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  • Quantification of oxidative stress biomarkers: development of a method by ultra performance liquid chromatography
    Publication . Rodrigues, Liliana da Silva; Araújo, Helena Caldeira; Câmara, José de Sousa
    Thiol and purine compounds are widely distributed in nature. They are involved in physiological processes, such as, homeostasis and redox signalling, and disturbances of these functions are the basis of many human diseases. Therefore, there has been a great effort to better understand the metabolomics of these compounds. The goal of the present work was to optimize an Ultra-High Performance Liquid Chromatography method for simultaneous detection and quantification of some thiol, and purine compounds in cells lysates and biological fluids. For this purpose, an UPLC® system, equipped with an HSS T3 column, with fluorescence and photodiode array detection was used. Preliminary experiences to optimize chromatographic separation of standard compounds were accomplished and involved testing of conditions for sample pre-analytical treatments, mobile phase compositions and detection conditions. A good resolution and separation was obtained for the standards tested. The thiols Glutathione, Cysteine and Homocysteine eluted at 0.655  0.005, 2.189  0.000 and 3.752  0.001 minutes. Intra-day precision of the method was 12.05, 9.87 and 9.06% for areas. Farther, inter-day precision for the retention times were 29.49, 1.63 and 2.24% and for areas were 41.55, 28.28 and 29.46%, respectively. Purines Adenosine and Inosine eluted at 2.249  0.001 and 2.584  0.005 minutes. Inter-day precision for the retention times were 0.06 and 0.01% and for areas were 6.99 and 6.63%, respectively. Linearity was tested in a range of concentrations from 5 to 100 M for thiols and 25 to 500 M for purines, with good results. Additionally, the detection and quantification limits were too high. Unfortunately, our analyses do not shown intra or inter-day precision. Therefore, the validation was not completed because reproducibility was inconsistent due to the mechanical failure of the chromatographic equipment used. However, we concluded that the identification of these analytes is possible with the methods established.
    2016-11-23Master thesis Open access Show more