Determination of thiocyanate in exhaled breath condensate

Thiocyanate is a heme peroxidase substrate that scavenges oxidants produced during inflammation and regulates host defense. In cystic fibrosis (CF) patients, increased airway thiocyanate levels are associated with improved lung function. Research on airway thiocyanate is limited, however, because convenient non-invasive airway sampling methods, such as exhaled breath condensate (EBC), yield low concentrations that are difficult to detect with available assays. In the present study, we developed a method for the determination of thiocyanate in dilute samples using isotope dilution headspace gas chromatography-coupled high-resolution, accurate-mass mass spectrometry (GC-HRMS). The method reliably quantified as little as 4 pmol thiocyanate in EBC and could detect even lower amounts. We successfully measured thiocyanate in EBC from seven healthy donors, with a mean +/- SD of 27 +/- 16 nM and a median inter-assay coefficient of variation of 10.4% over six months. The method was applied to other biological fluids (plasma from the same visit as EBC donation; bronchoalveolar lavage fluid [BALF] from infants with CF; and healthy adult mouse BALF), giving reliable quantification of samples ranging from 10 nM to 100 mu M. Thiocyanate concentrations in fluids besides EBC were (from lowest to highest): 0.73 +/- 0.39 mu M in BALF of healthy adult mice (n= 6); 1.4 +/- 1.4 mu M in BALF from infants with CF (n= 24); 46 +/- 22 mu M in the plasma of adult volunteers (n= 7). These results demonstrate the utility of this new method for clinical determination of thiocyanate in EBC and other biological fluids.