LC-MS/MS quantitative determination of Tetrapterys mucronata alkaloids, a plant occasionally used in ayahuasca preparation.

INTRODUCTION: Tetrapterys mucronata Cav. (Malpighiaceae) is a plant used in some regions of Brazil in the preparation of ayahuasca. OBJECTIVE: To determine the content of the main tryptamine alkaloids in the stem bark of T. mucronata Cav. and assess their possible toxic and hallucinogenic properties based on the doses found in a water decoction that mimics the ayahuasca preparation. METHODS: Four alkaloids previously described for their toxic and hallucinogenic properties were quantitated by multiple reaction monitoring HPLC combined with electrospray ionisation and tandem MS (HPLC-ESI/MS/MS) in the water decoction and ethanolic extracts from the bark of T. mucronata. RESULTS: Exhaustive extraction of the stem barks with ethanol revealed the following alkaloid levels: bufotenine (1) 3.26 ± 0.31 mg/g, 5-methoxy-N-methyltryptamine (2) 0.88 ± 0.08 mg/g, 5-methoxy-bufotenine (3) 3.07 ± 0.22 mg/g and 2-methyl-6-methoxy-1,2,3,4-tetrahydro-ß-carboline (4) 0.14 ± 0.004 mg/g. The water decoction presented slightly lower levels, ranging between 2.32 ± 0.14, 0.50 ± 0.04, 1.53 ± 0.09 and 0.10 ± 0.01 mg/g for (1), (2), (3) and (4) respectively. CONCLUSIONS: The HPLC-ESI/MS/MS quantitation revealed significant alkaloid levels, in particular for bufotenine and 5-methoxy-bufotenine. As such compounds are known for their toxic and hallucinogenic properties, these results indicate that the consumption of this plant as an ingredient in ayahuasca preparations may present a risk to consumers.

Analysis of psilocin, bufotenine and LSD in hair.

A method for the simultaneous extraction of the hallucinogens psilocin, bufotenine, lysergic acid diethylamide (LSD) as well as iso-LSD, nor-LSD and O-H-LSD from hair with hydrochloride acid and methanol is presented. Clean-up of the hair extracts is performed with solid phase extraction using a mixed-mode cation exchanger. Extracts are measured with liquid chromatography coupled with electrospray tandem mass spectrometry. The method was successfully validated according to the guidelines of the 'Society of Toxicological and Forensic Chemistry' (GTFCh). To obtain reference material hair was soaked in a solution of the analytes in dimethyl sulfoxide/methanol to allow incorporation into the hair. These fortified hair samples were used for method development and can be employed as quality controls.

Determination of psilocin, bufotenine, LSD and its metabolites in serum, plasma and urine by SPE-LC-MS/MS.

A validated method for the simultaneous determination of psilocin, bufotenine, lysergic acid diethylamide and its metabolites in serum, plasma and urine using liquid chromatography-electrospray ionization/tandem mass spectrometry was developed. During the solid-phase extraction procedure with polymeric mixed-mode cation exchange columns, the unstable analytes were protected by ascorbic acid, drying with nitrogen and exclusion of light. The limits of detection and quantitation for all analytes were low. Recovery was ≥86 % for all analytes and no significant matrix effects were observed. Interday and intraday imprecisions at different concentrations ranged from 1.1 to 8.2 % relative standard deviation, bias was within ±5.3 %. Processed samples were stable in the autosampler for at least 2 days. Furthermore, freeze/thaw and long-term stability were investigated. The method was successfully applied to authentic serum and urine samples.

Elevated urine levels of bufotenine in patients with autistic spectrum disorders and schizophrenia.

