- Email: [email protected]
MS
Journal of Chromatography B, 978–979 (2015) 62–69
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
The analysis of linear and monomethylalkanes in exhaled breath samples by GC × GC-FID and GC–MS/MS Alexandra Hengerics Szabó a , Peter Podolec a , Viktória Ferenczy a , Róbert Kubinec a,∗ , Jaroslav Blaˇsko a , Ladislav Soják a , Renáta Górová a , Gabriela Addová a , Ivan Ostrovsky´ a , c,d ˇ ˇ Jozef Viˇsnovsk y´ a,b , Václav Bierhanzl c , Radomír Cabala , Anton Amann e,f a
Institute of Chemistry, Faculty of Natural Sciences, Comenius University, Mlynská dolina CH-2, 842 15 Bratislava, Slovakia SynthCluster, s.r.o., Komenského 1439, 900 01 Modra, Slovakia c Department of Analytical Chemistry, Faculty of Science, Charles University in Prague, Albertov 6, 128 43 Prague 2, Czech Republic d Institute of Forensic Medicine and Toxicology, General University Hospital in Prague, U Nemocnice 2, 128 08 Prague 2, Czech Republic e Univ.-Clinic of Anesthesia and Intensive Care, Innsbruck Medical University, Anichstrasse 35, A-6020 Innsbruck, Austria f Breath Research Institute of the University of Innsbruck, Rathausplatz 4, A-6850 Dornbirn, Austria b
a r t i c l e
i n f o
Article history: Received 8 August 2014 Accepted 24 November 2014 Available online 3 December 2014 Keywords: Exhaled breath INCAT Monomethylalkanes n-Alkanes Needle trap
a b s t r a c t A new arrangement of the INCAT (inside needle capillary adsorption trap) device with Carbopack X and Carboxen 1000 as sorbent materials was applied for sampling, preconcentration and injection of C6 C19 n-alkanes and their monomethyl analogs in exhaled breath samples. For the analysis both GC–MS/MS and GC × GC-FID techniques were used. Identification of the analytes was based on standards, measured retention indices and selective SRM transitions of the individual isomers. The GC–MS/MS detection limits were in the range from 2.1 pg for n-tetradecane to 86 pg for 5-methyloctadecane. The GC × GC-FID detection limits ranged from 19 pg for n-dodecane to 110 pg for 3-methyloctane. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The analysis of the compounds in exhaled breath can provide insight into different biochemical processes in the healthy and the diseased human body [1,2]. Breath constituents are composed of both volatile [3] and non-volatile compounds [4–6], that together form an “exhaled metabolome.” A large number of compounds have been associated with different diseases, such as: ketones in ketoacidosis for diabetics [7–12], aldehydes in lung cancer patients [7–11], compounds associated with tuberculosis infection [12], and exhaled nitrate and nitric oxide in nonspecific pulmonary inflammation and/or asthma [10,13–15]. Alkanes in exhaled breath have also been proposed as endogenous marker compounds [3,7,8,12]. Their analysis is of medical importance, since it may lead to non-invasive clinical tests for carcinomas, oxidative stress and heart transplant rejection [16]. In a study of the relation of age and the presence of methylalkanes in exhaled
∗ Corresponding author. Tel.: +421 2 602 96 330; fax: +421 2 602 96 337. E-mail address: [email protected] (R. Kubinec). http://dx.doi.org/10.1016/j.jchromb.2014.11.026 1570-0232/© 2014 Elsevier B.V. All rights reserved.
breath [17], 22 different C6 C17 normal and monomethylalkanes were proposed as marker compounds of oxidative stress, the latter being considered as a pathologic mechanism in aging and several diseases. Phillips et al. [7] suggest that oxidative stress degrades membrane polyunsaturated fatty acids, thus producing n-alkanes and methylalkanes which are excreted in exhaled breath and which are considered to vary with the extent of oxidative stress. Phillips et al. [8] proposed the following compounds for the detection of lung cancer: butane, 3-methyltridecane, 7-methyltridecane, 4-methyloctane, 3-methylhexane, heptane, 2-methylhexane, pentane, and 5-methyldecane. For the detection of heart transplant rejection he proposed 2-methylpropane, 5-methyloctadecane, 6-methyloctadecane, 2methylpentadecane, octane, 2-methylheptane, 3-methylundecane, 2-methyloctadecane, and 2-methylhexadecane [18]. In addition to endogenously produced VOCs, 13 C- or 14 C-labeled precursor compounds are used for breath tests. An example is 13 Clabeled urea, used in gastroenterology tests for detection of Helicobacter pylori bacterial infections associated with stomach ulcers [19,20]. Other examples for precursors are aminopyridine and 14 C-ethanol for testing of impaired liver function [21,22] and 13 C-dextromethorphan for testing of the CYP2D6 activity [23].
A. Hengerics Szabó et al. / J. Chromatogr. B 978–979 (2015) 62–69
Sample preparation is the cornerstone of the chemical analysis. Preconcentration is the crucial step when volatile organic compounds (VOCs) often occurring in concentrations as low as ppb or ppt are to be determined. Needle trap devices (NTDs) represent promising tools for a robust and reproducible sample preparation, combining advantages of SPE and SPME [24,25]. One of their greatest advantages is that no additional equipment, apart from a heated GC injector, is necessary for application [26]. Several applications of single layer packed sorbents such as Carboxen and divinylbenzene have been described in the field of environmental monitoring (e.g. analysis of BTEX or higher alkanes) [27–30]. For the analysis of more complex samples containing compounds with a wide range of polarity and volatility single-bed NTDs are insufficient and therefore multibed NTDs are needed. Trefz et al. recently proved the applicability of multibed NTDs and expansive flow technique in medical breath analysis [31–33]. INCAT (inside needle capillary adsorption trap) is a needle trap device. Its new arrangement was developed by Kubinec et al. [28,34,35]. This device is more robust than most needle trap devices or SPME fibers and gives comparable results [35]. The aim of this work was the development of an INCAT device enabling the analysis of non-polar compounds with a wide range of volatility. Linear and monomethylalkanes were chosen for this study as they are considered as breath markers of various diseases. Many articles are focused on the analysis of a wide range of breath volatiles using various analytical techniques, from which GC–MS and GC-FID are still the most common. There is, however only a limited number of articles about the GC × GC analysis of exhaled breath samples [32,36–40]. Also, the identification of compounds present in breath samples is very often based only on spectral library match or on similar retention characteristics with standards, which approach might not be sufficient. The aim of the manuscript is not the analysis of all compounds present in breath samples, but the analysis and identification of the selected n-alkanes and monomethylalkanes. The separation and identification of monomethylalkanes in a broad range of carbon atoms is problematic due to the multicomponent character and close retention of isomers with methyl-branching near the middle of the carbon chain. The lack of standard reference materials and poor reproducibility of published retention data are also critical points. This manuscript is focused on the separation, identification and quantification of n-alkanes and monomethylalkanes using GC × GC-FID and GC–MS/MS techniques which can help in the future identification of these analytes in exhaled breath samples. It does not deal with the possible medical impact of the analytes, but is focused on their reliable analysis in exhaled breath. The article deals with the analysis of alkanes and monomethylalkanes which although are presented in scientific articles as markers of various diseases, their identification might not be adequate if only based on spectral library match or similar retention characteristics with standards.
63
o.d./0.9 mm i.d. diameter by sintering at 680 ◦ C for 45 min in a laboratory furnace. Glass beads were obtained from BDH (Poole, England), laboratory furnace CLASIC 1313 from CLASIC CZ s.r.o. ˇ Czech Republic). MTB sampling valves were purchased (Revnice, from Hamilton (Reno, Nevada, USA).
2.2. Sample preparation Monomethylalkanes C9 C19 as a model mixture were prepared from n-alkanes mixture C8 C18 by methylene insertion reaction [41] in an apparatus by Glastrup [42] by using gaseous diazomethane and UV radiation. The C8 C18 n-alkanes used for methylene insertion reaction were obtained from Supelco (Bellefonte, PA, USA) and C6 C7 n-alkanes were obtained from Sigma–Aldrich (Germany). The standard gas mixtures were prepared similarly as by Mochalski et al. [43]. Primary standards were prepared in 1 L glass bulbs (Supelco, Canada). Prior to use the bulbs were cleaned with methanol and dried at 80 ◦ C. Then the bulb was evacuated using a vacuum membrane pump and 1 L of a liquid analyte was injected into the bulb through a rubber septum. The bulb was heated to 70 ◦ C for 30 min to ensure the evaporation. Gas mixtures obtained through evaporation of liquid standards were appropriately diluted in synthetic breath (78% N2 , 15% O2 , 4% CO2 , 3% H2 O) in 3 L Tedlar bags (Supelco, Bellefonte, USA) to obtain the desired concentration levels of analytes for further analyses. Calibration standards were prepared by this method with analyte concentrations 1; 2; 5; 10; 20; 50; 100; 500 and 1000 pg L−1 . Samples of exhaled breath of consenting person were collected in 3 L gas-sampling Tedlar bags. Tedlar bags were chosen for sample collection due to their reusability, low background and high stability for collected hydrocarbons with no relevant losses even when stored up to a week [43]. Our goal was to find more of the C6 C19 alkanes and C9 C19 monomethylalkanes in an exhaled breath sample. More individuals were selected for analyses, but the samples of the chosen individual (a 47-year-old heavy smoker with a triple coronary artery bypass) contained the highest content of monomethylalkanes, detected by GC × GC-FID technique. All samples were collected after the volunteer had left a period of at least 1 h without consuming any food or beverages. Before sample collection, the Tedlar bags were flushed three times with the exhaled breath samples of the individuals. For each exhaled breath sample, 1000 mL of breath previously collected in Tedlar bag was pulled through the sorption trap using membrane vacuum pump KNF LAB (KNF, Neuberger, Freiburg, Germany). The extraction of analytes with INCAT device from the samples collected in Tedlar bags was performed at oven temperature of 40 ◦ C to avoid water condensation in the breath samples during sampling. Each sample was analyzed three times.
2.3. Needle trap devices 2. Experimental 2.1. Materials Chromosorb W with 20% SE-54 (meshes 60–80), Carbopack X (meshes 40–60, specific surface area 250 m2 g−1 ) and Carboxen 1000 (meshes 60–80, specific surface area 1200 m2 g−1 ) as packing materials for needle trap devices were obtained from Supelco (Bellefonte, USA). Stainless steel needles (cannula) 90 mm long with an outer/inner diameter of 1.3 mm/1.1 mm, and 1.1 mm/0.9 mm were from Nissho (Osaka, Japan). Frits of 5 mm length were prepared from glass beads (0.125–0.2 mm) filled in a needle of 1.1 mm
Needle trap devices developed in our laboratory based on our previous work [28] were investigated. The needle traps were packed with 39 mm length of the chosen sorbents, which were locked into position by frits with sintered glass beads. Sorbents were packed in 13 mm long layers into the INCAT devices. The novelty of the developed INCAT devices lie in the use of adsorbents with different adsorption strengths in series, allowing the analysis of a much wider range of volatile analytes than the previously published INCAT devices. Prior to first usage all INCAT devices were conditioned in the heated injection port of a gas chromatograph for 2 h at temperature of 280 ◦ C under a permanent helium flow of 3.5 mL min−1 .
