This paper critically reviews 69 studies (1955–2010) reporting endogenous N,N‑dimethyltryptamine, 5‑hydroxy‑DMT and 5‑methoxy‑DMT in human blood, urine and cerebrospinal fluid, evaluating the analytical methods and identification criteria used. The authors conclude that many early reports suffer from methodological shortcomings and inconsistent quantitation, and they offer recommendations to strengthen future research on endogenous psychedelic tryptamines.
Three indole alkaloids that possess differing degrees of psychotropic/psychedelic activity have been reported as endogenous substances in humans; N,N‐dimethyltryptamine (DMT), 5‐hydroxy‐DMT (bufotenine, HDMT), and 5‐methoxy‐DMT (MDMT). We have undertaken a critical review of 69 published studies reporting the detection or detection and quantitation of these compounds in human body fluids. In reviewing this literature, we address the methods applied and the criteria used in the determination of the presence of DMT, MDMT, and HDMT. The review provides a historical perspective of the research conducted from 1955 to 2010, summarizing the findings for the individual compounds in blood, urine, and/or cerebrospinal fluid. A critique of the data is offered that addresses the strengths and weaknesses of the methods and approaches to date. The review also discusses the shortcomings of the existing data in light of more recent findings and how these may be overcome. Suggestions for the future directions of endogenous psychedelics research are offered. Copyright © 2012 John Wiley & Sons, Ltd.
Barker and colleagues review the long-standing question of whether three tryptamine derivatives—N,N-dimethyltryptamine (DMT), 5-hydroxy-DMT (bufotenine, HDMT), and 5-methoxy-DMT (MDMT)—are endogenous in humans. Earlier research used a variety of analytical approaches as instrumentation evolved, and interest in these compounds has been driven both by their psychedelic properties and by recent findings that DMT interacts with molecular targets such as the sigma-1 receptor and trace amine receptors. The potential physiological or pathophysiological roles of these compounds have been discussed in relation to altered states of consciousness, psychiatric conditions (notably psychosis and schizophrenia), dreams, religious experiences and ayahuasca use, but no definitive biological role has been established. This review sets out to critically evaluate 69 published studies from 1955 to 2010 that report detection or quantitation of DMT, HDMT and MDMT in human body fluids (blood, urine and cerebrospinal fluid). The investigators examine the analytical methods and identification criteria applied across studies, summarise reported detection rates and concentration ranges, and highlight methodological strengths and weaknesses with the aim of clarifying which findings are reliable and which require further confirmation. They also outline questions for future research, including the origin, metabolism and optimal sampling strategies for these compounds.
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Hayashi, T., Vaupel, D. B., Su, T. P. · Science Signaling (2009)
McKenna, D. · ACS Chemical Neuroscience (2004)
Strassman, R. J., Qualls, C .R. · JAMA Psychiatry (1994)
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This paper is a narrative critical review rather than a systematic review with a fully enumerated protocol. The study selection comprises 69 publications identified primarily through searches of SciFinder (Chemical Abstracts) and PubMed; the review covers reports published between 1955 and 2010. The investigators included studies that reported detection and/or quantitation of DMT, HDMT or MDMT in human biological fluids (urine, blood, cerebrospinal fluid). The authors note that additional older or obscure reports may have been inaccessible or untranslated and therefore excluded. In appraising the literature, the reviewers focused on the analytical methods used, the criteria applied for compound identification (for example Rf or retention time matching, colour reactions, detector response, mass spectral matching), and the reporting of assay performance characteristics such as limits of detection, recovery, specificity and reproducibility. The review traces methodological evolution from early paper and thin-layer chromatography with colour reagents through gas chromatography (various detectors) to modern liquid chromatography–tandem mass spectrometry (LC-MS/MS). Where numerical data were provided in the source studies, the reviewers compiled counts of individuals tested and numbers positive or negative for each analyte in each biological matrix.
Scope of the literature: Sixty-nine studies were examined. The collected reports span a range of analytical technologies and clinical populations, with many studies comparing psychiatric patients (predominantly people with schizophrenia) to control subjects. HDMT (bufotenine): Fifty-one studies assessed urine for HDMT, collectively analysing urine from 1,912 individuals (1,249 patients and 663 controls). Using studies that reported presence/absence, HDMT was detected in 71% of patients (886 of 1,249) and 55% of controls (363 of 663), yielding an overall positive rate of approximately 65%. The reviewers note that about seven studies hydrolysed conjugates and estimated roughly 50% of urinary HDMT is excreted as a glucuronide; the remaining studies measured free HDMT only. Reported urinary concentrations varied widely depending on reporting units, with ranges (in common units) from 1 to 62.8 mg/24 h and 0.48 to 218 ng/ml. Four studies examined blood for HDMT (240 individuals total: 166 patients, 74 controls); 4 patients (2.4%) and 18 controls (24%) were reported positive, a combined blood positive rate of 9% (22 positives, 218 negatives). Blood