Mauricio Yonamine

Solid-phase micro-extraction-gas chromatography-mass spectrometry and headspace-gas chromatography of tetrahydrocannabinol, amphetamine, methamphetamine, cocaine and ethanol in saliva samples

In the present work, a method was developed aiming at the serial detection of tetrahydrocannabinol (THC), amphetamine, methamphetamine, cocaine and ethanol in saliva. Saliva samples were submitted to an initial headspace procedure for ethanol determination by gas chromatography/flame ionization detector (GC-FID). After this step, two consecutive solid-phase micro-extractions (SPME) were carried out: THC was extracted by submersing a polydimethylsiloxane fiber (100 micro m) in the vial for 20 min; amphetamine, methamphetamine and cocaine were subsequently extracted after alkalinization. Derivatization of the amphetamines was carried out directly in the solution by adding 2 micro l of butylchloroformate. Gas chromatography-mass spectrometry (GC-MS) was used to identify the analytes in selected ion monitoring (SIM) mode. Confidence parameters of validation of the method were: recovery, linearity, intra- and inter-assay precision as well as limits of detection and quantification of the analytes. The limits of quantification (LOQ) obtained were: ethanol (0.010 g/l); amphetamine (5.0 ng/ml); methamphetamine (0.5 ng/ml); cocaine (5 ng/ml) and THC (5 ng/ml). The method proved to be highly precise (coefficient of variation<8%) for all detected substances.

Amphetamine, cocaine and cannabinoids use among truck drivers on the roads in the State of Sao Paulo, Brazil

Drugs are important risk factors for traffic accidents. In Brazil, truck drivers report using amphetamines to maintain their extensive work schedule and stay awake. These drugs can be obtained without prescription easily on Brazilian roads. The use of these stimulants can result in health problems and can be associated with traffic accidents. There are Brazilian studies that show that drivers use drugs. However, these studies are questionnaire-based and do not always reflect real-life situations. The purpose of this study was to demonstrate the prevalence of drug use by truck drivers on the roads of Sao Paulo State, Brazil, during 2009. Drivers of large trucks were randomly stopped by police officers on the interstate roads during morning hours. After being informed of the goals of the study, the drivers gave written informed consent before providing a urine sample. In addition, a questionnaire concerning sociodemographic characteristics and health information was administered. Urine samples were screened for amphetamines, cocaine, and cannabinoids by immunoassay and the confirmation was performed using gas chromatography-mass spectrometry (GC-MS). Of the 488 drivers stopped, 456 (93.4%) provided urine samples, and 9.3% of them (n=42) tested positive for drugs. Amphetamines were the most commonly found (n=26) drug, representing 61.9% of the positive samples. Ten cases tested positive for cocaine (23.8%), and five for cannabinoids (11.9%). All drivers were male with a mean age of 40 ± 10.8 years, and 29.3% of them reported some health problem (diabetes, high blood pressure and/or stress). A high incidence of truck drivers who tested positive for drug use was found, among other reported health problems. Thus, there is an evident need to promote a healthier lifestyle among professional drivers and a need for preventive measures aimed at controlling the use of drugs by truck drivers in Brazil.

Environmental modulation of ethanol-induced locomotor activity : Correlation with neuronal activity in distinct brain regions of adolescent and adult Swiss mice

Drug abuse is a concerning health problem in adults and has been recognized as a major problem in adolescents. Induction of immediate-early genes (IEG), such as c-Fos or Egr-1, is used to identify brain areas that become activated in response to various stimuli, including addictive drugs. It is known that the environment can alter the response to drugs of abuse. Accordingly, environmental cues may trigger drug-seeking behavior when the drug is repeatedly administered in a given environment. The goal of this study was first to examine for age differences in context-dependent sensitization and then evaluate IEG expression in different brain regions. For this, groups of mice received i.p. ethanol (2.0 g/kg) or saline in the test apparatus, while other groups received the solutions in the home cage, for 15 days. One week after this treatment phase, mice were challenged with ethanol injection. Acutely, ethanol increased both locomotor activity and IEG expression in different brain regions, indistinctly, in adolescent and adult mice. However, adults exhibited a typical context-dependent behavioral sensitization following repeated ethanol treatment, while adolescent mice presented gradually smaller locomotion across treatment, when ethanol was administered in a paired regimen with environment. Conversely, ethanol-treated adolescents expressed context-independent behavioral sensitization. Overall, repeated ethanol administration desensitized IEG expression in both adolescent and adult mice, but this effect was greatest in the nucleus accumbens and prefrontal cortex of adolescents treated in the context-dependent paradigm. These results suggest developmental differences in the sensitivity to the conditioned and unconditioned locomotor effects of ethanol.

