Carolina Dizioli Rodrigues de Oliveira

Gas chromatographic analysis of dimethyltryptamine and β-carboline alkaloids in ayahuasca, an amazonian psychoactive plant beverage

INTRODUCTION Ayahuasca is obtained by infusing the pounded stems of Banisteriopsis caapi in combination with the leaves of Psychotria viridis. P. viridis is rich in the psychedelic indole N,N-dimethyltryptamine, whereas B. caapi contains substantial amounts of beta-carboline alkaloids, mainly harmine, harmaline and tetrahydroharmine, which are monoamine-oxidase inhibitors. Because of differences in composition in ayahuasca preparations, a method to measure their main active constituents is needed. OBJECTIVE To develop a gas chromatographic method for the simultaneous determination of dimethyltryptamine and the main beta-carbolines found in ayahuasca preparations. METHODOLOGY The alkaloids were extracted by means of solid phase extraction (C(18)) and detected by gas chromatography with nitrogen/phosphorous detector. RESULTS The lower limit of quantification (LLOQ) was 0.02 mg/mL for all analytes. The calibration curves were linear over a concentration range of 0.02-4.0 mg/mL (r(2 )> 0.99). The method was also precise (RSD < 10%). CONCLUSION A simple gas chromatographic method to determine the main alkaloids found in ayahuasca was developed and validated. The method can be useful to estimate administered doses in animals and humans for further pharmacological and toxicological investigations of ayahuasca.

Recent Advances in Chromatographic Methods to Detect Drugs of Abuse in Alternative Biological Matrices

In recent years, many studies have been developed with the aim of improving drug detection in both conventional specimens and alternative biological matrices. A large number of drug abuse studies, forensic toxicology analyses, drugs in the workplace and even in doping control in sports activities related to drug detection in biological samples have been reported in the literature. The interest in the development and optimization of analytical techniques to detect drugs of abuse in different specimens is explained by the several possi- bilities and information that they can provide. Conventional samples such as urine and blood and more recently, saliva and sweat, are of fundamental importance whenever recent exposure to drugs is under investigation. Human keratinized tissues such as hair and nails are especially important for obtaining data of chronic long-term exposure with the great advantage of being collected in a non-invasive way. Meconium can be a useful biological sample to evaluate fetal drug exposure following maternal drug use. This paper reviews chroma- tographic procedures for determination of amphetamines, cannabinoids, opiates, nicotine, cocaine and alcohol in alternative biological matrices. Gas chromatographic and liquid chromatographic procedures with different detectors (including mass spectrometry) and sample preparation techniques such as liquid-liquid extraction (LLE), solid phase extraction (SPE) and solid phase microextraction (SPME) were considered.

Maternal and developmental toxicity of ayahuasca in Wistar rats

INTRODUCTION Ayahuasca is a psychotropic plant beverage initially used by shamans throughout the Amazon region during traditional religious cult. In recent years, ayahuasca has also been used in ceremonies of a number of modern syncretic religious groups, including pregnant women. However, no documented study has been performed to evaluate the risk of developmental toxicity of ayahuasca. METHODS In the present work, maternal and developmental toxicity was evaluated in Wistar rats. Ayahuasca was administered to pregnant rats in three different doses [the equivalent typical dose (TD) administered to humans, five-fold TD and 10-fold TD] during the gestational period (6-20 days). RESULTS Dams treated with the highest ayahuasca dose showed maternal toxicity with decrease of weight gain and food intake. Visceral fetal findings were observed in all treatment groups. Skeletal findings were observed in the intermediate- and high-dose groups. The fetuses deriving from the highest dose group also presented a decrease in body weight. CONCLUSIONS From these results, it is possible to conclude that there is a risk of maternal and developmental toxicity following ayahuasca exposure and that the level of toxicity appears to be dose-dependent.

Development and practical application of accelerated solvent extraction for the isolation of cocaine/crack biomarkers in meconium samples.

A method using accelerated solvent extraction (ASE) for the isolation of cocaine/crack biomarkers in meconium samples, followed by solid phase extraction (SPE) and the simultaneous quantification by gas chromatography-mass spectrometry (GC-MS) was developed and validated. Initially, meconium samples were submitted to an ASE procedure, which was followed by SPE with Bond Elut Certify I cartridges. The analytes were derivatizated with PFP/PFPA and analyzed by GC-MS. The limits of detection (LOD) were between 11 and 17ng/g for all analytes. The limits of quantification (LOQ) were 30ng/g for anhydroecgonine methyl ester, and 20ng/g for cocaine, benzoylecgonine, ecgonine methyl ester and cocaethylene. Linearity ranged from the LOQ to 1500ng/g for all analytes, with a coefficients of determination greater than 0.991, except for m-hydroxybenzoylecgonine, which was only qualitatively detected. Precision and accuracy were evaluated at three concentration levels. For all analytes, inter-assay precision ranged from 3.2 to 18.1%, and intra-assay precision did not exceed 12.7%. The accuracy results were between 84.5 and 114.2% and the average recovery ranged from 17 to 84%. The method was applied to 342 meconium samples randomly collected in the University Hospital-University of São Paulo (HU-USP), Brazil. Cocaine biomarkers were detected in 19 samples, which represent 5.6% of exposure prevalence. Significantly lower birth weight, length and head circumference were found for the exposed newborns compared with the non-exposed group. This is the first report in which ASE was used as a sample preparation technique to extract cocaine biomarkers from a complex biological matrix such as meconium samples. The advantages of the developed method are the smaller demand for organic solvents and the minor sample handling, which allows a faster and accurate procedure, appropriate to confirm fetal exposure to cocaine/crack.