concentrations reported ranged from 22 pg/ml (HPLC-radioimmunoassay) to 40 ng/ml (direct fluorescence assay). No study in the reviewed set reported HDMT in cerebrospinal fluid (CSF). DMT: Twenty-nine studies assessed urine for DMT (861 individuals: 635 patients, 226 controls). Across these reports, 43% of patients (276 of 635) and 64% of controls (145 of 226) were reported positive; overall, 421 of 861 individuals (49%) were positive in urine. Urinary DMT concentrations reported in various units ranged from 0.02 to about 43 mg/24 h (with one mean ± SD quoted as 42.98 ± 8.6 mg/24 h) and from 0.16 to 19 ng/ml. Eleven studies examined blood (417 individuals: 300 patients, 117 controls) and reported 44 patient positives (15%) and 28 control positives (24%), for an overall blood positive rate of 17% (72 positive, 345 negative). Blood concentration ranges reported were 51 pg/ml to 55 ng/ml. Four studies assayed CSF (136 individuals: 82 patients, 54 controls); 34 patients and 22 controls were positive (41% positive overall), and reported CSF concentrations ranged from 0.12 to 100 ng/ml. MDMT (5-methoxy-DMT): Nine studies examined urine for MDMT (113 individuals: 94 patients, 19 controls) and found 2 patients positive (2%) and 2 controls positive (10.5%). Two studies analysed blood (39 individuals: 36 patients, 3 controls), reporting 20 patient positives (51%) and no positive controls; a single blood concentration of 2.0 ng/ml was cited (HPLC-radioimmunoassay). Four studies examined CSF (136 individuals: 83 patients, 53 controls); 28 patients and 12 controls were reported positive (29% overall). One study reported mean combined DMT plus MDMT concentrations in CSF of approximately 1,400 ng/ml for patients and 230 ng/ml for controls but with very large standard deviations. Analytical-method findings and trends: Early studies (1950s–early 1970s) relied heavily on paper chromatography, TLC and non-specific detection (colour reagents, UV, fluorescence) with identification based on Rf or Rt matching and colour reactions; such methods lack structural confirmation and are prone to interference. The reviewers document several methodological artefacts that could produce false positives or overestimates, including acetone forming adducts with primary amines, and reactions of extracted indoleamines with methylene chloride producing quaternary salts. From 1973 onwards the application of mass spectrometry—including isotope-dilution approaches, GC‑MS with total ion spectra, and selected ion monitoring—provided the first methodologically robust identifications of DMT and HDMT in human samples. The authors note that all studies using MS-based methods since 1973 have reported confirmation of one or more of these compounds in human body fluids. MDMT, however, lacks unequivocal MS confirmation in blood or urine; only two MS-based CSF positives were reported and both by the same group. The most recent analytical advances employ LC-MS/MS, which allow low pg/ml sensitivity and confirmation using parent ions, fragment ions and ion ratios.
Barker and colleagues conclude that DMT and HDMT are most likely endogenous in humans, reasoning that the preponderance of mass spectrometric evidence provides strong confirmation. By contrast, the evidence for endogenous MDMT is less compelling: MS-based positive reports for MDMT are limited to two CSF findings from a single group and there are no MS-confirmed detections of MDMT in blood or urine in the reviewed literature. The reviewers emphasise that many early reports probably contained misidentifications or overestimated concentrations because of nonspecific assays, lack of structural confirmation, incomplete reporting of assay performance (limits of detection, recoveries), and methodological artefacts (solvent-derived products and co-extracted compounds). They point out that peripheral concentrations of these tryptamines are often very low, their half-lives are measured in minutes, and they are excellent substrates for monoamine oxidase A (MAO-A), which rapidly degrades parent compounds to indoleacetic acids. These factors make detection in blood and urine challenging and may explain intermittent detectability in serial sampling. Studies that pre-treated subjects with MAO inhibitors generally increased detectable parent compound concentrations, but even then parent compounds remained at low levels. To improve detection and interpretation, the authors recommend future studies employ modern, fully validated mass spectrometric methods (ideally LC-MS/MS) with sensitivity at or below about 1.0 pg/ml. They further suggest assessing N-oxide metabolites—which are not substrates for MAO-A and retain the parent structure—because N-oxides may accumulate and be easier to detect; data from ayahuasca administration indicate DMT N-oxide levels can exceed parent DMT in blood and urine. Other technical recommendations include enzymatic hydrolysis to free conjugated HDMT and more frequent or prolonged sampling to address intermittent excretion. The reviewers also highlight critical gaps in knowledge about sites of synthesis: mapping of indole-N-methyltransferase (INMT) expression has suggested peripheral sites (adrenal, lung) but tissue-level confirmation and functional studies are needed, and brain INMT data remain inconclusive. Barker and colleagues advocate combining sensitive biochemical assays with molecular and cellular approaches (including tissue assays and genetic models such as an INMT knockout) to identify synthesis locations and to elucidate physiological roles. They close by calling for comprehensive studies that monitor parent compounds, N-oxides and conjugates across blood, urine, CSF and relevant tissues, ideally with MAO inhibition where appropriate, to clarify the endogenous biology of these psychedelic tryptamines.