OBJECTIVE: Previous studies have suggested that the endogeneous psychotomimetic molecule bufotenine (N-N-dimethyl-5-idroxytryptamine) may play a role in the pathogenesis of severe mental disorders. The potential association of bufotenine with the clinical features of autism and schizophrenia is not entirely understood. In this study, we measured urinary levels of bufotenine in subjects with autistic spectrum disorder (ASD), schizophrenia and healthy comparison subjects free of psychiatric symptoms. We also sought to assess whether urine concentrations of this molecule may be associated with the clinical characteristics of psychiatric patients. DESIGN: Urine bufotenine levels were measured using a high-performance liquid chromatography-mass spectrometry (HPLC-MS) assay in young adults with severe ASD (n=15), patients with schizophrenia (n=15), and healthy control subjects (n=18). The Vineland Adaptive Behavior Scale was used to measure adaptive behaviors in ASD individuals. The Brief Psychiatric Rating Scale (BPRS) was used for patients with schizophrenia. RESULTS: Urine bufotenine levels were significantly higher in ASD subjects (3.30 +/- 0.49 microg/L, p<0.05) and patients with schizophrenia (4.39 +/- 0.43 microg/L, p<0.001) compared with controls (1.53 +/- 0.30 microg/L). Among patients with ASD, there was a significant positive correlation between urine bufotenine and hyperactivity scores on the Vineland Adaptive Behavior Scale (r=0.479, p<0.05). No other associations were detected. CONCLUSIONS: Our results indicate that elevated urine levels of the endogeneous psychotomimetic molecule bufotenine may play a role in ASD and schizophrenia, and can be correlated with hyperactivity scores in autism.

Identification of 5-hydroxy-tryptamine (bufotenine) in takini (Brosimumacutifolium Huber subsp. acutifolium C.C. Berg, Moraceae), a shamanic potion used in the Guiana Plateau.

This paper is the first thorough analysis of takini, a hallucinogen used by the shamans of several peoples in Suriname, French Guiana, and the region east of the Para in Brazil. The drug is contained in the latex of the Brosimum acutifolium tree, and until now, its psychotropic properties appeared inconsistent with the more general medicinal uses of the tree in the surrounding region. Our chemical and botanical studies reveal that the active ingredient of takini is bufotenine; and that this compound is only contained in the subspecies Brosimum acutifolium Huber subsp. acutifolium C.C. Berg that is found in the same area of the eastern Guianas.

Efficacy of digoxin specific Fab fragments (Digibind) in the treatment of toad venom poisoning.

Chan Su, a traditional Chinese medication, and Love Stone, a topical aphrodisiac, are both made from dried venom of the toad bufo bufo gargarizans and contain bufalin, cinobufotalin, cinobufagin, and other cardioactive steroids of the bufadienolide class. Deaths have occurred following ingestion of these products and the clinical course resembles digoxin toxicity. The purpose of this study was to determine the efficacy of digoxin specific Fab fragments in treating Chan Su poisoning. An ethanolic extract was prepared from Chan Su. Digoxin specific Fab fragments were reconstituted in normal saline to a concentration of 80 mg/ml. An approximate LD90 dose was determined in preliminary experiments. Mice were then randomly divided into a treatment group of 15 mice and a control group of 30 mice. The treatment group was pretreated with 20 ml/kg of digoxin specific Fab fragment solution by intraperitoneal injection at t = 0, followed by 10 ml/kg of digoxin specific Fab fragments intraperitoneal at t = 30 min. The control group was pretreated with equal volumes of intraperitoneal normal saline at the same times. Immediately following the 30 min injection, both groups were given the estimated LD90 dose of Chan Su extract by subcutaneous injection. An endpoint of survival at 6 h was chosen after preliminary results showed that all deaths occurred in the first 4 h. All 30 of the control mice had seizures followed by death compared to 11 seizures and 7deaths in the 15 treatment mice. These results were statistically significant by Fisher's exact test (p = 0.00003 for mortality and p = 0.009 for seizures). Digoxin specific Fab fragments are effective in the treatment of Chan Su poisoning in mice and may be effective for poisoning by other cardioactive steroids of the bufadienolide class.

