Forensic Toxicology https://doi.org/10.1007/s11419-017-0394-5

CASE REPORT

Ayahuasca and Kambo intoxication after alternative natural therapy for depression, confirmed by mass spectrometry Damila Rodrigues de Morais1 · Rafael Lanaro2,3 · Ingrid Lopes Barbosa1 · Jandyson Machado Santos1 · Kelly Francisco Cunha4 · Vinicius Veri Hernandes1 · Elias Paulo Tessaro1 · Cezar Silvino Gomes5 · Marcos Nogueira Eberlin1 · Jose Luiz Costa2,6 Received: 16 August 2017 / Accepted: 3 November 2017 © Japanese Association of Forensic Toxicology and Springer Japan KK, part of Springer Nature 2017

Abstract Purpose  We present a case report about an acute intoxication episode after an oral administration of Ayahuasca and dermal exposure of Kambo for treatment of depression. The clinical features observed were hallucination, agitation, tremors of extremities, oral paresthesia, skin lesions and seizures. Diazepam was administered by the emergency service and was effective in controlling hallucination, but failed to control agitation and seizures. Methods  Patient biological fluids (urine and serum) and the samples of Ayahuasca and Kambo were submitted to toxicological analysis using liquid-liquid extraction followed by liquid chromatography–tandem mass spectrometry and high-resolution electrospray ionization-mass spectrometry. Results  The main active compounds present in Ayahuasca, N,N-dimethyltryptamine, harmine, harmaline and tetrahydroharmine were found and quantified in the different samples, confirming the use by the patient. In Kambo secretion used in the ritual, we were able to find sixteen potently active peptides: adenoregulin, bombesin, bombesinnona peptide, bradykinin -phe(8)-psi-CH2NH-arg(9)-, caerulein, deltorphin, neurokinin B, phyllomedusin, phyllocaerulein, phyllokinin, phyllolitorin, preprotachykinin B (50–79), ranatachykinin A, sauvagine, T-kinin and urechistachykinin II. Conclusions  The patient was discharged the day after exposure without any sequel. Clinical and toxicological analysis indicated that the symptoms presented by the patient occurred due to a joint action produced by the substances identified in both materials. To the best of our knowledge, this is the first case involving probable intoxication by simultaneous administration of Ayahuasca and Kambo. Keywords  Ayahuasca · Kambo · Depression treatment · N,N-Dimethyltryptamine · Adenoregulin · High-resolution ESI-MS

Introduction * Jose Luiz Costa [email protected] 1



ThoMSon Mass Spectrometry Laboratory, University of Campinas (UNICAMP), Campinas, SP, Brazil

2



Poison Control Center, University of Campinas (UNICAMP), Campinas, SP, Brazil

3

Department of Pharmacology, Faculty of Medical Sciences, University of Campinas (UNICAMP), Campinas, SP, Brazil

4

Faculty of Medical Sciences, University of Campinas (UNICAMP), Campinas, SP, Brazil

5

Brazilian Federal Police, Natal, RN, Brazil

6

Faculty of Pharmaceutical Sciences, University of Campinas (UNICAMP), Campinas, SP, Brazil



Depression is a serious and common psychiatric disorder, which contributes to mortality and morbidity [1]. Approximately 350 million people are affected by depression worldwide [2]. This illness is characterized by intense suffering in personal, occupational, and social life, and in the worst cases can lead to suicide or suicide attempts [3, 4]. According to Osório et al. [5], depression etiology is unknown but some theories, such as the monoamine hypothesis, suggest that an imbalance in cerebral dopamine, norepinephrine and serotonin neurotransmitters is responsible for depressive symptoms. The monoamine hypothesis is the leading hypothesis behind pharmacotherapy treatment for depression with different kinds of medicines such as fluoxetine (selective

