MS and Needle Trap

19 Feb 2010 - ... done on a Varian Star 3400 CX gas chromatograph equipped with a Varian ... A Varian VF-5 ms capillary column (30 m, 0.25 mm, 0.5 μm...
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Anal. Chem. 2010, 82, 2541–2551

Automated Needle Trap Heart-Cut GC/MS and Needle Trap Comprehensive Two-Dimensional GC/TOF-MS for Breath Gas Analysis in the Clinical Environment Maren Mieth,† Jochen K. Schubert,† Thomas Gro¨ger,‡ Bastian Sabel,† Sabine Kischkel,† Patricia Fuchs,† Dietmar Hein,§ Ralf Zimmermann,‡,| and Wolfram Miekisch*,† Department of Anesthesiology and Intensive Care Medicine, University Rostock, Schillingallee 35, 18057 Rostock, Germany, Institute of Ecological Chemistry, Helmholtz Zentrum Muenchen, Research Center for Environmental Health, Ingolsta¨dter Landstrasse 1, 85764 Neuherberg, Germany, PAS Technology Deutschland GmbH, Richard-Wagner-Strasse 10, 99441 Magdala, Germany, and Analytical Chemistry, Institute of Chemistry, University Rostock, Albert-Einstein-Strasse 3a 18051 Rostock, Germany This study was intended to evaluate low-volume (20 mL) multibed needle trap (NTD) sampling combined with heart-cut gas chromatography/mass spectrometry (GC/ MS) and comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry (GC × GC/TOFMS) for trace gas analysis under clinical conditions. NTDs, high-throughput automatic desorption and separation systems, were tested in vitro and within a study in 11 patients undergoing cardiac surgery with respect to reproducibility, reliability, and clinical applicability. NTDheart-cut GC/MS analysis of standard mixtures containing different volatile organic compounds (VOCs) yielded relative standard deviations (RSDs) from 4.0% to 18.5%. Substance adsorption was stable for 1 day if NTDs were closed on both ends and was stable for approximately 7.8 h when NTD tip ends had to be left open during autosampler storage. Even in the presence of high concentrations of contaminants linearity of heart-cut GC/MS was conserved. In patients’ breath potential biomarkers could be determined even in the presence of very high concentrations of sevoflurane. Profiles of blood-borne biomarkers, intravenous drugs, and clinical contaminants were characterized. Comprehensive GC × GC/TOF-MS may be used as a screening tool for new biomarkers, if patterns are generated from deconvoluted normalized areas. Needle trap sampling in combination with hyphenated chromatographic techniques can thus be used to provide well-tailored solutions for complex problems occurring in clinical breath analysis. Modern analytical science offers unique opportunities for practical applications in various fields. Innovative diagnostic * To whom correspondence should be addressed. E-mail: wolfram.miekisch@ uni-rostock.de. Phone: +49-381-494-5955. Fax: +49-381-494-5942. † Department of Anesthesiology and Intensive Care Medicine, University Rostock. ‡ Helmholtz Zentrum Muenchen. § PAS Technology Deutschland GmbH. | Institute of Chemistry, University Rostock. 10.1021/ac100061k  2010 American Chemical Society Published on Web 02/19/2010

