Internal Standard Addition System for Online Breath Analysis

Breath analysis with secondary electrospray ionization (SESI) coupled to mass spectrometry (MS) is a sensitive method for breath metabolomics. To enable quantitative assessments using SESI-MS, a system was developed to introduce controlled amounts of gases into breath samples and carry out standard addition experiments. The system combines gas standard generation through controlled evaporation, humidification, breath dilution, and standard injection with the help of mass-flow controllers. The system can also dilute breath, which affects the signal of the detected components. This response can be used to filter out contaminating compounds in an untargeted metabolomics workflow. The system’s quantitative capabilities have been shown through standard addition of pyridine and butyric acid into breath in real time. This system can improve the quality and robustness of breath data.


Table S1 Acquisition Parameters S3
Table S2 m/z values of the standards S4 Table S3 Henry's constants S5 Table S4 Gas concentrations S6 Table S5 Fit parameters of linear regression of the butyric acid standard addition without the last point

S7
Table S6 Fit parameters of linear regression of the butyric acid standard addition including the last point

S8
Table S7 Fit parameters of linear regression of the pyridine standard addition without the last point

S9
Table S8 Fit parameters of linear regression of the pyridine standard addition including the last point S10 Table S9 Butyric acid calibration curve fits S11 Table S10 Pyridine calibration curve fits S12     If a feature's intensity does not fall with increasing dilution, it can be removed.This step does not consider features, which could increase in intensity due to reduced ion suppression (Figure 2).
Figure S1Schematics of the standard addition system S13 Figure S2 Feature intensity clustering with the individual features visible

Figure S1 .
Figure S1.a) Representation of Figure 1a with selected distances inserted.The distances are in mm.The tubing is omitted for clarity.b) Different side-way views of the system.

Figure S2 .
Figure S2.Feature traces (red) and their reaction to dilution are plotted within their respective cluster.The median of all traces within a cluster is depicted as a black line.One feature trace corresponds to the average trace of one subject.

Figure S3 .
Figure S3.Example feature filtering workflow exploiting the dilution capabilities of the system.The first step involves the removal of features that possess higher intensities during the nitrogen flow set at 6 Lmin -1 .This nitrogen flow is seen as the baseline.The second step involves the removal of features whose intensities behave atypically with increasing dilution.

Figure S4 .
Figure S4.Standard addition curves recorded for pyridine and butyric acid with three subjects.The data points with the highest concentration were excluded from the linear fit depicted in Figure 3 due to the outliers observed.

Figure S5 .
Figure S5.a) Slopes of the standard addition curves according to subject.b) Calibration curve slopes obtained on different days.The slopes are given with their uncertainty.

Figure S6 .
Figure S6.Boxplot of the obtained slopes of the standard addition and calibration curves.The median values are depicted as well as the 25 th and 75 th with the interquartile ranges and outliers.While for pyridine the slope values between calibration and standard addition differ to a slight degree, the values for butyric acid differ heavily.

Table S1 .
Chosen window sizes for the acquisition with the Orbitrap Q-Exactive.For positive and negative ion mode measurements for the breath dilution experiments, the windows were chosen according to literature.1

Table S2 .
Exact and detected m/z values of the utilized gas standards.

Table S3 .
Henry's constants 2 used for the used standards and the concentrations of the solutions injected into the evaporation chambers.For the deuterated analoga, the same constants as the non-deuterated compounds were taken.

Table S4 .
Gas concentrations, which were added in the standard addition experiments listed in increasing order.

Table S9 .
Parameters for the linear regression of the calibration curves (a*concentration+b)

Table S10 .
Parameters for the linear regression of the calibration curves (a*concentration+b)