Daniel Bourquin

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).

Renal excretion profiles of psilocin following oral administration of psilocybin: a controlled study in man

In a clinical study eight volunteers received psilocybin (PY) in psychoactive oral doses of 212+/-25 microg/kg body weight. To investigate the elimination kinetics of psilocin (PI), the first metabolite of PY, urine was collected for 24 h and PI concentrations were determined by high-performance liquid chromatography with column switching and electrochemical detection (HPLC-ECD). Sample workup included protection of the unstable PI with ascorbic acid, freeze-drying, and extraction with methanol. Peak PI concentrations up to 870 microg/l were measured in urine samples from the 2-4 h collection interval. The PI excretion rate in this period was 55.5+/-33.8 microg/h. The limit of quantitation (10 microg/L) was usually reached 24 h after drug administration. Within 24 h, 3.4+/-0.9% of the applied dose of PY was excreted as free PI. Addition of beta-glucuronidase to urine samples and incubation for 5 h at 40 degrees C led to twofold higher PI concentrations, although 18+/-7% of the amount of unconjugated PI was decomposed during incubation. We conclude that in humans PI is partially excreted as PI-O-glucuronide and that enzymatic hydrolysis extends the time of detectability for PI in urine samples.

Pharmacodynamics and pharmacokinetics of intravenously, orally and rectally administered diacetylmorphine in opioid dependents, a two-patient pilot study within a heroin-assisted treatment program.

OBJECTIVE The pharmacokinetics and pharmacodynamics of high-dose intravenous (i.v.), oral and rectal diacetylmorphine (diamorphine, heroin, DAM) preparations were compared. METHOD Two heroin-dependent patients participating in a heroin-assisted treatment program received single or repeated doses of 200 - 690 mg DAM i.v., orally (capsules, controlled-release tablets) and rectally. Plasma and urine profiles of DAM and metabolites were monitored by high-performance liquid chromatography and gas chromatography mass spectrometry, flash and high effects by visual analog scaling (VAS). RESULTS DAM was only detectable in plasma after i.v. administration. With a t 1/2 beta of 1.3 - 2.2 min it was rapidly desacetylated to 6-acetylmorphine which was further metabolized to morphine and its 3- and 6-O-glucuronide. Morphine-3-glucuronide was the dominating metabolite in plasma and urine independent of the administration route. Oral and rectal doses and dosage intervals were adequate to produce flash and high effects without any cardiovascular and respiratory side-effects nor withdrawal symptoms. CONCLUSIONS Oral and rectal DAM should further be tested and validated on a wider patient group for the non-invasive, long-term application of high-dose DAM within heroin-assisted treatment programs as alternative to the harmful i.v. application.

Determination of morphine-3-glucuronide in human urine by capillary zone electrophoresis and micellar electrokinetic capillary chromatography.

Attempts to determine morphine-3-glucuronide (MO3G) by high-performance capillary electrophoresis and micellar electrokinetic capillary chromatography are reported. Using direct injection of urine, it was possible to achieve a limit of detection of about 20 micrograms/ml, which is poor compared with high-performance liquid chromatography and immunoassays. However, employing sample extraction with C8 cartridges, the presence of MO3G in urines that tested positive for opioids using a commercial enzyme-multiplied immunoassay technique could be successfully confirmed. The limit of detection with unambiguous identification of MO3G via spectral analysis was about 1 microgram/ml.

High-performance liquid chromatographic monitoring of intravenously administered diacetylmorphine and morphine and their metabolites in human plasma

A rapid and selective reversed-phase high-performance liquid chromatographic assay with gradient elution and diode-array detection for diacetylmorphine, morphine, codeine, and their free and glucuronidated metabolites in plasma, was developed. After addition of ethylmorphine as internal standard the plasma samples were extracted using C18 ODS-2 solid-phase columns with a recovery better than 80%. The limit of quantitation using an injection volume of 2 microl was 25 ng/ml for each compound. The intra- and inter-day precision was better than 5%. The described method cannot only be used for pharmacokinetic studies but also for intoxication cases to monitor a wide range of opiates.

