Comparative study of sample preparation procedures to determine the main compounds in ayahuasca beverages by QuEChERS and high-performance liquid chromatography analysis.

INTRODUCTION: Ayahuasca is a psychoactive drink originally consumed by indigenous people of the Amazon. The lack of regulation of this drink leads to uncontrolled consumption, and it is often consumed in religious contexts. OBJECTIVE: The aim of this work is to compare three miniaturised extraction techniques for extracting the main ayahuasca compounds from beverages. METHODOLOGY: Three sample pretreatment techniques were evaluated (dispersive liquid-liquid microextraction [DLLME], microextraction by packed sorbent [MEPS] and QuEChERS [Quick, Easy, Cheap, Effective, Rugged and Safe]) for the simultaneous extraction of N,N-dimethyltryptamine (DMT), tetrahydroharmine (THH), harmine, harmaline, harmol and harmalol from ayahuasca beverage samples. Then, the most promising technique (QuEChERS) was chosen to pre-concentrate the analytes, subsequently detected by high-performance liquid chromatography coupled to a diode array detector (HPLC-DAD). RESULTS: The procedure was optimised, with the final conditions being 500 µL of extractor solvent, 85 mg of primary secondary amine (PSA) and 4 s of vortexing. The analytical method was validated, showing to be linear between 0.16 and 10 µg/mL for ß-carbolines and between 0.016 and 1 µg/mL for DMT, with coefficients of determination (R2) between 0.9968 and 0.9993. The limit of detection (LOD) and lower limit of quantification (LLOQ) were 0.16 µg/mL for all compounds, except for DMT (0.016 µg/mL) and extraction efficiencies varied between 60.2% and 88.0%. CONCLUSION: The analytical methodology proved to be accurate and precise, with good linearity, LODs and LLOQs. This method has been fully validated and successfully applied to ayahuasca beverage samples.

In Vitro Study of the Bioavailability and Bioaccessibility of the Main Compounds Present in Ayahuasca Beverages.

Ayahuasca is a psychoactive beverage that contains the psychoactive compound N,N-dimethyltryptamine and ß-carboline alkaloids. This study aims at determining in vitro the bioavailability and bioaccessibility of the main compounds present in decoctions of four individual plants, in a commercial mixture and in four mixtures of two individual plants used in the preparation of Ayahuasca. The samples were subjected to an in vitro digestion process, and the Caco-2 cell line was used as an absorption model. The integrity and permeability of the cell monolayer were evaluated, as well as the cytotoxicity of the extracts. After digestion and cell incubation, the compounds absorbed by the cell monolayer were quantified by high-performance liquid chromatography coupled to a diode array detector. The results showed that compounds such as N,N-dimethyltryptamine, Harmine, Harmaline, Harmol, Harmalol and Tetrahydroharmine were released from the matrix during the in vitro digestion process, becoming bioaccessible. Similarly, some of these compounds, after being incubated with the cell monolayer, were absorbed, becoming bioavailable. The extracts did not show cytotoxicity after cell incubation, and the integrity and permeability of the cell monolayer were not compromised.

Correction: Simão, A.Y., et al. Evaluation of the Cytotoxicity of Ayahuasca Beverages. Molecules 2020, 25, 5594.

This paper [...].

Evaluation of the Cytotoxicity of Ayahuasca Beverages.

Ayahuasca is a beverage consumed at shamanic ceremonies and currently has gained popularity on recreational scenarios. It contains beta-carboline alkaloids and N,N-dimethyltryptamine, which possesses hallucinogenic effects. Only a few studies have elicited the psychoactive effects and the dose of such compounds on neurological dopaminergic cells or animals. In this work, we aimed to study the cytotoxic effects of these compounds present in ayahuasca beverages and on five different teas (Banisteriopsis caapi, Psychotria viridis, Peganum harmala, Mimosa tenuiflora and Dc Ab (commercial name)) preparations on dopaminergic immortalized cell lines. Moreover, a characterization of the derivative alkaloids was also performed. All the extracts were characterized by chromatographic systems and the effect of those compounds in cell viability and total protein levels were analyzed in N27 dopaminergic neurons cell line. This is the first article where cytotoxicity of ayahuasca tea is studied on neurological dopaminergic cells. Overall, results showed that both cell viability and protein contents decreased when cells were exposed to the individual compounds, as well as to the teas and to the two mixtures based on the traditional ayahuasca beverages.