64
A. Hengerics Szabó et al. / J. Chromatogr. B 978–979 (2015) 62–69
2.4. GC × GC-FID and GC–MS/MS conditions 2.4.1. GC × GC-FID A Thermo Electron TRACE GC Ultra (TRACE 2DGC) was used to perform comprehensive two-dimensional GC. The TRACE 2DGC system was provided with the dual-stage CO2 jet modulator, as proposed by Beens et al. [44] driven by a dedicated electronics which ensured the synchronization between the valves activation and the signal acquisition. A fast FID was used as detector, capable of producing a digital signal at a sampling rate up to 300 Hz. An injection system with modified metal liner (based on previous work of Jurdáková et al. [28] and optimized for Thermo Trace GC) to minimize dead volumes inside the injection system was used. The liner was made from stainless steel concerning good thermal conductivity and inert surface of this material. It is composed of two metal parts, which are connected by thread, and viton tubing between the two metal parts as a seal. Using this modified liner the carrier gas flows directly through the needle trap device by the side hole and no additional source of inert gas is needed to displace analytes from device. The separation of the mixture of n-alkanes and monomethylalkanes requires the use of a high resolution capillary column. The first dimension column was a 100 m long capillary column with an internal diameter of 0.32 mm and a phase thickness of 0.25 m of methylsilicone OV-1 as a stationary phase (SPB-1, Supelco). It was connected through a deactivated press-fit to a Carbowax second dimension column (FFAP, Hewlett Packard), 1 m long with an internal diameter of 0.2 mm and a phase thickness of 0.33 m. The oven temperature was programmed at 2 ◦ C min−1 from 40 ◦ C to 210 ◦ C, which was held for 10 min. The carrier gas (helium) was supplied at 3.5 mL min−1 during the whole analysis. The injector was set to a temperature of 280 ◦ C operating in splitless mode and the FID detector was set to a temperature of 230 ◦ C. Samples were injected in splitless mode which was held on during the whole analysis time. The GC × GC analyses were carried out with a modulation cycle time of 3 s. Data acquisition and data handling were performed with a software version for GC × GC (HyperChrom) based on Thermo Electron Chrom-Card Data System. 2.4.2. GC–MS/MS The GC–MS/MS analyses were carried out with a Trace GC Ultra gas chromatograph equipped with a split–splitless injector and a TSQ Quantum XLS mass spectrometer (Thermo Fisher, Austin, TX, USA). The temperatures of the injector and the MS-transfer line were 280 ◦ C and 240 ◦ C, respectively. Gas chromatographic conditions were identical to the GC × GC settings, but the separation was performed using only the first dimension column (100 m long SPB-1). The MS/MS conditions were as follows: ion source temperature 200 ◦ C, electron energy 70 eV, emission current 50 A. Selected reaction monitoring (SRM) conditions were experimentally optimized for the selected analytes. The used SRM transitions and collision energies are listed in Table 1.
Fig. 1. Scheme of the INCAT device. N, stainless steel needle; F, frit made of sintered glass beads; A1–A3, layers of adsorbents; H, side hole; T, silicone and PTFE sealing; V, sampling valve.
where x1 stands for the selected analyte (monomethylalkane), n is the number of carbon atoms in the n-alkane eluting before this analyte and n + 1 the number of carbon atoms in the n-alkane eluting after it, tr stands for retention time and I for the retention index. As the response of the MS detector is different for different analytes, response factors for monomethylalkanes were also calculated according to the equation: RF =
Ax cn , An cx
where Ax is the peak area of the monomethylalkane, cx is its concentration, An stands for the peak area of the n-alkane with the same number of carbon atoms and cn is its concentration. 3. Results and discussion 3.1. Needle trap devices A new arrangement of INCAT device was developed for the extraction and preconcentration of n-alkanes and monomethylalkanes with a wide range of volatility. The device consists of a stainless steel needle filled with layers of adsorbents which are fixed into position by frits with sintered glass beads, a side hole for the better desorption of analytes in the injection port of GC by carrier gas, silicone and PTFE sealing as a side hole cover during sampling and a sampling valve. The scheme of INCAT device is shown in Fig. 1. The composition of sorbent layers for each INCAT device is listed in Table 2. INCAT devices were tested by the analysis of a mixture of n-alkanes C6 C18 . Fig. 2 shows the dependencies of the desorption curves of C6 C18 n-alkanes at amount of 500 pg for INCAT devices with different composition of sorbent layers. As it can be seen from Fig. 2, each prepared INCAT device shows different sorption characteristics for the analyzed mixture of nalkanes. The INCAT device containing three layers of Carbopack
2.5. Method validation Limits of detection and quantification (LOD and LOQ) were determined according to the IUPAC guidelines, by means of the signal to noise ratio (S/N). Noise was determined experimentally from the blank samples. LOD was defined as S/N of 3 and LOQ as S/N of 10. Retention indices of all selected analytes were calculated according to the equation: I(x1 ) = 100
tr (x1 ) − tr (n) + 100n, tr (n + 1) − tr (n)
Fig. 2. Relative abundance of C6 C18 n-alkanes after desorption from the prepared INCAT devices packed with different sorbent layers (devices are labeled according to Table 2).
A. Hengerics Szabó et al. / J. Chromatogr. B 978–979 (2015) 62–69 Table 1 Retention times, SRM transitions and collision energies used for the analysis of C6
C19 n-alkanes and C9
65
C19 monomethylalkanes.
Peak number
Compound
Retention time (min)
SRM transition
Collision energy (V)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77
Hexane Heptane Octane 4-Methyloctane 2-Methyloctane 3-Methyloctane Nonane 5-Methylnonane 4-Methylnonane 2-Methylnonane 3-Methylnonane Decane 5-Methyldecane 4-Methyldecane 2-Methyldecane 3-Methyldecane Undecane 6-Methylundecane 5-Methylundecane 4-Methylundecane 2-Methylundecane 3-Methylundecane Dodecane 6-Methyldodecane 5-Methyldodecane 4-Methyldodecane 2-Methyldodecane 3-Methyldodecane Tridecane 7-Methyltridecane 6-Methyltridecane 5-Methyltridecane 4-Methyltridecane 2-Methyltridecane 3-Methyltridecane Tetradecane 7-Methyltetradecane 6-Methyltetradecane 5-Methyltetradecane 4-Methyltetradecane 2-Methyltetradecane 3-Methyltetradecane Pentadecane 8-Methylpentadecane 7-Methylpentadecane 6-Methylpentadecane 5-Methylpentadecane 4-Methylpentadecane 2-Methylpentadecane 3-Methylpentadecane Hexadecane 8-Methylhexadecane 7-Methylhexadecane 6-Methylhexadecane 5-Methylhexadecane 4-Methylhexadecane 2-Methylhexadecane 3-Methylhexadecane Heptadecane 9-Methylheptadecane 8-Methylheptadecane 7-Methylheptadecane 6-Methylheptadecane 5-Methylheptadecane 4-Methylheptadecane 2-Methylheptadecane 3-Methylheptadecane Octadecane 9-Methyloctadecane 8-Methyloctadecane 7-Methyloctadecane 6-Methyloctadecane 5-Methyloctadecane 4-Methyloctadecane 2-Methyloctadecane 3-Methyloctadecane Nonadecane
5.21 8.02 12.96 16.41 16.46 16.85 18.58 22.62 22.73 22.91 23.36 25.35 29.49 29.71 29.97 30.38 32.47 36.38 36.47 36.74 37.05 37.50 39.52 43.16 43.29 43.60 43.92 44.34 46.29 49.60 49.64 49.83 50.13 50.46 50.88 52.73 55.76 55.86 56.04 56.36 56.68 57.09 58.83 61.59 61.63 61.74 61.93 62.26 62.59 62.96 64.62 67.17 67.20 67.35 67.55 67.86 68.17 68.58 70.12 72.49 72.52 72.54 72.68 72.91 73.22 73.50 73.90 75.36 77.53 77.55 77.62 77.78 77.99 78.30 78.57 78.86 80.35
86 → 57 100 → 57 114 → 85 70 → 55 84 → 55 70 → 55 128 → 57 84 → 55 70 → 55 98 → 69 142 → 71 142 → 57 98 → 69 70 → 55 112 → 55 98 → 69 156 → 56 98 → 56 112 → 55 126 → 55 126 → 55 112 → 55 170 → 57 98 → 56 84 → 55 140 → 56 140 → 69 140 → 69 184 → 70 112 → 55 98 → 56 84 → 55 154 → 69 154 → 69 140 → 69 198 → 57 112 → 70 98 → 69 84 → 55 168 → 83 168 → 83 154 → 69 212 → 57 126 → 55 140 → 69 126 → 55 168 → 69 182 → 69 182 → 69 168 → 69 226 → 57 126 → 56 112 → 55 98 → 69 182 → 69 196 → 96 196 → 96 182 → 69 240 → 57 126 → 55 112 → 55 168 → 69 182 → 69 196 → 70 210 → 97 210 → 97 196 → 70 254 → 71 140 → 69 126 → 55 112 → 55 196 → 69 210 → 83 224 → 83 224 → 83 112 → 55 268 → 71
10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 15 10 10 15 10 10 10 10 10 10 10 10 10 15 15 20 20 10 15 15 10 10 15 15 15 10 20 15 15 15 10
66
A. Hengerics Szabó et al. / J. Chromatogr. B 978–979 (2015) 62–69
Table 2 The composition of sorbent layers in the INCAT devices. INCAT device
Sorbent layer 1
Sorbent layer 2
Sorbent layer 3
A B C D E
Carbopack X Carboxen 1000 Chromosorb W Carbopack X Chromosorb W
Carbopack X Carboxen 1000 Chromosorb W Carbopack X Carbopack X
Carbopack X Carboxen 1000 Chromosorb W Carboxen 1000 Carboxen 1000
X (A) allows the analysis of n-alkanes C7 and even high water content of the samples does not interfere with the analysis, but its sorption capacity is limited. The INCAT device containing three layers of Carboxen 1000 (B) captures alkanes quantitatively, but does not allow the desorption of higher alkanes at the given conditions. The INCAT device composed of three layers of Chromosorb W (C) is not able to capture hydrocarbons C12 , but all the higher ones can be quantitatively desorbed. The INCAT device with two layers of Carbopack X and one layer of Carboxen 1000 (D) appears to be suitable for the sorption of a wide range of n-alkanes, as it allows the adsorption of the lower hydrocarbons as well as the desorption of higher alkanes up to C18 . The INCAT device filled with three different sorbent beds (E), namely, Chromosorb W with 20% SE-54, Carbopack X and Carboxen 1000, was capable of the sorption of a wide range of analytes and had better sorption characteristics for higher alkanes C12 , but its background was too high, caused by the volatilization of siloxanes from the silicone stationary phase SE-54, making thus the identification and quantification of n-alkanes and monomethylalkanes at trace levels impossible. Therefore, sample analyses were carried out using INCAT device with two layers of Carbopack X and one layer of Carboxen 1000 (D), which had comparable sorption-desorption characteristics as device “E”, though the desorption of higher alkanes was incomplete. This device had very low background and enabled the analysis of the whole studied range of analytes. 3.2. Method validation The limits of detection, limits of quantification, retention indices and response factors of the used methods for the analysis of C6 C19 n-alkanes and C9 C19 monomethylalkanes are listed in Table 3. The separation was achieved using a 100 m long capillary column to obtain the maximal possible separation of monomethylalkanes with similar physico-chemical properties. The separation of C12 monomethylalkanes with methyl-branching near the middle of the carbon chain is the most problematic, as by GC × GC separation with 3 s modulation time, resolution on the first column is reduced. Limits of GC × GC-FID detection were in the range from 19 pg for dodecane to 110 pg for 3-methyloctane. The differences among the GC × GC-FID detection limits are lower than in MS/MS detection due to the equality of the response factors of the individual isomers. A significant decrease in detection limits was observed only for incompletely separated monomethylalkanes, especially with methyl-branching in the middle of the carbon chain. Limits of GC–MS/MS detection were in the range from 2.1 pg for n-tetradecane to 86 pg for 5-methyloctadecane. The wide range of detection limits is the result of the different intensity of the fragments chosen for SRM transitions. Especially for monomethylalkanes, where these fragments often represent less than 1% of the relative response and some of the selected SRM transitions for monomethylalkanes have a relatively low selectivity. For the analysis of linear n-alkanes, SRM transitions from the molecular ion to ions 57 or 71 were used, which provide a higher response. Retention indices were calculated according to the GC–MS/MS measurements where higher separation efficiency and complete separation was achieved by means of selective SRM transitions
Fig. 3. GC × GC-FID chromatogram of separation of the standard mixture of C16 monomethylalkanes and n-alkane (peaks are numbered according to Table 1).