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Fig. 1  Skin lesions on the leg of the patient after Kambo with a fire stick (a). The stick with secretion of Kambo applied to the patient (b)

serotonin reuptake inhibitor), isoniazid (monoamine oxidase inhibitor), amitriptyline (tricyclic antidepressant), mirtazapine (tetracyclic antidepressant), and other antidepressants. Other kinds of treatments for depression, also scientifically recognized, include psychotherapy (a controversial method with a great variety of alternatives including cognitive behavioral therapy, hypnosis therapy and Gestalt-hypnosis therapy), electroconvulsive therapy and repetitive transcranial magnetic stimulation [1, 6]. Pharmacotherapy and psychotherapy are both known as first-line treatments for depression, but have demonstrated efficacy only for about the half number of depressed individuals that have ever sought professional care [4]. Both treatments also present other problems such as poor compliance with medication, because medicines are not always available or affordable, and long durations or delays in treatment are common [1]. There are, therefore, compelling reasons for many people who suffer from depression to consider alternative natural therapies such as physical activity, hobbies, use of phytotherapy, acupuncture, and religious and spiritual relief [1, 7]. Ayahuasca (a blend of Banisteriopsis caapi vine and the Psychotriaviridis leaf) traditionally used by indigenous religious populations of the Amazon as a natural and spiritual medicine [8] has been previously reported to display antidepressive potential [5, 7, 9, 10]. The main components of Ayahuasca are N,N-dimethyltryptamine (DMT) and some β-carboline alkaloids, such as harmine, harmaline and tetrahydroharmine. These molecules are structurally similar to serotonin and reversible inhibitors of mitochondrial monoamine oxidase, which is responsible for the oxidation of neurotransmitters, resulting in elevated levels of brain serotonin, which enhances a sensation of well-being [5, 7, 11–13]. Because DMT and other active compounds present in Ayahuasca exert synergic psychoactive effects [7], there is a possibility that simultaneous use of Ayahuasca and other natural medicines can lead to health problems, such as intoxication. In Brazil, to avoid health and social problems, Ayahuasca beverage consumption has been regulated since 1986 [14, 15], while ensuring the freedom of religious

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practices by groups such as Santo Daime and União do Vegetal (UDV). According to Leban et al. [16], the use of a frog Phyllomedusa bicolor dermis secretion is also widely known and employed in religious practices and rituals to purify the body, to improve luck in hunting and to treat various diseases. In the ceremony, the secretion of the frog, known as frog vaccine or Kambo, which contains bioactive peptides (phyllocaerulein, phyllomedusin, phyllokinin, dermorphins, deltorphins, adenoregulin) is rubbed into the subject’s burned skin. Phyllocaerulein has a potent action on the gastrointestinal system, and phyllomedusin is an active vasodilator peptide that excites neurons and evokes behavioral responses [17, 18]. Dermorphins present an analgesic effect, and both phyllomedusin and phyllokinin increase the permeability of the blood-brain barrier, which facilitates the access of other peptides to the brain [19]. The present work reports an acute intoxication episode after simultaneous use of Ayahuasca and Kambo for treatment of depression. For the toxicological analyses, a previously developed liquid chromatography–tandem mass spectrometry (LC–MS/MS) method [12] was used to simultaneously identify and quantify Ayahuasca samples (biological fluids and brown tea), and ultra-high resolution electrospray ionization-mass spectrometry (ESI-MS) fingerprinting was also used to characterize the Kambo sample.

Case report A 37-year-old woman was admitted to emergency service (ES) presenting visual hallucination, agitation, tremors of extremities, oral paresthesia, seizures, nausea, vomiting, sweating and five skin lesions on the leg (Fig. 1a). It was reported by her parents that she submitted to an alternative treatment for depression at a spiritualist center where she ingested approximately 100 mL of a brown tea and received five subcutaneous applications of a secretion present on a stick (Fig. 1b) called frog vaccine. The main reason for this

Forensic Toxicology

treatment was the patient’s fear of adverse reactions caused by antidepressants. Diazepam was administered by ES and effectively controlled the hallucinations but failed to control agitation and seizures. The patient was asymptomatic after 6 h and discharged within 24 h without any sequel. The brown tea and the frog vaccine were suspected to be Ayahuasca and Kambo, respectively. The healer who works at the spiritualist center was reached to confirm that he had, indeed, collected, produced and administered the described materials. He also believed that the symptoms presented were a spiritual manifestation, part of the cleaning ritual, and should not be a cause for concern. Urine and blood samples were collected from the patient and submitted to toxicological analysis, together with the brown tea and the stick containing the secretion.