Determination of methadone and EDDP in oral fluid using the dried saliva spots sampling approach and gas chromatography-tandem mass spectrometry.

The present work describes the development and validation of a novel approach to determine methadone (MTD) and its main metabolite (EDDP) in oral fluid samples, using the dried saliva spots (DSS) sampling approach and gas chromatography-tandem mass spectrometry (GC-MS/MS). Oral fluid samples (50 µL) were applied into Whatman™ 903 protein saver filter paper cards and were allowed to dry overnight. The extraction was carried out by immersion of the spot in 1 mL of isopropyl alcohol with agitation for 1 min. Afterwards, the extract was centrifuged for 15 min at 3500 rpm and the supernatant evaporated to dryness and reconstituted with 50 µL of methanol. The procedure was considered linear in the range of 10 to 250 ng/mL for both compounds, with determination coefficients greater than 0.99. Intra- and inter-day precision and accuracy revealed coefficients of variation (CVs) lower than 15% at the studied concentrations, with mean relative errors within ± 15% of the nominal concentrations. Recoveries ranged from 45 to 74%. The limits of detection and quantification were 5 and 10 ng/mL respectively for both analytes. All studied parameters complied with the defined criteria and the method enabled the successful determination of MTD and EDDP in oral fluid samples from patients undergoing opiate substitution/maintenance therapy.

Peculiar surface behavior of some ionic liquids based on active pharmaceutical ingredients.

The ionic liquids based on biologically active cations and anions, commonly designated by ionic liquids based on active pharmaceutical ingredients (ILs-APIs), are interesting compounds for use in pharmaceutical applications. Lidocaine docusate, ranitidine docusate, and didecyldimethylammonium ibuprofen are examples of promising ILs-APIs that were recently synthesized. They were submitted to biological testing and calorimetric measurements, but nothing is known about their surface properties. In this work, we measured the surface tension and the contact angles on both hydrophilic and hydrophobic surfaces in a temperature range as wide as possible. Based on the wettability data, the polarity fractions were estimated using the Fowkes theory. The peculiar surface behavior observed was tentatively attributed to the presence of mesophases.

Ayahuasca Beverages: Phytochemical Analysis and Biological Properties.

Ayahuasca is a psychoactive beverage, originally consumed by indigenous Amazon tribes, of which consumption has been increasing worldwide. The aim of this study was to evaluate the phytochemical profile, as well as the antioxidant, anti-inflammatory and antimicrobial properties of decoctions of four individual plants, a commercial mixture and four mixtures of two individual plants used in the Ayahuasca preparation. For this purpose, a phytochemical characterization was performed, determining the content of flavonoids, total phenolic compounds, and analyzing the phenolic profile. Besides, 48 secondary metabolites were investigated by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q/TOF-MS) and their concentration estimated with real standards when present. The antioxidant activity was evaluated by both the ß-carotene bleaching test and DPPH free radical scavenging assay, and the anti-inflammatory activity was determined by a protein denaturation method. Finally, the antimicrobial properties were evaluated using the disc diffusion assay, resazurin microtiter method, anti-quorum sensing and anti-biofilm activity assays. The obtained results showed that, in general, the samples have a high content of phenolic compounds and flavonoids with noticeable differences, reflecting on remarkable antioxidant and anti-inflammatory activities. Significant antimicrobial properties were also observed, with emphasis on the effect of B. caapi and P. harmala on planktonic and biofilm cells of A. baumannii, inhibiting both the biofilm formation and the production of violacein pigment.

Novel synthetic opioids - toxicological aspects and analysis.

Over the past few years, there has been an emerging number of new psychoactive drugs. These drugs are frequently mentioned as "legal highs", "herbal highs", "bath salts" and "research chemicals". They are mostly sold and advertised on online forums and on the dark web. The emerging new psychoactive substances are designed to mimic the effects of psychoactive groups, which are often abused drugs. Novel synthetic opioids are a new trend in this context and represent an alarming threat to public health. Given the wide number of fatalities related to these compounds reported within the last few years, it is an important task to accurately identify these compounds in biologic matrices in order to administer an effective treatment and reverse the respiratory depression caused by opioid related substances. Clinicians dealing with fentanyl intoxication cases should consider that it could, in fact, be a fentanyl analogue. For this reason, it is a helpful recommendation to include synthetic opioids in the routine toxicological screening procedures, including analysis in alternative matrices, if available, to investigate poly-drug use and possible tolerance to opioids. To address this public health problem, better international collaboration, effective legislation, effective investigation, control of suspicious "research chemicals" online forums and continuous community alertness are required. This article aims to review diverse reported fatalities associated with new synthetic opioids describing them in terms of pharmacology, metabolism, posology, available forms, as well as their toxic effects, highlighting the sample procedures and analytical techniques available for their detection and quantification in biological matrices.