Three indole alkaloids that possess differing degrees of psychotropic/ psychedelic activity have been reported as endogenous substances in humans. These compounds, all metabolites of tryptophan, are N,N-dimethyltryptamine (DMT, 1, Figure), 5-hydroxy-DMT (bufotenine, HDMT, 2), and 5-methoxy-DMT (MDMT, 3). Their presence has been reported in human cerebrospinal fluid (CSF), urine, and/or blood utilizing either paper and/or thin layer chromatography (TLC), direct ultraviolet (UV) or fluorescence (Fl) measurements, gas chromatography (GC) using various sensors (nitrogen-phosphorous detector (NPD); electron capture detector (ECD); mass spectrometry detector (MSD)), high-performance liquid chromatography (HPLC) using UV and/or Fl detection, HPLCradioimmunoassay, HPLC-electrochemical detection, and liquid chromatography-tandem mass spectrometry (LC-MS/ MS) (Tables, references ). Indeed, the review of the 55-year history of the development of methodology for the analysis of these compounds shows how closely it has paralleled the evolution of analytical technology itself, with each researcher seeking more specific and sensitive techniques. A renewed interest in these compounds as naturally occurring substances in humans has occurred, in part, due to DMT's recent characterization as an endogenous substrate for the ubiquitous sigma 1 receptorand for its possible action at trace amine receptors.In both cases, the roles of DMT and the receptors themselves in regulating some aspect(s) of human physiology are poorly understood. Given their known psychedelic effects, there remains an interest in their possible role in naturally occurring altered states of consciousness, such as psychosis, dreams, creativity and imagination, religious phenomena, and even near-death experiences.Although the vast majority of research into the presence of these compounds sought their role in mental illness, no definitive conclusions yet exist. A determination of the role of these compounds in humans awaits further research, much of which awaits the development of adequate analytical methodology. Interest in DMT has also increased because of the burgeoning use and popularity of the religious sacrament ayahuasca which contains DMT and several harmala alkaloids, which serve to make DMT orally active. Ayahuasca tourism in South America and the establishment of syncretic churches using ayahuasca as a sacramenthave stimulated research into the mechanisms of its effects and its possible use as a therapeutic.The resumption of human research characterizing DMT's psychopharmacologyand the ongoing use of pure DMT for therapeutic and recreational purposes have also focused interest on this and related psychedelics. The dimethylated-tryptamines (DMTs) increasing visibility within medical, non-medical, religious and/or recreational contextsreinforce the importance of determining their endogenous role. This review addresses several fundamental issues regarding these three endogenous psychedelics. For example, are DMT, HDMT, and/or MDMT truly present in humans?Early criticisms of reports of endogenous psychedelics were directed at the fact that rather non-specific chemical tests were being applied, double-blind analyses were not always being performed, and dietary or medication sources were not always adequately ruled out as responsible for the identifications.Further, it was claimed that possible artifacts produced from the extraction solvents and conditions of analysis may have led to misidentification of the DMTs in some early studiesand, more recently, that the use of halogenated solvents in the analysis may have affected their detection.Biological factors that may have affected the detectabilty of these compounds in the periphery were also acknowledged, which included their rapid metabolism.Finally, there have been concerns that the studies searching for their presence and an association with specific clinical disorders have failed to understand and fully characterize their metabolism or monitor their metabolites.To address these issues, we have undertaken a critical review of 69 published studies reporting the detection or detection and quantitation of these compounds in human body fluids. In reviewing this literature, we address the methods applied and the criteria used in the determination of the presence of DMT, MDMT, and HDMT. We begin with the original report of the presence of bufotenin (HDMT) in human urine in 1955 using paper chromatographyand end with the most recent report concerning the presence of bufotenin (HDMT) in human urine using LC-MS/MS.We will be addressing the following questions: How valid were early studies regarding the presence and/or quantities of these compounds in human cerebrospinal fluid (CSF), blood and/or urine? Were the analytical methodologies and the identification criteria adequate? Are they truly there? When present, are they of dietary origin? When and where in the human body are they produced? Can we influence their detection in biological samples by pharmacologically inhibiting their metabolism by monoamine oxidase (MAO)? How does turnover rate and metabolism of these substances influence their detectabilty? Have the precursors and/or metabolites of these compounds been adequately monitored? Is monitoring these compounds in biological samples such as CSF, blood and/or urine the best, or even most practical way to determine their role? What will such data tell us about the function of these compounds? Where does the research on endogenous psychedelics go from here?
The search for endogenous psychedelics soon followed the discovery of the psychedelic effects of mescaline and lysergic acid diethylamide (LSD) in humans. Observations of these effects gave rise to hypotheses that they were related to the symptomology observed in a heterogeneous group of mental disorders, especially psychoseseither mania or schizophrenia.It was proposed that schizophrenics may biochemically produce similar compounds as 'schizotoxins'.A search for mescaline-like compounds proved unrewarding,but in studies examining urine samples for serotoninlike compounds, researchers reported in 1955and 1956,the presence of 5-hydroxy-N,N-DMT (HDMT, bufotenin) in humans. Subsequently, Axelrodreported the presence of an enzyme capable of N-methylating indole-ethylamines and producing DMTs. Following these reports, attention began to focus in earnest on the possible endogenous formation of the indole-ethylamine psychedelics. During the next 50 years, many studies reported finding DMT, HDMT, and/or MDMT in human CSF, urine, and/or blood. Most of these studies sought differences in levels between controls and psychiatric, especially psychotic, patients. Some studies claimed higher concentrations and significant