title: A new and fast methodology to assess oxidative damage in cardiovascular diseases risk development through eVol-MEPS-UHPLC analysis of four urinary biomarkers.
abstract: In this work, a new, fast and reliable methodology using a digitally controlled microextraction by packed sorbent (eVol(®)-MEPS) followed by ultra-high pressure liquid chromatography (UHPLC) analysis with photodiodes (PDA) detection, was developed to establish the urinary profile levels of four putative oxidative stress biomarkers (OSBs) in healthy subjects and patients evidencing cardiovascular diseases (CVDs). This data was used to verify the suitability of the selected OSBs (uric acid-UAc, malondialdehyde-MDA, 5-(hydroxymethyl)uracil-5-HMUra and 8-hydroxy-2'-deoxyguanosine-8-oxodG) as potential biomarkers of CVDs progression. Important parameters affecting the efficiency of the extraction process were optimized, particularly stationary phase selection, pH influence, sample volume, number of extraction cycles and washing and elution volumes. The experimental conditions that allowed the best extraction efficiency, expressed in terms of total area of the target analytes and data reproducibility, includes a 10 times dilution and pH adjustment of the urine samples to 6.0, followed by a gradient elution through the C8 adsorbent with 5 times 50 µL of 0.01% formic acid and 3×50 µL of 20% methanol in 0.01% formic acid. The chromatographic separation of the target analytes was performed with a HSS T3 column (100 mm × 2.1 mm, 1.7 µm in particle size) using 0.01% formic acid 20% methanol at 250 µL min(-1). The methodology was validated in terms of selectivity, linearity, instrumental limit of detection (LOD), method limit of quantification (LOQ), matrix effect, accuracy and precision (intra-and inter-day). Good results were obtained in terms of selectivity and linearity (r(2)>0.9906), as well as the LOD and LOQ, whose values were low, ranging from 0.00005 to 0.72 µg mL(-1) and 0.00023 to 2.31 µg mL(-1), respectively. The recovery results (91.1-123.0%), intra-day (1.0-8.3%), inter-day precision (4.6-6.3%) and the matrix effect (60.1-110.3%) of eVol(®)-MEPS/UHPLC-PDA method were also very satisfactory. Finally, the application of the methodology to the determination of target biomarkers in normal subjects and CVDs patients' revealed that the DNA adducts 5-HMUra and 8-oxodG levels are much more abundant in CVDs patients while no statistic differences were obtain for MDA and UAc. This result points to the importance of 5-HMUra and 8-oxodG as biomarkers of CVDs risk progression and further epidemiological studies are needed to explore the importance of this correlation.
authors.affiliation: CQM-Centro de Química da Madeira, Universidade da Madeira, Campus Universitário da Penteada, 9000-390 Funchal, Portugal; Centro Competências de Ciências Exactas e da Engenharia, Universidade da Madeira, Campus Universitário da Penteada, 9000-390 Funchal, Portugal.
authors.forename: Berta
authors.initials: B
authors.lastname: Mendes
authors.forename: Pedro
authors.initials: P
authors.lastname: Silva
authors.forename: Isabel
authors.initials: I
authors.lastname: Mendonça
authors.forename: Jorge
authors.initials: J
authors.lastname: Pereira
authors.forename: José S
authors.initials: JS
authors.lastname: Câmara
mesh_terms: D000080242:8-Hydroxy-2'-Deoxyguanosine; D000328:Adult; D015415:Biomarkers / Q000652:urine; D002138:Calibration; D016022:Case-Control Studies; D002851:Chromatography, High Pressure Liquid / Q000379:methods* / Q000592:standards; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000652:urine; D018450:Disease Progression; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006937:Hypercholesterolemia / Q000175:diagnosis / Q000652:urine*; D006973:Hypertension / Q000175:diagnosis / Q000652:urine*; D057230:Limit of Detection; D008297:Male; D008315:Malondialdehyde / Q000652:urine; D008875:Middle Aged; D018384:Oxidative Stress; D010432:Pentoxyl / Q000031:analogs & derivatives* / Q000652:urine; D015203:Reproducibility of Results; D012307:Risk Factors; D052617:Solid Phase Microextraction; D014527:Uric Acid / Q000652:urine
chemical_list: D015415:Biomarkers; D014527:Uric Acid; C008055:5-hydroxymethyluracil; D008315:Malondialdehyde; D010432:Pentoxyl; D000080242:8-Hydroxy-2'-Deoxyguanosine; D003849:Deoxyguanosine
keywords: Cardiovascular diseases (CVDs); Microextraction by packed sorbent (MEPS); Oxidative stress biomarkers (OSBs); Ultra high pressure liquid chromatography (UHPLC); Urinary metabolomic profile