses than DMT when in the presence of identical harmaline doses.

Case History

A 25-year-old white male went camping with family and friends in a national park. According to these individuals, he drank some type of “herbal tonics” and went to sleep. He was found dead the next morning. Subsequent investigation indicated that the decedent ingested a preparation from a South American tree bark, “ooasca” (sic), and approximately 4 h later, he ingested tryptamines.

An autopsy was performed the day after the body was discovered. External examination identified the presence of lividity that was fixed on the posterior surface of the body, except in areas exposed to pressure. The body was in full rigor. No remarkable external findings were noted. Internal examinations, both gross and microscopic, were unremarkable except for some tissue congestion and edema. Specimens were sent to the laboratory for toxicologic analysis.

Experimental

Materials

DMT was obtained from the Drug Enforcement Administration central laboratory (Atlanta, GA). 5-MeO-DMT was purchased over the internet from JMAR Chemical Corporation. Harmine hydrochloride, ammonium formate, 5-fluorotryptamine, and sodium borate decahydrate were obtained from Sigma-Aldrich (St. Louis, MO). Harmaline hydrochloride was purchased from Acros Organics (Morris Plains, NJ), and sodium borohydride was purchased from Fisher Scientific (Pittsburgh, PA). All solvents were high-performance liquid chromatography (HPLC) grade and were purchased from Fisher Scientific.

Preparation of tetrahydroharmine

The synthesis, of THH was based on a previously reported method (13). Harmaline HCl (500 mg) was added to 50 mL of methanol in a 0°C ice bath. The methanol was acidified by the addition of 4M HCI, and sodium borohydride was slowly added until the solution turned pale yellow and gas was no longer formed. After 30 min, the solution was alkalinized (pH > 10) by the addition of 1N NaOH. The solution was extracted twice with 10 mL of n-butyl chloride, and the solvent was transferred to a 100-mL beaker to evaporate at room temperature. The resulting crude tetrahydroharmine crystals were dissolved in methanol, and 100-µL portions were injected into an Agilent 1100 HPLC (Palo Alto, CA). The instrument consisted of a vacuum degasser, quaternary pump, well-plate autosampler, column compartment, diode-array detector (DAD), and fraction collector. An isocratic mobile phase of 25% acetonitrile, 75% 0.02M ammonium formate was used at a flow rate of 0.8 mL/min. The separation column was a Phenomenex Prodigy-ODS (150 x 4.6 mm, dP = 5 µm) maintained at 35°C. Time-based collections of the tetrahydroharmine peak were made, and fractions were pooled after each injection. Pooled fractions were alkalinized with ammonium hydroxide and extracted into n-butyl chloride. The solvent was evaporated, and the crystals were assayed for purity using the aforementioned HPLC-DAD apparatus. The chromatographic purity of the tetrahydroharmine was 87.4%, and its identity was verified using the full scan mass spectrometry parameters listed herein.

Sample preparation

Blood (central and peripheral), urine, gastric contents, bile, kidney, brain, and liver were collected at autopsy and stored, unpreserved, at -15°C. Standard curves were prepared in blood and urine at 0.010, 0.025, 0.050, 0.10, 0.50, 1.0, and 2.5 mg/L for DMT, 5-MeO-DMT, and harmine. Separate standard curves were prepared for THH and harmaline due to the lower purity of these standard reference materials.

A 5-fluorotryptamine internal standard was prepared at a concentration of 0.01 mg/mL in methanol. One-milliliter volumes of blood, urine, bile, and gastric were initially assayed, but final quantitation was based on respective dilutions of each. Tissue samples (0.5 g) were homogenized in 3 mL of saturated sodium borate buffer using a Brinkmann PT3000 tissue homogenizer (Westbury, NY). One-milliliter aliquots of homogenate were extracted.

Samples and/or their dilutions were added to clean, labeled 16 x 100-mm tubes, and 2 mL saturated sodium borate was added. Fifty microliters of the 5-fluorotryptamine internal standard solution was added to each tube along with 2 mL of n-butyl chloride. The tubes were capped and mixed for 10 min on an orbital mixer. After centrifuging the tubes for 10 min at 3500 rpm, the solvent was transferred to 10-mL conical tubes, a drop of 1% HCI in methanol was added, and the extracts were evaporated to dryness under nitrogen at 40°C. The sample residue was reconstituted with 100 µL of HPLC mobile phase (75% 0.02M ammonium formate/25% acetonitrile) and transferred to autosampler vials. Two microliters was injected for analysis.

Instrumentation

Biological extracts were analyzed using an Agilent 1100 LC-mass selective detector (SL) equipped with an orthogonal electrospray ionization interface. Separation was performed using an XTerra® MS-C18 column (100 x 3.0 mm, dP = 3.5 µm, Waters, Milford, MA) held at 35°C. The mobile phase consisted of 0.02M ammonium formate (75%) and acetonitrile (25%) at a flow rate of 0.4 mL/min.