Treatment of toad venom poisoning with digoxin-specific Fab fragments.

Toxicity from toad venom poisoning is similar to digoxin toxicity and carries a high mortality rate. We report on six previously healthy men who developed vomiting and bradycardia after ingesting a purported topical aphrodisiac. Each patient had positive apparent digoxin levels and the first four patients died of cardiac dysrhythmias. The last two patients recovered following treatment with digoxin Fab fragments. We analyzed samples of the purported aphrodisiac and found that it was identical to Chan Su, a Chinese medication made from toad venom. To our knowledge, this is the first reported use of digoxin Fab fragments to treat toad venom poisoning.

Serotonin in body fluids: characterization of human plasmatic and cerebrospinal fluid pools by means of a new HPLC method.

A new HPLC technique for the analysis of picomolar amounts of serotonin (5HT) in plasma and cerebrospinal fluid (CSF) is described. Bufotenin is used as internal standard. Detection is achieved electrochemically or fluorimetrically. The detection limit can be estimated as 50 pg 5HT/mL of either fluid (0.3 picomolar). The method is used to characterize a non-particulate pool of 5HT which is clearly distinct of the platelet pool. Administration of parachlorophenylalanine (PCPA) 300 mg/kg to rats leads to a 90% reduction in the plasmatic pool whereas platelet 5HT is only slightly decreased (3rd day after PCPA) or even increased (7th day after PCPA). Human concentration (n = 15) of 5HT in plasma is 2.6 +/- 0.9 ng/mL (X +/- S.D.). The application of the method to CSF of neurological patients reveals 5HT concentrations ranging from 93 to 962 pg/mL.

Extraction of pineal and uterine tissue at different pH values: a preliminary report on the occurrence of a few groups of compounds in both tissues.

Extraction of pineal glands has been performed at different pH values. After extraction at pH +/- 4.9, 'porphyrin'-containing protein did not occur in the extracts, while at pH +/- 6.3 these proteins had passed over partly into the extract; at pH +/- 6.8 the proteins could no longer be detected in the residues, but a partial breakdown had taken place. The activity of pineal extracts depends on the method of extraction. In the watery extracts (pH +/- 6.3) both of pineal and of uterine tissue the presence could be shown among others of: porphyrins bound to protein, the indole compound 'bufotenine' also bound to protein, 'kynurenine', a 'basic peptide' containing amines and 'flavoproteins'. The hypothesis is proposed that in uterine endometrium, tryptophan can be metabolized in at least two pathways, one leading to kynurenine and one to bufotenine. Formation of bufotenine, probably a physiologically important compound, might be influenced by the oestrous cycle and conversely.

[Extraction of serotonin, bufotenin, 5-methoxytryptamine and 5-methoxy-N,N-dimethyltryptamine and their fluorometric determination with o-phthaldialdehyde]. / Extraktion von Serotonin, Bufotenin, 5-Methoxytryptamin und 5-Methoxy-N,N-Dimethyltryptamin und ihre fluorimetrische Bestimmung mit o-Phthaldialdehyd.

For the investigated indolamines, the recovery following extraction with n-butanol and acetic acid ethyl ester from alkaline solution amounted to 80-90%. Using benzene only the two 5-methoxyindolamines were transferred from the alkaline medium into the organic phase. The reaction of the o-phthaldialdehyde with substituted indolamines was optimized in regard to higher fluorimetric detection sensitivity. The lower limit of determination was 2.0 ng for 5HT and 5OHDMT, and 1.0 ng for 5MeOT and 5MeODMT.

Quantitative assay of the N-methylated metabolites of tryptamine and serotonin by gas chromatography mass spectrometry as applied to the determination of lung indoleethylamine N-methyltransferase activity.