Materials and methods Chemicals Reference standards of harmine and harmaline were purchased from Sigma-Aldrich (Steinheim, Germany); DMT from THC Pharm (Frankfurt, Germany). Tetrahydroharmine was synthesized as described previously by Callaway et al. [20], and DMT-d6 was synthesized by a modified procedure based on the selective dimethylation method described by Pires et al. [21] and Giumanini et al. [22]. HPLC grade methanol was from Merck (Darmstadt, Germany), and water was purified by a Milli-Q gradient system (Millipore, Milford, MA, USA). Stock solutions (1.0 mg/mL) of DMT, harmine, harmaline, and tetrahydroharmine were prepared in methanol and stored at – 20 °C.

Toxicological analysis The toxicological analysis in biological samples (serum and urine), brown tea (Ayahuasca) and secretion (Kambo) were performed in the Poison Control Center laboratory and in the ThoMSon Mass Spectrometry Laboratory, both at University of Campinas (UNICAMP), by gas chromatograpy–tandem mass spectrometry (GC–MS/MS), LC–MS/MS and highresolution ESI-MS (Kambo confirmation), and included the detection, identification and quantification of the substances presented in the materials collected. Screening analysis in urine was done by GC–MS/MS (GCMS-TQ8040 Shimadzu, Kyoto, Japan) using a systematic toxicological analysis method developed by Maurer et al. [23]. The procedure was based on acid hydrolysis, liquid-liquid extraction and acetylation under microwave radiation; 2 µL was injected into the gas chromatograpy–mass spectrometry (GC–MS) system. The full-scan data files

obtained by the GC–MS analysis were analyzed by AMDIS in the simple mode.

Ayahuasca samples For Ayahuasca analytes (dimethyltryptamine, harmine, harmaline and tetrahydroharmine), urine and serum collected from the patient were immediately submitted to toxicological analysis, together with the beverage. At first, 200 µL of each sample was extracted in separate Eppendorf tubes, after addition of 50 µL of internal standard (IS) (DMT-d6, 250 ng/mL), 1 mL of methyl tert-butyl ether and 100 µL of saturated sodium tetraborate and vortexing for 5 min each. The mixtures were centrifuged for 5 min at 12,000 rpm at room temperature. The organic layer was transferred to a separate tube and was evaporated at 40 °C under a gentle stream of nitrogen. After it dried completely, the residue was reconstituted in 200 µL of methanol. The solution was transferred to LC vials, and 10 µL was submitted the LC–MS/MS (LCMS-8040 Shimadzu, Kyoto, Japan) analysis, and the target analytes (dimethyltryptamine, harmine, harmaline, tetrahydroharmine) were evaluated according to Oliveira et al. [12]. Each mobile phase consisted of 2 mM ammonium formate + 0.1% formic acid in water (solvent A) or methanol (solvent B). The analytes were separated using a C18 column (Atlantis-T3, 150 × 4 mm, particle size 3 µm; Waters, Dublin, Ireland), and the oven was operated at 40 °C. The chromatography was carried out using a gradient elution, where solvent B was maintained at 10% for the first 0.5 min, followed by linear increase to 95% from 0.5 to 4.5 min and held from 4.5 to 7.0 min. From 7.0 to 7.1 min it was decreased back to 10% B and held until 12 min for equilibration. The flow rate was kept constant at 0.3 mL/min. The separated analytes were identified and quantified with the triple quadrupole mass spectrometer operating in the multiple reaction monitoring (MRM) mode via positive ion ESI. The applied ESI(+) conditions were: capillary voltage 3.5 kV, desolvation temperature 250 °C, heat block temperature 400 °C, drying gas (­ N2) flow 15 L/min, nebulizing gas ­(N2) flow 3 L/min and collision gas (Ar) 230 kPa. MRM transitions and collision energies were established for each analyte. Data were acquired and treated using the software Labsolution (version 5.53 SP2, Shimadzu).

