Oxidative Stress Biomarker Levels of Simulated Artificial Body Fluids: Comparative HPLC-PDA and LC-MS/MS Approaches: Significance of Raw PDA and MS/MS Data for Chemometric Discrimination.

HPLC-PDA, LC-MS/MS methods were developed for simultaneous determination of a group of oxidative stress biomarkers (OSBs); 2dA, 2dC, 2dU, 3NLT, 5HMU and 8OHdG in 10 simulated artificial body fluids. O-phosphoric acid and methanol composed mobile phases A and B for gradient elution in HPLC-PDA using ODS-2 column. Linearity obtained for 1.0×10-6-1.0×10-4M range. LODs were 1.73×10-6, 1.19×10-6, 2.59×10-6, 1.40×10-6, 2.21×10-6 and 4.07×10-6M for 2dU, 8OHdG, 2dA, 2dC, 5HMU and 3NLT, respectively. LOQs were 5.29×10-6, 4.02×10-6, 6.82×10-6, 4.02×10-6, 6.82×10-6 and 9.92×10-6M. About 10 mM aqueous ammonium acetate solution and methanol containing 0.1% (v/v) formic acid composed mobile phases A and B for gradient elution in LC-MS/MS. Linearity obtained for 1.0×10-8-1.0×10-6M range. LODs were 2.88×10-10, 1.01×10-8, 3.38×10-9, 1.36×10-7, 1.81×10-7 and 1.40×10-8M for 2dU, 8OHdG, 2dA, 2dC, 5HMU and 3NLT, respectively. LOQs were 9.37×10-10, 3.22×10-8, 1.91×10-8, 4.53×10-7, 5.90×10-7 and 2.18×10-8M. Both methods were validated using ICH Q2(R1) guideline. Specificity, linearity, range, accuracy, precision, reproducibility, LOD, LOQ and recovery were achieved. Chemometric analysis was performed on raw PDA and MS data to check their significance for discrimination of OSBs. Sets of single and triple quadrupole fragmentations were evaluated for principle component analysis. Chosen number of PCs successfully distinguished OSBs of interest.