Neurobehavioral, reflexological and physical development of Wistar rat offspring exposed to ayahuasca during pregnancy and lactation

Ayahuasca is a hallucinogenic beverage prepared by the decoction of plants native to the Amazon Basin region. The beverage has been used throughout the world by members of some syncretic religious movements. Despite the recent legalization of ayahuasca in Brazil for religious purposes, there is little pre-clinical and clinical information attesting to its safety, particularly in relation to the use during pregnancy. The aim of the current work was to determine the effects of perinatal exposure to ayahuasca (from the 6th day of pregnancy to the 10th day of lactation) on physical, reflexology and neurobehavioral parameters of the Wistar rat offspring. The offspring showed no statistically significant changes in the physical and reflexology parameters evaluated. However, in adult rats, perinatally exposed to ayahuasca, an increase in frequency of entries in open arms in elevated plus-maze test, a decrease in total time of interaction in social interaction test, a decrease in time of latency for the animal to start swimming and a decrease of the minimum convulsant dose induced by pentylenetetrazol were observed. In conclusion, our results showed that the use of ayahuasca by mothers during pregnancy and lactation reduced the general anxiety and social motivation of the rat offspring. Besides, it promoted a higher sensitivity for initiation and spread of seizure activity.

Anhydroecgonine methyl ester, a cocaine pyrolysis product, may contribute to cocaine behavioral sensitization.

Crack cocaine has a high potential to induce cocaine addiction and its smoke contains cocaines pyrolysis product anhydroecgonine methyl ester (AEME), a partial agonist at M1- and M3-muscarinic acetylcholine receptor and an antagonist at the remaining subtypes. No reports have assessed AEMEs role in addiction. Adult male Wistar rats were intraperitoneally administered with saline, 3mg/kg AEME, 15mg/kg cocaine, or a cocaine-AEME combination on every other day during a period of 9 days. After a 7-days withdrawal period, a challenge injection of the respective drugs was performed on the 17th day. The locomotor activity was evaluated on days 1, 3, 5, 7, 9 and 17, as well as dopamine levels (9th day) and dopaminergic receptors proteins (D1R and D2R on the 17th day) in the caudate-putamen (CPu) and nucleus accumbens (NAc). AEME was not able to induce the expression of behavioral sensitization, but it substantially potentiates cocaine-effects, with cocaine-AEME combination presenting higher expression than cocaine alone. An increase in the dopamine levels in the CPu in all non-saline groups was observed, with the highest levels in the cocaine-AEME group. There was a decrease in D1R protein level in this brain region only for cocaine and cocaine-AEME groups. In the NAc, an increase in the dopamine levels was only observed for cocaine and cocaine-AEME groups, with no changes in both D1R and D2R protein levels. These behavioral and neurochemical data indicate that AEME alone does not elicit behavioral sensitization but it significantly potentiates cocaine effects when co-administered, resulting in dopamine increase in CPu and NAc, brain regions where dopamine release is mediated by cholinergic activity.

Accelerated Solvent Extraction for Gas Chromatographic Analysis of Nicotine and Cotinine in Meconium Samples

Adverse effects associated with smoking during pregnancy are well documented. Although self-report surveys on drug consumption during pregnancy have been improved with new interviewing techniques, underreporting is still a concern. Therefore, a series of biological markers and specimens to diagnose fetal exposure to tobacco have been studied. In the present study, an analytical method was developed to detect nicotine and cotinine (the main nicotine metabolite) in meconium samples. Accelerated solvent extraction (ASE) followed by solid-phase extraction (SPE) were used as sample preparation techniques. The analytes were detected by gas-chromatography with nitrogen-phosphorus detection. The limits of detection were 3.0 and 30 ng/g for cotinine and nicotine, respectively. The method showed good linearity (r(2) > 0.98) in the concentration range studied (LOQ-500 ng/g). The intraday precision, given by the RSD of the method, was less than 15% for cotinine and nicotine. The method proved to be fast, practical, and sensitive. Smaller volumes of organic solvents are necessary compared to other chromatographic methods published in the scientific literature. This is the first report in which ASE was used as sample preparation technique in methods to detect xenobiotics in meconium.

Development of a simple HPLC-DAD multi-analyte procedure and its application in cases evaluated by the Poison Control Center of São Paulo, Brazil

This work describes a simple approach to overcome challenges in emergency toxicological analysis, using liquid-liquid extraction and high-performance liquid chromatography coupled with a diode-array detector (HPLC-DAD). A rapid procedure has been developed, for the extraction and detection of 19 analytes from the following drug classes: analgesics, benzodiazepines, antidepressants, anticonvulsants and drugs of abuse. These substances are relevant in the context of emergency toxicology in Brazil. The method has been validated according to international guidelines by establishing parameters such as lower limit of quantification, sensitivity, linearity, accuracy and precision. The intra and inter-day precision values, at the lowest concentration levels, have always been less than 20% considering its relative standard deviation. As for accuracy values, these have also been satisfactory (above 81.3%). This method was successfully applied in 201 blood samples from patients with suspected poisoning of the Poison Control Center of São Paulo (PCC-SP), Brazil. Finally, the developed method has shown to be relevant for emergency toxicology due to its high sensitivity and it could be also very useful in both fields of clinical and forensic toxicology.

Blood Doping: Risks to Athletes’ Health and Strategies for Detection

Blood doping has been defined as the misuse of substances or certain techniques to optimize oxygen delivery to muscles with the aim to increase performance in sports activities. It includes blood transfusion, administration of erythropoiesis-stimulating agents or blood substitutes, and gene manipulations. The main reasons for the widespread use of blood doping include: its availability for athletes (erythropoiesis-stimulating agents and blood transfusions), its efficiency in improving performance, and its difficult detection. This article reviews and discusses the blood doping substances and methods used for in sports, the adverse effects related to this practice, and current strategies for its detection.