Development of a LC–MS/MS Method to Analyze 5-Methoxy-N,N-Dimethyltryptamine and Bufotenine: Application to Pharmacokinetic Study

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated to simultaneously measure the psychedelic drug 5-MeO-DMT and its active metabolite bufotenine in mouse serum. Using a simple protein precipitation and 9-minute run, the assay was linear over a wide concentration range with high precision and accuracy. After injecting mice with 2 mg/kg of 5-MeO-DMT, the systemic exposure to bufotenine was about 1/14 that of the parent drug. Because bufotenine binds the 5-HT2A receptor with roughly ten times higher affinity than 5-MeO-DMT, the metabolite may substantially contribute to the drug's overall pharmacological and toxic effects.

Study at a glance

Abstract

INTRODUCTION: 5-Methoxy-N,N-dimethyltryptamine (5-MeO-DMT) is a psychoactive indolealkylamine substance that has been used for recreational purpose and may lead to fatal toxicity. While 5-MeO-DMT is mainly inactivated via deamination, it is O-demethylated to an active metabolite, bufotenine. Quantitation of 5-MeO-DMT and bufotenine is essential to understand the exposure to and the effects of drug and metabolite. This study, therefore, aimed to develop and validate a LC-MS/MS method for simultaneous analysis of 5-MeO-DMT and bufotenine in mouse serum. METHODS: A simple protein precipitation method coupled with an optimal gradient elution was used for sample preparation and separation. Detection of 5-MeO-DMT and bufotenine was accomplished using multiple reaction monitoring of m/z 219.2→174.2 and 205.2→160.2, respectively, in the positive ion mode. 5-Methyl-N,N-dimethyltrypamine (m/z 203.2→158.3) was used as internal standard for quantification. Accuracy and precision were determined after the analyses of quality control samples. Validated assay was then employed to determine drug and metabolite concentrations in serum samples collected from mice at different time points after intraperitoneal administration of 5-MeO-DMT (2 mg/kg). RESULTS: With a total run time of 9 min, 5-MeO-DMT and bufotenine were eluted at 2.8 and 5.6 min, respectively. The assay was linear over the range 0.90-5,890 ng/mL (1.12-7,360 pg on-column) for 5-MeO-DMT and 2.52-5,510 ng/mL (3.14-6,890 pg) for bufotenine. Intra- and inter-day precision and accuracy were within 15% for both analytes. The recovery of each analyte from 20 µL of serum containing 8.08, 72.7 and 655 ng/mL of 5-MeO-DMT and 7.56, 68.1 and 613 ng/mL of bufotenine was more than 75%. Pharmacokinetic analysis revealed that the systemic exposure (area under the curve) to metabolite bufotenine was about 1/14 of that to 5-MeO-DMT. CONCLUSION: This LC-MS/MS method is a sensitive and reliable assay for quantitation of blood 5-MeO-DMT and bufotenine. Given the fact that bufotenine acts on 5-HT(2A) receptor with an affinity about 10-fold higher than 5-MeO-DMT, the active metabolite bufotenine may significantly contribute to the apparent pharmacological and toxicological effects of 5-MeO-DMT.