Quantitative determination of potential urine biomarkers of respiratory illnesses using new targeted metabolomic approach

Western Economic Diversification Canada, AllerGen NCE, Saskatchewan Health Research FoundationPeer ReviewedThe diagnosis of asthma and chronic obstructive pulmonary disease (COPD) can be challenging due to the overlap in their clinical presentations in some patients. There is a need for a more objective clinical test that can be routinely used in primary care settings. Through an untargeted 1H NMR urine metabolomic approach, we identified a set of endogenous metabolites as potential biomarkers for the differentiation of asthma and COPD. A subset of these potential biomarkers contains 7 highly polar metabolites of diverse physicochemical properties. To the best of our knowledge, there is no liquid chromatography-tandem mass spectrometry (LC-MS/MS) method that evaluated more than two of the target metabolites in a single analytical run. The target metabolites belong to the families of monosaccharides, organic acids, amino acids, quaternary ammonium compounds and nucleic acids, rendering hydrophilic interaction liquid chromatography (HILIC) an ideal technology for their quantification. Since a clinical decision is to be made from patients data, a fully validated analytical method is required for biomarker validation. Method validation for endogenous metabolites is a daunting task since current guidelines were designed for exogenous compounds. As such, innovative approaches were adopted to meet the validation requirements. Herein, we describe a sensitive HILIC-MS/MS method for the quantification of the 7 endogenous urinary metabolites. Detection was achieved in the multiple reaction monitoring (MRM) mode with polarity switching, using quadrupole-linear ion trap instrument (QTRAP 6500) as well as single ion monitoring in the negative-ion mode. The method was fully validated according to the regulatory guidelines. Linearity was established between 6 and 21000 ng/mL and quality control samples demonstrated acceptable intra- and inter-day accuracy (85.7%-112%), intra- and inter-day precision (CV%