Addressing a major interference in the quantification of psilocin in mouse plasma: Development of a validated liquid chromatography tandem mass spectrometry method.

Psilocybin is a psychedelic compound found in some hallucinogenic "magic mushrooms". Psilocin is the active metabolite of Psilocybin, and it is the subject of several studies for the treatment of psychological disorders, such as anxiety, depression, and post-traumatic stress disorder. As such, the pharmacokinetic properties of psilocin should be evaluated to ensure its safety and efficacy as part of the drug development process. Based on the previously published studies, reversed-phase liquid chromatography (LC) was tested for psilocin quantification. The analysis, however, showed a major interference in mouse plasma that was not, to the best of our knowledge, reported previously. We, therefore, aimed to identify and separate the interference, using various chromatographic columns, mobile phase conditions, and mass spectrometers (MS) instruments. Chromatographic separation was achieved on an ultra high performance liquid chromatography (UHPLC) system, and a quadrupole-linear ion trap equipped with an electrospray ionization (ESI) source was used in positive ion mode with multiple reaction monitoring (MRM). Several chromatographic conditions and column chemistries, including C-18 and Phenyl-hexyl were initially tested, and failed to separate the interference. Exact mass measurement and MS/MS analysis were used to determine the structure of the interfering compound, which was confirmed to be tryptophan. Using the identified structure of the interfering compound, a fast and reliable hydrophilic interaction liquid chromatography (HILIC)-MS/MS method was developed and validated, that was capable of separating psilocin from the interference while achieving a 0.5 ng/ml lower limit of quantification (LLOQ). The validated method was successfully applied to a pharmacokinetic study where psilocin was orally administered to C57BL/6 mouse subjects. Psilocin concentration in all the analyzed mouse plasma samples was successfully determined.

Development and validation of an LC-MS/MS method for the bioanalysis of psilocybin's main metabolites, psilocin and 4-hydroxyindole-3-acetic acid, in human plasma.

Psilocin is the active metabolite of psilocybin, a serotonergic psychedelic substance. It is used recreationally and investigated in substance-assisted psychotherapy. The pharmacokinetic properties of psilocin are only partially characterized. Therefore, we developed and validated a rapid LC-MS/MS method to quantify psilocin and its metabolite 4-hydroxyindole-3-acetic acid (4-HIAA) in human plasma. Plasma samples were processed by protein precipitation using methanol. The injected sample was mixed with water in front of a C18 analytical column to increase retention of the analytes. Psilocin and 4-HIAA were detected by multiple reaction monitoring (MRM) in positive and negative electrospray ionisation mode, respectively. An inter-assay accuracy of 100-109% and precision of ≤8.7% was recorded over three validation runs. The recovery was near to complete (≥94.7%) and importantly, consistent over different concentration levels and plasma batches (CV%: ≤4.1%). The plasma matrix caused negligible ion suppression and endogenous interferences could be separated from the analytes. Psilocin and 4-HIAA plasma samples could be thawed and re-frozen for three cycles, kept at room temperature for 8 h or 1 month at -20 °C without showing degradation (≤10%). The linear range (R ≥ 0.998) of the method covered plasma concentrations observed in humans following a common therapeutic oral dose of 25 mg psilocybin and was therefore able to assess the pharmacokinetics of psilocin and 4-HIAA. The LC-MS/MS method was convenient and reliable for measuring psilocin and 4-HIAA in plasma and will facilitate the clinical development of psilocybin.

Exposure-Response Analysis to Assess the Concentration-QTc Relationship of Psilocybin/Psilocin.

Psilocybin is being developed for treating major depressive disorder. Psilocybin is readily dephosphorylated to psilocin upon absorption. The potential for psilocin proarrhythmic effect was assessed using a concentration-QTc interval (C-QTc) analysis from an open-label single ascending dose study of psilocybin. Psilocybin doses ranged from 0.3 to 0.6 mg/kg. This trial showed a significant but shallow C-QTc relationship. At the clinical dose of 25 mg, the mean psilocin maximum concentration is 18.7 ng/mL, and the associated mean (upper 90% confidence interval of mean) QTcF change is 2.1 (6.6) milliseconds. Given the short half-life of psilocin of about 4 hours, there would be no accumulation after monthly oral doses used in clinical trials. The upper limit of the 90% confidence interval of the model-predicted mean ΔQTcF crossed 10 milliseconds at a psilocin concentration of 31.1 ng/mL. At a supraclinical psilocin maximum concentration of about 60 ng/mL, ΔQTcF remains low, with a mean (upper limit of the 90% confidence interval) of 9.1 (17.9) milliseconds. This analysis enabled the characterization of the C-QTc relationship and prediction of QTc prolongation at the expected clinical and possible higher psilocybin doses.

