In this study, we compared the adsorption capabilities of six different SPE cartridges containing different polymer-based adsorbent materials for VOC contained in the exhaled breath of dairy cows. To the best of our knowledge, this is the first study of its kind.

The average VOC numbers differed only slightly depending on the type of SPE cartridge used (table 2). The XAW SPE cartridges detected the highest average compound numbers, followed by HLB, XCW, HRX, ENV, and HRP. We used only polymer-based SPE cartridges, each with a column volume of 3 ml and an adsorbent weight of 200 mg to sample exhaled breath for 4 min. All SPE were used in parallel, resulting in a total air flow rate of 9.29 l min−1. In addition, we applied a uniform solvent for elution after VOC sampling. Therefore, the strongest effects on the specificity for VOC detection would arise from the adsorbent material, together with the SPE mode, chemical bond types, particle size, and shape. Table 1 shows that the differences in the number of VOCs detected are due to the SPE mode and their chemical bond type but not due to the difference in flow rate or specific surface area of the SPE.

Table 2. The total number of GC-MS peaks in exhaled breath from five dairy cows using six different solid-phase extraction (SPE) cartridges.

 Solid-phase extraction (SPE) cartridges ENVHRPHLBHRXXAWXCWNumber of VOCs detected1216 ± 49.11174 ± 40.91242 ± 32.21227 ± 39.41312 ± 83.91229 ± 75.1

Total number of GC-MS peaks per cartridge observed after sampling exhaled breath from five dairy cows (shown as mean ± SD). ENV: Bond Elut ENV (polystyrene-divinylbenzene polymer); HRX: Chromabond HRX (hydrophobic spherical polystyrene-divinylbenzene copolymer); HRP: Chromabond HRP (hydrophobic polystyrene-divinylbenzene copolymer); HLB: Chromabond HLB (hydrophilic-lipophilic balanced N-vinylpyrrolidone-divinylbenzene); XCW: Chromabond HR-XCW (hydrophobic spherical polystyrene-divinylbenzene copolymer); XAW: Chromabond HR-XAW (hydrophobic spherical polystyrene-divinylbenzene copolymer with secondary weak anion exchange); XCW: Chromabond HR-XCW (hydrophobic spherical polystyrene-divinylbenzene copolymer with weak cation exchange).

For all six SPE cartridge types, the level 3 VOCs detected were those from the chemical classes (in decreasing order of VOC numbers detected) of alkenes, followed by alkanes, esters, ketones, alcohols, aldehydes, amines, nitriles, ethers, amides, carboxylic acids, alkynes, azoles, terpenes, pyridines, and sulfur-containing compounds (table 3). These chemical compound groups were detectable in the exhaled breath of all five cows and with each SPE cartridge used. The SPE cartridges can generally be classified into normal phase, reversed phase, ion exchange (anion and cation-exchange), and mixed-mode (combination of reversed phase and ion exchange) [29]. All SPE cartridges used in our study had reversed phase functionality. The wide range of detection of acidic, basic, and neutral compounds results from both the hydrophobic and hydrophilic parts of the SPE adsorbent materials used, which impart an amphiphilic character [19]. The hydrophilic character is induced by polar functional groups, such as polystyrene or polyvinylpyrrolidone, that contribute to the interaction with the polar functional groups of the VOCs, while the hydrophobic divinylbenzene part allows π–π interactions with the aromatic functional groups of VOCs [19, 30]. Therefore, all SPE cartridges were able to detect all chemical compound groups. In addition to the reversed phase character, the XAW and XCW cartridges had a weak anion and cation exchange, respectively, giving them a mixed-mode character.

Table 3. Volatile organic compounds (VOCs) in various chemical compound classes identified at level three after sampling from exhaled breath of five dairy cows using six different solid-phase extraction (SPE) cartridges (presented as mean ± standard deviation).