Detection limits obtained by GC–MS/MS are lower than those obtained by GC × GC-FID, except for 3-methyldecane, 6-methyltetradecane, 6- and 3-methylpentadecane, 6- and 3methylhexadecane, 7- and 3-methylheptadecane, which effect could be caused by the lower selectivity of the SRM transitions chosen for these compounds. Retention indices of selected monomethylalkanes show a good correlation with published indices [16] with a maximum difference of three index units, what was caused by the different temperature gradient during separation. Response factors vary from 0.024 for 3-methylhexadecane to 9.3 for 3-methyloctadecane. Differences in response factors were caused by the different sensitivity of the individual SRM transitions. Therefore, the quantification of monomethylalkanes requires the use of internal standards or the knowledge of their response factors compared to the corresponding n-alkane.
3.3. Real samples Fig. 3 shows the chromatogram of separation of the standard mixture of monomethyl- and n-alkanes C16 by GC × GC-FID. From the chromatogram is obvious, that isomers with methyl-branching near the middle of the carbon chain could not be fully separated even by the use of a high-resolution capillary column. Fig. 4 shows a GC × GC-FID chromatogram of separation of breath volatiles of a 47year-old person with a triple coronary artery bypass, who smokes 40 cigarettes a day. As can be seen from this chromatogram, the GC × GC system in the second dimension allows the separation of alkanes, resp. isoalkanes from the possible sample contaminants as siloxanes arising from the volatilization of the stationary phase of the capillary column being responsible for the column drift, the siloxanes released from the septum in the GC injector and from the sealing septum of the Tedlar sampling bags. It also allows the separation of more polar breath components, as alkenes, e.g. compound signed in Fig. 4 as C, representing hexadec-1-ene, and other polar compounds as alcohols, aldehydes, ketones or aromatic compounds. In this exhaled breath sample we identified compounds eluting in the retention times of monomethylalkanes as the corresponding monomethylalkanes and their concentration in the sample was calculated according to the standard mixtures.
A. Hengerics Szabó et al. / J. Chromatogr. B 978–979 (2015) 62–69 Table 3 Limits of detection and quantification (LOD and LOQ), retention indices and response factors of C6 Peak no.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 a
Not focusing.
Compound name
Hexane Heptane Octane 4-Methyloctane 2-Methyloctane 3-Methyloctane Nonane 5-Methylnonane 4-Methylnonane 2-Methylnonane 3-Methylnonane Decane 5-Methyldecane 4-Methyldecane 2-Methyldecane 3-Methyldecane Undecane 6-Methylundecane 5-Methylundecane 4-Methylundecane 2-Methylundecane 3-Methylundecane Dodecane 6-Methyldodecane 5-Methyldodecane 4-Methyldodecane 2-Methyldodecane 3-Methyldodecane Tridecane 7-Methyltridecane 6-Methyltridecane 5-Methyltridecane 4-Methyltridecane 2-Methyltridecane 3-Methyltridecane Tetradecane 7-Methyltetradecane 6-Methyltetradecane 5-Methyltetradecane 4-Methyltetradecane 2-Methyltetradecane 3-Methyltetradecane Pentadecane 8-Methylpentadecane 7-Methylpentadecane 6-Methylpentadecane 5-Methylpentadecane 4-Methylpentadecane 2-Methylpentadecane 3-Methylpentadecane Hexadecane 8-Methylhexadecane 7-Methylhexadecane 6-Methylhexadecane 5-Methylhexadecane 4-Methylhexadecane 2-Methylhexadecane 3-Methylhexadecane Heptadecane 9-Methylheptadecane 8-Methylheptadecane 7-Methylheptadecane 6-Methylheptadecane 5-Methylheptadecane 4-Methylheptadecane 2-Methylheptadecane 3-Methylheptadecane Octadecane 9-Methyloctadecane 8-Methyloctadecane 7-Methyloctadecane 6-Methyloctadecane 5-Methyloctadecane 4-Methyloctadecane 2-Methyloctadecane 3-Methyloctadecane Nonadecane
GC × GC-FID
C19 n-alkanes and C9
GC–MS/MS
LOD (pg)
LOQ (pg)
LOD (pg)
LOQ (pg)
n.f.a n.f.a 45 83 68 110 40 57 71 52 60 34 34 30 21 23 22 37 36 41 31 25 19 29 33 32 24 30 22 44 39 40 39 24 26 25 42 41 46 39 40 33 33 38 39 37 38 30 30 28 32 35 29 46 54 42 30 33 33 56 56 46 69 60 59 66 56 36 78 82 79 81 86 85 74 80 47
n.f.a n.f.a 150 270 220 360 130 190 230 170 200 110 89 170 120 77 72 120 120 140 100 84 64 98 110 110 81 100 72 150 130 130 130 79 87 82 140 140 150 130 130 110 110 130 130 120 130 99 98 94 105 97 100 150 180 140 97 110 110 190 190 120 180 160 160 170 150 120 260 270 260 270 290 280 250 270 155
3.0 2.8 2.8 49 42 81 2.6 32 10 20 11 3.8 13 18 12 24 3.5 11 24 9.8 8.4 17 5.8 20 16 12 11 19 3.4 20 33 25 22 18 21 2.1 35 45 18 14 12 23 2.7 26 28 45 29 19 19 39 2.2 29 29 53 28 23 16 36 4.4 16 45 79 67 30 29 41 59 4.4 34 35 34 77 86 48 53 6.5 6.6
9.9 9.4 9.3 160 140 270 8.9 110 34 67 38 13 43 61 39 81 12 36 81 33 28 56 19 68 54 40 35 63 11 67 110 82 72 61 69 7.0 120 150 60.0 47 41 76 8.9 86 92 150 95 62 64 130 7.4 98 96 180 92 77 52 120 15 55 150 260 220 100 97 140 200 15 110 120 110 260 290 160 180 22 22
67
C19 monomethylalkanes. Retention index
Response factor
600.0 700.0 800.0 861.4 862.3 869.2 900.0 959.7 961.3 963.9 970.6 1000.0 1058.1 1061.2 1064.9 1070.6 1100.0 1155.5 1156.7 1160.6 1165.0 1171.3 1200.0 1253.8 1255.7 1260.3 1265.0 1271.2 1300.0 1351.4 1352.0 1355.0 1359.6 1364.8 1371.3 1400.0 1449.7 1451.3 1454.3 1459.5 1464.8 1471.5 1500.0 1547.7 1548.4 1550.3 1553.5 1559.2 1565.0 1571.3 1600.0 1646.4 1646.9 1649.6 1653.3 1658.9 1664.5 1672.0 1700.0 1745.2 1745.8 1746.2 1748.9 1753.2 1759.2 1764.5 1772.1 1800.0 1843.5 1843.9 1845.3 1848.5 1852.7 1858.9 1864.3 1870.1 1900.0
1.0 1.0 1.0 7.8 3.7 2.1 1.0 0.41 0.44 0.13 0.16 1.0 4.6 8.0 3.8 2.6 1.0 0.24 0.13 0.11 0.13 0.069 1.0 2.6 3.7 1.1 1.4 0.26 1.0 0.28 0.18 0.29 0.065 0.083 0.036 1.0 0.14 0.16 0.40 0.046 0.055 0.035 1.0 0.20 0.092 0.026 0.050 0.032 0.031 0.037 1.0 0.14 0.36 0.18 0.077 0.057 0.086 0.024 1.0 0.52 0.29 0.029 0.033 0.049 0.047 0.033 0.15 1.0 0.58 1.4 1.6 0.12 0.066 0.067 0.058 9.3 1.0
68
A. Hengerics Szabó et al. / J. Chromatogr. B 978–979 (2015) 62–69
Table 4 Concentrations of the n-alkanes and monomethylalkanes and relative standard deviations calculated from three measurements in exhaled breath samples of a 47-year-old smoker with a triple coronary artery bypass.
a
Compound number
Compound name
GC × GC-FID amount (pg)
RSD (%)
GC–MS/MS amount (pg)
RSD (%)
12 23 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 66 67 68
Decane Dodecane Pentadecane 8-Methylpentadecane 7-Methylpentadecane 6-Methylpentadecane 5-Methylpentadecane 4-Methylpentadecane 2-Methylpentadecane 3-Methylpentadecane Hexadecane 8-Methylhexadecane 7-Methylhexadecane 6-Methylhexadecane 5-Methylhexadecane 4-Methylhexadecane 2-Methylhexadecane 3-Methylhexadecane Heptadecane 2-Methylheptadecane 3-Methylheptadecane Octadecane
40 48 70 40 72 60 63 79 79 85 450 60 75 83 85 87 95 100 113 44 46 78
19 25 16 27 29 25 15 19 15 13 10 28 25 29 14 22 12 9 19 15 11 29
21 26 63 n.d.a n.d. n.d. n.d. n.d. n.d. n.d. 420 n.d. n.d. n.d. n.d. n.d. n.d. n.d. 104 n.d. n.d. 75
23 21 17
12
15
24
Not detected.
Fig. 4. GC × GC-FID chromatogram of separation of C16 monomethylalkanes and n-alkane found in an exhaled breath sample of a 47-year-old smoker with a triple coronary artery bypass (peaks are numbered according to Table 1). A, siloxanes from column bleeding; B, contamination with siloxanes from septum; C, hexadec-1-ene.
