title: Validation of a sensitive LC/MSMS method for chloronucleoside analysis in biological matrixes and its applications.
abstract: Myeloperoxidase promotes several kinds of damage and is involved in the development of various diseases (as atherosclerosis and cancers). An example of these damage is the chlorination of nucleic acids, which is considered as a specific marker of the MPO activity on those acids. This study aimed to develop and validate a method to analyze oxidized and MPO-specific chlorinated nucleosides in biological matrixes (cells, tissues and plasma). Although a lot of methods to quantify oxidized or chlorinated nucleosides have already been established, none of them took into account all these derivatives together. The new method used a Triple Quadrupole mass spectrometer fitted with a Jet Stream electrospray ionization source. This approach has two advantages compared with existing LC/MSMS analyses: it includes MPO-induced modifications in a unique analysis and obtains a better sensitivity. Our optimized method reached LOQs of 1.50pg and 1.42pg respectively for oxoG and oxo(d)G, being 4 times more sensitive than previous methods, and LOQs of 1.39pg, 1.30pg and 63.4 fg respectively for 5-chlorocytidine, 5-chloro-2'-deoxycytidine and 8-chloroguanosine. Developed method is also 25 times more sensitive for chloroguanosine than the best existing method. Nevertheless, this method is not specific enough for 8-chloro-(2'-deoxy)adenosine analysis. Examples of applications demonstrate the interest of this validated method. Indeed analysis of plasma from healthy donors highlighted exclusively the presence of 5-chlorocytidine (1.0±0.2nM) whereas analysis of treated endothelial cells by HOCl showed chlorination of guanosine and cytidine in cytoplasmic pools and chlorination of (deoxy)cytidine in DNA and RNA. In conclusion, this study shows that 5-chloro-2'-deoxycytidine, 5-chlorocytidine and 8-chloroguanosine are good markers allowing us to detect the MPO activity in biological fluids. The robust, specific and sensitive developed method enables future studies on MPO implications in human diseases.
authors.affiliation: Laboratory of Organic Pharmaceutical Chemistry, Faculty of Pharmacy, Université Libre de Bruxelles, Campus Plaine CP205/5, Boulevard du Triomphe, B-1050 Bruxelles, Belgium. Electronic address: cnoyon@ulb.ac.be.
authors.forename: Caroline
authors.initials: C
authors.lastname: Noyon
authors.affiliation: Laboratory of Organic Pharmaceutical Chemistry, Faculty of Pharmacy, Université Libre de Bruxelles, Campus Plaine CP205/5, Boulevard du Triomphe, B-1050 Bruxelles, Belgium; Analytical Platform of the Faculty of Pharmacy, Université Libre de Bruxelles, Campus Plaine CP205/5, Boulevard du Triomphe, B-1050 Bruxelles, Belgium. Electronic address: cedric.delporte@ulb.ac.be.
authors.forename: Cédric
authors.initials: C
authors.lastname: Delporte
authors.affiliation: Laboratory of Organic Pharmaceutical Chemistry, Faculty of Pharmacy, Université Libre de Bruxelles, Campus Plaine CP205/5, Boulevard du Triomphe, B-1050 Bruxelles, Belgium. Electronic address: damien.dufour@ulb.ac.be.
authors.forename: Damien
authors.initials: D
authors.lastname: Dufour
authors.affiliation: Laboratory of Organic Pharmaceutical Chemistry, Faculty of Pharmacy, Université Libre de Bruxelles, Campus Plaine CP205/5, Boulevard du Triomphe, B-1050 Bruxelles, Belgium; Analytical Platform of the Faculty of Pharmacy, Université Libre de Bruxelles, Campus Plaine CP205/5, Boulevard du Triomphe, B-1050 Bruxelles, Belgium. Electronic address: mecortes@ulb.ac.be.
authors.forename: Melissa
authors.initials: M
authors.lastname: Cortese
authors.affiliation: Laboratory of Experimental Medicine, CHU de Charleroi, A. Vésale Hospital, Université Libre de Bruxelles, Rue de Gozée 706, B-6110 Montigny-le-Tilleul, Belgium. Electronic address: Alexandre.ROUSSEAU@chu-charleroi.be.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Rousseau
authors.affiliation: Laboratory of Inflammation Molecular Biology (Laboratory of Molecular Parasitology), IBMM, Faculty of Sciences, Université Libre de Bruxelles, Rue des Prof. Jeener et Brachet 12, B-6041 Gosselies, Belgium. Electronic address: ppoelvoo@ulb.ac.be.
authors.forename: Philippe
authors.initials: P
authors.lastname: Poelvoorde
authors.affiliation: Laboratory of Organic Pharmaceutical Chemistry, Faculty of Pharmacy, Université Libre de Bruxelles, Campus Plaine CP205/5, Boulevard du Triomphe, B-1050 Bruxelles, Belgium. Electronic address: jneve@ulb.ac.be.
authors.forename: Jean
authors.initials: J
authors.lastname: Nève
authors.affiliation: Laboratory of Inflammation Molecular Biology (Laboratory of Molecular Parasitology), IBMM, Faculty of Sciences, Université Libre de Bruxelles, Rue des Prof. Jeener et Brachet 12, B-6041 Gosselies, Belgium. Electronic address: luc.vanhamme@ulb.ac.be.
authors.forename: Luc
authors.initials: L
authors.lastname: Vanhamme
authors.affiliation: Laboratory of Experimental Medicine, CHU de Charleroi, A. Vésale Hospital, Université Libre de Bruxelles, Rue de Gozée 706, B-6110 Montigny-le-Tilleul, Belgium. Electronic address: karim.zouaoui@chu-charleroi.be.
authors.forename: Karim
authors.initials: K
authors.lastname: Zouaoui Boudjeltia
authors.affiliation: Department of Urology, Erasme Hospital, Université Libre de Bruxelles, Route de Lennik 808, B-1070 Brussels, Belgium. Electronic address: Thierry.Roumeguere@erasme.ulb.ac.be.
authors.forename: Thierry
authors.initials: T
authors.lastname: Roumeguère
authors.affiliation: Laboratory of Organic Pharmaceutical Chemistry, Faculty of Pharmacy, Université Libre de Bruxelles, Campus Plaine CP205/5, Boulevard du Triomphe, B-1050 Bruxelles, Belgium; Analytical Platform of the Faculty of Pharmacy, Université Libre de Bruxelles, Campus Plaine CP205/5, Boulevard du Triomphe, B-1050 Bruxelles, Belgium. Electronic address: pvantwer@ulb.ac.be.
authors.forename: Pierre
authors.initials: P
authors.lastname: Van Antwerpen
mesh_terms: D002853:Chromatography, Liquid; D003841:Deoxycytidine / Q000031:analogs & derivatives; D006151:Guanosine / Q000031:analogs & derivatives; D009195:Peroxidase; D053719:Tandem Mass Spectrometry
chemical_list: D003841:Deoxycytidine; D006151:Guanosine; C043808:8-chloroguanosine; C035826:5-chloro-2'-deoxycytidine; D009195:Peroxidase
keywords: 5-chloro-(2′-deoxy)cytidine; 8-chloroguanosine; DNA; LC/MSMS; RNA; myeloperoxidase