Solid-phase extraction cartridgesChemical compound groupENVHRPHLBHRXXAWXCWAldehydes10.40 ± 4.3410.20 ± 2.7712.60 ± 2.8813.00 ± 3.399.80 ± 3.569.60 ± 2.30Alcohols23.20 ± 4.3214.40 ± 2.7018.60 ± 4.8817.40 ± 4.169.80 ± 3.0521.20 ± 3.83Alkanes63.80 ± 7.6668.60 ± 6.3564.40 ± 4.1064.20 ± 5.6365.20 ± 5.4061.80 ± 7.33Alkenes85.20 ± 10.8586.80 ± 5.4077.40 ± 2.6182.80 ± 4.1583.40 ± 8.7382.80 ± 4.97Alkynes3.40 ± 1.342.20 ± 1.104.00 ± 1.224.20 ± 1.304.00 ± 1.004.00 ± 1.22Azoles2.40 ± 1.674.20 ± 1.793.20 ± 1.302.60 ± 1.822.80 ± 0.842.20 ± 0.84Amides3.20 ± 2.284.40 ± 1.825.60 ± 1.523.60 ± 1.145.60 ± 2.703.60 ± 1.95Amines8.20 ± 3.905.60 ± 2.707.20 ± 2.399.20 ± 4.718.80 ± 4.387.40 ± 2.70Carboxylic acids4.60 ± 0.553.60 ± 1.144.80 ± 2.174.60 ± 1.143.20 ± 1.303.60 ± 1.52Esters27.60 ± 4.3928.40 ± 5.5036.40 ± 5.7334.40 ± 3.7132.20 ± 8.1729.20 ± 2.68Ethers6.20 ± 1.794.80 ± 0.845.00 ± 1.225.60 ± 1.674.60 ± 1.825.60 ± 1.52Ketones21.20 ± 3.6319.20 ± 3.2723.60 ± 3.2923.80 ± 2.7731.40 ± 11.2624.20 ± 4.21Nitriles7.20 ± 2.685.40 ± 2.196.80 ± 1.928.00 ± 2.356.40 ± 3.787.60 ± 1.82Pyridines1.00 ± 1.001.40 ± 1.142.80 ± 1.481.00 ± 1.411.00 ± 1.221.40 ± 1.34Sulfur compounds0.80 ± 0.840.80 ± 0.451.80 ± 0.842.60 ± 1.140.60 ± 0.892.00 ± 1.22Terpenes1.60 ± 0.552.20 ± 1.101.00 ± 0.712.60 ± 1.672.20 ± 0.452.00 ± 0.71Others19.40 ± 4.2216.20 ± 3.1116.60 ± 3.4418.00 ± 5.1019.40 ± 5.1820.20 ± 2.17

Compound identification (match factor >50%) using the National Institute of Standards and Technology NIST/EPA/NIH mass spectral library (NIST17). Number of volatile organic compounds (VOCs) within chemical groups shown as mean ± standard deviation (SD) from sampling of five cows per SPE cartridge used. ENV: Bond Elut ENV (polystyrene-divinylbenzene polymer); HRX: Chromabond HRX (hydrophobic spherical polystyrene-divinylbenzene copolymer); HRP: Chromabond HRP (hydrophobic polystyrene-divinylbenzene-copolymer); HLB: Chromabond HLB (hydrophilic-lipophilic balanced N-vinylpyrrolidone-divinylbenzene); XCW: Chromabond HR-XCW (hydrophobic spherical polystyrene-divinylbenzene copolymer); XAW: Chromabond HR-XAW (hydrophobic spherical polystyrene-divinylbenzene copolymer with secondary weak anion exchange); XCW: Chromabond HR-XCW (hydrophobic spherical polystyrene-divinylbenzene copolymer with weak cation exchange).