methods in medicine using ultratrace analysis of volatile biomarker or drugs in human breath may be an example.1-11 When trace gas analysis is to be translated into potentially life saving diagnostic procedures the challenge will be to combine reliable, fast, and ideally on-site sampling and preconcentration techniques with highly efficacious and fast separation and detection methods. Recently our group suggested customized needle trap devices (NTD) for sampling and preconcentration of breath gas.12 Needle traps are a promising alternative to solid-phase extraction (SPE) and solid-phase microextraction (SPME) preconcentration techniques as they offer significant advantages in terms of sensitivity, stability, on-site applicability, and speed. This is especially important for clinical studies when a high number of samples have to be processed in a short time. Beyond sampling, other crucial issues in clinical breath analysis are speed of analysis and effective separation of breath components. Chromatographic separation may become extremely difficult when exogenous contaminants are present in concentrations that might be orders of magnitudes higher than those of the compounds of interest. One possibility to improve separation of breath gas components and to speed up analysis is the application of heart-cut gas (1) Mukhopadhyay, R. Anal. Chem. 2004, 76, 273A–276A. (2) Thelen, S.; Miekisch, W.; Halmer, D.; Schubert, J.; Hering, P.; Murtz, M. Anal. Chem. 2008, 80, 2768–2773. (3) Sanchez, J. M.; Sacks, R. D. Anal. Chem. 2006, 78, 3046–3054. (4) Libardoni, M.; Stevens, P. T.; Waite, J. H.; Sacks, R. J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 2006, 842, 13–21. (5) Azad, M. A.; Ohira, S.; Toda, K. Anal. Chem. 2006, 78, 6252–6259. (6) Almstrand, A. C.; Ljungstrom, E.; Lausmaa, J.; Bake, B.; Sjovall, P.; Olin, A. C. Anal. Chem. 2009, 81, 662–668. (7) Ohira, S.; Li, J.; Lonneman, W. A.; Dasgupta, P. K.; Toda, K. Anal. Chem. 2007, 79, 2641–2649. (8) Pleil, J. D. J. Toxicol. Environ. Health, Part B 2008, 11, 613–629. (9) Miekisch, W.; Schubert, J. K.; Noeldge-Schomburg, G. F. Clin. Chim. Acta 2004, 347, 25–39. (10) Buszewski, B.; Kesy, M.; Ligor, T.; Amann, A. Biomed. Chromatogr. 2007, 21, 553–566. (11) Amann, A.; Spanel, P.; Smith, D. Mini-Rev. Med. Chem. 2007, 7, 115–129. (12) Mieth, M.; Kischkel, S.; Schubert, J. K.; Hein, D.; Miekisch, W. Anal. Chem. 2009, 81, 5851–5857.