Method validation The LC–MS/MS method used for alkaloid quantification in the three different matrices (serum, urine and Ayahuasca) was validated based on international guidelines for illicit drug analysis [24] with respect to limit of detection (LOD) and limit of quantification (LOQ), linearity, recovery rates, imprecision, and accuracy. The LOD and LOQ represent the

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Forensic Toxicology

lowest concentration of the substance under evaluation that can be detected and quantified using a certain experimental procedure. These parameters were calculated based on the signal-to-noise (S/N) ratios (S/N = 3 for LOD, S/N = 10 for LOQ). The calibration curves were constructed with blank biological matrices by spiking different concentrations of reference standards with IS prior to extraction in order to obtain the final concentrations in the calibration ranges from 5 to 200 ng/mL for urine and from 0.3 to 200 ng/mL for serum. The curve for Ayahuasca was constructed with methanol by spiking reference standards with IS in order to obtain the final concentrations in calibration range from 0.3 to 200 ng/mL. All curves were constructed by plotting the peak area ratio (analyte standard area/IS area) versus analyte concentration using the least-square linear regression method (1/x2 weighting) and coefficient of determination (r2). Accuracy and imprecision were determined using quality control (QC) samples prepared by spiking 1 and 150 ng/ mL (low and high QCs) of each alkaloid for serum and Ayahuasca matrices, and by spiking 15 and 150 ng/mL (low and high QCs) of each alkaloid for urine matrix. Three replicates of QC samples were run in the same batch together with calibration standards, and the calibration curve was then obtained and the QC concentrations determined. The accuracy was determined dividing the mean concentration for analytical runs (intraday n = 3; interday n = 9) by the expected concentration, and expressed as percentage. Accuracy values in the range of 80–120% were considered acceptable. The intraday and interday imprecisions were obtained by calculating the relative standard deviation (%RSD) for each mean concentration (n = 3 and n = 9, respectively), and one-way analysis of variance (ANOVA) was performed on each QC concentration to assess potentially significant interday variability at p < 0.05. Imprecision values with %RSD less than 20% were considered acceptable. Recovery rates were assessed in the blank biological matrices by spiking reference standards with IS prior to extraction to obtain the final concentration of 1 and 150 ng/mL of each alkaloid in serum, and 15 and 150 ng/mL of each alkaloid in urine. In Ayahuasca, recovery rates were assessed by spiking standards with IS in order to obtain the final concentration of 1 and 150 ng/mL of each alkaloid. The recovery rates were considered acceptable above 80%. The matrix effect was also studied, because it can affect the ionization of analytes, mainly when working with electrospray ionization. The method specificity was assessed for endogenous interference by analyzing blank serum and urine. Potential exogenous interferences by 28 commonly encountered drugs of abuse and medications were also assessed by fortifying them at 500 ng/mL into low QC and negative (only IS) samples. No interference can be judged if all analytes in the low QC are quantified within ± 20% of target concentration together with acceptable qualifier/quantifier MRM ratios

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and no peaks in the negative samples satisfy LOD criteria. Dilution integrity was determined for urine matrix by comparison of a standard solution of 1000 ng/mL against two different dilution ratios, 1:10 and 1:20 (v/v), and results were expressed as percentages.

Kambo samples Two samples were tested to confirm the use of Kambo by the patient: the stick containing the frog’s waxy secretion (K1) and a sample of the poison used for fingerprinting comparison (K2).1 For these analyses, high-resolution ESI-MS was performed using a Q-Exactive system (Thermo Fischer Scientific, Bremen, Germany) comprised of a heated ESI probe source and a hybrid quadrupole-Orbitrap mass analyzer. The sample extract (1 mg) was diluted in 1 mL of water/acetonitrile (1:1, v/v) mixture, and the mass spectra were acquired in the positive or negative ion modes. The extract solution and the reference poison were analyzed by direct infusion at a flow rate of 10 µL/min. The experimental parameters for the high-resolution ESI-MS analyses were as follows: spray voltage 3.5 (positive mode) and –3.4 kV (negative mode), capillary temperatures 280 (positive mode) and 320 °C (negative mode), and s-lens 100 V. The analyses were performed in the range of m/z 300–2000.

