Abstract
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.
Highlights
Oxidative stress results from an imbalance between the generation of reactive oxygen species (ROS) and antioxidant defences
The selection of the best sorbent was based on extraction efficiency, determined by the relative peak area, and reproducibility
The lowest extraction efficiency was obtained by using M1 and SIL sorbents, respectively
Summary
Oxidative stress results from an imbalance between the generation of reactive oxygen species (ROS) and antioxidant defences. Oxidative stress is known to cause damage to biomolecules, namely membrane lipids, DNA, proteins and sugars, a condition known as oxidative damage This damage is harmful to DNA when there are mutations in tumour suppressor genes that are not corrected, triggering critical initial events in carcinogenesis [3]. Over one hundred of such oxidatively modified DNA forms have been characterised in vitro [5], but only about 20 identified in vivo [4] and found to be involved in the induction of signal transduction pathways, replication errors and genomic instability and transcription induction or arrest [6] These results are strongly supported by, for example, the high levels of oxidative lesions in cancer tissues, and reduced cancer incidence in populations with high dietary antioxidant intake [7].
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