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chromatography (GC).13,14 Selected bands with overlapping compound peaks can be transferred from the first column to a second one for further separation.15 Another option to enhance separation capacity and selectivity of GC is the comprehensive coupling of one or more additional separation dimensions.15-17 Phillips et al. demonstrated that comprehensive two-dimensional gas chromatography (GC × GC) generates peak capacities which could be equal to the product of the peak capacities of the two individual separation systems.18 GC × GC has been successfully used for many applications in biology and medicine,19-23 petrochemistry,17,24,25 and food chemistry.26,27 Libardoni et al. and Sanchez and Sacks used comprehensive GC × GC with high volumes of breath gas preconcentrated by means of SPE and found 150 and 250 different substances in the breath gas of healthy controls.3,4 However, these separation systems have not yet been tested either in a clinical environment or with small volumes of breath gas. This study was intended to evaluate the use of multibed NTDs for controlled alveolar sampling and preconcentration combined with high-performance separation systems with respect to applicability under clinical conditions. For this purpose, an autosampler28 was adapted to the fast expanded flow technique. In addition, a heart-cut GC/MS method using microfluidic dean switch technology was applied to eliminate the impact of very high concentrations of disinfectants or volatile anesthetics on the separation. In a pilot study exhaled breath from 11 patients undergoing cardiac surgery was analyzed by means of NTD-heartcut GC/MS. In addition, composition of breath samples was assessed by means of comprehensive GC × GC/time-of-flight mass spectrometry (TOF-MS). EXPERIMENTAL SECTION Chemicals and Materials. Reference substances C1-C6 standard mixture, acetone, 2-methylbutane, and 1,2-dichloroethane were obtained from Sigma-Aldrich (Steinheim, Germany). Alde(13) Marriott, P.; Dunn, M.; Shellie, R.; Morrison, P. Anal. Chem. 2003, 75, 5532–5538. (14) Marriott, P. J.; Haglund, P.; Ong, R. C. Clin. Chim. Acta 2003, 328, 1–19. (15) Mondello, L.; Tranchida, P. Q.; Dugo, P.; Dugo, G. Mass Spectrom. Rev. 2008, 27, 101–124. (16) Watson, N. E.; Siegler, W. C.; Hoggard, J. C.; Synovec, R. E. Anal. Chem. 2007, 79, 8270–8280. (17) Mohler, R. E.; Dombek, K. M.; Hoggard, J. C.; Pierce, K. M.; Young, E. T.; Synovec, R. E. Analyst 2007, 132, 756–767. (18) Phillips, J. B.; Beens, J. J. Chromatogr., A 1999, 856, 331–347. (19) Li, X.; Xu, Z. L.; Lu, X.; Yang, X. H.; Yin, P. Y.; Kong, H. W.; Yu, Y.; Xu, G. W. Anal. Chim. Acta 2009, 633, 257–262. (20) Snow, N. H. Adv. Chromatogr., 2007, 45, 215–243. (21) Song, S. M.; Marriott, P.; Kotsos, A.; Drummer, O. H.; Wynne, P. Forensic Sci. Int. 2004, 143, 87–101. (22) Tranchida, P. Q.; Costa, R.; Donato, P.; Sciarrone, D.; Ragonese, C.; Dugo, P.; Dugo, G.; Mondello, L. J. Sep. Sci. 2008, 31, 3347–3351. (23) Welthagen, W.; Shellie, R. A.; Spranger, J.; Ristow, M.; Zimmermann, R.; Fiehn, O. Metabolomics 2005, 1, 65–73. (24) Mao, D.; Lookman, R.; Van de Weghe, H.; Weltens, R.; Vanermen, G.; De Brucker, N.; Diels, L. Environ. Sci. Technol. 2009, 43, 7651–7657. (25) Vendeuvre, C.; Ruiz-Guerrero, R.; Bertoncini, F.; Duval, L.; Thiebaut, D. Oil Gas Sci. Technol. 2007, 62, 43–55. (26) Focant, J. F.; Eppe, G.; Scippo, M. L.; Massart, A. C.; Pirard, C.; MaghuinRogister, G.; De Pauw, E. J. Chromatogr., A 2005, 1086, 45–60. (27) Tranchida, P. Q.; Giannino, A.; Mondello, M.; Sciarrone, D.; Dugo, P.; Dugo, G.; Mondello, L. J. Sep. Sci. 2008, 31, 1797–1802. (28) Gong, Y.; Eom, I. Y.; Lou, D. W.; Hein, D.; Pawliszyn, J. Anal. Chem. 2008, 80, 7275–7282.