Results and discussion Method validation Table 1 shows the validation results by the LC–MS/MS method for alkaloid (harmine, harmaline and tetrahydroharmine) parameters in different matrices (serum, urine and Ayahuasca). The LOD and LOQ for DMT, harmine, harmaline and tetrahydroharmine were of 5 and 15 ng/mL for urine, 0.3 and 1 ng/mL for serum, and 0.3 and 1 ng/mL for Ayahuasca. The method was also found to be linear from 5 to 200 ng/ mL for urine, from 0.3 to 200 ng/mL for serum and 0.3 to 200 ng/mL Ayahuasca for all four alkaloids (DMT, harmine, harmaline and tetrahydroharmine) with 1/x2 weighted linear regression. The imprecisions (intraday and interday %RSD) and accuracy bias (%) in three days were satisfactory at both QC concentrations, with no more than 20% RSD. The recovery rates were higher than 80%, and considered acceptable. DMT, harmine, harmaline and tetrahydroharmine showed 1

 The sample collection was approved by the “Sistema de Autorização e Informação em Biodiversidade” (Sisbio), process number 37974-1 and responsible professor Dr. Moisés Barbosa de Souza— Center of Biological and Nature Sciences/Federal University of Acre—UFAC, Rio Branco-Acre-Brazil.

Forensic Toxicology Table 1  Validation parameters (imprecision, accuracy, recovery rate and matrix effect) for the alkaloids (harmine, harmaline, tetrahydroharmine and N,N-dimethyltryptamine) in different matrices (serum, urine and Ayahuasca) Analyte

Matrix

Harmine

Serum  Low QC (1 ng/mL)  High QC (150 ng/mL) Urine  Low QC (15 ng/mL)  High QC (150 ng/mL) Ayahuasca  Low QC (1 ng/mL)  High QC (150 ng/mL) Serum  Low QC (1 ng/mL)  High QC (150 ng/mL) Urine  Low QC (15 ng/mL)  High QC (150 ng/mL) Ayahuasca  Low QC (1 ng/mL)  High QC (150 ng/mL) Serum  Low QC (1 ng/mL)  High QC (150 ng/mL) Urine  Low QC (15 ng/mL)  High QC (150 ng/mL) Ayahuasca  Low QC (1 ng/mL)  High QC (150 ng/mL) Serum  Low QC (1 ng/mL)  High QC (150 ng/mL) Urine  Low QC (15 ng/mL)  High QC (150 ng/mL) Ayahuasca  Low QC (1 ng/mL)  High QC (150 ng/mL)

Harmaline

Tetrahydroharmine

N,N-dimethyltryptamine

Intraday imprecision (%) (n = 3)

Interday imprecision (%) (n = 9)

Accuracy bias (%) (n = 9)

Recovery rate (%) (n = 6)

Matrix effect (%) (n = 6)

5.2 4.9

7.0 4.5

−8.9 −0.6

92.0 91.1

−8.4 34.1

4.3 3.9

6.0 9.3

6.4 −7.4

93.2 97.5

7.5 −2.4

3.6 2.3

5.1 13.2

−7.8 −9.2

93.5 –

– –

5.2 4.7

5.7 4.1

−10.0 −0.2

86.8 87.0

−4.4 33.5

2.5 4.2

4.8 9.6

5.0 –6.5

92.3 90.4

3.0 –2.7

10.8 2.3

10.4 12.5

−7.8 −3.3

110 –

– –

7.2 6.1

8.7 5.5

3.3 2.1

97.2 90.0

−10.2 43.4

4.9 4.6

8.1 10.9

3.5 −5.7

96.4 80.4

−0.7 −4.9

9.4 2.0

11.7 18.1

0.0 3.7

95.3 –

– –

6.8 3.8

6.8 4.3

−2.2 2.8

99.1 95.1

−11.9 33.1

2.1 4.1

3.7 11.1

2.8 −4.5

96.0 88.0

−0.2 −4.3

10.1 2.5

12.5 16.2

−6.7 1.7

95.2 –

– –

QC quality control

maximal matrix effects in serum at high QC. No interference was observed when LOQ was analyzed in the presence of 500 ng/mL of other drugs of abuse and pharmaceuticals. Dilution integrity for the urine matrix were acceptable with RSDs from 10 to 20% for all analytes.