Marijuana as Doping in Sports

A high incidence of positive cases for cannabinoids, in analyses for doping control in sports, has been observed since the International Olympic Committee (IOC) included them in the 1989 list of prohibited drugs under the title of classes of prohibited substances in certain circumstances. Where the rules of sports federations so provide, tests are conducted for marijuana, hashish or any other cannabis product exposure by means of urinalysis of 11-nor-delta-9-tetrahydro-cannabinol-9-carboxylic acid (carboxy-THC) the main metabolite of delta-9-tetrahydrocannabinol (THC). Concentrations >15 ng/mL (cut-off value) in confirmatory analytical procedures are considered doping. Cannabis is an illicit drug in several countries and has received much attention in the media for its potential therapeutic uses and the efforts to legalise its use.Studies have demonstrated that the use of cannabinoids can reduce anxiety, but it does not have ergogenic potential in sports activities. An increase in heart rate and blood pressure, decline of cardiac output and reduced psychomotor activity are some of the pharmacological effects of THC that will determine a decrease in athletic performance. An ergolytic activity of cannabis products has been observed in athletes of several different sport categories. In Brazil, analyses for doping control in sports, performed in our laboratories, have detected positive cases for carboxy-THC in urine samples of soccer, volleyball, cycling and other athletes.It is our intention to discuss in this article some points that may discourage individuals from using cannabis products during sports activities, even in the so-called permitted circumstances defined by the IOC and some sports federations.

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.

Drug Use by Truck Drivers in Brazil

In Brazil, those who are suspected of driving under the influence of drugs are tested only for ethanol. Professional drivers, especially truck drivers, use stimulant drugs to prevent sleeping during long-distance driving. Surveys on the patterns of use of illicit drugs in the workplace have rarely been conducted in Brazil, in spite of the high costs and the potential risk to public health. Since 1996, the authors have been compiling the results of tests, performed in their laboratories, for drugs in urine samples from truck drivers. The drugs analyzed were: amphetamine, methamphetamine, cannabinoids and cocaine. Urine samples (728) were collected in three out of the five geographical regions of Brazil: southeast (517 samples), northeast (161 samples) and south (50 samples). Fluorescence polarization immunoassay and capillary gas chromatography/mass spectrometry were utilized for the urinalyses. The results obtained were as follows: 41 samples (5.63% of the total) tested positive for the drugs being studied. The frequency of positivity of samples was quite similar for the three regions: 6% in the south, 6% in the southeast and 4.35% in the northeast. However, distribution of the drugs in the samples showed regional variations. Results such as those that we have obtained can provide an estimation of the extent of drug use by truck drivers in Brazil.

Environmental enrichment blocks ethanol‐induced locomotor sensitization and decreases BDNF levels in the prefrontal cortex in mice

The use of addictive drugs can lead to long‐term neuroplastic changes in the brain, including behavioral sensitization, a phenomenon related to addiction. Environmental enrichment (EE) is a strategy used to study the effect of environment on the response to several manipulations, including treatment with addictive drugs. Brain‐derived neurotrophic factor (BDNF) has been associated with behaviors related to ethanol addiction. The aim of the present study was to evaluate the effects of EE on ethanol‐induced behavioral sensitization and BDNF expression. Mice were exposed to EE and then repeatedly treated with a low dose (1.8 g/kg) of ethanol. Another group of mice was first subjected to repeated ethanol treatment according to the behavioral sensitization protocol and then exposed to EE. Environmental enrichment prevented the development of ethanol‐induced behavioral sensitization and blocked behavioral sensitization in sensitized mice. Both repeated ethanol and EE decreased BDNF levels in the prefrontal cortex but not in the hippocampus. However, BDNF levels were lower in ethanol‐treated mice exposed to EE. These findings suggest that EE can act on the mechanisms implicated in behavioral sensitization, a model for drug‐induced neuroplasticity and relapse. Additionally, EE alters BDNF levels, which regulate addiction‐related behaviors.