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hyde standard mixture was purchased from Ionimed Analytik (Innsbruck, Austria). 2,2,4,6,6-Pentamethylheptane was purchased from Chemos GmbH (Regenstauf, Germany). Nitrogen of purity 5.0 (i.e., 99.999%) was purchased from Linde (Pullach, Germany). Carboxen 1000, Carbopack X, and Tenax as packing material for the NTDs were purchased from Supelco (Bellefonte, PA). The 60 mm luer lock needles and Teflon caps were purchased from PAS Technology (Magdala, Germany). Epoxy glue was obtained from UHU (Buehl, Germany), 1 mL single-use sampling luer syringes were purchased from Transoject (Neumuenster, Germany), gastight syringes were purchased from Hamilton (Bonaduz, Switzerland), and 0.1 L gas bulbs were from Supelco (Bellefonte, PA). Needle Trap Devices. For preparing the NTDs 22 gauge stainless steel needles (60 mm × 0.41 mm i.d., 0.72 mm o.d.) were used. The adsorbent material was held inside the needle by means of a spiral plug on the luer lock side and by means of epoxy glue on the tip side. The packing material (seen from the spiral plug to the tip) consisted of 10 mm Carboxen 1000 (mesh size 60/80), 10 mm Carbopack X (mesh size 60/80), and 10 mm Tenax (mesh size 35/60). More information on needles and manufacturing procedures has already been published in recent literature.28-31 Prior to the first usage NTDs were conditioned in a special custom-made heating device (PAS Technology, Magdala, Germany) at 300 °C under a permanent helium flow (1 bar front pressure) for at least 20 h to eliminate contaminations from the manufacturing process. Afterward, both ends of the needles were sealed with Teflon caps. After longer storage the NTDs were conditioned again for 30 min in the heating device. Standard Gas Mixtures and Sample Preparation. Gas mixtures obtained through evaporation of liquid standards and commercially available gas standards were appropriately diluted with nitrogen in Tedlar bags (SKC Inc., Eighty Four, PA) to obtain the desired concentration levels. The needle traps were connected to a 1 mL syringe and pierced through the septum of the Tedlar bags. Loading of the needle was done by moving the plunger of the syringe manually up while the needle was in the bag and down after the needle had been removed from the bag. Moving the plunger up and down once was defined as one sampling cycle. A gas mixture containing 35 nmol/L of 2,3-dimethyl-1,3butadiene in nitrogen was prepared in a Tedlar bag and was used as an internal standard (IS). Before they were used for gas sampling the needles were loaded with the IS during two sampling cycles. NTD Autosampler. An NTD autosampler was optimized for the fast expanded flow technique.12,29 Needles were transported through a solenoid to the injector and adjusted by means of a pneumatic claw (Figure 1). The standard septum retainer nut of the injector was replaced by one with a bigger cone to ensure that the needle hits the injector safely and in a reproducible way. The whole length of the needle (60 mm) was then inserted into the GC injector through the septum inlay.29 The needle was kept in the injector for 20 s. When the needles had been loaded with the samples they were closed immediately at the luer lock (29) Eom, I. Y.; Pawliszyn, J. J. Sep. Sci. 2008, 31, 2283–2287. (30) Eom, I. Y.; Tugulea, A. M.; Pawliszyn, J. J. Chromatogr., A 2008, 11961197, 3–9. (31) Wang, A.; Fang, F.; Pawliszyn, J. J. Chromatogr., A 2005, 1072, 127–135.

Figure 1. Schematic drawing of the needle transport in the autosampler: (a) solenoid; (b) magnetic cap with Teflon inlay; (c) luer lock connection of the needle; (d) pneumatic claw; (e) needle trap; (f) injector nut with a bigger cone.

end by means of a magnetic cap with a Teflon inlay and stored in a sample rack. During storage in the sample rack of the autosampler the tip ends had to be left open. NTD-GC/MS. Analyses for testing storage stability of breath gas on different needles were done on a Varian Star 3400 CX gas chromatograph equipped with a Varian Saturn 2000 mass spectrometer. A Varian CP Pora Bond Q (25 m, 0.32 mm, 5 µm film thickness) capillary column was installed. The temperature program worked as follows: 90 °C for 6 min; 15 °C/min to 120 °C for 1 min; 10 °C/min to 140 °C for 7 min; 15 °C/min to 260 °C for 6 min. The front line pressure was 15 psi. The NTDs were desorbed manually. NTD-Heart-Cut GC/MS. An Agilent 7890A gas chromatograph equipped with a dean switch device was used for the separation of volatile organic compounds desorbed from the NTDs. Detection of the substances was achieved by a flame ionization detector in the first dimension and an Agilent 5975C inert XL MSD with triple axis detector in the second dimension. A Varian VF-5 ms capillary column (30 m, 0.25 mm, 0.5 µm film thickness) was used as the primary column and a Varian VF-624 ms capillary column (30 m, 0.32 mm, 1.8 µm film thickness) was used as the secondary column. The secondary column was directly connected to the MSD. As a restrictor between the dean switch device and flame ionization detector a deactivated empty fusedsilica capillary column (0.85 m, 1.8 µm i.d.) was installed. The temperature of the injector was 300 °C. A 0.8 mm i.d. SPME inlet liner (Supelco, Bellefonte, PA) was used inside the injector. The valve switching program for heart cutting worked as follows: the valve switched on at 3.37 min, switched off at 3.73 min, and finally switched on at 4.1 min. The optimized oven temperature program used in the patient study and for testing the interneedle variation was programmed as follows: 40 °C for 6 min, then 5 °C/min to

