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

Abstract

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.

Figure 1

The effect of pH on purine and urea signals was determined using purine and urea standards diluted to a final concentration of 10 μM of ATP, AMP, and adenosine, and 500 μM urea in ammonium formate or ammonium acetate buffers equilibrated to various pH. A volume of 5 μL of each standard solution was injected into the mass spectrometer and the area of the resulting peak determined, with each sample measured in triplicate. Signals for all compounds were detected, but the ATP signal was weaker and more pH dependent that those from other purines or urea. All data in this figure and others are presented as mean ± standard deviation.

Figure 2

LC/MS/MS analysis of analyte and isotopically labeled internal standards of urea, adenosine, and AMP. Standards were run on a 2 × 150 mm C18 column using 0.1% formic acid and methanol gradients, and mass spectrometric detection using SRM was performed as described in the text. Retention times for urea, AMP, and adenosine were 1.8, 2.6, and 3.2 min, respectively, and were identical for analyte and isotopically labeled standards.

Figure 3

Standard curves for detection of urea, adenosine, and AMP were determined using LC/MS/MS. Detection was linear over four orders of magnitude, with r² values as indicated. Concentrations of the injected analyte standards are given; nM for adenosine and AMP and μM for urea.

Figure 4

Concentrations of urea, adenosine, and AMP were measured in EBC from three healthy controls and seven children with CF. (A) Urea, adenosine, and AMP levels were not different between groups, although values for urea in particular were variable. Note that the measured values are after lyophilization and reconstitution, and are 20-fold higher than in native EBC. (B) Using purine to urea ratios to control for dilution revealed higher AMP/urea levels in CF than in control, consistent with expectations.