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01.12.2017 | Research | Ausgabe 1/2017 Open Access

Respiratory Research 1/2017

Determining the presence of asthma-related molecules and salivary contamination in exhaled breath condensate

Respiratory Research > Ausgabe 1/2017
Charmion Cruickshank-Quinn, Michael Armstrong, Roger Powell, Joe Gomez, Marc Elie, Nichole Reisdorph
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​s12931-017-0538-5) contains supplementary material, which is available to authorized users.



Researchers investigating lung diseases, such as asthma, have questioned whether certain compounds previously reported in exhaled breath condensate (EBC) originate from saliva contamination. Moreover, despite its increasing use in ‘omics profiling studies, the constituents of EBC remain largely uncharacterized. The present study aims to define the usefulness of EBC in investigating lung disease by comparing EBC, saliva, and saliva-contaminated EBC using targeted and untargeted mass spectrometry and the potential of metabolite loss from adsorption to EBC sample collection tubes.


Liquid chromatography mass spectrometry (LC-MS) was used to analyze samples from 133 individuals from three different cohorts. Levels of amino acids and eicosanoids, two classes of molecules previously reported in EBC and saliva, were measured using targeted LC-MS. Cohort 1 was used to examine contamination of EBC by saliva. Samples from Cohort 1 consisted of clean EBC, saliva-contaminated EBC, and clean saliva from 13 healthy volunteers; samples were analyzed using untargeted LC-MS. Cohort 2 was used to compare eicosanoid levels from matched EBC and saliva collected from 107 asthmatic subjects. Samples were analyzed using both targeted and untargeted LC-MS. Cohort 3 samples consisted of clean-EBC collected from 13 subjects, including smokers and non-smokers, and were used to independently confirm findings; samples were analyzed using targeted LC-MS, untargeted LC-MS, and proteomics. In addition to human samples, an in-house developed nebulizing system was used to determine the potential for EBC samples to be contaminated by saliva.


Out of the 400 metabolites detected in both EBC and saliva, 77 were specific to EBC; however, EBC samples were concentrated 20-fold to achieve this level of sensitivity. Amino acid concentrations ranged from 196 pg/mL – 4 μg/mL (clean EBC), 1.98 ng/mL – 6 μg/mL (saliva-contaminated EBC), and 13.84 ng/mL – 1256 mg/mL (saliva). Eicosanoid concentration ranges were an order of magnitude lower; 10 pg/mL – 76.5 ng/mL (clean EBC), 10 pg/mL – 898 ng/mL (saliva-contaminated EBC), and 2.54 ng/mL – 272.9 mg/mL (saliva). Although the sample size of the replication cohort (Cohort 3) did not allow for statistical comparisons, two proteins and 19 eicosanoids were detected in smoker vs. non-smoker clean-EBC.


We conclude that metabolites are present and detectable in EBC using LC-MS; however, a large starting volume of sample is required.
Additional file 1: Targeted mass spectrometry parameters for eicosanoid analysis. MRM parameters, retention times, associated internal standards, and ionization modes used for lipid mediators by LC-MS/MS. IS: internal standard. (PDF 23 kb)
Additional file 2: Peak areas of selected amino acids and eicosanoids detected in EBC and/or saliva samples. (A) Peak areas of selected eicosanoids using untargeted metabolomics; (B) Peak areas of selected amino acids in spiked water (green), clean-EBC (black), saliva-EBC (blue), and saliva (red) using untargeted metabolomics. A control water sample which was spiked with known concentrations of amino acids and eicosanoids was used to confirm the identities of these compounds in the saliva and EBC samples using exact mass, isotope ratios and retention time. (C) Peak area of LTB4 in internal standard, EBC and saliva using targeted analysis. (D) Peak area of LTE4 in internal standard and EBC using targeted analysis. Peak areas were extracted using MassHunter Quantitative Analysis software (Agilent). y-axis: mass spectral counts; x-axis: retention time. Starting volumes for untargeted metabolomics was 11.5 mL (clean-EBC) and 7.5 mL (saliva-EBC) with final volume of 20 μL and injection volume of 5 μL. Starting volume for targeted analysis was 1 mL saliva or EBC with an injection volume of 100 μL. (TIF 1669 kb)
Additional file 3: Separation of PGF2α isomers in spiked control water. Samples were injected onto an SB-AQ analytical column. Since the four isomers could not be differentiated using untargeted analysis, multiple reaction monitoring (MRM) using a triple quadrupole mass spectrometer (QQQ-MS) with a C18 column was used to determine their elution order. (TIF 710 kb)
Additional file 4: Molecular formula annotated metabolites and unannotated metabolites detected in exhaled breath condensate (EBC). These 37 out of 77 unique compounds were not matched to a database compound. Samples were analyzed in positive and negative ionization mode using LC-MS untargeted metabolomics on an SB-AQ analytical column. + indicates detected in positive ionization mode, - indicates detected in negative ionization mode. (PDF 38 kb)
Additional file 5: Tandem MS fragmentation patterns for six EBC metabolites. The mass spectral fragment peaks in red indicate the experimental results. The peaks in blue indicate the database matches based on standards. (PDF 36 kb)
Additional file 6: Putatively identified eicosanoids in EBC. 13 mL of pooled EBC from 107 asthmatic subjects was lyophilized, reconstituted in 20 μL of buffer, and analyzed using LC-MS based metabolomics. Metabolite peaks were extracted using Profinder and MassHunter software using exact mass and isotope ratios (Agilent). Detected peaks are indicated by single colored lines. Database isotope pattern and distribution is indicated by a circled red box. Matches with multiple adducts are indicated. (PDF 55 kb)
Additional file 7: Metabolite annotations and tandem MS fragmentation patterns of compound detected in EBC. EBC was collected from four groups of volunteers: healthy smokers, healthy non-smokers, non-smokers with nasal congestion, and non-smokers with the common cold. Samples were pooled, lyophilized, reconstituted in 20 μL of buffer, and analyzed using LC-MS based metabolomics. Metabolite peaks were extracted with Mass Hunter Profinder software (Agilent) using exact mass and isotope ratios. Tandem MS was performed, spectra was exported to NIST MS Search v2.2, and matched to the NIST14 Mass Spectral library. Fragments in red indicate EBC sample, fragments in blue indicate NIST standard reference spectra. (PDF 545 kb)
Additional file 8: Targeted eicosanoid analysis of EBC. EBC was collected from four groups of volunteers: healthy smokers, healthy non-smokers, non-smokers with nasal congestion, and non-smokers with the common cold. Samples were pooled, lyophilized, reconstituted in LC-MS buffer, and analyzed using targeted LC-MS on a triple quadruple mass spectrometer. (PDF 40 kb)
Additional file 9: Pathway analysis based on sample type. The compounds which were detected in each sample type were mapped to KEGG pathways using the online freeware pathway analysis software MBROLE. The compound names were based on database annotations using exact mass, isotope ratios and/or MSMS. Only pathways with hits ≥ 2 are listed. (PDF 278 kb)
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