60 °C for 0.5 min, then 50 °C/min to 250 °C for 5.7 min. The flow rate was kept constant at 0.9 mL/min for the primary column. For the secondary column the flow rate was 1.9 mL/min for 13.5 min, then 90 mL/min to 2.1 mL/min for the rest of the run time. NTD-Comprehensive GC × GC/MS. For the comprehensive two-dimensional separation approach a LECO Pegasus III GC × GC/TOF-MS System consisting of a LECO Pegasus III GC/ TOF-MS, an Agilent 6890 GC, a ZOEX KT 2001 jet modulator (Thermal Modulation), and an Atas Optic 3 injector was used. Mass range for the TOF-MS was set to 35-400 m/z; acquisition speed of the TOF-MS was 100 Hz. The GC was equipped with a nonpolar 5% phenyl phase in the first dimension (30 m, 0.25 mm i.d., 0.25 µm film of 5% phenyl-poly(silphenylene-siloxane) (BPX5)) and a poly(ethylene glycol) phase in the second dimension (1 m, 0.1 mm i.d., 0.1 µm film of poly(ethylene glycol) (PEG) in a sol-gel matrix (SolGel-WAX, custom-made design)). The column material was purchased from SGE Analytical Science, (Victoria, Australia). Since retention indexes on the nonpolar phases used on the first dimension were known for many of the target analytes identification of all compounds was based on retention indexes and corresponding mass spectra. The second column was chosen because of its good selectivity for oxygencontaining and aromatic species. The injector head pressure was set to 250 kPa (over) and was raised simultaneously to the oven temperature to an end pressure of 450 kPa (over). Before the GC run started the oven temperature was equilibrated to 20 °C by means of CO2 cooling. This temperature was held for 5 min after injection followed by a temperature ramp of 5 °C/min to 280 °C. The final temperature was also held for 5 min. The injection of the NTDs was performed manually at 300 °C. Sampling and injection for GC × GC was done manually. Analytical Chemistry, Vol. 82, No. 6, March 15, 2010

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Interneedle Variation and Stability. For characterization of the package quality flow resistance of the needles was controlled. For that purpose, the luer lock end of the needle was connected to a nitrogen gas supply. Then, the flow rates through the needle were determined at 2 bar front pressure using a soap film bubble flow meter at room temperature. After 29 needles had been loaded with the IS, 20 mL of a standard mixture containing 2.2 nmol/L C1-C6 mixture and 2.2 nmol/L butanal and hexanal, 2.1 nmol/L pentanal, 2.0 nmol/L heptanal, 1.8 nmol/L octanal, 1.7 nmol/L nonanal, and 1.3 nmol/L decanal was sampled through 20 sampling cycles onto each needle. The needles were closed with the autosampler caps and stored in an autosampler rack and were analyzed by means of heart-cut GC/MS. Results were interpreted in two ways: (1) To investigate the stability of substance adsorption when needles were stored in a sample tray, measurements were grouped into five time sections. The needles were stored for 0-138 min in the first section, 138-304 min in the second section, 304-469 min in the third section, 469-634 min in the fourth section, and 634-772 min in the fifth section. (2) To investigate the influence of different flow resistances caused by the packing material onto sensitivity, the needles from the first three time sections were sorted according to the flow rates which were obtained under 2 bar front pressure. The first group had flow rates from 75 to 100 mL/min (mean ) 87.2 mL/min; n ) 7) and the second group from 120 to 150 mL/min (mean ) 135 mL/min; n ) 8). Repeatability of Alveolar Breath Sampling and Effect of Storage. Sampling of breath gas by means of NTDs was first tested in healthy volunteers. For breath gas sampling from spontaneously breathing volunteers a sampling device was used as recently described.32 It consisted of a mouth piece, single-use plastic T-pieces as an artificial dead space, and a CO2-measuring cuvette. Alveolar sampling under CO2 control was performed in a similar way as described for the in vivo sampling from mechanically ventilated patients. Reproducibility of manual sampling was investigated by loading seven needles with alveolar gas from a healthy volunteer. Before sampling all needles were loaded with the IS. Four needles were loaded with breath gas through 20 fast sampling cycles. One fast sampling cycle did not take longer than 1.3 s. Three needles were loaded through 20 slow sampling cycles. The plunger was kept in the upper position for a short time before it was moved down again. A slow sampling cycle was done in 1.9 s. Samples were analyzed by means of heart-cut GC/MS. For investigating the storage stability of breath gas on the needles six different NTDs were loaded through 15 sampling cycles with alveolar gas from a healthy volunteer. Two samples were analyzed within 90 min after sampling, and four NTDs were analyzed 1 day later. Samples were analyzed by means of GC/ MS. Linearity and Quality Control. For calibration gas mixtures containing volatiles in seven different concentration levels in a range from 0.05 to 7.14 nmol/L for n-aldehydes, 0.05-5.35 nmol/L (32) Miekisch, W.; Kischkel, S.; Sawacki, A.; Liebau, T.; Mieth, M.; Schubert, J. K. J. Breath Res. 2008, 2, 026007.

