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Tears biofluid screening of potential biomarkers for health clinical diagnosis
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Resumo(s)
A investigação das lágrimas, juntamente com a medicina preventiva, o diagnóstico clínico e a espectrometria de massa em tandem, são uma fronteira essencial na evolução do desenvolvimento de terapias personalizadas. A recolha de lágrimas é um procedimento não invasivo que oferece vantagens significativas para a identificação de biomarcadores de doenças.
O objetivo deste estudo foi identificar e quantificar compostos presentes nas lágrimas
utilizando cromatografia líquida com deteção por espectrometria de massa em “tandem” (LC-MS/MS). Neste contexto foi desenvolvida uma metodologia analítica que consistiu em duas vertentes, amostragem e deteção. Foram selecionados onze biomarcadores e foram recolhidas amostras reais de vinte e quatro voluntários saudáveis do sexo feminino e masculino com idades entre os 18 e 28 anos, utilizando o método de amostragem com fitas de Schirmer para avaliação da composição destes biomarcadores nas lágrimas.
O desenvolvimento das condições experimentais envolveu a otimização de parâmetros
cruciais, como a escolha do solvente e da coluna de cromatografia. Foram selecionadas a mistura metanol/H2O (50%/50%) e a coluna C18. As amostras em branco foram utilizadas para
otimizar o processo de extração das lágrimas, permitindo identificar o método de extração
mais adequado para minimizar as interferências durante as análises LC-MS/MS.
A validação do método incluiu a avaliação dos parâmetros analíticos. A linearidade, o
limite de deteção (LOD) e o limite de quantificação (LOQ) foram validados, com todos os compostos mostrando um R2 > 0.9900. A precisão esteve dentro dos critérios de aceitação (coeficiente de variação < 15%), enquanto o rendimento esteve dentro do intervalo analítico de referência (70% a 110%), demonstrando que os resultados analíticos não necessitam de correção.
A análise das amostras de lágrimas dos voluntários confirmou a presença dos seguintes
compostos: Acetilcolina (ACh), Epinefrina (E), Norepinefrina (NE), Serotonina (5-HT), Ácido
Glutâmico (Glu), Tirosina (Tyr), Triptofano (Trp), Ácido Ascórbico (AsA), Ácido Láctico (Lac)
e Cortisol (Cort). Foi possível identificar a Dopamina (DA) na forma dos seus metabolitos,
ácido 3,4-dihidroxifenilacético (DOPAC) e 3-metoxitiramina (3-MT). A análise estatística demonstrou uma correlação significativa entre os compostos ACh/AsA e Trp/Tyr, com valores de p < 0.05.
Em conclusão, este estudo contribui para o avanço da pesquisa de biomarcadores nas
lágrimas através de LC-MS/MS, abrindo caminho para o desenvolvimento de novas abordagens na deteção precoce e no tratamento de doenças, sendo uma contribuição para o diagnóstico clínico.
Tear research, together with preventive medicine, clinical diagnostics and tandem mass spectrometry, is an essential frontier in the evolution of the development of personalised therapies. Tear collection is a non-invasive procedure that offers significant advantages for identifying disease biomarkers. The aim of this study was to identify and quantify compounds present in tears using liquid chromatography with tandem mass spectrometry detection (LC-MS/MS). In this context, an analytical methodology was developed which consisted of two parts: sampling and detection. Eleven biomarkers were selected and real samples were taken from twenty-four healthy male and female volunteers aged between 18 and 28, using the Schirmer strip sampling method to assess the composition of these biomarkers in tears. The development of the experimental conditions involved the optimisation of crucial parameters, such as the choice of the solvent and chromatography column. The MeOH/H2O mixture (50%/50%) and the C18 column were selected. The blank samples were used to optimise the tear extraction process, allowing the most suitable extraction method to be identified in order to minimise the interference during LC-MS/MS analysis. The validation of the method included evaluation of the analytical parameters. Linearity, limit of detection (LOD) and limit of quantification (LOQ) were validated, with all compounds showing an R2 > 0.9900. Precision was within the acceptance criteria (coefficient of variation <15%), while recovery was within the analytical reference range (70% to 110%), demonstrating that the analytical results do not require correction. Analysis of the volunteers' tear samples confirmed the presence of the following compounds Acetylcholine (ACh), Epinephrine (E), Norepinephrine (NE), Serotonin (5-HT), Glutamic acid (Glu), Tyrosine (Tyr), Tryptophan (Trp), Ascorbic Acid (AsA), Lactic Acid (Lac) and Cortisol (Cort). Dopamine (DA) was identified in the form of its metabolites, 3,4-dihydroxy-phenylacetic acid (DOPAC) and 3-methoxytyramine (3-MT). Statistical analysis showed a significant correlation between the ACh/AsA and Trp/Tyr compounds, with p-values < 0.05. In conclusion, this study contributes to the advancement of the search for biomarkers in tears using the LC-MS/MS, paving the way for the development of new approaches to the early detection and treatment of diseases, as well as contributing to clinical diagnosis.
Tear research, together with preventive medicine, clinical diagnostics and tandem mass spectrometry, is an essential frontier in the evolution of the development of personalised therapies. Tear collection is a non-invasive procedure that offers significant advantages for identifying disease biomarkers. The aim of this study was to identify and quantify compounds present in tears using liquid chromatography with tandem mass spectrometry detection (LC-MS/MS). In this context, an analytical methodology was developed which consisted of two parts: sampling and detection. Eleven biomarkers were selected and real samples were taken from twenty-four healthy male and female volunteers aged between 18 and 28, using the Schirmer strip sampling method to assess the composition of these biomarkers in tears. The development of the experimental conditions involved the optimisation of crucial parameters, such as the choice of the solvent and chromatography column. The MeOH/H2O mixture (50%/50%) and the C18 column were selected. The blank samples were used to optimise the tear extraction process, allowing the most suitable extraction method to be identified in order to minimise the interference during LC-MS/MS analysis. The validation of the method included evaluation of the analytical parameters. Linearity, limit of detection (LOD) and limit of quantification (LOQ) were validated, with all compounds showing an R2 > 0.9900. Precision was within the acceptance criteria (coefficient of variation <15%), while recovery was within the analytical reference range (70% to 110%), demonstrating that the analytical results do not require correction. Analysis of the volunteers' tear samples confirmed the presence of the following compounds Acetylcholine (ACh), Epinephrine (E), Norepinephrine (NE), Serotonin (5-HT), Glutamic acid (Glu), Tyrosine (Tyr), Tryptophan (Trp), Ascorbic Acid (AsA), Lactic Acid (Lac) and Cortisol (Cort). Dopamine (DA) was identified in the form of its metabolites, 3,4-dihydroxy-phenylacetic acid (DOPAC) and 3-methoxytyramine (3-MT). Statistical analysis showed a significant correlation between the ACh/AsA and Trp/Tyr compounds, with p-values < 0.05. In conclusion, this study contributes to the advancement of the search for biomarkers in tears using the LC-MS/MS, paving the way for the development of new approaches to the early detection and treatment of diseases, as well as contributing to clinical diagnosis.
Descrição
Palavras-chave
Tears biomarkers LC-MS/MS clinical diagnosis