1H NMR determination of β-N-methylamino-L-alanine (L-BMAA) in environmental and biological samples.

A nuclear magnetic resonance (1H NMR) method for the determination of beta-N-methylamino-L-alanine (L-BMAA) in environmental aqueous samples was developed and validated. L-BMAA is a neurotoxic modified amino acid that can be produced by cyanobacteria in aqueous environments. This toxin was extracted from samples by means of solid-phase extraction (SPE) and identified and quantified by 1H NMR without further derivatization steps. The lower limit of quantification (LLOQ) was 5 microg/mL. Good inter and intra-assay precision was also observed (relative standard deviation <8.5%) with the use of 4-nitro-DL-phenylalanine as an internal standard (IS). This method of 1H NMR analysis is not time consuming and can be readily utilized to monitor L-BMAA and confirm its presence in environmental and biological samples.

Determination of low levels of benzodiazepines and their metabolites in urine by hollow-fiber liquid-phase microextraction (LPME) and gas chromatography–mass spectrometry (GC–MS)

In this study, it is shown a method for the determination of benzodiazepines and their main metabolites in urine samples by hollow-fiber liquid-phase microextraction (LPME) in the three-phase mode. Initially, the hydrolysis step was performed using 100 μL of sodium acetate 2.0 mol/L buffer solution (pH 4.5), 25 μL of β-glucuronidase enzyme and incubation for 90 min at 55 °C. In parallel with hydrolysis, the LPME fiber (9 cm) was prepared. Its pores were filled with a mixture of dihexyl ether: 1-nonanol (9:1). Afterwards, a solution of 3.0 mol/L of HCl was introduced into the lumen of the fiber (acceptor phase). After hydrolysis, the fiber was submersed in the alkalinized urine (pH 10) containing 10% NaCl. Samples were then submitted to orbital shaking (2400 rpm) for 90 min. The acceptor phase was later withdrawn from the fiber, dried and the residue derivatized with trifluoroacetic anhydride (TFAA) for 10 min at 60 °C with further addition of N-methyl-N-tert-butyldimethylsilyltrifluoroacetamide containing 1% tert-butyldimethylchlorosilane (MTBSTFA) for 45 min at 90 °C followed by determination by gas chromatography-mass spectrometry (GC-MS). The calibration curves obtained showed linearity over the specified range, with a similar sensitivity to traditional techniques and a higher detection capability compared to most of the miniaturized methods described in the literature. The method has been developed and successfully validated and applied to urine samples from real cases of benzodiazepines intake.

Analysis of cocaine and its adulterants in drugs for international trafficking seized by the Brazilian Federal Police.

Here, gas chromatography with nitrogen phosphorous detector (GC-NPD) method was developed and validated for the quantification of cocaine and adulterants (caffeine, 4-dimethylaminoantipyrine, levamisole, lidocaine and phenacetin) in illicit samples. The method was based on direct dilution of samples in methanol, sonication for 5 min and centrifugation. After appropriate dilution, an aliquot was injected into GC-MS in order to identify the active compounds and into GC-NPD for the analytes quantification. Bupivacaine was used as an internal standard. The method showed to be precise, accurate and linear over a range of 0.5-100% (weight/weight percentages) for all analytes, except phenacetin which showed a linear range between 2% and 100%. The method was successfully applied to 54 samples seized by the Brazilian Federal Police in the International Airport of Sao Paulo and mailing services during the year 2011. All the samples were associated with international trafficking and were apprehended while leaving the country. The purity of cocaine ranged from 16.5% to 91.4%. Cocaine was the only detected active compound in 29.6% of total samples. Among the identified cutting agents, levamisole was the most abundant (55.6% of the total samples) and relative concentrations (weight/weight percentages) ranged from 0.7% to 23%. Lidocaine, caffeine, phenacetin and 4-dimethylaminoantipyrine were also identified in these samples in minor concentrations. In contrast with what we initially hypothesized, drugs intended to international trafficking did not present high cocaine purity and most of the samples were laced with adulterants before leaving Brazil.