A UPLC-MS/MS method for the determination of oxidative stress biomarkers in amniotic fluid

Oxidative stress in the fetal period is associated with preterm birth as well as short and long-term adverse clinical outcomes. Here, an Ultra-Performance Liquid Chromatography-tandem Mass Spectrometry (UPLC-MS/MS) method for the simultaneous quantification of biomarkers of oxidative stress-derived damage to proteins and DNA in amniotic fluid (AF) samples is presented. Appropriate accuracy and precision levels, as well as sensitivity with limits of detection in the low nanomolar (<2 nM) range were achieved. The analytical method was applied to a set of AF samples and reference ranges of the biomarker panel are presented. Median concentrations of biomarkers of protein oxidation (ortho-, 3-chloro-, and 3-nitrotyrosine) and their precursors (para-tyrosine and phenylalanine) ranged between 0.6 and 3 nM and 23 and 30 μM, respectively, while levels of a biomarker of DNA-oxidation (8-hydroxydeoxyguanosine, 8OHdG) and its precursor (2'-deoxyguanosine) were found to be 0.18 and 3 nM, respectively. Detection frequencies of all metabolites were 100% with exception of 3-chlorotyrosine (3Cl-Tyr) and 8OHdG, that were only detected in 8% of samples. The developed method may be applied in research studies focusing on oxidative stress-related complications during pregnancy.