Psychedelic effects of psilocybin correlate with serotonin 2A receptor occupancy and plasma psilocin levels.

The main psychedelic component of magic mushrooms is psilocybin, which shows promise as a treatment for depression and other mental disorders. Psychedelic effects are believed to emerge through stimulation of serotonin 2A receptors (5-HT2ARs) by psilocybin's active metabolite, psilocin. We here report for the first time the relationship between intensity of psychedelic effects, cerebral 5-HT2AR occupancy and plasma levels of psilocin in humans. Eight healthy volunteers underwent positron emission tomography (PET) scans with the 5-HT2AR agonist radioligand [11C]Cimbi-36: one at baseline and one or two additional scans on the same day after a single oral intake of psilocybin (3-30 mg). 5-HT2AR occupancy was calculated as the percent change in cerebral 5-HT2AR binding relative to baseline. Subjective psychedelic intensity and plasma psilocin levels were measured during the scans. Relations between subjective intensity, 5-HT2AR occupancy, and plasma psilocin levels were modeled using non-linear regression. Psilocybin intake resulted in dose-related 5-HT2AR occupancies up to 72%; plasma psilocin levels and 5-HT2AR occupancy conformed to a single-site binding model. Subjective intensity was correlated with both 5-HT2AR occupancy and psilocin levels as well as questionnaire scores. We report for the first time that intake of psilocybin leads to significant 5-HT2AR occupancy in the human brain, and that both psilocin plasma levels and 5-HT2AR occupancy are closely associated with subjective intensity ratings, strongly supporting that stimulation of 5-HT2AR is a key determinant for the psychedelic experience. Important for clinical studies, psilocin time-concentration curves varied but psilocin levels were closely associated with psychedelic experience.

[Suicide under the influence of "magic mushrooms"]. / Suizid unter Psilocin-Einfluss.

Psilocybin/psilocin from so-called psychoactive mushrooms causes hallucinogenic effects. Especially for people with mental or psychiatric disorders ingestion of magic mushrooms may result in horror trips combined with the intention of self-destruction and suicidal thoughts. Automutilation after consumption of hallucinogenic mushrooms has already been described. Our case report demonstrates the suicide of a man by self-inflicted cut and stab injuries. A causal connection between suicidal behaviour and previous ingestion of psychoactive mushrooms is discussed.

Identification of novel psychoactive drug use in Sweden based on laboratory analysis--initial experiences from the STRIDA project.

AIM: The study aimed to collect information concerning the increasing use of new psychoactive substances, commonly sold through online shops as 'Internet drugs' or 'legal highs', or in terms of masked products such as 'bath salts' and 'plant food'. METHODS: The Karolinska Institutet and Karolinska University Laboratory and the Swedish Poisons Information Centre have initiated a project called 'STRIDA' aiming to monitor the occurrence and trends of new psychoactive substances in Sweden, and collect information about their clinical symptoms, toxicity and associated health risks. A liquid chromatographic-tandem mass spectrometric (LC-MS/MS) multi-component method has been developed, currently allowing for the determination of > 80 novel psychoactive compounds or metabolites thereof. This study focused mainly on the particular drug substances identified and the population demographics of the initial STRIDA cases. RESULTS: In urine and/or blood samples obtained from 103 consecutive cases of admitted or suspected recreational drug intoxications in mostly young subjects (78% were ≤ 25 years, and 81% were males) presenting at emergency departments all over the country, psychoactive substances were detected in 82%. The substances comprised synthetic cannabinoids ('Spice'; JWH analogues), substituted cathinones ('bath salts'; e.g. butylone, MDPV and methylone) and tryptamines (4-HO-MET), plant-based substances (mitragynine and psilocin), as well as conventional drugs-of-abuse. In 44% of the cases, more than one new psychoactive substance, or a mixture of new and/or conventional drugs were detected. CONCLUSION: The initial results of the STRIDA project have documented use of a broad variety of new psychoactive substances among mainly young people all over Sweden.