Fig. 5 shows the chromatogram of separation of the standard mixture of monomethylpentadecanes and n-hexadecane by GC–MS/MS. As can be seen from this chromatogram, all isomers of C16 monomethylalkanes can be identified and quantified using the selected SRM transitions. In the case of the analysis of the exhaled breath sample by this method, the specific SRM transitions were used for each monomethylalkane to ensure the identification of the targeted components of exhaled breath. The exhaled breath sample contained only n-hexadecane, and no monomethylpentadecanes were detected as was assumed from the literature and the GC × GCFID analyses. The amounts of the detected monomethyl- and n-alkanes in the exhaled breath samples of a 47-year-old smoker with a triple coronary artery bypass, together with the relative standard
Fig. 5. GC–MS/MS chromatogram of separation of the standard mixture of C16 monomethylalkanes and n-alkane (peaks are numbered according to Table 1).
deviations of the measurements calculated from three analyses are listed in Table 4. As we can see from Table 4, by GC–MS/MS no monomethylalkanes were detected despite the low limits of the detection and selective transitions. Therefore it is obvious that the GC × GC-FID results were false-positive and the compounds identified by GC × GC-FID as monomethylalkanes were most probably substances with similar elution properties, but with a different mass fragmentation pattern from monomethylalkanes,
A. Hengerics Szabó et al. / J. Chromatogr. B 978–979 (2015) 62–69
and therefore they were not detected by the developed GC–MS/MS method. However, the presence of n-alkanes was confirmed by both techniques and the obtained amounts show a good agreement. The relatively high RSD of the measurements was most probably caused by the variability of the breath volume pushed through the INCAT device. The results of the analyses indicate that identification of monomethylalkanes in exhaled breath samples based only on matching retention characteristics might not be proven by other, more selective techniques. 4. Conclusion A new multibed arrangement of INCAT device with two layers of Carbopack X and one layer of Carboxen 1000 as sorbent materials was developed for sampling and preconcentration of non-polar analytes with a wide range of volatility in gaseous samples. The device was used for the analysis of C6 C19 nalkanes and C9 C19 monomethylalkanes in an exhaled breath sample. The article was aimed at the analysis of all C6 C19 alkanes and C9 C19 monomethylalkanes by GC techniques in exhaled breath samples. For the analysis of the selected analytes both GC × GC-FID and GC–MS/MS techniques were used as the complete separation of monomethylalkanes by GC × GC was not achieved even if using a high-resolution capillary column. However, the use of the Carbowax column in the second dimension of GC × GC enabled the complete separation of alkanes from other coeluting sample components (alkenes, aromatic hydrocarbons, etc.). GC–MS/MS enabled the separation of all monomethylalkanes because of the selective SRM transitions used for each isomer. The amount of nalkanes detected in the exhaled breath samples was similar by both techniques (GC × GC-FID and GC–MS/MS), but monomethylalkanes were detected only by GC × GC. This was most probably caused by the presence of hydrocarbons with more methyl groups eluting in the area of the selected monomethylalkanes what explains their absence in the GC–MS/MS spectra with MS/MS transitions optimized for the selected monomethylalkanes. Most articles about the analysis of exhaled breath samples rely only on spectral library match by which method the identification of isomers cannot be performed. Another mode of identification of monomethylalkanes in these articles is based on the correlation of retention characteristics of the analytes with standards. The authors have not found articles where all C6 C19 alkanes and C9 C19 monomethylalkanes were used as standards as most of these are not commercially available. This article shows that the identification based only on retention characteristics can lead to false-positive interpretations as by a highly selective method (GC–MS/MS) the identification might not be confirmed. The obtained results might prove that identification based only on spectral library match or matching retention times can lead to misinterpretation of results. The results of this manuscript also suggest, that monomethylalkanes as markers of diseases should be confirmed by highly selective identification techniques, as GC–MS/MS. Acknowledgements This publication is the result of the project implementation: ITMS 26240220071, ITMS 26240220086 and ITMS 26240220007 supported by the Research & Development Operational Program funded by the ERDF. Research was carried out within the framework of the Specific University Research (SVV260084). Work was also supported by the Slovak Research and Development Agency
69
under the contract numbers APVV-0840-11, APVV-0416-10 and APVV-0061-11. References ˇ [1] T.S. Wang, P. Spanˇ el, D. Smith, in: A. Amann, D. Smith (Eds.), Breath Analysis for Clinical Diagnosis and Therapeutic Monitoring, World Scientific, Singapore, 2005, pp. 479–490. ˇ [2] A. Amann, P. Spanˇ el, D. Smith, Mini Rev. Med. Chem. 7 (2007) 115–129. [3] W. Miekisch, J.K. Schubert, G.F.E. Noeldge-Schomburg, Clin. Chim. Acta 347 (2004) 25–39. [4] S.A. Kharitonov, P.J. Barnes, Biomarkers 7 (2002) 1–32. [5] T.M. Dwyer, Lung 182 (2004) 241–250. [6] L.M. Gonzalez-Reche, A. Kucharczyk, A.K. Musiol, T. Kraus, Rapid Commun. Mass Spectrom. 20 (2006) 2747–2752. [7] M. Phillips, K. Gleeson, J.M.B. Hughes, J. Grennberg, R.N. Cataneo, L. Baker, W.P. McVay, Lancet 353 (1999) 1930–1933. [8] M. Phillips, R.N. Cataneo, A.R. Cummin, A.J. Gagliardi, K. Gleeson, J. Greenberg, R.A. Maxfield, W.N. Rom, Chest 123 (2003) 2115–2123. [9] C.H. Deng, X.M. Zhang, N. Li, J. Chromatogr. B 808 (2004) 269–277. [10] J. Choi, L.A. Hoffman, G.W. Rodway, J.M. Sethi, Biol. Res. Nurs. 7 (2006) 241–255. [11] M. McCulloch, T. Jezierski, M. Broffman, A. Hubbard, K. Turner, T. Janecki, Integr. Cancer Ther. 5 (2006) 30–39. [12] M. Phillips, R.N. Cataneo, R. Condos, G.A.R. Erickson, J. Greenberg, V. La Bombardi, M.I. Munawar, O. Tietje, Tuberculosis 87 (2007) 44–52. [13] J. Chladkova, I. Krcmova, J. Chladek, P. Cap, S. Micuda, Y. Hanzalkova, Respiration 73 (2006) 173–179. [14] C. Gessner, S. Hammerschmidt, H. Kuhn, G. Hoheisel, A. Gillissen, U. Sack, H. Wirtz, Resp. Med. 101 (2007) 2271–2278. [15] C.E. Davis, M.J. Bogan, S. Sankaran, M.A. Molina, B.R. Loyola, W. Zhao, W.H. Benner, M. Schivo, G.R. Farquar, N.J. Kenyon, M. Frank, IEEE Sens. J. 10 (2010) 114–122. [16] Zˇ . Krkoˇsová, R. Kubinec, L. Soják, A. Amann, J. Chromatogr. A 1179 (2008) 59–68. [17] M. Phillips, R.N. Cataneo, J. Greenberg, R. Gunawardena, A. Naidu, F. RahbariOskoui, J. Lab. Clin. Med. 136 (2000) 243–249. [18] M. Phillips, J.P. Boehmer, R.N. Cataneo, T. Cheema, H.J. Eisen, J.T. Fallon, P.E. Fisher, A. Gass, J. Greenberg, J. Kobashigawa, D. Mancini, B. Rayburn, M.J. Zucker, J. Heart Lung Transplant. 23 (2004) 701–708. [19] G.D. Bell, J. Weil, G. Harrison, A. Morden, P.H. Jones, P.W. Gant, J.E. Trowell, A.K. Yoong, T.K. Daneshmend, R.F. Logan, Lancet 1 (1987) 1367–1368. [20] D.Y. Graham, D.J. Evans, L.C. Alpert, P.D. Klein, D.G. Evans, A.R. Opekun, T.W. Boutton, Lancet 1 (1987) 1174–1177. [21] B. Dordoni, R.P. Thompson, R. Williams, Gut 16 (1975) 400. [22] R. Platzer, I. Gikalov, A. Kupfer, Schweiz. Med. Wochenschr. 106 (1976) 317–318. [23] A. Modak, J. Breath Res. 3 (2009) 040201. [24] H.L. Lord, W. Zhan, J. Pawliszyn, Anal. Chim. Acta 677 (2010) 3–18. [25] W. Filipiak, A. Filipiak, C. Ager, H. Wiesenhofer, A. Amann, J. Breath Res. 6 (2012) 027107. [26] I.Y. Eom, J. Pawliszyn, J. Sep. Sci. 31 (2008) 2283–2287. [27] I.Y. Eom, V.H. Niri, J. Pawliszyn, J. Chromatogr. A 1196–1197 (2008) 10–14. ´ L. [28] H. Jurdáková, R. Kubinec, M. Jurˇciˇsinová, Z. Krkoˇsová, J. Blaˇsko, I. Ostrovsky, Soják, V.G. Berezkin, J. Chromatogr. A 1194 (2008) 161–164. [29] V.H. Niri, I.Y. Eom, F.R. Kermani, J. Pawliszyn, J. Sep. Sci. 32 (2009) 1075–1080. [30] J.A. Koziel, M. Odziemkowski, J. Pawliszyn, Anal. Chem. 73 (2001) 47–54. [31] M. Mieth, S. Kischkel, J.K. Schubert, D. Hein, W. Miekisch, Anal. Chem. 81 (2009) 5851–5857. [32] M. Mieth, J.K. Schubert, T. Gröger, B. Sabel, S. Kischkel, P. Fuchs, D. Hein, R. Zimmermann, W. Miekisch, Anal. Chem. 82 (2010) 2541–2551. [33] P. Trefz, S. Kischkel, D. Hein, E.S. James, J.K. Schubert, W. Miekisch, J. Chromatogr. A 1219 (2012) 29–38. [34] R. Kubinec, V.G. Berezkin, R. Górová, G. Addová, H. Mraˇcnová, L. Soják, J. Chromatogr. B 800 (2004) 295–301. ˇ cík, I. Ostrovsky, ´ L. Soják, V. Berezkin, [35] P. Pˇrikryl, R. Kubinec, H. Jurdáková, J. Sevˇ Chromatographia 64 (2006) 65–70. [36] M. Caldeira, R. Perestrelo, A.S. Barros, M.J. Bilelo, A. Morête, J.S. Câmara, S.M. Rocha, J. Chromatogr. A 1254 (2012) 87–97. [37] M. Phillips, R.N. Cataneo, A. Chaturvedi, P.D. Kaplan, M. Libardoni, M. Mundada, U. Patel, X. Zhang, PLOS ONE 8 (2013) e75274, 1–7. [38] M. Libardoni, P.T. Stevens, J.H. Waite, R. Sacks, J. Chromatogr. B 842 (2006) 13–21. [39] J.M. Sanchez, R.D. Sacks, Anal. Chem. 75 (2003) 2231–2236. [40] J.M. Sanchez, R.D. Sacks, Anal. Chem. 78 (2006) 3046–3054. [41] M.C. Simmons, D.B. Richardson, I. Dvoretsky, in: R.P.W. Scott (Ed.), Gas Chromatography, Butterworth, London, 1960, p. 211. [42] J. Glastrup, J. Chromatogr. A 827 (1988) 133–136. [43] P. Mochalski, J. King, K. Unterkofler, A. Amann, Analyst 138 (2013) 1405–1418. [44] J. Beens, M. Adahchour, R.J.J. Vreuls, K. van Altena, U.A.T. Brinkman, J. Chromatogr. A 919 (2001) 127–132.
