A mass spectrometric method to simultaneously measure a biomarker and dilution marker in exhaled breath condensate

Exhaled breath condensate (EBC) collection is a simple and non-invasive method to sample airway secretions, but analysis is limited by extensive and variable dilution of airway secretions within the condensate. To overcome this limitation, we developed a sensitive and specific liquid chromatography/tandem mass spectrometry (LC/MS/MS) method to simultaneously detect adenyl purines as biomarkers of inflammation and urea as a dilution marker in EBC. Separation prior to mass spectrometry was achieved using a C18 column with methanol and formic acid as the mobile phase, and characteristic precursor to product ion transitions of m/z 268 to 136 (for adenosine), m/z 348 to 136 (for AMP), and m/z 61 to 44 (for urea) were monitored for quantification. To correct for matrix effects, isotopically labeled adenosine, AMP, and urea were used as internal standards. Using these methods, we detected urea and the adenyl purines adenosine and AMP in EBC from seven subjects with cystic fibrosis (CF) and seven healthy controls and found that the AMP/urea ratio was elevated in the CF samples. These results demonstrate that mass spectrometry can be used successfully in EBC analysis to simultaneously detect a biomarker for airway inflammation and control for variable dilution.