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.

A validated method for quantitation of psilocin in plasma by LC-MS/MS and study of stability.

A liquid chromatography-electrospray ionization/tandem mass spectrometry method for the quantitation of psilocin in plasma is presented. Sample workup was performed with mixed-mode solid-phase extraction using ascorbic acid and nitrogen for drying to protect the unstable analyte. Calibration curves were linear from 2 to 100 ng/mL, and no selectivity problems occurred. The limit of detection was 0.1 ng/mL, and the limit of quantitation was 0.34 ng/mL. Recovery was >86% and matrix effects were <110%. Both were reproducible. Interday and intraday precisions at different concentrations were 1.5-4.3% relative standard deviation, bias within ±9%. Processed samples were stable in the autosampler for at least 26 h. Furthermore, the stability of psilocin in blood stored at different temperatures over various periods of time was investigated. Samples stored at room temperature showed a continuous decrease of analyte leading to a loss of about 90% after 1 week. Storage in the fridge improved sample stability significantly. Freezing of blood samples led to a not reproducible loss of psilocin.

Determining the pharmacokinetics of psilocin in rat plasma using ultra-performance liquid chromatography coupled with a photodiode array detector after orally administering an extract of Gymnopilus spectabilis.

This study established ultra-performance liquid chromatography coupled with a photodiode array detector for determining psilocin and its pharmacokinetics in rat plasma after orally administering an extract of Gymnopilus spectabilis. The extract was separated on an ODS C18 column (2.3 µm, 100 mm × 2.1 mm I.D.) by gradient elution with (A) water containing 50mM AcONH(4) and (B) acetonitrile. The wavelength was set at 265 nm and the injection volume was 10 µL. Under these conditions, the calibration curve was linear over the concentration range 0.2-20 µg/mL with a correlation coefficient of r(2)=0.9992. The inter- and intraday precision levels were less than 7% and the accuracies (%) were within the range 92.0-102.5%. The method was sufficiently valid to be applied to a pharmacokinetics study of psilocin in rat plasma. The pharmacokinetic parameters of psilocin in rat plasma after the oral administration of a G. spectabilis extract were as follows: C(max), 0.43 ± 0.12 µg/mL; T(max), 90 ± 2.1 min; AUC(0→t), 1238.3 ± 96.4 (µg/mL) min; and T(1/2), 117.3 ± 40.3 min.

Development of a psilocin immunoassay for serum and blood samples.

After the immunisation of rabbits with a psilocin-specific immunogen, polyclonal antisera were obtained. With these antisera a competitive, heterogeneous radioimmunoassay for the detection of psilocin was developed. As tracer a derivative of psilocin was synthesised, which contained a tritiated CH(3) group. The antisera showed a specific reaction with psilocin. The cross-reactivity of structurally related endogenous substances like serotonin, tryptophan and tyrosine was below 0.01%. Also common drugs of abuse (Delta(9)-tetrahydrocannabinol, cocaine, morphine, amphetamine) showed negligible cross-reactivity (0.01-2%). Only tricyclic neuroleptics with a (dimethylamino)ethyl side-chain showed some cross-reactivity (20%). Spiked serum and blood samples were analysed with this new immunoassay and the results obtained were compared with the values measured with a validated GC-MS method.

Detection of psilocin in body fluids.

Active compounds of some mushrooms e.g. Psilocybe cubensis, Paneolus subalteatus or Stropharia coronilla, the psychotropic agents psilocybin and psilocin, have hallucinogenic effects. In one case of 'magic mushroom' intake, we had to analyse blood and urine. Psilocin was detected in the urine with REMEDi HS. Most of the psilocin was excreted as the glucuronide. Therefore an enzymatic hydrolysis should be the first step in analysis. Free psilocin was determined at a concentration of 0.23 mg/l while the total amount was 1.76 mg/l urine. The concentration of psilocin in serum was too low for detection with REMEDi HS. We proved a GC-MS-method with d(3)-morphine as internal standard and silylation with MSTFA. Similarly to urine, most of the psilocin in serum was found in the conjugated form. The concentration of free psilocin was 0.018 mg/l, that of total psilocin, 0.052 mg/l serum.