differences in levels between the groups; some reported not finding the compounds at all in either patients or controls. It is of interest to note that in its original conception, the schizotoxin hypothesis proposed that the formation of an endogenous psychedelic schizotoxin would be an aberration of metabolism and that 'normals' would not form such compounds.However, numerous studies subsequently reported finding one or more of these compounds in controls HNMT, HDMT 24-hour urine 10 ml portions, HCl; urease Evap, Acetone, evap, MeOH, evap, AlO3 column 1956 RodnightHNMT, HDMT Fischer et al.HDMT 1 L of urine NaHCO3 sat., butanol, evap, acetone 1961 Fischer et al.HDMT PerryHDMT; DMT 24 or 48 hour urine; ext vol 500 mg creatinine Amberlite CG-120, CG-50; ethanol-acetone ppt 1963 Sprince et al.DMT, HDMT 24 hour urine pH 10, ethyl ether-butanone ext, evap, acetone 1963 Perry and SchroederHDMT 24-36 hour urine; ext vol 250-350 mg creatinine Amberlite CG-120, CG-50; ethanol-acetone ppt 1965 Franzen and Gross [11] DMT, HDMT blood and urine (24 hour) Extensive multi-step extraction, ppt and clean-up 1965 SiegelHDMT fresh urine, 100 ml pH 10, ethyl ether ext, evap, acetone 1965 Nishimura and GjessingHDMT fresh urine vol 500- HellerHDMT TanimukaiHNMT, HDMT, NMT, DMT, MDMTHDMT 100 ml urine NaCO3, ether ext, evap, acetone 1968 Faurbye and PindHDMT 24 hour urine, hydrolyzed at pH1.6 column chromatography, sublimation, paper/TLC 1969 Sireix and MariniHDMT 100 ml fresh urine Fischer et al.HDMT, glucuronide Narasimhachari and HimwichDMT, HDMT Oon and RodnightDMT, NMT Raisanen and KarkkainenDMT, HDMT HNMT, HDMT paper chromatography (1 system), color reaction, bioassay ND Rf and color (1 system) 1956 RodnightHNMT, HDMT paper chromatography (3 systems), color reaction, bioassay HDMT paper chromatography and auto-radiographs ND Rf and color (1 system), radioactive spot 1962 Perry et al.HDMT, conjugate paper chromatography (2-D), color reaction ND Rf and color (2-D) 1963 Brune et al.HDMT; DMT paper chromatography (2-D), color reaction 20 ng/ml Rf and color (2-D) 1963 PerryHDMT; DMT 2-D paper chromatography, color reaction 1963 Perry and SchroederHDMT paper chromatography (3 systems)
Rf and color (3 systems) 1965 Franzen and Gross [11] DMT, HDMT Fluorescence 2 ng/ml Fluoresence reading 1965 Siegel [12] HDMT TLC (1 system), color reaction 0.1 mg/100 ml Rf and color (1 system) 1965 Nishimura and GjessingHDMT 1966 HellerHDMT paper chromatography (2-D), color reaction ND Rf and color (2-D) 1967 Fischer and SpatzHDMT paper chromatography (2-D), color reaction 1967 TanimukaiHNMT, HDMT, NMT, DMT, paper and TLC (2-D); color reaction; GC-FID of HDMTHDMT paper chromatography (2-D), color reaction ND Rf and color (2-D) 1968 Faurbye and PindHDMT paper chromatography and TLC, color reaction >0.7 mg/24 hour Rf and color (paper and 2-D TLC) 1969 Sireix and MariniHDMT UV; paper chromatography, color reaction ND Rf and color (2-D) 1969 Spatz et al. [26] HDMT UV of diazo-deriv; paper chromatography, color reaction ND UV; Rf and color 1970 Fischer and SpatzHDMT UV; TLC, color reaction ND UV; Rf and color 1970 Saavedra and Udabe [28] HDMT UV; TLC, color reaction ND UV; Rf and color 1970 Tanimukai et al. [29] HNMT, HDMT, DMT, MDMT paper and TLC (2-D); color reaction; GC-FID of HDMT ND Rf and color (2-D paper, TLC); GC-RT 1970 Heller et al. [30] DMT, MDMT, HDMT GC-FID, TLC, and Spectrofluorometry 2 ng/ml GC-RT and TLC or spectrofluorometer 1971 Narsimhachari et al. [31] DMT, MDMT, HDMT TLC and GC-FID, verified with spectrofluorometer 5 mg/ml per 24hour for DMT TLC and GC-FID, spectrofluorometer 1971 Narasimhachari et al. [32] NMT, DMT, MDMT TLC and GC-FID, verified with spectrofluorometer 2 ng/ml TLC and/or GC-FID, spectrofluorometer 1971 Fischer et al. RaisanenHDMT TMS derivatives; GC/MS, multiple ion detection 0.1-0.15 ng/ml DMT; 0.25-0.3 ng/ml HDMT RT, molecular ions or fragments 1995 Karkkainen et al. [65] HDMT TMS derivatives; GC/MS, multiple ion detection 0.1-0.15 ng/ml DMT; 0.25-0.3 ng/ml HDMT RT, molecular ions or fragments 1995 Takeda et al. [66] HDMT, HNMTHNMT, HDMT 4 healthy adults pooled sample; 5-HNMT, HDMT ND 1956 RodnightHNMT, HDMT 11 HimwichDMT, HDMT 6 chronic schizophrenics HimwichDMT, HDMT
Drug Testing and Analysis as well as patients. Despite many such efforts, a definitive link has yet to be demonstrated between the blood and/or urine levels of these compounds and any psychiatric diagnosis.The earliest studies (1950s-1960s) in the search for endogenous psychedelics applied the technology available at the time. These were mainly paper and thin-layer chromatography (TLC) using different reagents as visualization (colour development) sprays, as well as comparing Rf values with spotted standards as the criteria for identification. In 1967, thin-layer spots were isolated and derivatized in an attempt to confirm their identification by gas-liquid-chromatography (GC) using a flame-ionization detector (FID).In this case, Rf values from TLC and relative retention (Rt) from GC that were consistent with known standards served as the confirmation criteria. Subsequent studies applied this technology utilizing other detectors, such as nitrogen-phosphorous, electron capture and, eventually, mass spectrometry (MS). In many of these studies, the sole criterion for identification was retention time compared to a reference standard. However, in the case of the early MS data, the presence of a single major fragment ion(m/z 58) or one or two minor ions,served as additional confirmation. Liquid chromatography with UV and fluorescence detection was also applied, with the collected peaks being confirmed by GC-MS in some cases. As the analytical technology evolved, so too did the methods applied to detect and measure the compounds of interest, with resultant gains in sensitivity, specificity, and validity. The most recent methods have applied LC-MS/MS technologies in combination with more stringent confirmation criteria.These criteria are based on specific protonated molecules, fragment ions and their ratios to one another, and on relative retention times. However, as the criteria have become more exacting and the specificity of the methodology has improved, detection of the endogenous psychedelics appears to have become less frequent and, where detection has occurred, at significantly lower concentrations than originally reported. Tables 1-3 are a compilation of 69 studies directed towards detecting or detecting and quantitating the three indole psychedelics -DMT, HDMT, and MDMTin human (patient and/or control) CSF, blood, and/or urine. The entries for each study were taken from copies of the original publications. In some cases, the published studies neglected to address the relevant analytical issues reviewed.
