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.

Development and validation of a multi-substance method for routine analysis of pesticides in post-mortem samples by Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry.

Pesticides play an important role in forensic toxicology and are usually classified as a single class of chemicals. Despite their commonly perceived unity, pesticides encompass a spectrum of compounds, including organophosphates, carbamates, pyrethroids or organochlorines, among others, each with varying degrees of toxicity. Pesticide analysis in post-mortem samples can be difficult due to the complexity of the samples and to the high toxicity of these compounds. The aim of this study was to develop and validate an easy to use, sensitive, and robust method, using ultra-performance liquid chromatography-tandem mass spectrometry to be incorporated in the routine flow for pesticide analysis in post-mortem blood samples. Described herein is a streamlined, expeditious, yet highly efficient method facilitating the screening, qualitative assessment, and quantitative confirmation of 15 pesticides, including acetamiprid, azinphos-ethyl, bendiocarb, carbofuran, chlorfenvinphos, dimethoate, imidaclopride, malathion, methiocarb, methomyl, parathion, pirimicarb, strychnine, tetrachlorvinphos, and thiacloprid in post-mortem blood, recognizing the pivotal role blood plays in forensic investigations. The developed method was linear from 10 to 200 ng/mL; limits of detection were between 1 and 10 ng/mL, depending on the compound; it was successfully evaluated a dilution ratio of 1-2, 5 and 10; and 8 substances showed maximum stability for the time interval studied. This UHPLC-MS/MS method is useful and a powerful tool in a toxicology lab because it is fast, simple, effective, and trustworthy. The results of this validation highlight the robustness of the analytical method, providing a valuable tool for the accurate and sensitive detection of pesticides in post-mortem blood. Poised for routine implementation, this method has already found success in suspected intoxication cases, promising to elevate the standards of forensic pesticide analysis.

Sensors in the Detection of Abused Substances in Forensic Contexts: A Comprehensive Review.

Forensic toxicology plays a pivotal role in elucidating the presence of drugs of abuse in both biological and solid samples, thereby aiding criminal investigations and public health initiatives. This review article explores the significance of sensor technologies in this field, focusing on diverse applications and their impact on the determination of drug abuse markers. This manuscript intends to review the transformative role of portable sensor technologies in detecting drugs of abuse in various samples. They offer precise, efficient, and real-time detection capabilities in both biological samples and solid substances. These sensors have become indispensable tools, with particular applications in various scenarios, including traffic stops, crime scenes, and workplace drug testing. The integration of portable sensor technologies in forensic toxicology is a remarkable advancement in the field. It has not only improved the speed and accuracy of drug abuse detection but has also extended the reach of forensic toxicology, making it more accessible and versatile. These advancements continue to shape forensic toxicology, ensuring swift, precise, and reliable results in criminal investigations and public health endeavours.

The Therapeutic Potential of Amphetamine-like Psychostimulants.

This review delves into the therapeutic applications of amphetamine-type stimulants such as lisdexamphetamine dimesylate, mixed amphetamine salts, 3,4-methylenedioxymethamphetamine (MDMA), dextroamphetamine, and phentermine. These compounds have been investigated for their potential in treating a range of psychiatric disorders, including attention deficit hyperactivity disorder (ADHD), drug dependence, post-traumatic stress disorder (PTSD), and obesity. Lisdexamphetamine dimesylate has shown promise in effectively treating ADHD symptoms in both children and adults. Additionally, it has been explored as a potential treatment for drug dependency and withdrawal, demonstrating encouraging results. Mixed amphetamine salts have also exhibited efficacy in reducing ADHD symptoms in adults. Future research should explore their potential use in treating bipolar disorder and cocaine dependence, considering the associated risks and benefits. MDMA-assisted psychotherapy has emerged as an innovative approach to treating PTSD, leading to sustained reductions in symptoms and even promoting post-traumatic growth. Furthermore, it has shown promise in managing anxiety related to life-threatening illnesses. Dextroamphetamine and phentermine have demonstrated efficacy in treating cocaine and opioid dependence, ADHD, and obesity. However, careful consideration and monitoring by medical professionals are essential due to the potential risks and benefits associated with them. In conclusion, amphetamine-type stimulants present a promising avenue for therapeutic interventions in various psychiatric conditions. Nevertheless, further research is necessary to comprehensively understand their mechanisms of action, dosage requirements, and long-term effects in different patient populations.

