Non-Targeted Breath Volatile Analysis

The subjects involved in the study breathed through a facemask connected to a valve of a resistance-free 1L plastic bag (Tedlar bag, SKC Ltd, Dorset, UK). One hour before sampling, eating and exercise were not allowed. The use of inhaled corticosteroids was stopped four weeks before the measurements except in 7 patients due to severe asthma symptoms. Children participated in the study were sampled randomly without division on healthy controls and children with wheezing. All samples from subjects taking part in this study were collected in the same room in order to prevent the appearance of a background bias. The plastic bags were emptied via pump with constant flow over a stainless-steel two-bed sorption tube, filled with carbograph 1TD/Carbopack X (Markes International, Llantrisant, Wales, UK) within 1 h after collection. The air-tight capped tubes were kept at room temperature until analysis (in average for three weeks). The capped tubes can be kept at room temperature up to six months without significant changes of VOCs profile. The bags were cleaned by filling and empting 2 times with nitrogen and reused for next measurements. In this study 1L of mixed breath (end-tidal and dead space air) was collected. Dead-space air comprises only a small part (30 ml) of the total sample of exhaled air collected and we have shown that the contribution of dead-space air to the total volume of whole breath does not lead to sensitivity issues in measuring VOCs by GC-tof-MS [28] (link).
The exhaled air samples were measured by means of GC-tof-MS [35] . All collected samples were measured randomly, i.e. the batch of 26 samples a random set of breath samples obtained from healthy controls and children with wheezing. The GC-tof-MS method applied here is a non-targeted GC-tof-MS method, i.e. no prior identification of the compounds was performed. All chromatographic conditions were optimized by us previously [28] (link) and consequently in consultation with the producer of our instrument and based on common chromatographic experience we chose a column and the temperature programming that were suitable to detect many different classes of volatile compounds and at the same time keep the best possible separation of the compounds at a high sensitivity and a high dynamic range. The detailed parameters of GC-tof-MS measurements are listed in Table 2.