• Sixty-nine studies were reviewed. Other studies that exist were either not accessible through current abstract search engines, were sufficiently obscure as not to be abstracted, or were not available in a translated form for inclusion in this analysis. Articles were obtained through SciFinder (Chem Abstracts Selects;) and PubMed () database searches.
• Fifty-one studies examined urine samples for HDMT (27 assayed urine for HDMT only). Taking into account the presence of the 5-hydroxyl group on HDMT, 7 studies specifically addressed the issue of the excretion of HDMT as a From these studies we know that approximately 50% of the total HDMT is excreted as a glucuronide conjugate. The remaining 44 studies did not conduct hydrolysis or enzyme treatment and thus did not determine the total amount of HDMT excreted but rather free HDMT alone. • Urine samples from 1912 individuals were assayed; 1249 patients (predominantly diagnosed with schizophrenia) and 663 controls. Among patients, 886 were positive for (71%) and 363 were negative. Among controls, 363 were positive for HDMT (55%) and 300 were negative. Thus, 1249 individuals were positive (65%) and 663 were negative. Most of the urine samples were obtained from 24-h collections with varying quantities of the total collection being used for analysis. However, many other studies only used morning or random samples, while a few used 8-or 12-h collections. Varying amounts of urine were used in the assays, based on volume or total mg creatinine. The range of extraction techniques is shown in Tableand the analytical approaches employed are shown in Table. One study examined and failed to find a diurnal variation in urine concentrations of HDMT,while another reported that HDMT excretion did not vary diurnally but rather was intermittent.Several studies examined dietary influences on detection of HDMT but none established a dietary source (Table). • Concentrations of HDMT were usually reported as mg/24 h while other studies reported concentrations as mg/g or mg/mg creatinine, nmol or pmol/ml or per 24 h, and ng/ml or mg/L. Using the most common methods of reporting, these studies demonstrated concentrations ranging from 1 to 62.8 mg/24 h, and from 0.48 to 218 ng/ml.
• Of the 69 studies, 4 examined blood for the presence of HDMT. • Blood samples from 240 individuals were examined: 166 patients and 74 controls. Plasma, serum, and whole blood were used. A single study provided 146 of these total samples; it used a limit of detection of 0.3 ng/ml and a 1.0 ml sample of plasma or serum for analyses. For all of the studies combined, 4 patients were positive for HDMT (2.4%) and 162 were negative. Eighteen controls were positive for HDMT (24%) and 56 were negative. Thus, a total of 22 individuals were positive for HDMT (9%) in blood and 218 were negative. One study reported higher concentrations of HDMT were obtained from extraction of whole blood compared to serum.• When concentrations were reported (rather than simply present or not present) the concentrations of HDMT in blood ranged from 22 pg/ml (HPLC-radioimmunoassay)to 40 ng/ml (direct fluorescence assay of extracts).HDMT: cerebrospinal fluid • None of the 69 studies examined CSF for HDMT.
• Of the 69 studies, 29 examined urine for DMT. • Urine samples from 861 individuals were examined: 635 patients and 226 controls. Among patients, 276 were positive for DMT (43%) and 359 were negative. Among controls, 145 were positive (64%) and 81 were negative. Thus, a total of 421 individuals were positive for DMT (49%) in urine and 440 were negative. Most of the urine samples were 24-h collections and analytical samples varied in volume. However, many also used morning or random samples, while a few used 8-or 12-h collections. Various amounts of the urine were used in the assays, based on a set volume of urine or that containing a predetermined amount of creatinine. The range of extraction techniques is shown in Tableand analytical approaches employed are shown in Table. Several studies examined dietary influences on detection of DMT and were uniformly negative (Table). One study reported that DMT and NMT (N-methyltryptamine; 4, Figure) concentrations in urine were stable when stored at À15 C for up to 90 days.• Concentrations of DMT were usually reported as mg/24 h while others used mg/g or mg/mg creatinine, nmol/ml or pmol/ml nmol/24 h, pmol/24 h, ng/ml or mg/L, etc. Concentrations ranged from 0.02 to 42.98 +/À 8.6 (SD) mg/24 h, and from 0.16 to19 ng/ml.