A specific and sensitive method is described for the identifcation and quantification of the N-mono- and dimethylated derivatives of tryptamine and serotonin by gas chromatography mass spectrometry. Deuterated analogues of the amines have been prepared for use as internal standards. The technique has been applied to the determination of indoleethylamine N-methyltransferase activity in rabbit and human lung. No interference from the beta-carboline formation or other side reactions between the substrates and the methyl donor was observed.

Reversed-phase high-pressure liquid chromatography of some tryptamine derivatives.

A quantitative reversed-phase high-pressure liquid chromatographic method is described for tryptamine, bufotenine, N-methyltryptamine and N,N-dimethyl tryptamine. All compounds were eluted with base-line separation within 15 min. The method is used to determine the tryptamine level in a leaf extract of Acacia podalyriaefolia A. Cunn. Satisfactory recoveries of tryptamine from plant material, and N,N-dimethyltryptamine from urine were obtained.

Determination of N,N-dimethylindolealkylamines in plasma, blood and urine extracts by radioimmunoassay and high pressure liquid chromatography.

A sensitive radioimmunoassay for N,N-dimethylindolealkylamine derivatives has been developed. It is possible to detect 200 to 700 femtomoles of 5-hydroxy-N,N-dimethyltryptamine, 5-methoxy-N,N-dimethyltryptamine or N,N-dimethyltryptamine in a given sample. Antibodies were produced in rabbits immunized with a conjugate prepared by reacting 5-hydroxy-N,N-dimethyltryptamine with a diazotized dl-p-amino-phenylalanine bovine serum albumin conjugate. For identification of immunoreactive material high pressure liquid chromatography was used to separate these compounds from each other and from known cross-reacting compounds found in physiological specimens. After chromatography, individual fractions were analyzed by the radioimmunoassay. This combination of high pressure liquid chromatography and radioimmunoassay has permitted the identification and quantification of these compounds in extracts of urine, plasma and whole blood from normal individuals.

New and uncommon indole- and imidazole-alkylamines in skins of amphibians from Australia and Papua New Guinea.

Extracts of the skin of some amphibians from Australia and Papua New Guinea contained, in addition to the usual 5-hydroxyindolealkylamines and histamine: a. two new, hitherto unknown indolealkylamines, i.e. O-sulphate of bufotenidine and 2-(3-indolyl) ethyltrimethylammonium, a quaternary ammonium base of tryptamine. The rare O-sulphate of bufotenine was also present, the occurrence of which had previously been demonstrated only in the skin of some South American toads; b. a series of uncommon imidazolealkylamines, such as N'-acetylhistamine, N'-methylhistamine, N',N'-dimethylhistamine, spinaceamine and 6-methylspinaceamine. It appears evident that amphibian skin continues to be an exceptionally rich source of aromatic amines.

Determination of serotonin and bufotenin as their dansyl derivatives.

A method is described which permits the determination of serotonin and bufotenin in the same tissue sample. It comprises the following steps: (a) tissue extraction with acetone-0.1 M hydrochloric acid (19:1); (b) reaction of the tissue extract with dansyl (Dns) chloride; (c) pre-separation of O-Dns-bufotenin from O,N-bis-Dns-serotonin and other Dns-amides on a small silica gel column (this step is dispensable if only serotonin or bufotonin is being determined); (d) TLC separation of O-Dns-bufotenin and O,N-bis-Dns-serotonin from other Dns derivatives; (e) quantitative evaluation of the separated compounds by fluorimetry for O-Dns-bufotenin and by fluorimetry or mass spectrometry for the serotonin derivative. The photometer response is linear within the range 0.1-300 nmole. With the mass spectrometric method, 2 pmole of O,N-bis-Dns-serotonin could be determined with a standard deivation of +/-9%. The recovery of the amines from tissue was better than 85%. Reserpine treatment of toads caused a concomitant decrease in serotonin and bufotenin in the brain, but not in the skin of the animals. Repletion of bufotenin in the brain occurs at a higher rate than the repletion of the serotonin pool.