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.

Estudo da estabilidade de novas substâncias psicoativas (NPS) em amostras de sangue seco em papel (DBS), e sua aplicação em toxicologia forense / Stability of new psychoactive substances (NPS) in dried blood spots (DBS), and its application in forensic toxicology

Resumo: Introdução: A classe de drogas de abuso conhecida como novas substâncias psicoativas ou, ainda, drogas desenhadas (do inglês new psychoactive substances (NPS) ou designer drugs) é composta por substâncias químicas obtidas a partir de alterações estruturais de substâncias ou mistura de substâncias psicoativas já existentes (e ilícitas), a fim de mimetizar e/ou potencializar os efeitos proporcionados por estas, com a vantagem de circundar a legislação antidrogas vigente. Dentro da classe das NPS, os grupos de maior destaque atualmente são das catinonas sintéticas, dos canabinóides sintéticos e dos NBOMes (feniletilaminas N-2-metoxibenzil substituídas). As catinonas sintéticas, vendidas pela internet como "sais de banho" ou bath salts, são substâncias estruturalmente relacionadas a catinona, um alcalóide presente no Catha edulis (Khat), com propriedades estimulantes. A classe dos NBOMes é derivada da classe dos alucinógenos 2C, a partir da adição de um grupo N-2-metoxibenzil na amina primária. Os NBOMes possuem ação agonista nos receptores de serotonina, especialmente do subtipo 5-HT2A, o que confere os efeitos alucinógenos. Objetivos: Desenvolver e validar métodos para identificação e quantificação de algumas das NPS de maior relevância em amostras de manchas de sangue seco em papel (do inglês dried blood spots, DBS) e comparar a estabilidade entre dois tipos de amostras (DBS e sangue total), em três diferentes temperaturas (ambiente, 4 °C e -20 °C) e períodos de armazenamento. Metodologia: As amostras de DBS e sangue total foram analisadas em cromatógrafo líquido acoplado à espectrômetro de massas com analisador de massas triplo quadrupolar (LC-MS/MS), utilizando os métodos primeiramente desenvolvidos e validados para cada classe de NPS (catinonas sintéticas e NBOMes). Resultados: A técnica de DBS apresentou um aumento significativo da estabilidade das NPS em comparação à técnica convencional de armazenamento de sangue total. Para os NBOMes, observou-se que os compostos eram estáveis no DBS por período de tempo de até 6 meses, nas três temperaturas estudadas, enquanto que no sangue total, os analitos tiveram uma queda maior que 20% da concentração em 15 ou 30 dias em temperatura ambiente e 180 dias para 4 °C. Para as catinonas, em temperatura ambiente, observou-se baixa estabilidade nas duas matrizes. Porém, para armazenamento à 4 °C, observou-se uma queda na concentração inicial maior que 20% com 60 e 90 dias para mefedrona e benzedrona em DBS, respetivamente, contra 45 e 25 dias. Catinonas que possuem grupamento metilenodioxi em sua estrutura, como a butilona e a pentilona, foram mais estáveis, independente da matriz, em comparação àquelas com grupamento alquila no anel aromático, como mefedrona e benzedrona. Conclusão: A técnica de DBS se mostrou vantajosa para análises forenses em comparação à técnica convencional de armazenamento de sangue total. Além de facilitar o armazenamento (por ocupar menos espaço), sua extração se torna mais rápida e facilitada, já que envolve menos etapas, além de tornar possível a identificação dos analitos de interesse por um maior período de tempo, podendo facilmente ser aplicada na rotina laboratorial(AU)

Abstract: Introduction: New psychoactive substances (NPS) or designer drugs is a class of drugs of abuse composed of chemical substances obtained from structural alterations of substances or mixture of existing psychoactive substances (and illicit), in order to mimic and/or maximize their effects, with the advantage of circumventing existing anti-drug legislation. Within the NPS class, the most prominent groups currently are synthetic cathinones, synthetic cannabinoids and NBOMes (phenylethylamines N-2-methoxybenzyl substituted). Synthetic cathinones, sold by the internet as "bath salts", are substances structurally related to cathinone, an alkaloid present in Catha edulis (Khat), with stimulant properties. The class of NBOMes is derived from the hallucinogen class 2C, from the addition of an N-2-methoxybenzyl group to the primary amine. NBOMes have agonist action at serotonin receptors, especially the 5-HT2A subtype, which confers the hallucinogenic effects. Objectives: To develop and validate methods to identify and quantify some of the most relevant NPS in dried blood spots (DBS) samples and to compare the stability between two matrixes (DBS and whole blood), at three different temperatures (ambient, 4 °C and -20 °C) and storage periods. Method: DBS and whole blood samples were analyzed using a liquid chromatography tandem mass spectrometer with a triple quadrupole analyzer (LC-MS/MS), using the developed and validated methods for each class of NPS (synthetic cathinones and NBOMes). Results: The DBS technique showed a significant increase in the NPS stability compared to the conventional whole blood storage technique. For the NBOMes, the compounds were found to be stable in DBS for a time period of up to 6 months, at the three temperatures studied, whereas in whole blood, the analytes had a decreased higher than 20% of the initial concentration in 15 or 30 days at room temperature and 180 days at 4 °C. For the cathinones, at room temperature, low stability was observed in both matrixes. However, for storage at 4 °C, the initial concentration decreased more than 20% at 60 and 90 days for mephedrone and benzedrone in DBS, respectively, against 45 and 25 days. Cathinones having methylenedioxy group in their structure, such as butylone and pentylone, were more stable, independent of the matrix, compared to those with alkyl group on the aromatic ring, such as mephedrone and benzedrone. Conclusion: The DBS technique proved to be advantageous for forensic analysis compared to the conventional storage technique. In addition to occupying less storage space, DBS extraction becomes faster and easier, since it involves fewer steps, besides to make possible to identify the analytes of interest for a longer period of time, which can easily be applied in the laboratory routine(AU)