Development of analytical method for the determination of methylphenidate, the analog ethylphenidate and their metabolite ritalinic acid in oral fluid samples by micro-QuEChERS and liquid chromatography-tandem mass spectrometry.

The aim of this study was to develop a quantitative method for the analysis of methylphenidate, the analog ethylphenidate and their metabolite ritalinic acid in oral fluid, using micro-QuEChERS extraction and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Oral fluid samples were collected with Quantisal™ device, extracted by micro-QuEChERS technique and analyzed by LC-MS/MS. The developed method met the validation criteria of Academy Standards Board (ASB) Standard Practices for Method Validation in Forensic Toxicology (Standard 036, 2019) with limits of detection and quantification of 0.5 ng/mL and calibration curve from 0.5 to 50 ng/mL. Within-run imprecision was greater than 18.7% while between-run imprecision was greater than 17.0 % for all analytes. Bias did not vary more than 7.7 %. No evidence of carryover was found. Stability studies presented satisfactory results for 24 h on autosampler (10 °C), after 3 cycles of freeze/thaw, 7 days on freezer (-20 °C) and until 7 days on refrigerator (4 °C) for methylphenidate. The validated method was further successfully applied to the analysis of 5 authentic oral fluid samples collected from volunteers at parties and music festivals from different cities in Brazil. Four samples had positive results for methylphenidate and ritalinic acid, and only one sample was positive for methylphenidate. Ethylphenidate was not detected in the samples. The method showed acceptable analytical performance and is environmentally friendly, requiring reduced use of solvents and reagents, with potential to be applied to clinical and forensic analyses.

Toxicological analyses: analytical method validation for prevention or diagnosis.

The need for reliable results in Toxicological Analysis is recognized and required worldwide. The analytical validation ensures that a method will provide trustworthy information about a particular sample when applied in accordance with a predefined protocol, being able to determine a specific analyte at a distinct concentration range for a well-defined purpose. The driving force for developing method validation for bioanalytical projects comes from the regulatory agencies. Thus, the approach of this work is to present theoretical and practical aspects of method validation based on the analysis objective, whether for prevention or diagnosis. Although various legislative bodies accept differing interpretations of requirements for validation, the process for applying validation criteria should be adaptable for each scientific intent or analytical purpose.

Validation of an analytical method for the simultaneous determination of 16 drugs and metabolites in hair in the context of driving license granting.

The use of psychoactive substances has been associated with increased risk for traffic accidents. Hair testing has become a routine practice in clinical and forensic toxicological laboratories, with a unique perspective in the investigation of drug consumption. The study aimed to develop and validate a UHPLC-MS/MS method for the determination of multiple drugs in hair, to be used for toxicological examination in driving license granting. Sample preparation was a one-step liquid extraction of milled hair with methanol, which was incubated for 15h at 50°C. The chromatographic separation was performed in a reversed phase column, with a run time of 2.2min. Measured compounds were cocaine, benzoylecgonine, norcocaine, anhydroecgonine methyl ester, cocaethylene, amphetamine, methamphetamine, methylenedioxyamphetamine, methylenedioxymethamphetamine, fenproporex, amfepramone, mazindol, codeine, morphine, 6-monoacetylmorphine, and tetrahydrocannabinol. The assay was linear for all substances (r>0.99), accurate (86.63-105.87 %), and precise, with a cv ranging from 1.9-13.5 % for intra-assay and 3.3-14.3 % for inter-assay. There was no significant carry over effect and the internal standard corrected matrix effect was minimal. The relative uncertainty percentages were below 9% for all the substances at cut-off values. The method was successfully applied to 50 hair samples from injured drivers, with 12% of positivity, including cocaine, MDMA and THC.

Evaluation of the Use of a Coffee Industry By-Product in a Cereal-Based Extruded Food Product.

The evaluation of by-products to be added to food products is complex, as the residues must be analyzed to demonstrate their potential use as safe foods, as well as to propose the appropriate process and product for recycling. Since coffee is a very popular beverage worldwide, the coffee industry is responsible for generating large amounts of by-products, which include the coffee silverskin (CS), the only by-product of the roasting process. In this work, its characterization and food safety were evaluated by chemical composition assays, microbiological determinations, aflatoxin measurements and acute toxicity tests. The results showed that CS is safe for use in food, in addition to providing dietary fiber, protein and bioactive compounds. An extruded cereal-based ready-to-eat food product was developed through an extreme vertices mixture design, producing an extruded food product being a source of protein and with a high fiber content. Up to 15% of CS was incorporated in the extruded product. This work contributes to the establishment of routes for the valorization of CS; nevertheless, further research is necessary to demonstrate the sustainability of this food industry by-product.

Minimalist strategies applied to analysis of forensic samples using elemental and molecular analytical techniques - A review.

