Development of an HPLC method with electrochemical detection of femtomoles of 8...

• Autor(es): Rebelo, Isabel A. ; Piedade, José António P. ; Oliveira-Brett, Ana Maria
• Data: 2004
• Identificador Persistente: http://hdl.handle.net/10316/5133
• Origem: Estudo Geral - Universidade de Coimbra
• Assunto(s): DNA damage; Oxidative stress; 8-oxoGua; 8-oxodGuo; 8-oxo-7,8-Dihydroguanine; 8-oxo-7,8-Dihydro-2'-deoxyguanosine; Biomarker; Uric acid; HPLC-ECD
• Tipo de Documento: Artigo
• Idioma: Inglês

Descrição

A selective method based on high performance liquid chromatography with electrochemical detection (HPLC-ECD) was developed to enable simultaneous detection of 8-oxo-7,8-dihydroguanine (8-oxoGua) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), products of DNA oxidative damage, in the presence of uric acid (UA), a strong interferent in their electrochemical detection. The method developed consists of HPLC isocratic elution with amperometric detection on a glassy carbon electrode, enabling a detection limit for 8-oxoGua and 8-oxodGuo lower than 1 nM in standard mixtures. Detection of low concentrations up to 25 nM of 8-oxoGua and 8-oxodGuo in the presence of UA in a 104-fold higher concentration was achieved after one-step solid phase extraction (SPE). The method was tested with urine samples and it was possible to detect and quantify the presence of 8-oxoGua, and to confirm that UA was eliminated after uricase degradation and SPE. The LOD found in urine samples was about 80 nM, a value higher than in standard mixtures, due to the increase of background current in the urine matrix. The results presented here contribute to the development of a methodological approach to simultaneous determination of 8-oxoGua and 8-oxodGuo in urine samples. http://www.sciencedirect.com/science/article/B6THP-4BG45K9-1/1/203c0cf340998db82825488fecf36e47