Solid Phase-Based Microextraction Techniques in Therapeutic Drug Monitoring.

Therapeutic drug monitoring is an established practice for a small group of drugs, particularly those presenting narrow therapeutic windows, for which there is a direct relationship between concentration and pharmacological effects at the site of action. Drug concentrations in biological fluids are used, in addition to other clinical observation measures, to assess the patient's status, since they are the support for therapy individualization and allow assessing adherence to therapy. Monitoring these drug classes is of great importance, as it minimizes the risk of medical interactions, as well as toxic effects. In addition, the quantification of these drugs through routine toxicological tests and the development of new monitoring methodologies are extremely relevant for public health and for the well-being of the patient, and it has implications in clinical and forensic situations. In this sense, the use of new extraction procedures that employ smaller volumes of sample and organic solvents, therefore considered miniaturized and green techniques, is of great interest in this field. From these, the use of fabric-phase extractions seems appealing. Noteworthy is the fact that SPME, which was the first of these miniaturized approaches to be used in the early '90s, is still the most used solventless procedure, providing solid and sound results. The main goal of this paper is to perform a critical review of sample preparation techniques based on solid-phase microextraction for drug detection in therapeutic monitoring situations.

Evaluation of Antipsychotic Drugs' Stability in Oral Fluid Samples.

Antipsychotics have narrow therapeutic windows, and their monitoring in biological fluids is therefore important; consequently, stability in those fluids must be investigated during method development and validation. This work evaluates the stability of chlorpromazine, levomepromazine, cyamemazine, clozapine, haloperidol, and quetiapine in oral fluid (OF) samples, using the dried saliva spots (DSS) sampling approach and gas chromatography coupled to tandem mass spectrometry. Since many parameters can influence the stability of the target analytes, design of experiments was adopted to check the crucial factors that affect that stability in a multivariate fashion. The studied parameters were the presence of preservatives at different concentrations, temperature, light, and time. It was possible to observe that antipsychotic stability improved when OF samples in DSS were stored at 4 °C, with a low ascorbic acid concentration, and in the absence of light. With these conditions, chlorpromazine and quetiapine were stable for 14 days, clozapine and haloperidol were stable for 28 days, levomepromazine remained stable for 44 days, and cyamemazine was stable for the entire monitored period (146 days). This is the first study that evaluates the stability of these antipsychotics in OF samples after application to DSS cards.

Analysis of Cannabinoids in Biological Specimens: An Update.

Cannabinoids are still the most consumed drugs of abuse worldwide. Despite being considered less harmful to human health, particularly if compared with opiates or cocaine, cannabis consumption has important medico-legal and public health consequences. For this reason, the development and optimization of sensitive analytical methods that allow the determination of these compounds in different biological specimens is important, involving relevant efforts from laboratories. This paper will discuss cannabis consumption; toxicokinetics, the most detected compounds in biological samples; and characteristics of the latter. In addition, a comprehensive review of extraction methods and analytical tools available for cannabinoid detection in selected biological specimens will be reviewed. Important issues such as pitfalls and cut-off values will be considered.

Synthesis of emerging cathinones and validation of a SPE GC-MS method for their simultaneous quantification in blood.