Forensic science is an emerging field driven by a number of factors, and the development of different methods of analyses, instruments, and techniques is of great help to experts in the field. Sampling and sample preparation in forensic cases are of utmost importance, and therefore, the methods for processing (or not) the samples are critical for acquiring accurate results. Some alternatives for attaining the minimalist concept, i.e. little or no sample treatment, are discussed in this review. For elemental analysis, analytical techniques, such as X-ray spectrometry, laser-ablation mass spectrometry, laser-induced breakdown spectrometry, inductively coupled plasma mass spectrometry and optical emission spectrometry, and Mössbauer spectrometry are overviewed. Molecular analysis, such as Raman spectroscopy, and ambient ionization mass spectrometry are discussed. Some representative examples are presented that involve in situ analysis, counterfeit bank notes and documents, post-mortem and bone analyses, and forensic analysis of drugs, glass, fingerprints, biological fluids and explosives.

Avaliação da presença de cocaína e anfetamina em amostras de sangue post mortem e de indivíduos vivos, utilizando técnica de microextração em fase líquida (HF-LPME) / Amphetamine, cocaine and tetrahydrocannabinol evaluation in blood samples of living people and post mortem blood samples using microextraction technique in liquid phase (HF-LPME)

Estima-se atualmente que mais de 5% da população mundial vem fazendo uso recreativo de algum tipo de substância psicoativa, sendo que o direito a esse uso é tema recorrente da sociedade contemporânea. Por apresentar riscos associados à saúde e a segurança das populações, o uso abusivo dessas substâncias tem instigado a toxicologia social na busca de respostas, com as quais se possa caracterizar, analisar e gerenciar esses riscos. Drogas de grande consumo no Brasil são a anfetamina, cocaína e Cannabis sativa. Esta tese desenvolveu uma nova metodologia para detectar e quantificar anfetamina, cocaína e tetrahidrocanabinol em sangue total, com uso de microextração em fase líquida via fibra de polipropileno (HF-LPME), seguida de cromatografia gasosa acoplada a espectrometria de massa (GC-MS). Trata-se de uma técnica que apresenta vantagens sobre as tradicionais, uma vez que demanda quantidades menores de solvente orgânico, diminuindo riscos e custos de processo. Também propôs um estudo com a aplicação dos métodos em 69 amostras de sangue de vivos e de post mortem, as quais foram obtidas por convênio com a superintendência da polícia técnica científica de São Paulo (SPTC/SP). Os métodos desenvolvidos foram validados de acordo com diretrizes internacionais de interesse forense. Como resultado da validação, os métodos desenvolvidos se mostraram precisos e exatos para anfetamina e cocaína. O limite de detecção da cocaína foi de 5 ng . mL-1 e o limite de quantificação de 10 ng . mL-1. Quanto a anfetamina, os limites de detecção e de quantificação foram de 5 ng . mL-1. A técnica de HF-LPME não foi aplicável ao tetraidrocanabinol (Δ9-THC). Como resultado da análise das amostras, 40% delas apresentaram resultados positivos para cocaína. Desses positivos, 35% foram oriundos das matrizes de sangue de vivos e 64% oriundos de sangue post mortem. Nenhuma delas apresentou resultado quantificável para anfetamina

It is currently estimated that more than 5% of the world's population has been doing recreational use of some kind of psychoactive substances and the legal right to such use is a recurring theme debated by contemporary society. Due to the risks associated with populations health and safety, the abusive use of these substances has been instigating by social toxicology to search for answers to characterize, analyze and manage these risks. Drugs of great consumption in Brazil are, amphetamine cocaine and marijuana. This thesis proposes to develop a new methodology to detect and quantify psychoactive drugs in whole blood with the use of liquid phase microextraction by polypropylene fiber (HFLPME), followed by gas chromatography coupled to mass spectrometry (GC-MS). It is a technique that presents advantages compared with traditional ones, because of the smaller amounts demands of organic solvent, reducing risks and process costs. It also proposes a study with 69 blood samples taken from living persons and post mortem blood samples, which were obtained by agreement with the Superintendency of São Paulo Scientific Technical Police (SPTC / SP). The methods developed were validated according to international guidelines of forensic interest. As a result of the validation, the methods developed were precise and accurate for amphetamine and cocaine. The limit of cocaine detection was 5 ng . mL-1 and the limit of quantification was 10 ng . mL-1. As for amphetamine, the limits of detection and quantification were 5 ng . mL-1. The HF-LPME technique was not applicable to tetrahydrocannabinol (Δ9-THC). As a result of the sample analysis, 40% of them presented positive results for cocaine. Of these, 35% were from blood samples taken from living persons and 64% from the post mortem blood samples. None of the samples presented quantifiable results for amphetamine

Acute and subchronic toxicity of the antitumor agent rhodium (II) citrate in Balb/c mice after intraperitoneal administration.