The number of level 3 VOCs within a particular chemical compound group differed between the SPE cartridges used (table 3). The greatest difference between the SPE cartridges was noted for the association with ketones, where the highest numbers of VOCs were detected using XAW. The XAW adsorbent cartridge has a mixed-mode character, which imparts a higher efficiency for binding VOCs from complex matrices compared with cartridges with only a reversed phase character [29]. Mixed-mode cartridges can also form ionic interactions (anion or cation exchange). In particular, the weak anion exchanging capacity of the XAW adsorbent cartridge allows interactions between the positively charged groups on the SPE adsorbent cartridge and the negatively ionized or ionizable groups of the VOCs (anionic parts) [19]. The higher sensitivity of XAW for capturing ketones may be due to the enhanced formation of anionic enols within the keto-enol tautomerism, reinforced by the alkalinity of bovine saliva (pH 8.55–8.90 [31]) within the humid matrix of the exhaled breath [32] (>95% relative humidity) [33]. Due to their lower charge compared to carboxylic acids, ketones can better elute from the surface of the SPE adsorbent material before reverting to the amplified keto form, which is enhanced by the neutral pH of the acetonitrile elution solvent. With the XAW cartridge, a greater number of carboxylic acids, which form stronger ionic interactions, could be detectable by lowering the pH during the elution process.

The use of the XCW cartridge, which has an additional weak cation exchange section, did not improve the detection of basic chemical compounds. Further investigation of the effect of elution pH on the detection of different compound groups is required, as strongly acidic or basic compounds might only be eluted by changing the pH. Other moderate differences between the detection of VOCs from specific chemical compound groups were observed using the ENV polymer, which showed the best specificity for alcoholic compounds, while HRP was best for alkanes, alkenes, and azoles; HLB was best for esters; and HRX was best for aldehydes. Alkanes and alkenes are not typically produced by physiological processes involving the organism's metabolism, but are environmental pollutants originating from vehicle exhaust, gasoline evaporation, biomass burning, the use of volatile chemical products (solvents, paints, pesticides, detergents, personal care products, etc) and vegetation emissions, and can also be contaminants in plants used as animal feed [34]. Very small differences were found between the different SPE cartridges for the detection of amides, amines, alkynes, carboxylic acids, ethers, nitriles, pyridines, sulfur-containing compounds, and terpenes.

In total, 176 specific VOCs were detected from the physiologically relevant compound groups of aldehydes, alcohols, azoles, amides, amines, carboxylic acids, esters, ethers, ketones, nitriles, pyridines, sulfur-containing compounds and terpenes (table 4). Differences in the detection of these VOCs were observed between the five cows and between the SPE cartridges used. This could reflect animal-specific metabolism. The largest number of specific VOCs was captured with the XAW cartridge (149), followed by ENV (118), HLB (117), HRP (115), HRX (114), and XCW (114). The different numbers indicate some differences in the detected VOCs among the six SPE cartridges used, indicating their different specificity for particular VOCs. More insight into the differences is provided by the chromatograms, which differed according to the SPE adsorbent cartridge in terms of the peaks detected (figure 3). The main differences, with more peaks detected, occurred at a retention time (RT) between 8 and 9 min with the XAW cartridge; RT 10–12 min with XAW and XCW; RT 18–19 min with HLB and XAW; RT 23–24 min and 26–27 min with HRP and XAW.

Figure 3. Comparison of chromatograms of exhaled breath from one dairy cow obtained using six different solid-phase extraction (SPE) cartridges. Differences in the peaks detected between the different chromatograms highlighted with black circles. ENV: Bond Elut ENV (polystyrene-divinylbenzene polymer); HRX: Chromabond HRX (hydrophobic spherical polystyrene-divinylbenzene copolymer); HRP: Chromabond HRP (hydrophobic polystyrene-divinylbenzene-copolymer); HLB: Chromabond HLB (hydrophilic-lipophilic balanced N-vinylpyrrolidone-divinylbenzene); XCW: Chromabond HR-XCW (hydrophobic spherical polystyrene-divinylbenzene copolymer); XAW: Chromabond HR-XAW (hydrophobic spherical polystyrene-divinylbenzene copolymer with secondary weak anion exchange); XCW: Chromabond HR-XCW (hydrophobic spherical polystyrene-divinylbenzene copolymer with weak cation exchange).