Table 2  Alkaloid levels in biological samples of patient and in Ayahuasca tea

Ayahuasca samples

Dimethyltryptamine Harmine Harmaline Tetrahydroharmine

Table 2 shows the quantification results of DMT and the β-carbolines (harmine, harmaline and tetrahydroharmine)

Alkaloid

Sample Patient’s serum Patient’s urine Brown tea (ng/mL) (ng/mL) (µg/mL) 3.2 12.3 0.3 182

488 60.6 49.4 567

678 597 132 1240

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Forensic Toxicology

in serum, urine (after 3 h exposure) and Ayahuasca samples. Diazepam was also found in the biological samples, because the ES administered it. The levels of DMT and β-carbolines found in the brown tea were similar to those obtained in the previous work and compatible with Ayahuasca use in religious contexts [11]. The patient’s symptoms such as visual hallucination, nausea, and vomiting seemed to be caused by serotonergic stimulation of the vagal pathways, the main mechanism of action after oral DMT exposure [11, 12].

Kambo samples Analysis of animal toxins generally poses an analytical challenge; therefore it was expected that toxicological analysis

by LC–MS/MS of urine and serum samples of the patient would result in no toxins found, likely due the low amounts of the peptides applied to the body or due to their rapid metabolism. The patient presented skin lesions on the leg and her companion had retained a secretion on a stick used in the ritual. To elucidate the case, we evaluated such drugs (K1) and the available sample of skin secretions of Phyllomedusa bicolor used in the Kambo ritual of Amazon natives (K2). K1 and K2 were then compared via high-resolution ESI-MS fingerprinting analysis in both the negative (Fig. 2) and positive (Fig. 3) ion modes. Table 3 summarizes the sixteen different peptides identified. Therefore,

Fig. 2  High-resolution electrospray ionization (−) [ESI(–)] mass spectra for Phyllomedusa bicolor secretion in K1 (a) and K2 (b) samples

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Forensic Toxicology

Fig. 3  High-resolution ESI (+) mass spectra for Phyllomedusa bicolor secretion in K1 (a) and K2 (b) samples

the patient was confirmed to be treated with the frog secretion. The peptides found in K1 were likely responsible for the patient`s symptoms such as seizures, nausea, vomiting and sweating, reported in another case report of Kambo intoxication [16], where phyllocaerulein was likely responsible for the last three symptoms [17, 18].

The tremors could be associated with the presence of bombesin [25], whereas neurokinin B could be associated with variations in the heart rate, blood pressure, as sweating and skin temperature [26]. The caerulein could cause falling blood pressure, sweating and tachycardia [27].

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Forensic Toxicology

Table 3  Identified peptides from a secretion in the stick (K1) and Phyllomedusa bicolor sample secretion (K2), their associated molecular formula, theoretical and experimental mass and error obtained from electrospray ionization (±) mass spectrometry data Ionization mode Peptide

Molecular formula

Theoretical mass (Da) Theoretical (m/z) Experimental (m/z)

Error (ppm)

ESI(−)

Adenoregulin

C142H242N40O42

3179.80303

1058.93434

ESI(+)

Bombesin dihydrochloride

C71H112Cl2N24O18S 1690.76841

564.58947

ESI(−)

Bombesinnona peptide

C47H71N15O11S

1053.51781

1052.51782

ESI(–)

Bradykinin, phe(8)-psiCH2NH-arg(9)-

C50H75N15O10

1045.58213

1044.58213

ESI(+)

Caerulein

C58H73N13O21S2

1351.44853

676.72427

ESI(−)