• Of the 69 studies, 11 examined blood for DMT. • Blood samples from 417 individuals were examined for the presence of DMT: 300 patients and 117 controls. Blood samples used were plasma, serum and/or whole blood. Among patients, 44 were positive (15%) and 256 were negative. A single study is responsible for 137 of these negative samples; the authorswho used a 1.0 ml sample of plasma or serumreported a limit of detection of 0.2 ng DMT/ml. Among controls, 28 were positive (24%) and 89 were negative. Thus, a total of 72 individuals were positive for DMT (17%) in blood and 345 were negative. The range of extraction methods used is shown in Tableand analytical approaches employed are shown in Table. One study demonstrated that higher concentrations of DMT were found by extracting whole blood rather than using plasma.One study demonstrated that there was no difference in DMT blood levels between venous and arterial blood.One study reported that DMT concentrations were stable in plasma when stored for 60 days at 6 C(Table). • When concentrations were reported (rather than simply present or not present), the concentrations of DMT in blood ranged from 51 pg/ml (HPLC-radioimmunoassay)to 55 ng/ml (direct fluorescence assay of extracts).DMT: cerebrospinal fluid • Of the 69 studies, 4 examined CSF for DMT. • CSF samples from 136 individuals were examined for the presence of DMT: 82 patients and 54 controls. Among patients, 34 were positive for DMT (41%) and 48 were negative. Among controls, 22 were positive (41%) and 32 were negative. Thus, 56 individuals were positive (41%) and 80 were negative. • Concentrations of DMT in CSF ranged from 0.12 to100 ng/ml (Table).
• Of the 69 studies, 9 examined urine for the presence of MDMT. • Urine samples from 113 individuals were examined: 94 patients and 19 controls. A single study was responsible for 65 of these samples.Combining all studies, two patients were positive for MDMT in urine (2%) and 92 were negative. Two controls were positive (10.5%) and 17 were negative. • The concentrations of MDMT in urine ranged from 0.3 1.3 ng/ml (HPLC-radioimmunoassay).MDMT: blood • Of the 69 studies, 2 examined blood for the presence of MDMT. • Blood samples from 39 individuals were examined: 36 patients and 3 controls. Among patients, 20 were positive (51%) and 16 were negative. None of the 3 controls was positive for MDMT (Table). • A single estimate of 2.0 ng/ml was reported by one study (HPLC-radioimmunoassay).MDMT: cerebrospinal fluid • Of the 69 studies, 4 examined CSF for MDMT. • CSF samples from 136 individuals were assayed: 83 patients and 53 controls. Among patients, 28 were positive (34%) and 55 were negative. Among controls, 12 were positive (23%) and 41 were negative. Thus, a total of 40 individuals were positive (29%) and 96 were negative. • Only one study reported concentrations of MDMT in CSF, in which case the mean combined concentrations of DMT and MDMT were approximately 1400 ng/ml for patients and 230 ng/ml for controls with quite large standard deviations (GC-FID).The above does not address the analytical methods' sensitivity and specificity, and assumes that all of the data as collected and reported are accurate, either in their detection or non-detection of the target analyte(s) or the concentrations observed. However, this is almost certainly not the case. As can be seen from Table, almost every study conducted between 1955 and 1972 used paper or TLC for detection, quantitation, and confirmation of one or more of these compounds. Several studies used multiple chromatographic conditions and detection reagents in attempting to 'confirm' their results. It is well-known, however, that paper chromatography is limited in specificity and sensitivity in that spots tend to be diffused and the mobility of the compounds of interest is influenced by the presence of other components and salts. TLC is somewhat better but is also susceptible to these same factors in addition to many other variables such as humidity. Other studies used 2-D chromatographic conditions and very sensitive and moderately specific detection reagents. Nevertheless, the criteria for detection relied on Rf values and colour reactions relative to standards (Table). There were no data regarding the structure of the detected compounds. Much of the literature acknowledged their limitations and qualified results by referring to the compounds detected as, for example, 'bufotenin-like'.In many studies, large volumes of urine were extracted and concentrated (Table), resulting in a final extract less than optimal for such analysis. For example, in order to precipitate salts and other compounds, acetone was often used in the final steps of sample purification. However, Tanimukai demonstrated that acetone forms adducts with primary amines co-extracted in the process leading to formation of compounds that behaved similarly to bufotenin, for example, on paper or TLC.Although there do not seem to be any published replications of Tanimukai's findings, they did lead to modification of many of the extraction procedures that were subsequently designed to fractionate tertiary from primary amines (Table). As can be seen from Table, the extraction methods employed were predominantly classical liquid-liquid extractions with appropriate pH adjustments or the use of ion exchange resins or packings. The earliest studies, and especially those extracting large volumes of urine, often used a combination of methods in sequence in an attempt to obtain an adequately purified and appropriate extract for paper or TLC analysis. Almost none of these studies reported analyte recoveries, however. The most recent methods have all employed ion exchange solid-phase extraction for the isolation of the target compounds from urine.In addition to methodological complications, misidentifications of compounds may also have occurred because both paper and TLC using colour reagents require a somewhat subjective interpretation. For example, Rodnightand Siegel et al.proposed that the substance detected by Bumpus and Paigewas tryptamine and not HDMT. Another potential problem, involving co-injection of extracted indoleethylamines in GC analyses using the solvent methylene chloride, was addressed by Brandt et al.These authors showed that the compounds of interest react with methylene chloride under such conditions, forming quaternary salts and analytical artifacts. Some early studies used more than one method for their analyses, increasing the likelihood that their identifications were accurate; for example, combining TLC and GC with packed column technology. However, the resolving power of packed column technology is low and individual 'peaks' were often broad humps, sometimes several minutes wide. Subsequent studies using capillary chromatography have consistently demonstrated that some peaks observed using packed columns were often a composite of several compounds. In addition, the flame ionization detector that many studies used also lacked specificity. Although these approaches used two different technologies, the technologies themselves were relatively non-specific and yielded equivocal results. Some investigators added, or used