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
The analysis of linear and monomethylalkanes in exhaled breath samples by GC × GC-FID and GC–MS/MS Alexandra Hengerics Szabó a , Peter Podolec a , Viktória Ferenczy a , Róbert Kubinec a,∗ , Jaroslav Blaˇsko a , Ladislav Soják a , Renáta Górová a , Gabriela Addová a , Ivan Ostrovsky´ a , c,d ˇ ˇ Jozef Viˇsnovsk y´ a,b , Václav Bierhanzl c , Radomír Cabala , Anton Amann e,f a
Institute of Chemistry, Faculty of Natural Sciences, Comenius University, Mlynská dolina CH-2, 842 15 Bratislava, Slovakia SynthCluster, s.r.o., Komenského 1439, 900 01 Modra, Slovakia c Department of Analytical Chemistry, Faculty of Science, Charles University in Prague, Albertov 6, 128 43 Prague 2, Czech Republic d Institute of Forensic Medicine and Toxicology, General University Hospital in Prague, U Nemocnice 2, 128 08 Prague 2, Czech Republic e Univ.-Clinic of Anesthesia and Intensive Care, Innsbruck Medical University, Anichstrasse 35, A-6020 Innsbruck, Austria f Breath Research Institute of the University of Innsbruck, Rathausplatz 4, A-6850 Dornbirn, Austria b
a r t i c l e
i n f o
Article history: Received 8 August 2014 Accepted 24 November 2014 Available online 3 December 2014 Keywords: Exhaled breath INCAT Monomethylalkanes n-Alkanes Needle trap
a b s t r a c t A new arrangement of the INCAT (inside needle capillary adsorption trap) device with Carbopack X and Carboxen 1000 as sorbent materials was applied for sampling, preconcentration and injection of C6 C19 n-alkanes and their monomethyl analogs in exhaled breath samples. For the analysis both GC–MS/MS and GC × GC-FID techniques were used. Identification of the analytes was based on standards, measured retention indices and selective SRM transitions of the individual isomers. The GC–MS/MS detection limits were in the range from 2.1 pg for n-tetradecane to 86 pg for 5-methyloctadecane. The GC × GC-FID detection limits ranged from 19 pg for n-dodecane to 110 pg for 3-methyloctane. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The analysis of the compounds in exhaled breath can provide insight into different biochemical processes in the healthy and the diseased human body [1,2]. Breath constituents are composed of both volatile [3] and non-volatile compounds [4–6], that together form an “exhaled metabolome.” A large number of compounds have been associated with different diseases, such as: ketones in ketoacidosis for diabetics [7–12], aldehydes in lung cancer patients [7–11], compounds associated with tuberculosis infection [12], and exhaled nitrate and nitric oxide in nonspecific pulmonary inflammation and/or asthma [10,13–15]. Alkanes in exhaled breath have also been proposed as endogenous marker compounds [3,7,8,12]. Their analysis is of medical importance, since it may lead to non-invasive clinical tests for carcinomas, oxidative stress and heart transplant rejection [16]. In a study of the relation of age and the presence of methylalkanes in exhaled
∗ Corresponding author. Tel.: +421 2 602 96 330; fax: +421 2 602 96 337. E-mail address: [email protected] (R. Kubinec). http://dx.doi.org/10.1016/j.jchromb.2014.11.026 1570-0232/© 2014 Elsevier B.V. All rights reserved.
breath [17], 22 different C6 C17 normal and monomethylalkanes were proposed as marker compounds of oxidative stress, the latter being considered as a pathologic mechanism in aging and several diseases. Phillips et al. [7] suggest that oxidative stress degrades membrane polyunsaturated fatty acids, thus producing n-alkanes and methylalkanes which are excreted in exhaled breath and which are considered to vary with the extent of oxidative stress. Phillips et al. [8] proposed the following compounds for the detection of lung cancer: butane, 3-methyltridecane, 7-methyltridecane, 4-methyloctane, 3-methylhexane, heptane, 2-methylhexane, pentane, and 5-methyldecane. For the detection of heart transplant rejection he proposed 2-methylpropane, 5-methyloctadecane, 6-methyloctadecane, 2methylpentadecane, octane, 2-methylheptane, 3-methylundecane, 2-methyloctadecane, and 2-methylhexadecane [18]. In addition to endogenously produced VOCs, 13 C- or 14 C-labeled precursor compounds are used for breath tests. An example is 13 Clabeled urea, used in gastroenterology tests for detection of Helicobacter pylori bacterial infections associated with stomach ulcers [19,20]. Other examples for precursors are aminopyridine and 14 C-ethanol for testing of impaired liver function [21,22] and 13 C-dextromethorphan for testing of the CYP2D6 activity [23].
A. Hengerics Szabó et al. / J. Chromatogr. B 978–979 (2015) 62–69
Sample preparation is the cornerstone of the chemical analysis. Preconcentration is the crucial step when volatile organic compounds (VOCs) often occurring in concentrations as low as ppb or ppt are to be determined. Needle trap devices (NTDs) represent promising tools for a robust and reproducible sample preparation, combining advantages of SPE and SPME [24,25]. One of their greatest advantages is that no additional equipment, apart from a heated GC injector, is necessary for application [26]. Several applications of single layer packed sorbents such as Carboxen and divinylbenzene have been described in the field of environmental monitoring (e.g. analysis of BTEX or higher alkanes) [27–30]. For the analysis of more complex samples containing compounds with a wide range of polarity and volatility single-bed NTDs are insufficient and therefore multibed NTDs are needed. Trefz et al. recently proved the applicability of multibed NTDs and expansive flow technique in medical breath analysis [31–33]. INCAT (inside needle capillary adsorption trap) is a needle trap device. Its new arrangement was developed by Kubinec et al. [28,34,35]. This device is more robust than most needle trap devices or SPME fibers and gives comparable results [35]. The aim of this work was the development of an INCAT device enabling the analysis of non-polar compounds with a wide range of volatility. Linear and monomethylalkanes were chosen for this study as they are considered as breath markers of various diseases. Many articles are focused on the analysis of a wide range of breath volatiles using various analytical techniques, from which GC–MS and GC-FID are still the most common. There is, however only a limited number of articles about the GC × GC analysis of exhaled breath samples [32,36–40]. Also, the identification of compounds present in breath samples is very often based only on spectral library match or on similar retention characteristics with standards, which approach might not be sufficient. The aim of the manuscript is not the analysis of all compounds present in breath samples, but the analysis and identification of the selected n-alkanes and monomethylalkanes. The separation and identification of monomethylalkanes in a broad range of carbon atoms is problematic due to the multicomponent character and close retention of isomers with methyl-branching near the middle of the carbon chain. The lack of standard reference materials and poor reproducibility of published retention data are also critical points. This manuscript is focused on the separation, identification and quantification of n-alkanes and monomethylalkanes using GC × GC-FID and GC–MS/MS techniques which can help in the future identification of these analytes in exhaled breath samples. It does not deal with the possible medical impact of the analytes, but is focused on their reliable analysis in exhaled breath. The article deals with the analysis of alkanes and monomethylalkanes which although are presented in scientific articles as markers of various diseases, their identification might not be adequate if only based on spectral library match or similar retention characteristics with standards.
63
o.d./0.9 mm i.d. diameter by sintering at 680 ◦ C for 45 min in a laboratory furnace. Glass beads were obtained from BDH (Poole, England), laboratory furnace CLASIC 1313 from CLASIC CZ s.r.o. ˇ Czech Republic). MTB sampling valves were purchased (Revnice, from Hamilton (Reno, Nevada, USA).
2.2. Sample preparation Monomethylalkanes C9 C19 as a model mixture were prepared from n-alkanes mixture C8 C18 by methylene insertion reaction [41] in an apparatus by Glastrup [42] by using gaseous diazomethane and UV radiation. The C8 C18 n-alkanes used for methylene insertion reaction were obtained from Supelco (Bellefonte, PA, USA) and C6 C7 n-alkanes were obtained from Sigma–Aldrich (Germany). The standard gas mixtures were prepared similarly as by Mochalski et al. [43]. Primary standards were prepared in 1 L glass bulbs (Supelco, Canada). Prior to use the bulbs were cleaned with methanol and dried at 80 ◦ C. Then the bulb was evacuated using a vacuum membrane pump and 1 L of a liquid analyte was injected into the bulb through a rubber septum. The bulb was heated to 70 ◦ C for 30 min to ensure the evaporation. Gas mixtures obtained through evaporation of liquid standards were appropriately diluted in synthetic breath (78% N2 , 15% O2 , 4% CO2 , 3% H2 O) in 3 L Tedlar bags (Supelco, Bellefonte, USA) to obtain the desired concentration levels of analytes for further analyses. Calibration standards were prepared by this method with analyte concentrations 1; 2; 5; 10; 20; 50; 100; 500 and 1000 pg L−1 . Samples of exhaled breath of consenting person were collected in 3 L gas-sampling Tedlar bags. Tedlar bags were chosen for sample collection due to their reusability, low background and high stability for collected hydrocarbons with no relevant losses even when stored up to a week [43]. Our goal was to find more of the C6 C19 alkanes and C9 C19 monomethylalkanes in an exhaled breath sample. More individuals were selected for analyses, but the samples of the chosen individual (a 47-year-old heavy smoker with a triple coronary artery bypass) contained the highest content of monomethylalkanes, detected by GC × GC-FID technique. All samples were collected after the volunteer had left a period of at least 1 h without consuming any food or beverages. Before sample collection, the Tedlar bags were flushed three times with the exhaled breath samples of the individuals. For each exhaled breath sample, 1000 mL of breath previously collected in Tedlar bag was pulled through the sorption trap using membrane vacuum pump KNF LAB (KNF, Neuberger, Freiburg, Germany). The extraction of analytes with INCAT device from the samples collected in Tedlar bags was performed at oven temperature of 40 ◦ C to avoid water condensation in the breath samples during sampling. Each sample was analyzed three times.
2.3. Needle trap devices 2. Experimental 2.1. Materials Chromosorb W with 20% SE-54 (meshes 60–80), Carbopack X (meshes 40–60, specific surface area 250 m2 g−1 ) and Carboxen 1000 (meshes 60–80, specific surface area 1200 m2 g−1 ) as packing materials for needle trap devices were obtained from Supelco (Bellefonte, USA). Stainless steel needles (cannula) 90 mm long with an outer/inner diameter of 1.3 mm/1.1 mm, and 1.1 mm/0.9 mm were from Nissho (Osaka, Japan). Frits of 5 mm length were prepared from glass beads (0.125–0.2 mm) filled in a needle of 1.1 mm
Needle trap devices developed in our laboratory based on our previous work [28] were investigated. The needle traps were packed with 39 mm length of the chosen sorbents, which were locked into position by frits with sintered glass beads. Sorbents were packed in 13 mm long layers into the INCAT devices. The novelty of the developed INCAT devices lie in the use of adsorbents with different adsorption strengths in series, allowing the analysis of a much wider range of volatile analytes than the previously published INCAT devices. Prior to first usage all INCAT devices were conditioned in the heated injection port of a gas chromatograph for 2 h at temperature of 280 ◦ C under a permanent helium flow of 3.5 mL min−1 .