Over the past 15 years, synthetic cathinones have emerged as an important class of new psychoactive substances (NPS) worldwide. The proliferation of these psychostimulants and their sought-after effects among recreational drug users pose a serious threat to public health and enormous challenges to forensic laboratories. For forensic institutions, it is essential to be one step ahead of covert laboratories, foreseeing the structural changes possible to introduce in the core skeleton of cathinones while maintaining their stimulating activity. In this manner, it is feasible to equip themselves with standards of possible new cathinones and validated analytical methods for their qualitative and quantitative detection. Therefore, the aim of the work herein described was to synthesize emerging cathinones based on the evolving patterns in the illicit drug market, and to develop an analytical method for their accurate determination in forensic situations. Five so far unreported cathinones [4'-methyl-N-dimethylbuphedrone (4-MDMB), 4'-methyl-N-ethylbuphedrone (4-MNEB), 4'-methyl-N-dimethylpentedrone (4-MDMP), 4'-methyl-N-dimethylhexedrone (4-MDMH), and 4'-methyl-N-diethylbuphedrone (4-MDEB)] and a sixth one, 4'-methyl-N-ethylpentedrone, already reported to EMCDDA and also known as 4-MEAP, were synthesized and fully characterized by nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS). An analytical method for the simultaneous quantification of these cathinones in blood, using solid phase extraction (SPE) combined with gas chromatography-mass spectrometry (GC-MS) was developed and validated. The results prove that this methodology is selective, linear, precise, and accurate. For all target cathinones, the extraction efficiency was higher than 73%, linearity was observed in the range of 10 (lower limit of quantification, LLOQ) to 800 ng/mL, with coefficients of determination higher than 0.99, and the limits of detection (LODs) were 5 ng/mL for all target cathinones. The stability of these cathinones in blood matrices is dependent on the storage conditions; 4-MNEB is the most stable compound and 4-MDMH is the least stable compound. The low limits obtained allow the detection of the compounds in situations where they are involved, even if present at low concentrations.

The Determination of Cannabinoids in Urine Samples Using Microextraction by Packed Sorbent and Gas Chromatography-Mass Spectrometry.

Cannabis is the most consumed illicit drug worldwide, and its legal status is a source of concern. This study proposes a rapid procedure for the simultaneous quantification of Δ9-tetrahydrocannabinol (THC), 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC), 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH), cannabidiol (CBD), and cannabinol (CBN) in urine samples. Microextraction by packed sorbent (MEPS) was used to pre-concentrate the analytes, which were detected by gas chromatography-mass spectrometry. The procedure was previously optimized, and the final conditions were: conditioning with 50 µL methanol and 50 µL of water, sample load with two draw-eject cycles, and washing with 310 µL of 0.1% formic acid in water with 5% isopropanol; the elution was made with 35 µL of 0.1% ammonium hydroxide in methanol. This fast extraction procedure allowed quantification in the ranges of 1-400 ng/mL for THC and CBD, 5-400 ng/mL for CBN and 11-OH-THC, and 10-400 ng/mL for THC-COOH with coefficients of determination higher than 0.99. The limits of quantification and detection were between 1 and 10 ng/mL using 0.25 mL of sample. The extraction efficiencies varied between 26 and 85%. This analytical method is the first allowing the for determination of cannabinoids in urine samples using MEPS, a fast, simple, and low-cost alternative to conventional techniques.

Analysis of opiates in urine using microextraction by packed sorbent and gas Chromatography- Tandem mass spectrometry.

Opiates recreational consumption has always been a concern in society, public health, and in clinical toxicology analysis. The aim of this study was to develop and fully validate an analytical method, which was simple and rapid for the determination of tramadol, codeine, morphine, 6- acetylcodeine, 6-monoacetylmorphine and fentanyl using gas chromatography coupled to tandem mass spectrometry. The procedure includes the use of microextraction by packed sorbent for sample clean-up. A mixed mode sorbent was used, allowing the minimal use of solvents. The method was validated in urine samples, with the ability to detect and quantify all analytes with satisfactory linearity (in the range of 1 - 1000 ng/mL for all analytes, except for fentanyl (10-1000 ng/mL)). Extraction efficiency varied from 17 to 107%, which did not impair sensitivity, taking into account the low LLOQs obtained (1 ng/ mL for all analytes; and 10 ng/mL for fentanyl). The developed procedure proved to be fast, selective, and accurate for use in routine analysis, with a low volume of sample (250 µL).