Determination of amphetamine-type stimulants in urine samples using microextraction by packed sorbent and gas chromatography-mass spectrometry.

The aim of this work was the development, optimization and full validation of a method applying microextraction by packed sorbent (MEPS) coupled to gas chromatography-mass spectrometry (GC-MS) to determine amphetamine (AMP), methamphetamine (MAMP), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxyethylmethamphetamine (MDMA), 3,4-methylenedioxy-N-methyl-α-ethylfenilethylamine (MBDB), and 3,4-methylenedioxy-N-ethylamphetamine (MDE) in urine samples. Using 200 µL of sample, the MEPS procedure was optimized concerning type of sorbent, sample dilution, number of strokes, activation of the ion exchange mechanism and composition of both washing and elution solvents. The method was fully validated according to the Food and Drug Administration and the Scientific Working Group of Forensic Toxicology guidelines for the validation of bioanalytical methods. The studied parameters included selectivity, calibration model and linearity, limits of detection and quantification, precision, accuracy, stability, dilution integrity and recoveries. Linearity was obtained in the range of 25-1000 ng/mL for MAMP, MBDB and MDE, 35-1000 ng/mL for AMP and MDMA, and 50-1000 ng/mL for MDA, with coefficients of determination (R2) >0.99 for all analytes. Both intra- and inter-day precision and accuracy were adequate, and coefficients of variation lower than 15% and mean relative errors (RE) within a range of ±15% of the theoretical concentrations were obtained for all compounds under study. Analyte recoveries ranged from 19 to 71%, allowing LLOQs ≤50 ng/mL.

Determination of opiates in whole blood using microextraction by packed sorbent and gas chromatography-tandem mass spectrometry.

In Portugal, and worldwide, the abuse of psychoactive substances is increasing, with a significant incidence of deaths related to their consumption. Opiates are one of the most prevalent group of substances in that context, and they are responsible for a significant impact of the mentioned harms. Therefore, it becomes necessary to equip labs with faster and effective methods to identify and quantify these substances. This work describes the development and validation of a novel analytical method for the simultaneous determination of morphine, codeine and 6-monoacetylmorphine in blood samples by gas chromatography-tandem mass spectrometry (GC-MS/MS), using microextraction by packed sorbent (MEPS) for sample preparation. Before the MEPS procedure, a precipitation step with acetonitrile was performed. The MEPS parameters were optimized using the fractional factorial planning (2k-1) and surface response methodology. The final optimized conditions were: number of strokes (20), amount of formic acid in the washing solution (3.36%), number of washes of the sorbent (1), amount of ammonium hydroxide in the elution solution (2.36%) and number of elution cycles (11). After the extraction procedure, the analytes were derivatized with MSTFA with 5% TMS. Using a sample volume of 250 µL, the method was validated according to internationally accepted standards. The method proved to be linear in the range of 5-1000 ng/mL with coefficients of determination greater than 0.99 for all analytes. Intra-and inter-day precision and accuracy were in accordance with the above-mentioned criteria, presenting coefficients of variation ≤15% and relative errors within a range of ± 15% of the theoretical concentration. The absolute recoveries ranged from 6 to 23%. The validated method was applied to the analysis of real samples, being an advantageous tool for the detection of those substances in blood. This is the first time that GCMS/MS with MEPS was used for the determination of these compounds in biological fluids.

Contactless decontamination of hair samples: cannabinoids.