Quantitation of psilocin in human plasma by high-performance liquid chromatography and electrochemical detection: comparison of liquid-liquid extraction with automated on-line solid-phase extraction.

Two modifications of the HPLC-ED method with respect to extraction procedure used have been developed for psilocin, the active metabolite of psilocybin, in human plasma using either liquid-liquid extraction (LLE) or automated on-line solid-phase extraction (on-line SPE). Each type of the sample preparation required a different HPLC system followed by electrochemical detection at 650 to 675 mV. The limit of quantitation of both modifications was 10 ng/ml psilocin. There was no significant difference observable between the LLE and the on-line SPE in terms of method standard deviation (LLE 1.82%, on-line SPE 1.13%) and the analytical results. However, the advantages of on-line SPE in addition to different selectivity were less manual effort, smaller plasma volumes of 400 microl (LLE 2 ml) and a recovery of psilocin in human plasma of nearly 100% (LLE 88%). In contrast to a previous procedure both methods were rapid, simple and reliable and yielded high plasma recoveries. They were used successfully in the quantitation of psilocin in plasma samples obtained from healthy volunteers after p.o. administration of 0.2 mg psilocybin per kg body mass. Plasma concentration curves and pharmacokinetic parameters were calculated.

Determination of psilocin and 4-hydroxyindole-3-acetic acid in plasma by HPLC-ECD and pharmacokinetic profiles of oral and intravenous psilocybin in man.

In order to investigate the pharmacokinetic properties of psilocybin (PY), the main psychoactive compound of Psilocybe mushrooms, high performance liquid chromatographic procedures with column-switching coupled with electrochemical detection (HPLC-ECD) for reliable quantitative determination of the PY metabolites psilocin (PI) and 4-hydroxyindole-3-acetic acid (4HIAA) in human plasma were established. Sample work-up includes protection of the highly unstable phenolic analytes with ascorbic acid, freeze-drying and in-vitro microdialysis. The data of two controlled clinical studies with healthy volunteers are presented. The subjects (N = 6 for both studies) received single oral PY doses of 0.224 +/- 0.02 mg/kg b.wt. (10-20 mg) and intravenous doses of 1 mg PY, respectively. Peak plasma levels of PI after oral administration of PY were measured after 105 +/- 37 min showing an average concentration of 8.2 +/- 2.8 ng PI/ml plasma. 4HIAA peak concentrations of 150 +/- 61 ng/ml plasma were found 113 +/- 41 min after ingestion of PY. After intravenous administration, a mean PI maximum plasma concentration of 12.9 +/- 5.6 ng/ml plasma was found 1.9 +/- 1.0 min after injection. The maximum plasma levels appearing within a very short period indicate a rapid dephosphorylation of PY also when administered systemically. 4HIAA was not detected after 1 mg of intravenous PY. Estimates for the absolute bioavailability of PI after oral administration of PY were 52.7 +/- 20% (N = 3).

Positron emission tomography and fluorodeoxyglucose studies of metabolic hyperfrontality and psychopathology in the psilocybin model of psychosis.

The effects of the indolehallucinogen psilocybin, a mixed 5-HT2 and 5-HT1 agonist, on regional cerebral glucose metabolism were investigated in 10 healthy volunteers with PET and [F-18]-fluorodeoxyglucose (FDG) prior to and following a 15- or 20-mg dose of psilocybin. Psychotomimetic doses of psilocybin were found to produce a global increase in cerebral metabolic rate of glucose (CMRglu) with significant and most marked increases in the frontomedial and frontolateral cortex (24.3%), anterior cingulate (24.9%), and temporomedial cortex (25.3%). Somewhat smaller increases of CMRglu were found in the basal ganglia (18.5%), and the smallest increases were found in the sensorimotor (14.7%) and occipital cortex (14.4%). The increases of CMRglu in the prefrontal cortex, anterior cingulate, temporomedial cortex, and putamen correlated positively with psychotic symptom formation, in particular with hallucinatory ego disintegration. The present data suggest that excessive 5-HT2 receptor activation results in a hyperfrontal metablic pattern that parallels comparable metabolic findings associated with acute psychotic episodes in chronic schizophrenics and contrasts with the hypofrontality in chronic schizophrenic patients.