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for n-alkanes, 0.07-6.87 nmol/L for isopentane, and 0.82-81.61 nmol/L for acetone were analyzed by means of NTD-heart-cut GC/MS. In order to simulate contamination and to test the efficacy of the NTD-heart-cut GC/MS system 72.9 nmol/L sevoflurane, 31.2 nmol/L 2-propanol, and 16.0 nmol/L isoprene were spiked into each calibration level. Limits of detection (LODs) for the NTD method were directly determined through repeated analysis of freshly preconditioned needles stored in a sample rack for different lengths of time. LODs for butanal and heptanal were determined through repeated analysis of nitrogen blanks (20 sampling cycles).33 LODs were 0.047 nmol/L for pentane, 0.069 nmol/L for isopentane, 0.04 nmol/L for hexane, 2.51 nmol/L for acetone, 0.04 nmol/L for butanal, 0.01 nmol/L for pentanal, 0.07 nmol/L for hexanal, and 0.06 nmol/L for heptanal. As a quality control for NTD measurements two nitrogen blanks and mixtures with seven calibration levels (without sevoflurane, 2-propanol, and isoprene) were analyzed between each patient sequence. In addition, a preconditioned needle was analyzed between NTD measurements every day. To confirm linearity at higher concentrations calibration curves were determined from analysis of seven concentration levels, from 43.5 to 4352.6 nmol/L for acetone, 2.5-2526.1 nmol/L for 1,2dichloroethane, and 2.1-2113.4 nmol/L for 2,2,4,6,6-pentamethylheptane. Study Design. The study was approved by the local ethics committee. After having given their informed consent 11 patients undergoing cardiac surgery with extracorporeal circulation (ECC) were enrolled into the study. Alveolar breath samples were taken after induction of anesthesia, after sternotomy, 5 min after end of ECC, and 30, 60, 90, 120, and 150 min after end of surgery. Preconcentration and sample preparation was done by means of handmade multibed NTDs (Carboxen 1000/Carbopack X/Tenax). Alveolar gas samples32,34-36 were withdrawn from the respiratory circuit under visual control of expired PCO2 in the way that gas collection only took place during the alveolar phase of expiration. For that purpose a sterilized stainless steel T-piece and the measuring cuvette of a fast-responding mainstream capnometer (Emma, MEDACX Ltd., Hayling Island, Hampshire, England) had been incorporated into the respiratory circuit near the endotracheal tube. Inspiratory samples were taken from the inspiratory limb of the ventilator tubing. Directly after having been conditioned and still under permanent helium flow the needles were pierced through septa of singleuse sterile luer lock plugs.12 Afterward both ends of the needles were closed with Teflon plugs for transport. For sampling the needles were equipped with a 1 mL single-use sterile syringe. The luer lock plug with the needle in it was connected to the respiratory circuit via the T-piece. The needle tip was positioned in the middle of the tubing diameter. The plunger of the syringe was moved up during the alveolar phase of the breathing cycle and down during the inspiratory phase. This procedure was repeated 20 times for each needle. At least two samples for each (33) Huber, W. Accredit. Qual. Assur. 2003, 8, 213–217. (34) van den Velde, S.; Quirynen, M.; van Hee, P.; van Steenberghe, D. Anal. Chem. 2007, 79, 3425–3429. (35) Birken, T.; Schubert, J.; Miekisch, W.; Noldge-Schomburg, G. Technol. Health Care 2006, 14, 499–506. (36) Schubert, J. K.; Miekisch, W.; Birken, T.; Geiger, K.; Noldge-Schomburg, G. F. Biomarkers 2005, 10, 138–152.