The positive, pseudomolecular ions of DMT (m/z 189), 5-MeO-DMT (m/z 219), 5-FT (ISTD, m/x 179), THH (m/z 217), harmaline (m/z 215), and harmine (m/z 213) were formed by electrospray ionization and selectively monitored for quantitation. Pneumatic-assisted nebulization utilized nitrogen at 30 psi, and 350°C nitrogen drying gas was used at a flow of 12 L/min. Full scan mass spectra were also acquired at three separate capillary exit voltages (100, 150, 200V) over the mass range m/z 100-400. The fragmentation patterns were compared to those of the drug standards for confirmatory identification.

Results

The heart blood and urine specimens from this case were tested for volatile substances and therapeutic and abused drugs. This included volatile testing for methanol, ethanol, acetone, and isopropanol by headspace gas chromatography (GC); acid/neutral drug testing by GC-nitrogen-phosphorus detection (NPD); alkaline drug testing by GC-NPD; morphine by radioimmunoassay; and acetaminophen and salicylate by color test. No ethanol or other volatile substances were detected in the case. Diphenhydramine was detected in the urine; no diphenhydramine was detected in the blood at a limit of quantitation of 0.05 mg/L. In addition, an unidentified peak was detected in both the blood and urine on the alkaline drug screen that eluted around chlorpheniramine. Subsequent mass spectral analysis tentatively identified the substance as 5-MeO-DMT (14). This was verified when an authentic drug standard was subjected to the same extraction, chromatographic, and mass spectral conditions.

Table I lists the distribution of the tryptamines and β-carbolines in the specimens, and Figure 1 shows representative chromatograms of the heart blood sample and a negative blood control. Quantitative values were calculated by multi-point, linear regression of the peak-area ratio of individual compounds to that of the 5-FT internal standard. Regression lines for the five analytes’ standard curves were between 0.989 and 0.999. The limits of detection, quantitation, and linearity for all drugs were 0.005, 0.01, and 2.5 mg/L, respectively. All of the analytes were identified in the specimens except for DMT, which was not present in the brain, kidney, or liver. In samples where DMT and 5-MeO-DMT were both measured, 5-MeO-DMT was present at higher concentrations. THH was present in higher amounts than both harmaline and harmine in all samples with the exception of the gastric contents, where the amount of harmine was 10-fold greater than that of THH.

Discussion

To the authors’ knowledge, this is the only reported case of death following ingestion of hallucinogenic tryptamines contained in an ayahuasca preparation. Morano et al. (15) reported a fatal case of ethyltryptamine intoxication in a 19-year-old female who consumed a beer allegedly containing Ecstasy. The heart blood concentration in this case was 5.6 mg/L. In addition, methamphetamine (0.12 mg/L) and amphetamine (0.05 mg/L) were detected. Warren (16) presented an acute nicotine intoxication resulting in death following ayahuasca ingestion during a healing ritual. Harmine and harmaline presence were reported in the ayahuasca brew.

In addition to fatalities from these preparations, there have also been several non-fatal intoxications from hallucinogenic tryptamine use. Meatherall and Sharma (17) published a case report of a 21-year-old male who ingested 5-methoxy-N,N-diisopropyltryptamine, also known as “Foxy.” A urine concentration of 1.7 mg/L was measured. A metabolite, 5-methoxy-indoleacetic acid (1.3 mg/L) was also detected. Within 4 h of oral ingestion, the hallucinations stopped and the patient was discharged from the hospital. Another nonfatal case of 5-methoxy-N,N-diisopropyltryptamine was reported by Vorce and Sklerov (18); the urine concentration was 0.229 mg/L. In addition, a monoisopropyl metabolite was tentatively identified. A single case of intoxication in a 17-year-old college student was reported after the ingestion of the extract of three Peganum harmala seeds and a combined smoked and snorted dose of 25-30 mg of 5-MeO-DMT (19). This was accompanied by emesis, hyperthermia, increased heart rate, and rhabdomyolysis. However, in this case, only harmine and harmaline were detected in urine.

Oral dosing of 15 volunteers with a traditional ayahuasca preparation was reported by Callaway et al. (13). Average doses, based on a 59-kg individual, were 28.8, 204, 24, and 128.4 mg for DMT, harmine, harmaline, and THH, respectively. The DMT plasma range reported for this trial was 0.011-0.025 mg/L, which is similar to both central and peripheral values for this case. Plasma carboline concentrations were given in the following ranges: THH, 0.049-0.134 mg/L; harmaline, <>References

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