Determination of 18β-glycyrrhetinic acid in biological fluids from humans and rats by solid-phase extraction and high-performance liquid chromatography

Abstract Methods have been developed and characterized allowing rapid isolation and quantification of 18β-glycyrrhetinic acid (GRA) in biological fluids from both humans and rats. Sample preparation includes extraction with urea—methanol for plasma samples, and solid-phase extraction (SPE) for urine and bile samples. Hydrolysis of GRA glucuronides in urine and bile was performed by treatment with β-glucuronidase. MGRA, the 3-O-methyl derivative of GRA was synthesized as an internal standard resistant to hydrolysis. High-performance liquid chromatography (HPLC) was performed with an isocratic system using methanol—water—acetic acid (83:16.8:0.2, v/v/v) as solvent on a Lichrocart RP-18 column at 30°C with ultraviolet detection. The methods allowed base line separation of GRA and MGRA from all biological fluids tested, with a detection limit of 0.15 mg/l. Validation of the methods included determination of recovery, accuracy and precision in plasma, bile and urine from humans and rats. The methods were further evaluated by investigating the pharmacokinetics of GRA in normal rats and in rats with a bile fistula. Following an intravenous dose of 10 mg/kg, the plasma concentration—time curve of GRA could be fitted to a one compartment model both in control and bile fistula rats. The elimination half life averaged 15.0 ± 2.2 versus 16.8 ± 2.4 min in control and bile fistula rats (difference not significant). Within 90 min following administration of GRA, urinary elimination of GRA and GRA glucuronides was less than 1% in both groups whereas biliary elimination averaged 51.3 ± 3.1%. The results show that the methods developed allow pharmacokinetic studies of GRA in humans and rats.

DIACETYLMORPHINE AND ITS METABOLITES IN PLASMA BY HPLC WITH DIODE-ARRAY AND ATMOSPHERIC PRESSURE IONIZATION MASS SPECTROMETRIC DETECTION

Based on reversed phase high performance liquid chromatography with gradient elution and diode-array detection (HPLC-DAD) a specific and sensitive assay for the quantitation of diacetylmorphine (heroin, DAM) and its primary and secondary metabolites 6-acetylmorphine, morphine, morphine-3-β-D-glucuronide, morphine-6-β-D-glucuronide and nor-morphine in plasma was developed. Confirmation of the peak assignment was performed by atmospheric pressure ionization-mass spectrometry (API-MS). Plasma samples were extracted on C18 ODS-2 columns using an automated solid-phase extraction (SPE) unit. The recovery was between 45 and 68%. The quantitation limit of DAM and its metabolites was 50 and 10 ng/mL, respectively. The inter-day precision was in the range of 4.0 to 17.6%. The described method was applied for pharmacokinetic studies which were part of the evaluation and validation of DAM preparations used within a heroin-assisted treatment program for dependent drug users.

Determination of phenylethylamines in hallucinogenic cactus species by high-performance liquid chromatography with photodiode-array detection

Abstract A high-performance liquid chromatographic procedure with photodiode-array detection has been developed to create phytochemical profiles of phenylalkylamine-containing cactus species. The basic methanolic cactus extracts with methoxamine as internal standard were separated on a 3-μm ODS column with acetonitrile—water—phosphoric acid—hexylamine as the mobile phase. Peak assignation was performed by on-line UV detection (190–300 nm) and by gas chromatography—mass spectrometry of the isolated compounds. The quantitation of mescaline was done at 205 nm. The excellent sensitivity (the detection limit of mescaline was 500 pg, corresponding to 0.002% in cactus material) allowed the analysis of milligram amounts of cactus material taken from living plants. The mescaline content of the psychotropic Peyote cactus Lophophora williamsii (Lem. ex Salm-Dyck) Coult. ranged from 680 to 1010 mg per 100 g. The morphologically very similar Lophophora diffusa (Croizat) Bravo could be differentiated by the absence of mescaline and the dominant alkaloid pellotine.

11C-radiolabeling of hallucinogenic psilocin, a potential radioligand for studying the role of serotonin receptors in psychotic symptom formation

The desmethyl compound, 4-hydroxy-N-methyltryptamine (4), was synthesized via a four-step reaction sequence starting from 4-benzyloxyindole (1). Psilocin (4-hydroxy-N,N-dimethyltryptamine), an indole hallucinogen, was labeled by N-monomethylation of the side chain using the classical methylating agent [ 11 C]CH 3 I in 20±5% (decay corrected from [ 11 C]CH 3 I) radiochemical yield. Specific activities obtained ranged from 900-2300Ci/mmol at EOS (end of synthesis). The radiosynthesis, semi-preparative HPLC and formulation were completed in an average time of 45 minutes.