64
A. Hengerics Szabó et al. / J. Chromatogr. B 978–979 (2015) 62–69
2.4. GC × GC-FID and GC–MS/MS conditions 2.4.1. GC × GC-FID A Thermo Electron TRACE GC Ultra (TRACE 2DGC) was used to perform comprehensive two-dimensional GC. The TRACE 2DGC system was provided with the dual-stage CO2 jet modulator, as proposed by Beens et al. [44] driven by a dedicated electronics which ensured the synchronization between the valves activation and the signal acquisition. A fast FID was used as detector, capable of producing a digital signal at a sampling rate up to 300 Hz. An injection system with modified metal liner (based on previous work of Jurdáková et al. [28] and optimized for Thermo Trace GC) to minimize dead volumes inside the injection system was used. The liner was made from stainless steel concerning good thermal conductivity and inert surface of this material. It is composed of two metal parts, which are connected by thread, and viton tubing between the two metal parts as a seal. Using this modified liner the carrier gas flows directly through the needle trap device by the side hole and no additional source of inert gas is needed to displace analytes from device. The separation of the mixture of n-alkanes and monomethylalkanes requires the use of a high resolution capillary column. The first dimension column was a 100 m long capillary column with an internal diameter of 0.32 mm and a phase thickness of 0.25 m of methylsilicone OV-1 as a stationary phase (SPB-1, Supelco). It was connected through a deactivated press-fit to a Carbowax second dimension column (FFAP, Hewlett Packard), 1 m long with an internal diameter of 0.2 mm and a phase thickness of 0.33 m. The oven temperature was programmed at 2 ◦ C min−1 from 40 ◦ C to 210 ◦ C, which was held for 10 min. The carrier gas (helium) was supplied at 3.5 mL min−1 during the whole analysis. The injector was set to a temperature of 280 ◦ C operating in splitless mode and the FID detector was set to a temperature of 230 ◦ C. Samples were injected in splitless mode which was held on during the whole analysis time. The GC × GC analyses were carried out with a modulation cycle time of 3 s. Data acquisition and data handling were performed with a software version for GC × GC (HyperChrom) based on Thermo Electron Chrom-Card Data System. 2.4.2. GC–MS/MS The GC–MS/MS analyses were carried out with a Trace GC Ultra gas chromatograph equipped with a split–splitless injector and a TSQ Quantum XLS mass spectrometer (Thermo Fisher, Austin, TX, USA). The temperatures of the injector and the MS-transfer line were 280 ◦ C and 240 ◦ C, respectively. Gas chromatographic conditions were identical to the GC × GC settings, but the separation was performed using only the first dimension column (100 m long SPB-1). The MS/MS conditions were as follows: ion source temperature 200 ◦ C, electron energy 70 eV, emission current 50 A. Selected reaction monitoring (SRM) conditions were experimentally optimized for the selected analytes. The used SRM transitions and collision energies are listed in Table 1.
Fig. 1. Scheme of the INCAT device. N, stainless steel needle; F, frit made of sintered glass beads; A1–A3, layers of adsorbents; H, side hole; T, silicone and PTFE sealing; V, sampling valve.
where x1 stands for the selected analyte (monomethylalkane), n is the number of carbon atoms in the n-alkane eluting before this analyte and n + 1 the number of carbon atoms in the n-alkane eluting after it, tr stands for retention time and I for the retention index. As the response of the MS detector is different for different analytes, response factors for monomethylalkanes were also calculated according to the equation: RF =
Ax cn , An cx
where Ax is the peak area of the monomethylalkane, cx is its concentration, An stands for the peak area of the n-alkane with the same number of carbon atoms and cn is its concentration. 3. Results and discussion 3.1. Needle trap devices A new arrangement of INCAT device was developed for the extraction and preconcentration of n-alkanes and monomethylalkanes with a wide range of volatility. The device consists of a stainless steel needle filled with layers of adsorbents which are fixed into position by frits with sintered glass beads, a side hole for the better desorption of analytes in the injection port of GC by carrier gas, silicone and PTFE sealing as a side hole cover during sampling and a sampling valve. The scheme of INCAT device is shown in Fig. 1. The composition of sorbent layers for each INCAT device is listed in Table 2. INCAT devices were tested by the analysis of a mixture of n-alkanes C6 C18 . Fig. 2 shows the dependencies of the desorption curves of C6 C18 n-alkanes at amount of 500 pg for INCAT devices with different composition of sorbent layers. As it can be seen from Fig. 2, each prepared INCAT device shows different sorption characteristics for the analyzed mixture of nalkanes. The INCAT device containing three layers of Carbopack
2.5. Method validation Limits of detection and quantification (LOD and LOQ) were determined according to the IUPAC guidelines, by means of the signal to noise ratio (S/N). Noise was determined experimentally from the blank samples. LOD was defined as S/N of 3 and LOQ as S/N of 10. Retention indices of all selected analytes were calculated according to the equation: I(x1 ) = 100
tr (x1 ) − tr (n) + 100n, tr (n + 1) − tr (n)
Fig. 2. Relative abundance of C6 C18 n-alkanes after desorption from the prepared INCAT devices packed with different sorbent layers (devices are labeled according to Table 2).
A. Hengerics Szabó et al. / J. Chromatogr. B 978–979 (2015) 62–69 Table 1 Retention times, SRM transitions and collision energies used for the analysis of C6
C19 n-alkanes and C9
65
C19 monomethylalkanes.
Peak number
Compound
Retention time (min)
SRM transition
Collision energy (V)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77
Hexane Heptane Octane 4-Methyloctane 2-Methyloctane 3-Methyloctane Nonane 5-Methylnonane 4-Methylnonane 2-Methylnonane 3-Methylnonane Decane 5-Methyldecane 4-Methyldecane 2-Methyldecane 3-Methyldecane Undecane 6-Methylundecane 5-Methylundecane 4-Methylundecane 2-Methylundecane 3-Methylundecane Dodecane 6-Methyldodecane 5-Methyldodecane 4-Methyldodecane 2-Methyldodecane 3-Methyldodecane Tridecane 7-Methyltridecane 6-Methyltridecane 5-Methyltridecane 4-Methyltridecane 2-Methyltridecane 3-Methyltridecane Tetradecane 7-Methyltetradecane 6-Methyltetradecane 5-Methyltetradecane 4-Methyltetradecane 2-Methyltetradecane 3-Methyltetradecane Pentadecane 8-Methylpentadecane 7-Methylpentadecane 6-Methylpentadecane 5-Methylpentadecane 4-Methylpentadecane 2-Methylpentadecane 3-Methylpentadecane Hexadecane 8-Methylhexadecane 7-Methylhexadecane 6-Methylhexadecane 5-Methylhexadecane 4-Methylhexadecane 2-Methylhexadecane 3-Methylhexadecane Heptadecane 9-Methylheptadecane 8-Methylheptadecane 7-Methylheptadecane 6-Methylheptadecane 5-Methylheptadecane 4-Methylheptadecane 2-Methylheptadecane 3-Methylheptadecane Octadecane 9-Methyloctadecane 8-Methyloctadecane 7-Methyloctadecane 6-Methyloctadecane 5-Methyloctadecane 4-Methyloctadecane 2-Methyloctadecane 3-Methyloctadecane Nonadecane
5.21 8.02 12.96 16.41 16.46 16.85 18.58 22.62 22.73 22.91 23.36 25.35 29.49 29.71 29.97 30.38 32.47 36.38 36.47 36.74 37.05 37.50 39.52 43.16 43.29 43.60 43.92 44.34 46.29 49.60 49.64 49.83 50.13 50.46 50.88 52.73 55.76 55.86 56.04 56.36 56.68 57.09 58.83 61.59 61.63 61.74 61.93 62.26 62.59 62.96 64.62 67.17 67.20 67.35 67.55 67.86 68.17 68.58 70.12 72.49 72.52 72.54 72.68 72.91 73.22 73.50 73.90 75.36 77.53 77.55 77.62 77.78 77.99 78.30 78.57 78.86 80.35
86 → 57 100 → 57 114 → 85 70 → 55 84 → 55 70 → 55 128 → 57 84 → 55 70 → 55 98 → 69 142 → 71 142 → 57 98 → 69 70 → 55 112 → 55 98 → 69 156 → 56 98 → 56 112 → 55 126 → 55 126 → 55 112 → 55 170 → 57 98 → 56 84 → 55 140 → 56 140 → 69 140 → 69 184 → 70 112 → 55 98 → 56 84 → 55 154 → 69 154 → 69 140 → 69 198 → 57 112 → 70 98 → 69 84 → 55 168 → 83 168 → 83 154 → 69 212 → 57 126 → 55 140 → 69 126 → 55 168 → 69 182 → 69 182 → 69 168 → 69 226 → 57 126 → 56 112 → 55 98 → 69 182 → 69 196 → 96 196 → 96 182 → 69 240 → 57 126 → 55 112 → 55 168 → 69 182 → 69 196 → 70 210 → 97 210 → 97 196 → 70 254 → 71 140 → 69 126 → 55 112 → 55 196 → 69 210 → 83 224 → 83 224 → 83 112 → 55 268 → 71
10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 15 10 10 15 10 10 10 10 10 10 10 10 10 15 15 20 20 10 15 15 10 10 15 15 15 10 20 15 15 15 10
66
A. Hengerics Szabó et al. / J. Chromatogr. B 978–979 (2015) 62–69
Table 2 The composition of sorbent layers in the INCAT devices. INCAT device
Sorbent layer 1
Sorbent layer 2
Sorbent layer 3
A B C D E
Carbopack X Carboxen 1000 Chromosorb W Carbopack X Chromosorb W
Carbopack X Carboxen 1000 Chromosorb W Carbopack X Carbopack X
Carbopack X Carboxen 1000 Chromosorb W Carboxen 1000 Carboxen 1000
X (A) allows the analysis of n-alkanes C7 and even high water content of the samples does not interfere with the analysis, but its sorption capacity is limited. The INCAT device containing three layers of Carboxen 1000 (B) captures alkanes quantitatively, but does not allow the desorption of higher alkanes at the given conditions. The INCAT device composed of three layers of Chromosorb W (C) is not able to capture hydrocarbons C12 , but all the higher ones can be quantitatively desorbed. The INCAT device with two layers of Carbopack X and one layer of Carboxen 1000 (D) appears to be suitable for the sorption of a wide range of n-alkanes, as it allows the adsorption of the lower hydrocarbons as well as the desorption of higher alkanes up to C18 . The INCAT device filled with three different sorbent beds (E), namely, Chromosorb W with 20% SE-54, Carbopack X and Carboxen 1000, was capable of the sorption of a wide range of analytes and had better sorption characteristics for higher alkanes C12 , but its background was too high, caused by the volatilization of siloxanes from the silicone stationary phase SE-54, making thus the identification and quantification of n-alkanes and monomethylalkanes at trace levels impossible. Therefore, sample analyses were carried out using INCAT device with two layers of Carbopack X and one layer of Carboxen 1000 (D), which had comparable sorption-desorption characteristics as device “E”, though the desorption of higher alkanes was incomplete. This device had very low background and enabled the analysis of the whole studied range of analytes. 3.2. Method validation The limits of detection, limits of quantification, retention indices and response factors of the used methods for the analysis of C6 C19 n-alkanes and C9 C19 monomethylalkanes are listed in Table 3. The separation was achieved using a 100 m long capillary column to obtain the maximal possible separation of monomethylalkanes with similar physico-chemical properties. The separation of C12 monomethylalkanes with methyl-branching near the middle of the carbon chain is the most problematic, as by GC × GC separation with 3 s modulation time, resolution on the first column is reduced. Limits of GC × GC-FID detection were in the range from 19 pg for dodecane to 110 pg for 3-methyloctane. The differences among the GC × GC-FID detection limits are lower than in MS/MS detection due to the equality of the response factors of the individual isomers. A significant decrease in detection limits was observed only for incompletely separated monomethylalkanes, especially with methyl-branching in the middle of the carbon chain. Limits of GC–MS/MS detection were in the range from 2.1 pg for n-tetradecane to 86 pg for 5-methyloctadecane. The wide range of detection limits is the result of the different intensity of the fragments chosen for SRM transitions. Especially for monomethylalkanes, where these fragments often represent less than 1% of the relative response and some of the selected SRM transitions for monomethylalkanes have a relatively low selectivity. For the analysis of linear n-alkanes, SRM transitions from the molecular ion to ions 57 or 71 were used, which provide a higher response. Retention indices were calculated according to the GC–MS/MS measurements where higher separation efficiency and complete separation was achieved by means of selective SRM transitions
Fig. 3. GC × GC-FID chromatogram of separation of the standard mixture of C16 monomethylalkanes and n-alkane (peaks are numbered according to Table 1).
