A Micro-Extraction Technique Using a New Digitally Controlled Syringe Combined with UHPLC for Assessment of Urinary Biomarkers of Oxidatively Damaged DNAReportar como inadecuado




A Micro-Extraction Technique Using a New Digitally Controlled Syringe Combined with UHPLC for Assessment of Urinary Biomarkers of Oxidatively Damaged DNA - Descarga este documento en PDF. Documentación en PDF para descargar gratis. Disponible también para leer online.

The formation of reactive oxygen species ROS within cells causes damage to biomolecules, including membrane lipids, DNA, proteins and sugars. An important type of oxidative damage is DNA base hydroxylation which leads to the formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine 8-oxodG and 5-hydroxymethyluracil 5-HMUra. Measurement of these biomarkers in urine is challenging, due to the low levels of the analytes and the matrix complexity. In order to simultaneously quantify 8-oxodG and 5-HMUra in human urine, a new, reliable and powerful strategy was optimised and validated. It is based on a semi-automatic microextraction by packed sorbent MEPS technique, using a new digitally controlled syringe eVol®, to enhance the extraction efficiency of the target metabolites, followed by a fast and sensitive ultrahigh pressure liquid chromatography UHPLC. The optimal methodological conditions involve loading of 250 µL urine sample 1∶10 dilution through a C8 sorbent in a MEPS syringe placed in the semi-automatic eVol® syringe followed by elution using 90 µL of 20% methanol in 0.01% formic acid solution. The obtained extract is directly analysed in the UHPLC system using a binary mobile phase composed of aqueous 0.1% formic acid and methanol in the isocratic elution mode 3.5 min total analysis time. The method was validated in terms of selectivity, linearity, limit of detection LOD, limit of quantification LOQ, extraction yield, accuracy, precision and matrix effect. Satisfactory results were obtained in terms of linearity r2 > 0.991 within the established concentration range. The LOD varied from 0.00005 to 0.04 µg mL−1 and the LOQ from 0.00023 to 0.13 µg mL−1. The extraction yields were between 80.1 and 82.2 %, while inter-day precision n = 3 days varied between 4.9 and 7.7 % and intra-day precision between 1.0 and 8.3 %. This approach presents as main advantages the ability to easily collect and store urine samples for further processing and the high sensitivity, reproducibility, and robustness of eVol®MEPS combined with UHPLC analysis, thus retrieving a fast and reliable assessment of oxidatively damaged DNA.



Autor: Berta Mendes, Pedro Silva, Fernando Aveiro, Jorge Pereira, José S. Câmara

Fuente: http://plos.srce.hr/



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