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Determinação do teor de canabinoides a partir de material vegetal por cromatografia líquida acoplada a um detetor de matriz de foto-díodos
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Authors
Abstract(s)
A importância da análise das plantas de canábis tem vindo crescer, nomeadamente para controlo de qualidade dentro dos produtos para uso recreacional, indústria medicinal, ou ainda para discriminação forense entre a tipologia de substância de abuso ou cânhamo. No presente estudo foi desenvolvido um método com recurso a cromatografia líquida de alta eficiência com deteção por fotodíodos (HPLC-DAD), destinado à análise quantitativa dos canabinoides principais da planta de canábis: Δ9 -tetrahidrocanabinol (Δ9 -THC) e canabidiol (CBD), respetivas formas ácidas, canabinol (CBN) e o Δ8 - tetrahidrocanabinol Δ8 -THC, em material vegetal da canábis.
A etapa de extração foi otimizada com recurso a desenhos experimentais, permitindo uma recuperação ~100% dos canabinoides. As amostras herbáceas foram extraídas com etanol:acetonitrilo (50:50), com recurso a ultrassons, e posterior deteção e quantificação dos canabinoides em HPLC-DAD, sendo apenas necessário utilizar 50 mg de amostra. Para os óleos da planta foi aplicada uma simples diluição em etanol.
A separação cromatográfica foi conseguida numa coluna de fase reversa, utilizando como eluente uma mistura de acetonitrilo/água contendo 0,1% ácido fórmico. Os compostos foram quantificados a 230 nm, em 30 minutos de corrida.
Os ensaios de validação do método mostraram que esta era linear (R2>0,997), sendo seletivo e sensível, com a precisão e veracidade adequados, bem como excelente estabilidade e repetibilidade entre análise de amostra real. Os principais canabinoides identificados (% p/p) em amostras reais foram o Δ9-THCA, CBD, CBDA, e o Δ9-THC.
A presente metodologia foi aplicada com sucesso a várias amostras reais ricas em THC ou CBD, demonstrando a sua eficácia em análises de rotina para permitir dar resposta à atual procura de serviços de análise desta planta e seus derivados.
The importance of the analysis of cannabis plants has been developing globally, namely for quality control within products for recreational use, medicinal industry, or even for forensic discrimination between the typology of substance of abuse or hemp, for industrial purposes. In the present study, a HPLC-DAD method was developed for the quantitative analysis for the main cannabinoids: Δ9-THC and CBD, their acid forms, CBN and Δ8 -THC, in cannabis plant material. Extraction was optimized using experimental design (DoE), granting ~100% recovery of the cannabinoids. Herbal samples were extracted with ethanol:acetonitrile (50:50) by ultrasonication, and subsequent detection and quantification of cannabinoids in HPLC-DAD, only requiring 50 mg of sample. For the plant oils a simple dilution in ethanol was necessary. Chromatographic separation was achieved on a reverse-phase C18 column, using an acetonitrile/water mixture containing 0.1% formic acid as eluent. The compounds were quantified at 230 nm in 30 minutes of run time. Method validation proved its linearity (R2>0.997), being selective and sensitive, with excellent results for precision and trueness, as well as excellent stability and repeatability. The main cannabinoids identified (% w/w) in actual samples were Δ9-THCA, CBD, CBDA, and Δ9-THC. The presented method was successfully applied to real samples, demonstrating its suitability in routine analyses to assist current demand for analysis services of this plant and its derivatives.
The importance of the analysis of cannabis plants has been developing globally, namely for quality control within products for recreational use, medicinal industry, or even for forensic discrimination between the typology of substance of abuse or hemp, for industrial purposes. In the present study, a HPLC-DAD method was developed for the quantitative analysis for the main cannabinoids: Δ9-THC and CBD, their acid forms, CBN and Δ8 -THC, in cannabis plant material. Extraction was optimized using experimental design (DoE), granting ~100% recovery of the cannabinoids. Herbal samples were extracted with ethanol:acetonitrile (50:50) by ultrasonication, and subsequent detection and quantification of cannabinoids in HPLC-DAD, only requiring 50 mg of sample. For the plant oils a simple dilution in ethanol was necessary. Chromatographic separation was achieved on a reverse-phase C18 column, using an acetonitrile/water mixture containing 0.1% formic acid as eluent. The compounds were quantified at 230 nm in 30 minutes of run time. Method validation proved its linearity (R2>0.997), being selective and sensitive, with excellent results for precision and trueness, as well as excellent stability and repeatability. The main cannabinoids identified (% w/w) in actual samples were Δ9-THCA, CBD, CBDA, and Δ9-THC. The presented method was successfully applied to real samples, demonstrating its suitability in routine analyses to assist current demand for analysis services of this plant and its derivatives.
Description
Dissertação para obtenção do grau de Mestre no Instituto Universitário Egas Moniz
Keywords
Canábis Fitocanabinoides Quantificação HPLC-DAD THC Desenho experimental Otimização Matrizes reais