Rapid detection and validated quantification of psychoactive compounds in complex plant matrices by direct analysis in real time‐high resolution mass spectrometry – Application to “Kava” psychoactive pepper products

Abstract

Rationale Classified by the UNODC as a top 20 plant of concern, Piper methysticum (also known as Kava) is being increasingly abused recreationally for its mind‐altering effects. It is of significant forensic relevance to establish methods to rapidly identifyand quantify psychoactive compounds, especially those yet to be scheduled ascontrolled substances and which have exhibited various noteworthy health concerns. Methods Direct analysis in real time high‐resolution mass spectrometry (DART‐HRMS) demonstrated the ability to detect a range of kavalactones in Pipermethysticum derived products and plant material with no sample preparation. Inaddition, a validated method using calibration curves developed with a deuteratedinternal standard was used for the quantification of the psychoactive moleculeyangonin in various products. Results DART‐HRMS detected the protonated masses of six major kavalactonesand three flavokavains in 18 commercial Kava products. A method consistent withFDA validation guidelines was established for the quantification of yangonin in thevarious complex matrices. Implementation of this method, with an LLOQ of 5 mg/mL, enabled successful quantification of yangonin in 16 Kava products.Concentrations for solid products ranged from 2.71 to 8.99 mg/g, while that forliquid products ranged from 1.03 to 4.59 mg/mL. Conclusions Rapid identification and quantification of psychoactive smallmolecules in plant material can be accomplished using a validated DART‐HRMSprotocol. This work illustrates an approach to qualitative and quantitative analysesof a wide variety of complex matrices derived from plants, and demonstrates thatthe commercially available products analyzed are P. methysticum derived and docontain psychoactive yangonin at quantifiable levels.

Detection and Quantification of Psychoactive N,N-Dimethyltryptamine in Ayahuasca Brews by Ambient Ionization High-Resolution Mass Spectrometry

“…The US Food and Drug Administration (FDA) Guidelines for the Development and Validation of Bioanalytical Methods have been used to validate DART-HRMS protocols to quantify the psychoactive molecules in commercial botanical products and plant preparations. − These guidelines include: (1) demonstration that the method is selective, sensitive, and reproducible; (2) development of standard curves using a minimum of six nonzero calibrators; (3) a blank calibrator and a zero calibrator (i.e., a blank calibrator with internal standard) that do not interfere with the analyte of interest and do not exceed 5% of the average responses in the curve calibrators and quality control (QC) standards; (4) the lowest point of the standard curve being the lower limit of quantification (LLOQ); and (5) utilization of QC standards at four levels (high, medium, low, and LLOQ) incorporated into the curve . Criteria that an analysis must meet to be declared validated are as follows: (1) nonzero calibrators should be within 15% of nominal (theoretical) concentrations, except for the LLOQ calibrator, which can be within 20% of its nominal value; (2) 75%, and a minimum of six nonzero calibrators are required to meet the previous guideline; (3) ≥67% of the QC standards should be within 15% of their nominal values; and (4) ≥50% of the QC standards at each level must be within 15% of their nominal values .…”

“…The dramatic rise in law enforcement encounters with ayahuasca and the plants from which it is made, particularly in a forensic context, have increased the demand for methods to assist forensic crime labs in the efficient analysis and identification of the plant ingredients, as well as the ayahuasca beverage itself. Although reports of quantification of psychoactive molecules for forensic purposes have become more prevalent with the increasing recreational use of the plants identified by the UNODC, − few reports involve the quantification of these molecules in plant-based complex mixtures. Various gas chromatography- (GC) ,,− and liquid chromatography-mass spectrometry (LC-MS) ,,,− methods have been developed for the analysis of ayahuasca and quantification of DMT in the plant material and brews.…”

Quantification of hordenine in a complex plant matrix by direct analysis in real time–high‐resolution mass spectrometry: Application to the “plant of concern” Sceletium tortuosum

“…DART-HRMS has successfully been used to quantify psychoactive materials in other plants of concern including N,N-dimethyltryptamine in ayahuasca brews, 19 yangonin in kava, 20 mitragynine in kratom, 21 and mescaline in various cacti, 22…”

“…With the extensive sample preparation and long run times per sample associated with previously used techniques, the ability to quantify the amount of hordenine present, specifically in various complex forms of S. tortuosum , using a rapid technique such as DART‐HRMS would be extremely beneficial. DART‐HRMS has successfully been used to quantify psychoactive materials in other plants of concern including N , N ‐dimethyltryptamine in ayahuasca brews, 19 yangonin in kava, 20 mitragynine in kratom, 21 and mescaline in various cacti, 22 thereby adding to the growing list of validated protocols that crime labs can use to quantify psychoactive compounds by DART‐HRMS. Herein, we describe the development of a validated protocol for the quantification of hordenine via DART‐HRMS and demonstrate its efficient application to the determination of hordenine levels in several commercially available S. tortuosum products.…”

Introducing “DoPP”: A Graphical User-Friendly Application for the Rapid Species Identification of Psychoactive Plant Materials and Quantification of Psychoactive Small Molecules Using DART-MS Data

“…Previous studies have shown that direct analysis in real time–high-resolution mass spectrometry (DART-HRMS), with minimal if any sample preparation required, reveals within a single analysis of the bulk material a range of detected molecules extending across the dielectric constant spectrum. − Furthermore, it has been shown that when analyzed by DART-HRMS, plants exhibit species-specific chemical signatures that can be utilized to predict the identities of species within a given genus, using advanced statistical analysis tools. ,− These findings imply the possibility that the application of machine learning tools to a library of DART-HRMS-derived species-specific chemical signatures might provide a mechanism to predict the species identity of plant material unknowns with a statistical level of certainty. Furthermore, this same instrumental technique can be used to quantify the psychoactive molecules present. − In principle, it could provide a more universal approach for the identification of new psychoactive materials, rather than relying on current conventional methods, which require nuanced method development that is also time- and resource-intensive. Importantly, the analysis can be conducted in less than 1 min per sample.…”