Isocratic and gradient HPLC method development and analytical method validation studies for the simultaneous determination of A group of oxidative stress biomarkers

Studies are ongoing for decades for the determination and monitoring of oxidative stress, as one of the basic reasons of various pathophysiological phenomenons. Detection and quantification of oxidative stress biomarkers, as measurable analytical parameters, in low quantities in matrix environments is an important topic of the recent years. During our thesis studies methods were developed for the simultaneous liquid chromatographic determination of a group of oxidative stress biomarkers and analytical method validation studies of the developed methods were performed in accordance to the ICH Q2(R1) validation guidance. The biomarkers determined were 8-hydroxy-2'-deoxyguanosine (8OHdG); 2'-deoksiuridin (2dU); 2'-deoxyadenosine (2dA); 2'-deoxycytidine (2dC); 5-hydroxymethyluracil (5HMU) and 3-nitrotyrosine (3NLT). The 5 different developed HPLC methods were HPLC-DAD methods in gradient conditions, assays were performed at 40OC coulumn temperature, in 0.3-0.7 mL/min flow rate range, by recording DAD spectrum in the range 200-450nm, by using C18 Thermo Hypersil Gold, 100x2,1mm, 3µm as the analytical column, mobile phases (MP) as; MPA: methanol and MPB: 10mM H3PO4 (pH 2.5), at 20µL loop volume and at 5OC rack temperature of the autosampler. Each developed method has its unique and optimized gradient programme. A solid phase extraction (SPE) method was also developed by using AccuBOND II ODS-C18 SPE cartidges for the quantifications done in the artificial body fluids prepared by spking desired amount of standards of the studied biomarkers. Keywords: 8-hydroxy-2'-deoxyguanosine (8OHdG); 2'-deoxyuridine (2dU); 2'-deoxyadenosine (2dA); 2'-deoxycytidine (2dC); 5-hydroxymethyluracil (5HMU);HPLC;LC-MS/MS;LC-MS.