exclusively, GC with ECD or NP detectors. While these detectors added sensitivityand in the case of NPD a degree of specificitythey continued to rely on Rt and detector response as their identification criteria. No structural data were generated. Other research teams used ultraviolet spectrometry and/or spectrofluorometry to detect and quantify the relevant compounds in extracted samples, either directly or after thin-layer or paper chromatography purification. However, the non-specificity of these methods also did not provide data regarding structural identity. For example, Siegeldemonstrated that the fluorescence method used by Franzen and Grossdid not actually measure a maxima from HDMT but instead the tail of the fluorescence spectrum of another compound. These findings bring into question studies that applied these and similar methods. Inconsistent findings in previous research suggest that sensitivity was also an issue. Data concerning extraction efficiency and recovery, limits of detection, specificity, reproducibility, storage stability, the use of double blind and replicate analyses, and other variables that are now basic requirements in assay research are lacking either altogether or in part in earlier studies. At best, some early papers point to other references for some of these data. However, we found direct comparisons of methods in positive or negative studies difficult to conduct. The first applications of mass spectrometry to the detection and quantitation of putative endogenous psychedelics in man occurred in 1973. Walker et al.and Wyatt et al.employed an isotope dilution method monitoring two ions to detect and quantitate DMT in blood. Soon thereafter, Narasimhachari and Himwich used GC-MS with single ion monitoring (m/z 58) to detect DMT from urine extracts.These latter authors also extracted sufficient material, using TLC for clean-up, to obtain a total ion mass spectrum of the detected substance, and demonstrated its identity with authentic DMT. These data were the first methodologically credible regarding DMT's presence in humans. Subsequent studies by these and other authors applied different MS capabilities for the detection, quantitation, and unequivocal confirmation of DMT and HDMT in humans. In 1974, Narasimhachari et al., providing a matching total ion spectrum of an extracted compound, reported the unequivocal identification of HDMT from human urine.In 1976, Rodnight et al.,using similar methods, published a matching total ion spectrum for DMT in human urine. Other MS techniques matched the retention time and protonated molecule ions (chemical ionization MS) for DMT and HDMT in urine.Additional studies detected, quantified, and confirmed the identity of DMT, NMT, and HDMT in human blood and urine using selected ion monitoring (SIM) of multiple fragment ions (Table). It is important to note that MDMT has yet to be unequivocally detected by any MS-based method in blood or urine. However, there are two reports of its presence in CSF using GC-MS/SIM.Continual improvement in MS technologies has greatly enhanced detection, sensitivity, and specificity of analytic studies searching for these compounds; for example, capillary chromatography for GC, and more advanced LC-mass spectrometers. This being the case, it is encouraging to note that all studies since 1973 using MS methodology have confirmed the presence of one or more of these compounds in human body fluids (Table). The most recent methods utilize LC-MS/MS which afford analyses and confirmation by several additional chemical processes; LC separation and matching of Rt, molecular ion matching, and fragment ion presence and ratio matching. This technique also allows for the detection of these compounds in the pg/ml range while providing unequivocal mass spectrometric confirmation of structural identity. Thus, while many early studies lacked today's more definitive technology, it is likely that many have been confirmed by later MS-based studies. On the other hand, most early studies that reported rather high concentrations on these compounds were most likely in error.
The answer to the question, 'Are the tryptamine psychedelic substances DMT, HDMT and MDMT present in the human body?' is most likely yes. We believe that the preponderance of the mass spectral evidence proves, to a scientific certainty, that DMT and HDMT are indeed endogenous and can be measured in human body fluids. The evidence is less compelling for MDMT where the only two MS-based positive studiesin CSFwere performed by the same research group. There is no mass spectral data on detection of MDMT in blood or urine. Thus, further studies are necessary to determine whether MDMT exists in humans. Similarly, there are no data on the possible presence of HDMT in CSF. This too requires examination. With respect to the paucity of data regarding endogenous MDMT, it should also be noted that HDMT is both a metabolite of and precursor for MDMT. The relationship of these two compounds may help explain why HDMT is so much more frequently detected than MDMT. Future studies will help explicate this relationship. As to the question, 'Were the analytical methodologies and the criteria for compound identification adequate?', the answer is less certain. Undoubtedly, some studies misidentified the target compounds or, at the minimum, greatly overestimated their concentrations. Are they of dietary origin? Many early studies attempted to determine if diet or gut bacteria were responsible for positive results. Sterilization of the gut with antibiotics or feeding subjects special diets had no effect on these studies' results. In addition, no evidence suggested that medication(s) played a role. More recently, however, Karkkainen et al.isolated significant quantities of HDMT from stool samples, and hypothesized that HDMT may be synthesized by cells of the intestinal epithelium or the kidney, but not by gut flora. When are these compounds produced? The very small numbers of studies that have looked for diurnal, circadian, or ultradian variations in levels of DMT or HDMT in humans have been negative. This may be due, in part, to too infrequent sampling times and inadequate assay methodologies. However, one longitudinal study and one assessing diurnal rhythms of DMT in human urine suggest that measurable concentrations occur only intermittently.The same is apparently true for HDMT.There are no comparable data available for MDMT. The two DMT studies cited were conducted in urine only and such analyses are probably best conducted in blood. They do stand, however, as examples of one of the possible further complications in understanding the source, role and function of these compounds. Where in the human body are they synthesized? The tissue source or sources of these compounds in humans remains unknown and, that being the case, we should not assume that monitoring blood, urine, or CSF will answer this question. DMT synthesis has been proposed to occur in adrenal and lung, where high levels of the enzyme responsible for its synthesisindole-Nmethyltransferase (INMT)have been reported.While these studies did not demonstrate high INMT levels in brain, the active transport of