Advances on Bioanalysis: Recent Approaches in the Determination of Biomarkers, Drugs of Abuse and Medicines.

New developments in instrumental approaches, for instance, hyphenated techniques, have allowed great advances in the bioanalytical field over the last half century, and there is no doubt that toxicology was one of the most improved areas [...].

An Update on the Implications of New Psychoactive Substances in Public Health.

The emergence of new psychoactive substances has earned a great deal of attention, and several reports of acute poisoning and deaths have been issued involving, for instance, synthetic opiates. In recent years, there have been profound alterations in the legislation concerning consumption, marketing, and synthesis of these compounds; rapid alert systems have also been subject to changes, and new substances and new markets, mainly through the internet, have appeared. Their effects and how they originate in consumers are still mostly unknown, primarily in what concerns chronic toxicity. This review intends to provide a detailed description of these substances from the point of view of consumption, toxicokinetics, and health consequences, including case reports on intoxications in order to help researchers and public health agents working daily in this area.

Optimization and validation of a procedure using the dried saliva spots approach for the determination of tobacco markers in oral fluid.

Exposure to tobacco smoke is one of the most common causes of premature death worldwide and is the cause of 8 million deaths annually. We have developed, optimized, and validated a procedure for the detection of nicotine, cotinine and trans-3-hydroxycotinine (biomarkers of tobacco exposure) in oral fluid using the dried saliva spots sampling approach and gas chromatography coupled to tandem mass spectrometry, thus allowing the distinction between active and passive smokers. For optimization, four parameters were evaluated, namely extraction solvent, extraction solvent volume, extraction time and spots drying time. During method validation, the parameters selectivity, linearity, precision and accuracy, recovery, stability, and dilution factor were assessed. Linearity was obtained for all target analytes in the concentration range of 10-200 ng/mL allowing the quantification of compounds up to 1000 ng/mL considering the dilution factor. The method recoveries ranged from 29.2% to 43.30% for nicotine, 66.60-89.10% for cotinine and 80.30-92.80% for trans-3-hydroxycotinine, while achieving intra-day, inter-day and intermediate precision and accuracy values never higher than 10.37% and ±6.62% respectively for all compounds. The herein described analytical method is the first to allow the determination of tobacco biomarkers in oral fluid using dried saliva spots, which is considered a sensitive, simple and low-cost alternative to conventional methods.

Stability of Cocaine, Opiates, and Metabolites in Dried Saliva Spots.

Drug abuse still represents a global problem, and it is associated with an increased risk of diseases, injuries, and deaths. Cocaine (COC) and opiates are the most abused drugs and account for a significant number of fatalities. Therefore, it is important to develop methods capable of effectively identifying and quantifying these substances. The present study aims to evaluate the long-term stability of COC, ecgonine methylester (EME), benzoylecgonine (BEG), cocaethylene (COET), norcocaine (NCOC), morphine (MOR), codeine (COD) and 6-monoacetylmorphine (6-MAM) in oral fluid samples. The analytes of interest were isolated from the matrix (50 µL) using the dried saliva spots (DSS) sampling approach and were subsequently analyzed by gas chromatography coupled with tandem mass spectrometry (GC-MS/MS). The parameters that could influence the stability of the target compounds were studied, and these were storage temperature, light, use of preservatives (and respective concentrations), and time. The effects of each parameter were evaluated using the design of experiments (DOE) approach. The stability of the target analytes was improved when the DSS were stored at room temperature, in the presence of light and using 1% sodium fluoride. The best conditions were then adopted for the DSS storage and long-term stability was assessed. COD was only stable for 1 day, EME was stable for 3 days, COC, COET, NCOC and 6-MAM were stable for 7 days, MOR for 14 days and BEG remained stable throughout the study (136 days). This is the first study that evaluates the stability of these compounds in oral fluid samples after application in DSS cards, and optimizes the conditions in order to improve their stability.