Deltorphin

C44H62N10O10S2

ESI(+)

Neurokinin B

C55H79N13O14S2

1209.53108

404.17703

ESI(−)

Phyllomedusin

C52H82O13N16S

1170.59680

1169.58842

ESI(−)

Phyllocaerulein

C54H67O19N11S2

1237.40561

1236.39723

ESI(−)

Phyllokinin

C65H93N17O14

1335.70879

1334.70879

ESI(+)

Phyllolitorin

C49H69N11O11S

1019.48987

1020.48987

ESI(−)

Preprotachykinin B (50–79) C143H243N37O46

3214.78129

1070.59376

ESI(+)

Ranatachykinin A

C60H92N16O15S

1308.66487

1309.66488

ESI(+)

Sauvagine

C202H346N56O63S

4596.53129

920.30626

ESI(+)

T-kinin

C59H89N17O14

1259.67749

418.89250

ESI(+)

Urechistachykinin II

C44H66N14O10S

982.48070

492.24035

K1 K2 K1 K2 K1 K2 K1 K2 K1 K2 K1 K2 K1 K2 K1 K2 K1 K2 K1 K2 K1 K2 K1 K2 K1 K2 K1 K2 K1 K2 K1 K2

954.409180

953.40918

K1 K2 K1 K2 K1 K2 K1 K2 K1 K2 K1 K2 K1 K2 K1 K2 K1 K2 K1 K2 K1 K2 K1 K2 K1 K2 K1 K2 K1 K2 K1 K2

1058.93706 1058.93469 564.58729 564.58929 1052.51643 1052.51235 1044.57764 1044.57733 676.72791 676.72737 ND 953.40523 404.17648 404.17649 1169.59119 1169.58972 1236.40051 1236.39929 1334.70292 1334.70278 1020.48438 1020.48563 1070.59398 1070.59329 1309.66599 1309.66577 920.30635 920.30835 ND 418.89203 492.23801 492.23741

2.565 −0.327 −3.867 0.324 −1.320 5.196 −4.302 4.599 5.380 −4.582 ND 4.143 −1.357 1.332 2.365 1.108 2.648 1.661 −4.400 4.505 −5.383 4.158 0.201 0.443 0.850 −0.682 0.098 −2.272 ND 1.115 −4.759 5.978

ND not detected

Conclusions According to the healer, in the routine procedures used in the “cleaning soul ritual”, habitual doses at different times are used for Ayahuasca and Kambo. Therefore, it would not usually be probable that the patient presented these symptoms. However, it was necessary to use both Ayahuasca and Kambo at the same time due to the highest level of depression presented by the patient in an attempt to “purify the spirit”. The symptoms presented by the patient were likely due to an additive or synergic effect produced by the combination of the active natural substances present in the two materials applied by the healer to his patient at the same time

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and by the different routes of administration. The toxicological analyses by high-resolution ESI-MS and LC–MS/MS were essential in confirming the ingestion of the Ayahuasca and the cutaneous application of the Kambo. The use of alternative therapies for depression is becoming more and more common, but the indiscriminate use of the bioactive substances such as the ones investigated in this case study, which have not yet been fully characterized in terms of their composition/amounts and neurological effects, may present serious health risks. Although there have been only a few case reports involving probable intoxication by Ayahuasca [28] or Kambo [16, 29], this is the first case involving both Ayahuasca and Kambo to our knowledge.

Forensic Toxicology Acknowledgements  The authors thank all the institutions, Coordination for Improvement of Personnel with Higher Education—CAPES (Process Number 23038.006844/2014-46), The National Council for Scientific and Technological Development—CNPq (Process Number 830525/1999-8) and São Paulo Research Foundation—FAPESP (Process Number 2015/10650-8 and 2016/23157-0) for the fellowships and the financial support.

Compliance with ethical standards  Conflict of interest  The authors declare no conflicts of interest. Ethical approval  The small amounts of blank human blood (to be prepared to serum) and urine samples were collected from healthy volunteers after obtaining the informed consent. This article does not contain any studies with animals performed by any of the authors.

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