Room temperature ionic liquids (ILs) have already been shown to provide efficient extraction media for several systems, and to capture volatile compounds, namely opiates. In this work, a novel, contactless, artefact-free extraction procedure for the removal of Δ9 -tetrahrydrocannabinol (THC) from the surface of human hair is presented. To prepare in vitro cannabinoids-contaminated hair, samples were flushed with hashish smoke for 7 h. The decontamination experiments were carried at 100 °C for 24 h, according to the procedure previously described. Fifty-three ILs were screened and presented decontamination efficiencies ranging from 0 to 96 %. Although the majority of the ILs presented efficiencies above 90%, the 1-ethanol-3-methyl tetrafluoroborate (96%) was chosen for further process optimization. The Design of Experiments results demonstrated that all studied variables were significant for the process and the obtained optimum conditions were: 100 °C, 13 h and 175 mg of IL. In the work of Perrotin-Brunel et al. (J. Mol. Struct. 2011, 987, 67), it is demonstrated that, at 100 °C, full conversion of tetrahydrocannabinolic acid (THCA) into THC is obtained after 60 min. Since our decontamination takes place over 13 h at 100 °C, full conversion of THCA into THC is expected. Additionally, our method was compared with the method proposed by Cairns et al. (Forensic Sci. Int. 2004, 145, 97), through the analysis of 15 in vitro contaminated hair samples. The results demonstrated that with our method a mean extraction efficiency of 11 % higher was obtained. Copyright © 2016 John Wiley & Sons, Ltd.

Development, optimization, and validation of a novel extraction procedure for the removal of opiates from human hair's surface.

Room temperature ionic liquids (ILs) have proved to be efficient extraction media for several systems, and their ability to capture volatile compounds from the atmosphere is well established. We report herein a contactless extraction procedure for the removal of opiate drugs from the surface of human hair. The compounds were chosen as a model drug, particularly due to their low volatility. Equal amounts of IL and hair (about 100 mg) were introduced in a customized Y-shaped vial, and the process occurred simply by heating. After testing several ILs, some of them (e.g. 1-methyl-3-ethanol-imidazolium tetrafluoroborate, phenyl-trimethyl-ammonium triflate or bis(dimethyl) diheptylguanidinium iodide) showed extraction efficiencies higher than 80% for the two studied compounds, morphine and 6-monoacetylmorphine. Using the design of experiments (DOE) approach as an optimization tool, and bearing in mind the hygroscopic properties of the ILs (in particular, 1-methyl-3-ethanol-imidazolium tetrafluoroborate), the process was optimized concerning the following variables: temperature (50-120 ºC), extraction time (8-24 h), IL amount (50-200 mg) and water content of the IL (0.01-60%). This study not only provided the optimum conditions for the process (120 ºC, 16 h, 100 mg of IL containing 40% of water), but has also showed that the water content of the IL represents the variable with the most significant effect on the extraction efficiency. Finally, we validated our method through the comparison of the results obtained by treating hair samples with the described procedure to those obtained using a standard washing method and criteria for positivity.

On the interfacial behavior of ionic liquids: surface tensions and contact angles.

In this work the liquid/vapour and the solid/liquid interfaces of a series of ionic liquids: 1-ethyl-3-methylpyridinium ethyl sulfate, [EMPy][EtSO4], 1-ethyl-3-methylimidazolium ethyl sulfate, [EMIM][EtSO4], 1-ethanol-3-methylimidazolium tetrafluoroborate, [C2OHMIM][BF4], 1-butyl-3-methylimidazolium tetrafluoroborate, [BMIM][BF4], and 1-octyl-3-methylimidazolium tetrafluoroborate, [OMIM][BF4], were investigated. The surface tension was measured in a wide temperature range, (298-453) K. The contact angles were determined on substrates of different polarities. Both on the polar (glass) and the non-polar substrates ((poly-(tetrafluoroethylene) and poly-(ethylene)), the liquids with maximum and minimum surface tensions lead, respectively, to the highest and the lowest contact angles. The dispersive, gamma(L)(d), and non-dispersive, gamma(L)(nd), components of the liquid surface tension, gamma(L), were calculated from the contact angles on the non-polar substrates using the Fowkes approach. The polarity fraction, gamma(L)(nd)/gamma(L), was compared with the polarity parameter, k, obtained from the fitting of the surface tension vs. temperature data to the Eötvös equation. Good agreement was found for the extreme cases: [OMIM][BF4] exhibits the lowest polarity and [BMIM][BF4], the highest. When compared with the polarity fractions of standard liquids considered as "polar" liquids, the ionic liquids studied may be considered as moderately polar.