Table 1. Reproducibility of Breath Sampling in a Healthy Volunteer with Different Times for One Sampling Cycle time for sampling cycle

1.3-1.9 s RSD; n)7

∼1.3 s percent of average (RSD; n ) 4)

∼1.9 s percent of average (RSD; n ) 3)

acetone 1,3-butadiene, 2,3-dimethylbenzene toluene hexanal pentanal

32.4% 21.1%

75.9 (21.0%) 101.8 (25.6%)

132.2 (4.8%) 97.5 (17.8%)

19.6% 24.6% 17.0% 15.9%

90.9 (23.7%) 84.6 (20.2%) 97.9 (9.5%) 98.4 (22.5%)

112.1 (7.5%) 120.5 (13.6%) 102.8 (26.0%) 102.1 (3.6%)

measurement were collected. Immediately after sampling had been accomplished the needles were closed by means of a magnetic cap with a Teflon inlay at the luer lock side and by means of Teflon plug at the tip end. RESULTS AND DISCUSSION Needle Traps for Breath Sampling and Preconcentration. Needle trap devices have been reported as a promising alternative to SPME- and SPE-based breath gas sampling and preconcentration, as they combine the advantages of these two methods and avoid most of the disadvantages.12 Due to the handmade manufacturing of the needle traps determination of flow rates through the needles showed highly varying results from 30 to 150 mL/ min. Variation in determination of substance concentrations due to different flow rates through the needles was lower than 20%. Relative standard deviations (RSDs) were 11.6-29.6% in low-flow needles, compared to 4.0-18.5% in high-flow needles. Due to the higher flow resistance it may have happened with low-flow needles that the plunger of the syringe was moved down before 1 mL had been completely drawn through the needle. Table 1 summarizes analogous results during manual breath sampling; with respect to clinical applicability, RSDs of the seven samples analyzed with seven different needles were sufficiently low. As flow resistance depends on the quality of packing, reproducibility of adsorption and preconcentration onto NTDs can be further improved by controlling and standardizing the packing process. To realize flow rates of 60 mL/min through the needles during 2 s sampling cycles only needles with flow rates g70 mL/min were used in the patient study. During sampling for the patient study the plunger was kept in the upper position until the end of expiration to enhance reproducibility of sampling. Sampling cycles of 1.9 s could be realized easily in healthy volunteers and in mechanically ventilated patients. Hence, the alveolar phase of every breath could be used for sampling, and performing 20 sampling cycles was possible in approximately 2 min. This short sampling time represents a great advantage in comparison to conventional SPE sampling. Storage of Breath Gas. A crucial issue in breath gas analysis is storage of breath samples. Substance recoveries after storage for 1 day were between 96.6% and 104.8% for isoprene, acetone, and pentanal, while propane showed a recovery of 55.6% and dimethyl sulfide was not detectable anymore after 1 day of storage. RSDs were 8.0% for isoprene, 34.9% for acetone, 10.6% for propane, and 22% for pentanal after 1 day of storage. The maximum acceptable length of storage depended on physicochemical