DMT across the blood-brain barriersuggests that peripheral synthesis may nevertheless affect central function. In addition, the mapping of INMT sites thus far has been based solely on INMT mRNA studies which only establish where active enzyme translation is occurring. However, recent studies by Cozzi et al.,an inducible enzyme. These molecular biological approaches, in combination with advances in assay methodology, may help finally characterize the biochemistry and physiology of these compounds in humans. The next questions -Can we influence the detection of endogenous psychedelics in humans by pre-treatment with MAO inhibitors? How does the turnover rate and metabolism of these substances influence their detectabilty? Have the precursors and/or metabolites of these compounds been adequately monitored?require synthesizing parallel lines of evidence. In humans, only a very small percentage of exogenously administered DMT is excreted in urine as the parent compound.This is also true for HDMTand MDMT.Despite this fact, every cited study monitored, without exception, only the parent compounds themselves in the various biological fluids examined. These compounds all have a very short half-lifea few minutesand blood levels are undetectable in less than an hour after administration. This rapid metabolism is due to their being excellent substrates for MAO-A. This enzyme's action on the psychedelic tryptamines results in the formation of their corresponding indoleacetic acids, which are indistinguishable from these same acids resulting from other better-known sources, such as tryptamine and serotonin. Several studies attempted to maximize detection of these substances by treating subjects with MAO inhibitors such as tranylcypromine and phenelzine (Table). In most cases, this did result in higher concentrations of the target compounds. Nevertheless, even with significant MAO inhibition, the concentrations of parent compounds remained quite small. This observation has, perhaps, a ready explanation: the other metabolic pathways for DMT, MDMT, and HDMT. Recognition and understanding of these compounds' pathways for degradation may afford an approach to circumventing the low concentrations of the parent compounds observed even after MAO inhibition. Sitaram et al.have shown that, in MAO-inhibited rats, metabolism of these psychoactive tryptamines is shifted away from MAO-A and indoleacetic acid formation to the N-oxidase and the respective N-oxides. However, no studies have yet pre-treated humans with MAO inhibitors and measured the parent compounds and their corresponding N-oxides. The advantage of such a study is that the N-oxide, as opposed to the indoleacetic acid, retains the original structure of the parent molecule, permitting a cumulative association. As a proof of concept, we, have measured blood and urine levels of DMT and its N-oxide (5, Figure) in humans administered a botanical preparation of DMT and MAO-A inhibiting harmala alkaloidsthe Amazonian brew ayahuasca.Concentrations of the N-oxide of DMT in these subjects were 3-4 times greater in blood, and 20 times greater in urine, than DMT itself. Therefore, monitoring the N-oxide metabolites rather than the parent compounds alone in MAO-inhibited humans may provide a substantial advantage in detecting and quantitating the endogenous psychedelic compounds. Several of the studies reviewed did examine samples for the corresponding NMT, which is both a precursor for and a metabolite of the three endogenous psychedelics (NMT, HNMT, MNMT). However, in humans administered ayahuasca NMT was only intermittently detected in blood and urine and concentrations were quite low (pg/ml).This also may be the result of a shift in metabolism of DMT to the N-oxide after MAO inhibition and suggests that monitoring NMT in vivo may not be necessary or possible. Nonetheless, several of the reviewed studies suggested that the corresponding NMT was detected (Table). That data must now also be in question. DMT-N-oxide is neither a substrate for MAO-A nor for N-demethylases. Since similar metabolic pathways exist for HDMT and MDMT, we suggest that MAO inhibition in humans will enhance detection and quantitation of these compounds in the periphery, especially if the N-oxide metabolites are monitored. Thus, we can respond to the questions 'Is monitoring these compounds in biological samples such as CSF, blood and/or urine the best, or even most practical way to determine their activity?' and 'What will such data tell us about the possible normal function of these compounds in humans?' Data regarding their peripheral dynamicsconcentrations, circadian variation, and metabolismas assessed by rigorous analytic methods applied to biological samples represent the most accessible approach to beginning to determine their possible role in human psychophysiology and should be pursued. Our last question is 'Where does the research on endogenous psychedelics go from here?' One avenue for future studies concerns the endogenous nature of MDMT. This review has illustrated the convincing evidence that DMT and HDMT are endogenous in humans. However, MDMT has not been reported in human blood or urine but is apparently present in CSF. However, CSF has not been examined for the presence of HDMT. We propose that future studies of CSF, blood (including whole blood where higher concentrations may be observed) and urine monitor all three compounds and their N-oxides using superior, fully validated mass spectrometric methodology. Pretreatment of study subjects with an MAO inhibitor should optimize results and may prove critical to such studies. A technical issue regarding HDMT analysis also must be considered in future studies. Assays for this compound should include an enzyme hydrolysis step to free conjugates that may be formed from both the parent compound and its N-oxide. Another area for future research concerns assay sensitivity. We believe it is necessary to improve sensitivity of assays of the parent compounds to 1.0 pg/ml or less. Given the possible intermittent presence of these compounds in the periphery, blood and urine analyses may require more frequent sampling and longer collection times. The search for endogenous psychedelic tryptamines should also turn towards other human tissues than blood, urine and CSF; that is, solid organs such as adrenal, brain, lung, pineal, retina, and other tissues in which INMT activity has been noted using molecular biology tools. The combination of assaying relevant compounds with cell and molecular biology approaches will provide the most detailed possible assessment of the location(s) of synthesis and, ultimately, the role of these compounds in human physiology. For example, mapping of INMT and its presence within certain cell types and locations should reveal its intracellular distribution and possible associations with various receptors. The introduction of an INMT knockout mouse to the research effort could greatly assist in understanding the role of this enzyme and, by inference, the endogenous psychedelics. With these tools in hand, the research that can be conducted may S. A. Barker
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