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Microextração adsortiva em barra em controlo antidopagem para a determinação qualitativa de cocaína e metabolitos em amostras de urina
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Durante os últimos anos, os laboratórios de controlo antidopagem têm registado um
aumento de resultados positivos para a classe dos estimulantes, destacando-se a cocaína como
uma das substâncias com maior incidência. A cocaína forma dois metabolitos principais, no corpo
humano, a ecgonina metil éster e a benzoilecgonina, bem como o cocaetileno aquando do
consumo conjunto de cocaína e álcool.
No presente trabalho foi desenvolvido um novo método analítico para identificar
qualitativamente a cocaína, ecgonina metil éster, cocaetileno e benzoilecgonina em urina humana.
Esta metodologia consiste na aplicação da microextração adsortiva em barra (BAμE), onde um
pequeno dispositivo analítico revestido com uma fase sorvente adequada, operando em modo de
flutuação e seguido de micro dessorção líquida (μLD), permite a extração dos analitos para
posterior análise por cromatografia gasosa acoplada à espetrometria de massa.
Na otimização do método BAμE-μLD e, após testar diferentes materiais sorventes, o
polímero P3 (Divinilbenzeno) foi o que apresentou os melhores resultados na extração dos
analitos em estudo. Após estudos sistemáticos de parâmetros suscetíveis de afetar a microextração
e retroextração das substâncias em estudo, verificou-se que o pH da matriz é determinante na
eficiência analítica da metodologia.
A validação da metodologia de acordo com critérios impostos pela Agência Mundial
Antidopagem, do inglês World Anti-Doping Agency (WADA), mostrou boa seletividade,
capacidade de identificação, limites de identificação (LOI) adequados (1 ng/mL a 10 ng/mL),
recuperações entre 39,4% e 71,1%, robustez, ausência de fenómenos de arrastamento e
estabilidade dos analitos no extrato final, à temperatura ambiente, até 144 horas após preparação.
O método provou, ainda, ser linear (0,9950 < r2 < 0,9994) na gama testada e com bons resultados
de exatidão, repetibilidade e precisão intermédia. A aplicação da presente metodologia a amostras
previamente analisadas pelo laboratório, revelou excelente concordância entre os resultados
reportados e os obtidos.
Em suma, a metodologia desenvolvida demostrou ser fit-for-purpose e, assim, uma forte
alternativa aos métodos utilizados em rotina.
Over the last years, anti-doping control laboratories have registered an increase of positive results for the class of stimulants, with cocaine presenting the highest incidence. Cocaine metabolizes, in the human body, into ecgonine methyl ester and benzoylecgonine, but also cocaethylene which results from the intake of cocaine together with alcohol. In the present work, it was developed a new analytical method to identify qualitatively cocaine, ecgonine methyl ester, cocaethylene and benzoylecgonine in human urine. This methodology uses bar adsorptive microextraction (BAμE), where a small analytical device coated with a suitable sorbent phase, operating in the floating mode and followed by liquid micro desorption (μLD), allows the analytes extraction for further gas chromatography-mass spectrometry analysis. During the optimization of BAμE-μLD and after testing different sorbent materials, the polymer P3 (Divinylbenzene) evidenced the best results for the extraction of the analytes in study. After systematic studies of the parameters which can influence the microextraction and liquid desorption of the substances of interest, it was observed that the matrix pH, strongly affects the analytical efficiency of the methodology. The methodology BAμE(P3)-μLD/GC-MS(SIM), validated according with World AntiDoping Agency (WADA) requirements, showed good selectivity, identification capacity, suitable identification limits (LOI) (1 ng/mL to 10 ng/mL), recoveries between 39,4% and 71,1%, robustness, carryover absence, as well as stability of the analytes in the final extract, at room temperature, up to 144 hours after extraction. The method still proved to be linear (0,9950 < r2 < 0,9994) in the tested range and presented good accuracy, repeatability and intermediate precision. The application of the present methodology to samples previously analyzed by the laboratory, showed excellent agreement between the reported and the obtained results. Therefore, the methodology developed showed to be fit-for-purpose and thus a strong alternative to the routine used methods.
Over the last years, anti-doping control laboratories have registered an increase of positive results for the class of stimulants, with cocaine presenting the highest incidence. Cocaine metabolizes, in the human body, into ecgonine methyl ester and benzoylecgonine, but also cocaethylene which results from the intake of cocaine together with alcohol. In the present work, it was developed a new analytical method to identify qualitatively cocaine, ecgonine methyl ester, cocaethylene and benzoylecgonine in human urine. This methodology uses bar adsorptive microextraction (BAμE), where a small analytical device coated with a suitable sorbent phase, operating in the floating mode and followed by liquid micro desorption (μLD), allows the analytes extraction for further gas chromatography-mass spectrometry analysis. During the optimization of BAμE-μLD and after testing different sorbent materials, the polymer P3 (Divinylbenzene) evidenced the best results for the extraction of the analytes in study. After systematic studies of the parameters which can influence the microextraction and liquid desorption of the substances of interest, it was observed that the matrix pH, strongly affects the analytical efficiency of the methodology. The methodology BAμE(P3)-μLD/GC-MS(SIM), validated according with World AntiDoping Agency (WADA) requirements, showed good selectivity, identification capacity, suitable identification limits (LOI) (1 ng/mL to 10 ng/mL), recoveries between 39,4% and 71,1%, robustness, carryover absence, as well as stability of the analytes in the final extract, at room temperature, up to 144 hours after extraction. The method still proved to be linear (0,9950 < r2 < 0,9994) in the tested range and presented good accuracy, repeatability and intermediate precision. The application of the present methodology to samples previously analyzed by the laboratory, showed excellent agreement between the reported and the obtained results. Therefore, the methodology developed showed to be fit-for-purpose and thus a strong alternative to the routine used methods.
Descrição
Tese de Mestrado, Química, 2022, Universidade de Lisboa, Faculdade de Ciências
Palavras-chave
Microextração adsortiva em barra (BAµE) Dopagem Cocaína GC-MS Urina Teses de mestrado - 2023