Investigation of different approaches for exhaled breath and tumor tissue analyses to identify lung cancer biomarkers

Abstract

Development of early noninvasive methods for lung cancer diagnosis is among the most promising technologies, especially using exhaled breath as an object of analysis. Simple sample collection combined with easy and quick sample preparation, as well as the long-term stability of the samples, make it an ideal choice for routine analysis. The conditions of exhaled breath analysis by preconcentrating volatile organic compounds (VOCs) in sorbent tubes, two-stage thermal desorption and gas-chromatographic determination with flame-ionization detection have been optimized. These conditions were applied to estimate differences in exhaled breath VOC profiles of lung cancer patients and healthy volunteers. The combination of statistical methods was used to evaluate the ability of VOCs and their ratios to classify lung cancer patients and healthy volunteers. The performance of diagnostic models on the test data set was greater than 90 % for both VOC peak areas and their ratios. Some of the exhaled breath samples were analyzed using gas chromatography coupled with mass spectrometry (GC-MS) to identify VOCs present in exhaled breath at lower concentration levels. To confirm the endogenous origin of VOCs found in exhaled breath, GC-MS analysis of tumor tissues was conducted. Some of the VOCs identified in exhaled breath were found in tumor tissues, but their frequency of occurrence was significantly lower than in the case of exhaled breath.

Keywords: Analytical chemistry; Biomedical engineering; Cancer research; Exhaled breath analysis; GC-FID; GC-MS; Lung cancer; Thermal desorption; Tumor tissue analysis; Volatile organic compounds.

Figure 1

Chromatograms of an exhaled air sample preconcentrated in a sorbent tube with the Tenax TA sorbent using various chromatographic columns (a – СR-5, b – HP FFAP, c – Equity TM 1701, d – CP-Porabond-Q; 1 – acetone, 2 – acetonitrile, 3 – hexane, 4 – isoprene, 5 – 2-porpanol, 6 – ethanol, 7 – benzene, 8 – toluene, 9 – ethyl ester, 10 – 2-butanone, 11 – pentanal).

Figure 2

Chromatograms of exhaled air samples obtained on the CP-Porabond-Q column using sorbent tubes with the following sorbents: a – Tenax TA, b – Porapak N, c – a combined sorbent, d – Chromosorb 106.

Figure 3

GC-MS chromatograms of exhaled breath sampled by using a – a Tedlar sampling bag or b – a Mylar sampling bag, with preconcentration immediately after filling and 20 h later (1 – acetonitrile, 2 – acetone, 3 – isopropanol, 4 – dimethyl sulfide, 5 – ethyl ester, 6 – isoprene, 7 – 2-butanone, 8 – hexane, 9 – benzene, 10 – heptane, 11 – toluene, 12 – hexanal, 13 – N,N-dimethylformamide, 14 – phenol).

Figure 4

GC-MS chromatograms of exhaled breath samples from: a – a lung cancer patient, b – a healthy volunteer (1 – acetaldehyde, 2 – ethanol, 3 – acetonitrile, 4 – acetone, 5 – 2-propanol, 6 – dimethyl sulfide, 7 – methyl acetate, 8 – ethyl ester, 9 – isoprene, 10–1,4-penthadiene, 11 – butanal, 12–2,3-butandione, 13 – 2-butanone, 14 – dimethyl carbonate, 15 – ethyl acetate, 16 – hexane, 17 – 3-methyl-3-penten-1-yne, 18 – benzene, 19 – 2-pentanone, 20 – pentanal, 21 – furane, 2,5-dimethyl, 22 – 1-methylthio-propane, 23 – 1-methylthio-propene, 24 – heptane, 25 – 1-pentanol, 26 – toluene, 27 – hexanal, 28 – ethylbenzene, 29 – m-xylene + p-xylene, 30 – 3-heptanone, 31 – 2-heptanone, 32– phenol, 33 – benzaldehyde, 34 –6-methyl-5-hepten-2-one, 35 – benzene, 1,4-dichloro-, 36 – octanal, 37 – 2-ethyl-1-hexanol, 38–1,2-nonadiene, 39–1,1-(1,4-phenylene)bis-ethanone, 40 – nonanal).

Figure 5

GC-FID chromatograms of exhaled breath samples from a – a lung cancer patient and b – a healthy volunteer.

Figure 6

GC-MS chromatograms of sarcoma with poorly differentiated cells tissue sample after a 10-min heating at 37 (a) and 50 °C (b). (1 – acetaldehyde, 2 – ethanol, 3 – acetonitrile, 4 – acetone, 5 – 2-propanol, 6 – sevoflurane, 7 – methyl acetate, 8 – ethyl ester, 9 – pentane, 10 – 1-propanol, 11 – hexafluoroisopropyl alcohol, 12 – methacrolein, 13 – methyl vinyl ketone, 14 – butanal, 15 – 2-butanone, 16 – dimethyl carbonate, 17 – ethyl acetate, 18 – hexane, 19 – 1-butanol, 20 – benzene, 21 – 1-pentene-3-one, 22 – 1-pentene-3-ol, 23 – 2-pentanone, 24 – pentanal, 25 – 3-methyl-3-buten-1-ol, 26 – heptane, 27 – 1-pentanol, 28 – (E)-2-pentenal, 29 – toluene, 30 – 2-methyl-pentanal, 31 – hexanal, 32 – butyl acetate, 33 – ethyl formate, 34 – cyclohexanone + o-xylene, 35 – m-xylene + p-xylene, 36 – 2-heptanone, 37 – heptanal, 38 – benzaldehyde, 39 – 2-pentyl-furan, 40 – octanal, 41 – 2-ethyl-1-hexanol, 42 – limonene, 43–1,2-nonandiene, 44 – nonanal, 45 – undecane).

Figure 7

GC-FID chromatograms of ambient air samples obtained from a – a hospital and b – a solvent-free room.