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Table 4. Volatile organic compounds (VOCs) from the physiologically relevant chemical groups (aldehydes, alcohols, azoles, amides, amines, carboxylic acids, esters, ethers, ketones, nitriles, pyridines, sulfur containing compounds and terpenes) detected using six different solid phase extraction (SPE) cartridges (highlighted in brackets with the number of cows, where these VOCs were detected) after sampling from exhaled breath of five dairy cows.

         SPE cartridgeVolatile organic compoundsBMRT (min)CAS numberm/zLevelMatch factorRI calcRI refENVHRXHRPHLBXAWXCW2-ButenalBM-0015.8534170-30-370190.2665657    (5) 2-Pentyn-4-oneBM-0026.0097299-55-082281.5669672 (5)(5)(5)(5)(5)Pyridine, 2-nitro-tBM-0036.30215009-91-378383.8675n.d.(5)(5)(5)(5)(5)(5)4,6-Heptadiyn-3-onetBM-0046.31429743-27-978380.8676n.d.(5)(5)(5)(5)(5)(5)UnknownBM-0056.669—574—683—(5)(5)(5)(5)(5)(5)UnknownBM-0066.688—784—684—(5)(5)(5)(5)(5)(5)2-Propanol, 1-methoxy-BM-0076.705107-98-275289684672(5)(5)(5)(5)(5)(5)4-Penten-2-olBM-0086.949625-31-071283.3690688(5)(5)(5)(5)(5)(5)AcetoinBM-0096.978513-86-088285.2690700(1) (1)   UnknownBM-0107.482—784—702—(5)(5)(5)(5)(5)(5)UnknownBM-0117.869498-60-2904—715—    (1) 2,4-DimethylfuranBM-0127.8833710-43-896289716703(5)(5)(5)(5)(5)(5)3,4-DimethylfuranBM-0137.88920843-07-696286.5716724(5)(5)(5)(5)(5)(5)PyrazineBM-0148.488290-37-980199.4736740    (5) 3-Penten-1-ol, (Z)-BM-0158.508764-38-568286.9737725(5)(5)(5)(5)(5)(5)3-Buten-1-ol, 3-methyl-BM-0168.511763-32-686285.8737734(5)   (5) 3-Penten-2-oneBM-0178.580625-33-284285.1739739    (5) UnknownBM-0188.605—844—740—    (5) UnknownBM-0198.686—704—743—(5)  (5)  UnknownBM-0208.723—864—744—    (5) UnknownBM-0218.972—984—752—    (5) 1,3,5-CycloheptatrieneBM-0229.743n.d.61287.6772765(5)(5)(5)(5)(5)(5)2-Butenal, 3-methyl-BM-0239.946107-86-884190785783    (5) UnknownBM-0249.958—914—786—(5)(5)(5)(5)(5)(5)UnknownBM-02510.279—1004—797—    (5) HexanalBM-02610.32166-25-182190.1798802 (5)    4-Pentenal,2,2-dimethyl-tBM-02710.3395497-67-683383.3799n.d.(5)(5)(5)(5)(5)(5)1-Hexyn-3-oltBM-02810.346105-31-783383.7799n.d.(5)(5)(5)(5)(5)(5)UnknownBM-02910.348—844—799—     (5)3.5-Dimethyl-1.6-heptadien-4-oltBM-03010.42519549-66-785384802n.d.(5)(5)(5)(5)(5)(5)Hydroperoxide, hexyltBM-03110.4384312-76-985390.2802n.d.(5)(5)(5)(5)(5)(5)Acetic acid, butyl esterBM-03210.753123-86-473287.8815812(5)(5) (5)(5)(5)UnknownBM-03310.919—1044—822—     (1)FurfuralBM-03411.23598-01-195281.4834835 (5)   (5)UnknownBM-03511.359—1294—839—    (1) 3-Hexen-2-oneBM-03611.413763-93-983190.3842845    (5)(5)2-Pentanone, 4-hydroxy-4-methyl-BM-03711.427123-42-283190842841    (5)(5)2-Pentanone, 3-methylenetBM-03811.4364359-77-798390.1842n.d.    (5)(5)UnknownBM-03911.863—