Detection limits obtained by GC–MS/MS are lower than those obtained by GC × GC-FID, except for 3-methyldecane, 6-methyltetradecane, 6- and 3-methylpentadecane, 6- and 3methylhexadecane, 7- and 3-methylheptadecane, which effect could be caused by the lower selectivity of the SRM transitions chosen for these compounds. Retention indices of selected monomethylalkanes show a good correlation with published indices [16] with a maximum difference of three index units, what was caused by the different temperature gradient during separation. Response factors vary from 0.024 for 3-methylhexadecane to 9.3 for 3-methyloctadecane. Differences in response factors were caused by the different sensitivity of the individual SRM transitions. Therefore, the quantification of monomethylalkanes requires the use of internal standards or the knowledge of their response factors compared to the corresponding n-alkane.
3.3. Real samples Fig. 3 shows the chromatogram of separation of the standard mixture of monomethyl- and n-alkanes C16 by GC × GC-FID. From the chromatogram is obvious, that isomers with methyl-branching near the middle of the carbon chain could not be fully separated even by the use of a high-resolution capillary column. Fig. 4 shows a GC × GC-FID chromatogram of separation of breath volatiles of a 47year-old person with a triple coronary artery bypass, who smokes 40 cigarettes a day. As can be seen from this chromatogram, the GC × GC system in the second dimension allows the separation of alkanes, resp. isoalkanes from the possible sample contaminants as siloxanes arising from the volatilization of the stationary phase of the capillary column being responsible for the column drift, the siloxanes released from the septum in the GC injector and from the sealing septum of the Tedlar sampling bags. It also allows the separation of more polar breath components, as alkenes, e.g. compound signed in Fig. 4 as C, representing hexadec-1-ene, and other polar compounds as alcohols, aldehydes, ketones or aromatic compounds. In this exhaled breath sample we identified compounds eluting in the retention times of monomethylalkanes as the corresponding monomethylalkanes and their concentration in the sample was calculated according to the standard mixtures.
A. Hengerics Szabó et al. / J. Chromatogr. B 978–979 (2015) 62–69 Table 3 Limits of detection and quantification (LOD and LOQ), retention indices and response factors of C6 Peak no.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 a
Not focusing.
Compound name
Hexane Heptane Octane 4-Methyloctane 2-Methyloctane 3-Methyloctane Nonane 5-Methylnonane 4-Methylnonane 2-Methylnonane 3-Methylnonane Decane 5-Methyldecane 4-Methyldecane 2-Methyldecane 3-Methyldecane Undecane 6-Methylundecane 5-Methylundecane 4-Methylundecane 2-Methylundecane 3-Methylundecane Dodecane 6-Methyldodecane 5-Methyldodecane 4-Methyldodecane 2-Methyldodecane 3-Methyldodecane Tridecane 7-Methyltridecane 6-Methyltridecane 5-Methyltridecane 4-Methyltridecane 2-Methyltridecane 3-Methyltridecane Tetradecane 7-Methyltetradecane 6-Methyltetradecane 5-Methyltetradecane 4-Methyltetradecane 2-Methyltetradecane 3-Methyltetradecane Pentadecane 8-Methylpentadecane 7-Methylpentadecane 6-Methylpentadecane 5-Methylpentadecane 4-Methylpentadecane 2-Methylpentadecane 3-Methylpentadecane Hexadecane 8-Methylhexadecane 7-Methylhexadecane 6-Methylhexadecane 5-Methylhexadecane 4-Methylhexadecane 2-Methylhexadecane 3-Methylhexadecane Heptadecane 9-Methylheptadecane 8-Methylheptadecane 7-Methylheptadecane 6-Methylheptadecane 5-Methylheptadecane 4-Methylheptadecane 2-Methylheptadecane 3-Methylheptadecane Octadecane 9-Methyloctadecane 8-Methyloctadecane 7-Methyloctadecane 6-Methyloctadecane 5-Methyloctadecane 4-Methyloctadecane 2-Methyloctadecane 3-Methyloctadecane Nonadecane
GC × GC-FID
C19 n-alkanes and C9
GC–MS/MS
LOD (pg)
LOQ (pg)
LOD (pg)
LOQ (pg)
n.f.a n.f.a 45 83 68 110 40 57 71 52 60 34 34 30 21 23 22 37 36 41 31 25 19 29 33 32 24 30 22 44 39 40 39 24 26 25 42 41 46 39 40 33 33 38 39 37 38 30 30 28 32 35 29 46 54 42 30 33 33 56 56 46 69 60 59 66 56 36 78 82 79 81 86 85 74 80 47
n.f.a n.f.a 150 270 220 360 130 190 230 170 200 110 89 170 120 77 72 120 120 140 100 84 64 98 110 110 81 100 72 150 130 130 130 79 87 82 140 140 150 130 130 110 110 130 130 120 130 99 98 94 105 97 100 150 180 140 97 110 110 190 190 120 180 160 160 170 150 120 260 270 260 270 290 280 250 270 155
3.0 2.8 2.8 49 42 81 2.6 32 10 20 11 3.8 13 18 12 24 3.5 11 24 9.8 8.4 17 5.8 20 16 12 11 19 3.4 20 33 25 22 18 21 2.1 35 45 18 14 12 23 2.7 26 28 45 29 19 19 39 2.2 29 29 53 28 23 16 36 4.4 16 45 79 67 30 29 41 59 4.4 34 35 34 77 86 48 53 6.5 6.6
9.9 9.4 9.3 160 140 270 8.9 110 34 67 38 13 43 61 39 81 12 36 81 33 28 56 19 68 54 40 35 63 11 67 110 82 72 61 69 7.0 120 150 60.0 47 41 76 8.9 86 92 150 95 62 64 130 7.4 98 96 180 92 77 52 120 15 55 150 260 220 100 97 140 200 15 110 120 110 260 290 160 180 22 22
67
C19 monomethylalkanes. Retention index
Response factor
600.0 700.0 800.0 861.4 862.3 869.2 900.0 959.7 961.3 963.9 970.6 1000.0 1058.1 1061.2 1064.9 1070.6 1100.0 1155.5 1156.7 1160.6 1165.0 1171.3 1200.0 1253.8 1255.7 1260.3 1265.0 1271.2 1300.0 1351.4 1352.0 1355.0 1359.6 1364.8 1371.3 1400.0 1449.7 1451.3 1454.3 1459.5 1464.8 1471.5 1500.0 1547.7 1548.4 1550.3 1553.5 1559.2 1565.0 1571.3 1600.0 1646.4 1646.9 1649.6 1653.3 1658.9 1664.5 1672.0 1700.0 1745.2 1745.8 1746.2 1748.9 1753.2 1759.2 1764.5 1772.1 1800.0 1843.5 1843.9 1845.3 1848.5 1852.7 1858.9 1864.3 1870.1 1900.0
1.0 1.0 1.0 7.8 3.7 2.1 1.0 0.41 0.44 0.13 0.16 1.0 4.6 8.0 3.8 2.6 1.0 0.24 0.13 0.11 0.13 0.069 1.0 2.6 3.7 1.1 1.4 0.26 1.0 0.28 0.18 0.29 0.065 0.083 0.036 1.0 0.14 0.16 0.40 0.046 0.055 0.035 1.0 0.20 0.092 0.026 0.050 0.032 0.031 0.037 1.0 0.14 0.36 0.18 0.077 0.057 0.086 0.024 1.0 0.52 0.29 0.029 0.033 0.049 0.047 0.033 0.15 1.0 0.58 1.4 1.6 0.12 0.066 0.067 0.058 9.3 1.0
68
A. Hengerics Szabó et al. / J. Chromatogr. B 978–979 (2015) 62–69
Table 4 Concentrations of the n-alkanes and monomethylalkanes and relative standard deviations calculated from three measurements in exhaled breath samples of a 47-year-old smoker with a triple coronary artery bypass.
a
Compound number
Compound name
GC × GC-FID amount (pg)
RSD (%)
GC–MS/MS amount (pg)
RSD (%)
12 23 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 66 67 68
Decane Dodecane Pentadecane 8-Methylpentadecane 7-Methylpentadecane 6-Methylpentadecane 5-Methylpentadecane 4-Methylpentadecane 2-Methylpentadecane 3-Methylpentadecane Hexadecane 8-Methylhexadecane 7-Methylhexadecane 6-Methylhexadecane 5-Methylhexadecane 4-Methylhexadecane 2-Methylhexadecane 3-Methylhexadecane Heptadecane 2-Methylheptadecane 3-Methylheptadecane Octadecane
40 48 70 40 72 60 63 79 79 85 450 60 75 83 85 87 95 100 113 44 46 78
19 25 16 27 29 25 15 19 15 13 10 28 25 29 14 22 12 9 19 15 11 29
21 26 63 n.d.a n.d. n.d. n.d. n.d. n.d. n.d. 420 n.d. n.d. n.d. n.d. n.d. n.d. n.d. 104 n.d. n.d. 75
23 21 17
12
15
24
Not detected.
Fig. 4. GC × GC-FID chromatogram of separation of C16 monomethylalkanes and n-alkane found in an exhaled breath sample of a 47-year-old smoker with a triple coronary artery bypass (peaks are numbered according to Table 1). A, siloxanes from column bleeding; B, contamination with siloxanes from septum; C, hexadec-1-ene.
