Bioanalysis and metabolic investigation of psychoactive substances

In Part I, four liquid chromatography–tandem mass spectrometry (LC–MS/MS) methods for the bioanalysis of psychoactive substances and their metabolites were developed and validated according to United States Food and Drug Administration (FDA) guidelines.
The first method was validated for the quantification of mescaline and its metabolites 3,4,5-trimethoxyphenylacetic acid (TMPAA), N-acetyl mescaline (NAM), and 3,5-dimethoxy-4-hydroxyphenethylamine (4-desmethyl mescaline). A linear range of 12.5 to 5,000 ng/mL for mescaline and TMPAA, and 1.25 to 500 ng/mL for NAM and 4-desmethyl mescaline was achieved. For optimal chromatographic separation, an Acquity Premier HSS T3 C18 column was employed. A single-step extraction procedure was implemented, enabling a non-laborious analysis of plasma samples with a runtime of 4.25 minutes per sample. The method satisfied all FDA validation criteria, including those on accuracy (85–106%), precision (coefficient of variation [CV] ≤ 7%), sensitivity and selectivity, matrix effect (92–100%), and extraction recovery (≥ 98%). Ultimately, the method was successfully employed for the analysis of mescaline, TMPAA, and NAM in pharmacokinetic samples from participants enrolled in a clinical phase I study. 4-desmethyl mescaline could not be selectively analyzed in pharmacokinetic samples due to interference and incomplete chromatographical separation with another metabolite, presumably 3,4-dimethoxy-5-hydroxyphenethylamine (3-desmethyl mescaline).
The second method was developed for the analysis of pharmacokinetic parameters in plasma samples from a clinical study involving diamorphine-dependent patients. The objective of the study was to investigate the feasibility of intranasal diamorphine administration as a component of diamorphine-assisted treatment. Analytes were separated using a Kinetex EVO C18 analytical column. The method is capable of quantifying the concentrations of diamorphine, 6-monoacetylmorphine (6-MAM), morphine, morphine-3-glucuronide (M3G), and morphine-6-glucuronide (M6G) in human plasma, spanning a linear range of 1 to 1,000 ng/mL. The total runtime for a single sample was four minutes. The method was demonstrated to be accurate (91–106%) and precise (CV ≤ 9%) while exhibiting a high extraction recovery (> 87%) and a negligible matrix effect (99–125%) for all analytes. No interferences with endogenous plasma compounds were observed and the method was successfully applied for the analysis of numerous clinical study samples.
In the third project, two methods for the quantification of racemic and chiral 3,4-methylenedioxymethamphetamine (MDMA) and its metabolite 3,4-methylenedioxyamphetamine (MDA) in human plasma were developed and validated. A linear range of 0.5–500 ng/mL was achieved for racemic MDMA and MDA analysis, while linear ranges of 0.5–1,000 ng/mL and 1–1,000 ng/mL were achieved for chiral MDMA and MDA, respectively. The achiral chromatographic separation of the compounds was performed using a Luna PFP(2) column and the chiral analysis was conducted with a Lux AMP column. The total run times were 4.25 and 6 minutes for achiral and chiral sample analysis, respectively. Both methods met the FDA validation guidelines criteria for accuracy, precision, selectivity, sensitivity, matrix effect, and extraction recovery. Finally, a subset of clinical plasma samples from a study involving R-, S-, and racemic MDMA was analyzed to demonstrate the method’s functionality.
In conclusion, four sensitive, non-laborious, highly reliable, and robust LC–MS/MS methods for the bioanalysis of mescaline, diamorphine, racemic MDMA, and chiral MDMA plus their respective metabolites were developed and validated. These methods are applicable in pharmacokinetic investigations in a clinical setting as well as for forensic studies.
Part II of this thesis presents an investigation of the metabolic pathways of psilocybin’s active metabolite psilocin. The primary focus was on the phase I metabolism of psilocin through the cytochrome P450 (CYP) system, as well as the influence of monoamine oxidases (MAOs). Phase II metabolism, with a particular focus on UDP-glucuronosyltransferases (UGTs), was also examined. To conduct a comprehensive analysis of psilocin’s metabolism, enzymatic in vitro assays were performed using human liver microsomes (HLM) and recombinant CYP, MAO, and UGT enzymes. Moreover, plasma samples from C57BL/6J mice and humans were analyzed following psilocybin administration to obtain in vivo data.
After a 4-hour incubation period, approximately 29% of psilocin was metabolized by HLM. Recombinant CYP2D6 and CYP3A4 enzymes demonstrated even higher metabolic activity, with nearly 100% and 40% of psilocin being metabolized, respectively. The metabolites 4-hydroxyindole-3-acetic acid (4-HIAA) and 4-hydroxytryptophol (4-HTP) were identified in the presence of HLM, but not after incubation of psilocin with recombinant CYP enzymes. Nevertheless, trace amounts of 4-HIAA and 4-HTP were generated by MAO-A from psilocin, thereby substantiating its involvement in this metabolic pathway. In contrast to the in vivo data, where conjugated psilocin is one of the main metabolites, UGT1A10 did not extensively conjugate psilocin in vitro.
Furthermore, two potential metabolites were identified. In vitro and in vivo analyses identified N-methyl-4-hydroxytryptamine (norpsilocin) and an oxidized metabolite of psilocin. The former was detected following incubation with CYP2D6 and in the plasma of mice, while the latter was identified in CYP2D6 incubations, in mice, and in humans. However, the investigation into the influence of the CYP2D6 genotype on psilocin degradation yielded no significant results. While norpsilocin has never been described as a psilocin metabolite in mice before, the exact structure of the oxidized metabolite remains to be elucidated.
In conclusion, the phase I enzymes CYP2D6, CYP3A4, and MAO-A are implicated in psilocin’s metabolism. The identification of putative norpsilocin in mice and oxidized psilocin in humans offers further insight into the metabolic pathway of psilocybin and could contribute to the safety and efficacy of psilocybin application.