This study aimed to investigate potential acute and subchronic toxicity of rhodium (II) citrate in female Balb/c mice after intraperitoneal injections. In the acute test, independent groups received five doses; the highest dose (107.5 mg/kg) was equivalent to 33 times that used in our previous reports. The other doses were chosen as proportions of the highest, being 80.7 (75%), 53.8 (50%), 26.9 (25%) or 13.8 mg/kg (12.5%). Animals were monitored over 38 days and no severe signs of toxicity were observed, according to mortality, monitoring of adverse symptoms, hematological, biochemical and genotoxic parameters. We conclude that the median lethal dose (LD50) could be greater than 107.5 mg/kg. In the subchronic test, five doses of Rh2Cit (80, 60, 40, 20 or 10 mg/kg) were evaluated and injections were conducted on alternate days, totaling five applications per animal. Paclitaxel (57.5 mg/kg) and saline solution were controls. Clinical observations, histopathology of liver, lung and kidneys and effects on hematological, biochemistry and genotoxic records indicated that Rh2Cit induced no severe toxic effects, even at an accumulated dose up to 400 mg/kg.We suggest Rh2Cit has great potential as an antitumor drug without presenting acute and subchronic toxicity.

Alternative methods in toxicity testing: the current approach

Alternative methods are being developed to reduce, refine, and replace (3Rs) animals used in experiments, aimed at protecting animal welfare. The present study reports alternative tests which are based on the principles of the 3Rs and the efforts made to validate these tests. In Europe, several methodologies have already been implemented, such as tests of irritability, cell viability, and phototoxicity as well as in vitro mathematical models together with the use of in silico tools. This is a complex process that spans from development to regulatory approval and subsequent adoption by various official entities. Within this regulatory framework is REACH, the European Community Regulation for chemicals and their safe use. In Brazil, the BraCVAM (Brazilian Center for the Validation of Alternative Methods) was recently established to validate alternative methods and stimulate incorporation of new methodologies. A new vision of toxicology is emerging for the 21st century (Tox-21), and the subsequent changes are shaping a new paradigm.

Métodos alternativos estão sendo desenvolvidos para a redução, o refinamento e a substituição (3R) do número de animais utilizados em experimentos, visando ao seu bem-estar. Esses testes alternativos baseiam-se no princípio dos 3R e esforços têm sido empregados para que sejam validados. Na Europa, diversas metodologias já foram implantadas tais como: testes de irritabilidade, testes de viabilidade celular, testes de fototoxicidade e modelos matemáticos in vitro, além do uso de ferramentas in silico. Esse é um processo complexo, que abrange desde o seu desenvolvimento até a aceitação regulatória e posterior adoção por diversas organizações oficiais. No contexto regulatório está o REACH, o Regulamento da Comunidade Européia, para produtos químicos e sua utilização segura. No Brasil, o BraCVAM (Centro Brasileiro de Validação de Métodos Alternativos) foi recentemente estabelecido para validação de métodos alternativos e estímulo à incorporação de novas metodologias. Uma nova visão de toxicologia vem surgindo para o século XXI (Tox-21) e as mudanças ocasionadas promoverão um novo paradigma.

Perfil de fármacos por espectrofotometria no ultravioleta / Drugs profile by using ultraviolet spectrophotometry

Intoxicações por medicamentos alcançam o primeiro lugar entre os agentes tóxicos envolvidos em atendimento médico em serviços de urgência e emergência no Brasil, sendo muitos dos casos relacionados às tentativas de suicídio ou envolvendo crianças e idosos que se expuseram a doses acima da terapêutica, resultando em concentrações tóxicas na corrente sangüínea. O diagnóstico pode ser realizado com o auxílio de exames complementares e análises específicas para determinação do agente tóxico. A triagem de fármacos é a mais freqüente dos testes toxicológicos solicitados, sendo o objetivo deste trabalho, analisar a utilização da espectrofotometria de varredura na faixa ultravioleta para identificação de fármacos. Foram analisados os espectros de absorção ultravioleta de 40fármacos de diferentes classes químicas. Os fármacos foram submetidos à varredura na faixa do ultravioleta e identificados os comprimentos de onda nos quais apresentavam picos de absorbância máxima. O procedimento utilizado explora a baixa seletividade doultravioleta, permitindo identificar grupos de medicamentos da mesma classe, que frequentemente apresentam o mesmo padrão de absorção, e que podem estar presentes no material biológico de forma rápida e simples, diminuindo o tempo de resposta do laboratório à solicitação médica.

Drug intoxications ranked first among toxic agents involved in medical toxicological emergency interviewed in Brazil general hospitals. The main circumstances were suicidal attempts and accidental ingestion by child and old-aged using medicines above therapeutically level, thus resulting in toxic blood levels. The diagnosis can be carried through with the help of complementary examination and specific analysis for the toxic agent determination. The inquiry of drugs is the most frequent of the toxicological tests, being the objective of this work, to analyze the use of the ultraviolet spectrophotometry band for the research of drugs. There where standardized 40 available drugs, prepared in methanol and carried through the ultraviolet spectrophotometry. The drugs had been identified through wavelengths in which the drugs showed maximum absorbance peaks. The used procedure exploited the relatively low selectivity of UV, allows identifying groups of drugs closely resemble of their parent compound that can be present in urine in a fast and simple way, thus, diminishing the laboratory time of reply to the medical request.