Fig. 5 shows the chromatogram of separation of the standard mixture of monomethylpentadecanes and n-hexadecane by GC–MS/MS. As can be seen from this chromatogram, all isomers of C16 monomethylalkanes can be identified and quantified using the selected SRM transitions. In the case of the analysis of the exhaled breath sample by this method, the specific SRM transitions were used for each monomethylalkane to ensure the identification of the targeted components of exhaled breath. The exhaled breath sample contained only n-hexadecane, and no monomethylpentadecanes were detected as was assumed from the literature and the GC × GCFID analyses. The amounts of the detected monomethyl- and n-alkanes in the exhaled breath samples of a 47-year-old smoker with a triple coronary artery bypass, together with the relative standard
Fig. 5. GC–MS/MS chromatogram of separation of the standard mixture of C16 monomethylalkanes and n-alkane (peaks are numbered according to Table 1).
deviations of the measurements calculated from three analyses are listed in Table 4. As we can see from Table 4, by GC–MS/MS no monomethylalkanes were detected despite the low limits of the detection and selective transitions. Therefore it is obvious that the GC × GC-FID results were false-positive and the compounds identified by GC × GC-FID as monomethylalkanes were most probably substances with similar elution properties, but with a different mass fragmentation pattern from monomethylalkanes,
A. Hengerics Szabó et al. / J. Chromatogr. B 978–979 (2015) 62–69
and therefore they were not detected by the developed GC–MS/MS method. However, the presence of n-alkanes was confirmed by both techniques and the obtained amounts show a good agreement. The relatively high RSD of the measurements was most probably caused by the variability of the breath volume pushed through the INCAT device. The results of the analyses indicate that identification of monomethylalkanes in exhaled breath samples based only on matching retention characteristics might not be proven by other, more selective techniques. 4. Conclusion A new multibed arrangement of INCAT device with two layers of Carbopack X and one layer of Carboxen 1000 as sorbent materials was developed for sampling and preconcentration of non-polar analytes with a wide range of volatility in gaseous samples. The device was used for the analysis of C6 C19 nalkanes and C9 C19 monomethylalkanes in an exhaled breath sample. The article was aimed at the analysis of all C6 C19 alkanes and C9 C19 monomethylalkanes by GC techniques in exhaled breath samples. For the analysis of the selected analytes both GC × GC-FID and GC–MS/MS techniques were used as the complete separation of monomethylalkanes by GC × GC was not achieved even if using a high-resolution capillary column. However, the use of the Carbowax column in the second dimension of GC × GC enabled the complete separation of alkanes from other coeluting sample components (alkenes, aromatic hydrocarbons, etc.). GC–MS/MS enabled the separation of all monomethylalkanes because of the selective SRM transitions used for each isomer. The amount of nalkanes detected in the exhaled breath samples was similar by both techniques (GC × GC-FID and GC–MS/MS), but monomethylalkanes were detected only by GC × GC. This was most probably caused by the presence of hydrocarbons with more methyl groups eluting in the area of the selected monomethylalkanes what explains their absence in the GC–MS/MS spectra with MS/MS transitions optimized for the selected monomethylalkanes. Most articles about the analysis of exhaled breath samples rely only on spectral library match by which method the identification of isomers cannot be performed. Another mode of identification of monomethylalkanes in these articles is based on the correlation of retention characteristics of the analytes with standards. The authors have not found articles where all C6 C19 alkanes and C9 C19 monomethylalkanes were used as standards as most of these are not commercially available. This article shows that the identification based only on retention characteristics can lead to false-positive interpretations as by a highly selective method (GC–MS/MS) the identification might not be confirmed. The obtained results might prove that identification based only on spectral library match or matching retention times can lead to misinterpretation of results. The results of this manuscript also suggest, that monomethylalkanes as markers of diseases should be confirmed by highly selective identification techniques, as GC–MS/MS. Acknowledgements This publication is the result of the project implementation: ITMS 26240220071, ITMS 26240220086 and ITMS 26240220007 supported by the Research & Development Operational Program funded by the ERDF. Research was carried out within the framework of the Specific University Research (SVV260084). Work was also supported by the Slovak Research and Development Agency
69
under the contract numbers APVV-0840-11, APVV-0416-10 and APVV-0061-11. References ˇ [1] T.S. Wang, P. Spanˇ el, D. Smith, in: A. Amann, D. Smith (Eds.), Breath Analysis for Clinical Diagnosis and Therapeutic Monitoring, World Scientific, Singapore, 2005, pp. 479–490. ˇ [2] A. Amann, P. Spanˇ el, D. Smith, Mini Rev. Med. Chem. 7 (2007) 115–129. [3] W. Miekisch, J.K. Schubert, G.F.E. Noeldge-Schomburg, Clin. Chim. Acta 347 (2004) 25–39. [4] S.A. Kharitonov, P.J. Barnes, Biomarkers 7 (2002) 1–32. [5] T.M. Dwyer, Lung 182 (2004) 241–250. [6] L.M. Gonzalez-Reche, A. Kucharczyk, A.K. Musiol, T. Kraus, Rapid Commun. Mass Spectrom. 20 (2006) 2747–2752. [7] M. Phillips, K. Gleeson, J.M.B. Hughes, J. Grennberg, R.N. Cataneo, L. Baker, W.P. McVay, Lancet 353 (1999) 1930–1933. [8] M. Phillips, R.N. Cataneo, A.R. Cummin, A.J. Gagliardi, K. Gleeson, J. Greenberg, R.A. Maxfield, W.N. Rom, Chest 123 (2003) 2115–2123. [9] C.H. Deng, X.M. Zhang, N. Li, J. Chromatogr. B 808 (2004) 269–277. [10] J. Choi, L.A. Hoffman, G.W. Rodway, J.M. Sethi, Biol. Res. Nurs. 7 (2006) 241–255. [11] M. McCulloch, T. Jezierski, M. Broffman, A. Hubbard, K. Turner, T. Janecki, Integr. Cancer Ther. 5 (2006) 30–39. [12] M. Phillips, R.N. Cataneo, R. Condos, G.A.R. Erickson, J. Greenberg, V. La Bombardi, M.I. Munawar, O. Tietje, Tuberculosis 87 (2007) 44–52. [13] J. Chladkova, I. Krcmova, J. Chladek, P. Cap, S. Micuda, Y. Hanzalkova, Respiration 73 (2006) 173–179. [14] C. Gessner, S. Hammerschmidt, H. Kuhn, G. Hoheisel, A. Gillissen, U. Sack, H. Wirtz, Resp. Med. 101 (2007) 2271–2278. [15] C.E. Davis, M.J. Bogan, S. Sankaran, M.A. Molina, B.R. Loyola, W. Zhao, W.H. Benner, M. Schivo, G.R. Farquar, N.J. Kenyon, M. Frank, IEEE Sens. J. 10 (2010) 114–122. [16] Zˇ . Krkoˇsová, R. Kubinec, L. Soják, A. Amann, J. Chromatogr. A 1179 (2008) 59–68. [17] M. Phillips, R.N. Cataneo, J. Greenberg, R. Gunawardena, A. Naidu, F. RahbariOskoui, J. Lab. Clin. Med. 136 (2000) 243–249. [18] M. Phillips, J.P. Boehmer, R.N. Cataneo, T. Cheema, H.J. Eisen, J.T. Fallon, P.E. Fisher, A. Gass, J. Greenberg, J. Kobashigawa, D. Mancini, B. Rayburn, M.J. Zucker, J. Heart Lung Transplant. 23 (2004) 701–708. [19] G.D. Bell, J. Weil, G. Harrison, A. Morden, P.H. Jones, P.W. Gant, J.E. Trowell, A.K. Yoong, T.K. Daneshmend, R.F. Logan, Lancet 1 (1987) 1367–1368. [20] D.Y. Graham, D.J. Evans, L.C. Alpert, P.D. Klein, D.G. Evans, A.R. Opekun, T.W. Boutton, Lancet 1 (1987) 1174–1177. [21] B. Dordoni, R.P. Thompson, R. Williams, Gut 16 (1975) 400. [22] R. Platzer, I. Gikalov, A. Kupfer, Schweiz. Med. Wochenschr. 106 (1976) 317–318. [23] A. Modak, J. Breath Res. 3 (2009) 040201. [24] H.L. Lord, W. Zhan, J. Pawliszyn, Anal. Chim. Acta 677 (2010) 3–18. [25] W. Filipiak, A. Filipiak, C. Ager, H. Wiesenhofer, A. Amann, J. Breath Res. 6 (2012) 027107. [26] I.Y. Eom, J. Pawliszyn, J. Sep. Sci. 31 (2008) 2283–2287. [27] I.Y. Eom, V.H. Niri, J. Pawliszyn, J. Chromatogr. A 1196–1197 (2008) 10–14. ´ L. [28] H. Jurdáková, R. Kubinec, M. Jurˇciˇsinová, Z. Krkoˇsová, J. Blaˇsko, I. Ostrovsky, Soják, V.G. Berezkin, J. Chromatogr. A 1194 (2008) 161–164. [29] V.H. Niri, I.Y. Eom, F.R. Kermani, J. Pawliszyn, J. Sep. Sci. 32 (2009) 1075–1080. [30] J.A. Koziel, M. Odziemkowski, J. Pawliszyn, Anal. Chem. 73 (2001) 47–54. [31] M. Mieth, S. Kischkel, J.K. Schubert, D. Hein, W. Miekisch, Anal. Chem. 81 (2009) 5851–5857. [32] M. Mieth, J.K. Schubert, T. Gröger, B. Sabel, S. Kischkel, P. Fuchs, D. Hein, R. Zimmermann, W. Miekisch, Anal. Chem. 82 (2010) 2541–2551. [33] P. Trefz, S. Kischkel, D. Hein, E.S. James, J.K. Schubert, W. Miekisch, J. Chromatogr. A 1219 (2012) 29–38. [34] R. Kubinec, V.G. Berezkin, R. Górová, G. Addová, H. Mraˇcnová, L. Soják, J. Chromatogr. B 800 (2004) 295–301. ˇ cík, I. Ostrovsky, ´ L. Soják, V. Berezkin, [35] P. Pˇrikryl, R. Kubinec, H. Jurdáková, J. Sevˇ Chromatographia 64 (2006) 65–70. [36] M. Caldeira, R. Perestrelo, A.S. Barros, M.J. Bilelo, A. Morête, J.S. Câmara, S.M. Rocha, J. Chromatogr. A 1254 (2012) 87–97. [37] M. Phillips, R.N. Cataneo, A. Chaturvedi, P.D. Kaplan, M. Libardoni, M. Mundada, U. Patel, X. Zhang, PLOS ONE 8 (2013) e75274, 1–7. [38] M. Libardoni, P.T. Stevens, J.H. Waite, R. Sacks, J. Chromatogr. B 842 (2006) 13–21. [39] J.M. Sanchez, R.D. Sacks, Anal. Chem. 75 (2003) 2231–2236. [40] J.M. Sanchez, R.D. Sacks, Anal. Chem. 78 (2006) 3046–3054. [41] M.C. Simmons, D.B. Richardson, I. Dvoretsky, in: R.P.W. Scott (Ed.), Gas Chromatography, Butterworth, London, 1960, p. 211. [42] J. Glastrup, J. Chromatogr. A 827 (1988) 133–136. [43] P. Mochalski, J. King, K. Unterkofler, A. Amann, Analyst 138 (2013) 1405–1418. [44] J. Beens, M. Adahchour, R.J.J. Vreuls, K. van Altena, U.A.T. Brinkman, J. Chromatogr. A 919 (2001) 127–132.