A new and fast methodology to assess oxidative damage in cardiovascular diseases risk development through eVol-MEPS–UHPLC analysis of four urinary biomarkers Berta Mendes a,b , Pedro Silva a,b , Isabel Mendonça c , Jorge Pereira a,b , José S. Câmara a,b,n a CQM—Centro de Química da Madeira, Universidade da Madeira, Campus Universitário da Penteada, 9000-390 Funchal, Portugal b Centro Competências de Ciências Exactas e da Engenharia, Universidade da Madeira, Campus Universitário da Penteada, 9000-390 Funchal, Portugal c Research Unit and Cardiology Department, Dr. Nélio Mendonça Hospital, Avda. Luís de Camões, no. 57, 9050-514 Funchal, Portugal article info Article history: Received 17 January 2013 Received in revised form 16 April 2013 Accepted 24 April 2013 Available online 3 May 2013 Keywords: Urinary metabolomic profile Oxidative stress biomarkers (OSBs) Microextraction by packed sorbent (MEPS) Ultra high pressure liquid chromatography (UHPLC) Cardiovascular diseases (CVDs) abstract In this work, a new, fast and reliable methodology using a digitally controlled microextraction by packed sorbent (eVol s -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 mL of 0.01% formic acid and 3  50 mL 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 mm in particle size) using 0.01% formic acid 20% methanol at 250 mL 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 40.9906), as well as the LOD and LOQ, whose values were low, ranging from 0.00005 to 0.72 mg mL -1 and 0.00023 to 2.31 mg 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 s -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. & 2013 Elsevier B.V. All rights reserved. 1. Introduction Cardiovascular diseases (CVDs) are the leading cause of death in humans. According to World Health Organization statistical data, 17.3 million people died from CVDs in 2008 and it is estimated that by 2030, this number will reach approximately 23.6 million deaths, mainly from heart disease and stroke [1]. There are several risk factors contributing for the CVDs develop- ment, but accumulating evidences points to the influence of oxidative stress damage, also known as oxidative damage, in key aspects of several disorders as cancer, different neurodegenerative diseases and particularly different CVDs [2–5]. Oxidative damage is produced whenever the antioxidant defenses are unable to cope with the deleterious effects caused by high levels of reactive oxygen species (ROS). These highly instable molecules can easily react and damage several cellular constituents, namely nucleic acids, proteins, and lipids [4,6]. This has driven several studies exploring the use of dietary antioxidants against oxidative damage effects and particularly in CVDs, the effect of antioxidants on Contents lists available at SciVerse ScienceDirect journal homepage: www.elsevier.com/locate/talanta Talanta 0039-9140/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.talanta.2013.04.064 n Corresponding author at: Centro Competências de Ciências Exactas e da Engenharia, Universidade da Madeira, Campus Universitário da Penteada, 9000- 390 Funchal, Portugal. Tel.: +351 291705112; fax: +351 291705149. E-mail address: jsc@uma.pt (J.S. Câmara). Talanta 116 (2013) 164–172