New Method for the Monitoring of Antidepressants in Oral Fluid Using Dried Spot Sampling.

The increase in the consumption of antidepressants is a public health problem worldwide, as these are a class of compounds widely used in the treatment of several illnesses, such as depression and anxiety. This work aimed to develop and optimize a method for the quantification of a number of antidepressants and their metabolites (fluoxetine, venlafaxine, O-desmethylvenlafaxine, citalopram, sertraline, and paroxetine) in 100 µL of oral fluid using the dried saliva spots (DSS) sampling approach and gas chromatography coupled with tandem mass spectrometry (GC-MS/MS). The method was validated, presenting linearity within the studied range, with detection and quantification limits ranging between 10 and 100 ng/mL, and coefficients of determination (R2) of at least 0.99 for all analytes. Recoveries were between approximately 13 and 46%. The analysis of precision and accuracy presented acceptable coefficients of variation and relative errors, considering the criteria usually accepted in the validation of bioanalytical procedures. The method herein described is the first to be reported using DSS for the extraction of antidepressants, proving to be a sensitive, simple, and fast alternative to conventional techniques, and capable of being routinely applied in clinical and forensic toxicology scenarios.

Trends in microextraction approaches for handling human hair extracts - A review.

The complementary role of hair in testing scenarios has expanded across the spectrum of toxicological and clinical monitoring investigations and, over the last 20 years, hair analysis has gained increasing attention and recognition. Moreover, a great deal of attention has been paid to the miniaturisation of extraction procedures, minimising/eliminating toxic organic solvents consumption, making them user-friendly and rapid, in addition to maximising extraction efficiency. The aim of this work is to provide a critical review of the advances observed over the last 5 years in the use of miniaturised approaches for sample clean-up and drug pre-concentration in hair analysis. There have been major improvements in some well-established microextraction approaches, such as liquid phase microextraction, mainly through the use of supramolecular and ionic liquids. In addition, new developments have also been reported in solid phase microextraction, driven by d-SPE applications. In the last 5 years, a total of 69 articles have been published using some type of microextraction technique for hair specimens, thus justifying the relevance of a critical review of innovations, improvements and trends related to these miniaturised approaches for sample preparation.

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

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Recent Developments in the Determination of Biomarkers of Tobacco Smoke Exposure in Biological Specimens: A Review.

Environmental tobacco smoke exposure (ETS) and smoking have been described as the most prevalent factors in the development of certain diseases worldwide. According to the World Health Organization, more than 8 million people die every year due to exposure to tobacco, around 7 million due to direct ETS and the remaining due to exposure to second-hand smoke. Both active and second-hand exposure can be measured and controlled using specific biomarkers of tobacco and its derivatives, allowing the development of more efficient public health policies. Exposure to these compounds can be measured using different methods (involving for instance liquid- or gas-chromatographic procedures) in a wide range of biological specimens to estimate the type and degree of tobacco exposure. In recent years, a lot of research has been carried out using different extraction methods and different analytical equipment; this way, liquid-liquid extraction, solid-phase extraction or even miniaturized procedures have been used, followed by chromatographic analysis coupled mainly to mass spectrometric detection. Through this type of methodologies, second-hand smokers can be distinguished from active smokers, and this is also valid for e-cigarettes and vapers, among others, using their specific biomarkers. This review will focus on recent developments in the determination of tobacco smoke biomarkers, including nicotine and other tobacco alkaloids, specific nitrosamines, polycyclic aromatic hydrocarbons, etc. The methods for their detection will be discussed in detail, as well as the potential use of threshold values to distinguish between types of exposure.