properties of the target substances. In the patient study, needles were not stored longer than 5 h before analysis. By using an IS effects of long storage times and insufficient needle closing could be monitored. Automated Desorption. Clinical studies usually require large numbers of analyses in a short time. In order to employ needle traps for such high-throughput purposes an NTD autosampler28 was optimized for the fast expanded flow technique. Up to 32 needle traps could be processed in one sequence (Figure 1). Figure 2 shows the stability of substance adsorption onto the needles during storage in the autosampler rack. Storing the needles in the sample rack was possible up to 469 min without relevant loss of substances. The RSD of the measurements (n ) 15) performed after 469 min of storage was 23.4% for 2,3-dimethyl1,3-butadiene (IS), 18.1% for pentane, 16.2% for hexane, 9.3% for pentanal, 8.8% for hexanal, and 41% for toluene. The variation was very low despite the large number of needles used for this experiment. Needles kept longer than 7.8 h in the sample tray before measurement showed an increased loss of analytes and an increased adsorption of contaminants such as toluene, respectively. Higher alkanes and aldehydes were stable on the needles over the whole time of the experiment. This demonstrates once again that adsorption stability depended on physicochemical properties of the substances. 2,3-Dimethyl-1,3-butadiene as IS was a good indicator for exaggerated storage times before measurement. 2,3-Dimethyl-1,3-butadiene is not found in human breath, and concentrations in ambient air are very low. Desorbed amounts remained stable up to 7.8 h of storage, and loss of substance beyond 7.8 h was high. In addition, the IS can be used to control completeness of desorption. For a complete and fast substance desorption the needles have to be tightly closed at their luer lock side. Even small leaks will cause decrease of intensity and reproducibility. Heart-Cut GC/MS. In the clinical environment contaminants such as disinfectants or volatile anesthetics are found in part-permillion concentrations in ambient air or in exhaled breath, respectively. The problem is further aggravated because reliable separation and identification of potential biomarkers have to be realized within a short time and in a large number of samples. To overcome these problems we employed a heart-cut GC/MS system based on dean switch technology. Due to its mode of action the linearity of the method was conserved even in the presence of high concentrations of sevoflurane and 2-propanol. Calibration curves determined with 72.9 nmol/L sevoflurane and 31.2 nmol/L 2-propanol on each level showed r2 ) 0.97 for butanal, r2 ) 0.98 for pentanal, r2 ) 0.99 for hexanal, r2 ) 0.99 for heptanal, r2 ) 0.91 for acetone, r2 ) 0.96 for hexane, r2 ) 0.93 for pentane, and r2 ) 0.97 for isopentane. This was even more remarkable as each concentration level was analyzed by means of different needles. Even concentrations in the part-per-million range of acetone, 1,2-dichloroethane, and 2,2,4,6,6-pentamethylheptane could be determined with good linearity (r2 ) 0.99 for acetone, r2 ) 0.95 for 1,2-dichloroethane, and r2 ) 0.99 for 2,2,4,6,6-pentamethylheptane). As a clinically relevant task, the volatile anesthetic sevoflurane was separated from the other compounds on the first column and a small section of the GC run was cut out and sent to the second Analytical Chemistry, Vol. 82, No. 6, March 15, 2010

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Figure 2. Stability of substance adsorption onto the needles after storage in an autosampler rack without a Teflon plug on the tip side. Needles were loaded with 2 mL of an internal standard (IS) (35 nmol/L 2,3-dimethyl-1,3-butadiene) and then with 20 mL of a standard mixture (2.2 nmol/L C1-C6 alkanes and C1-C10 aldehydes). The needles were closed with a magnetic cap with a Teflon inlay and stored in an autosampler rack. The measurements were grouped according to the duration of storage. Mean values of the normalized results for the (five) different durations of storage are shown; all measurements were normalized to the average value of the first time interval of storage (0-138 min): circle, 0-138 min (n ) 5); box, 138-304 min (n ) 5); asterisk, 304-469 min (n ) 5); diamond, 469-634 min (n ) 6); triangle, 634-772 min (n ) 5); *, normality test failed.

column for further separation. In this way it was possible to identify and quantify, e.g., pentane and isopentane in the presence of large amounts of sevoflurane. This is in accordance with results of application of heart-cut GC in other fields where the target compounds could be separated clearly and analyzed in short times.37,38 The temperature and cut program used in this study had a run time of 20 min. For monitoring of specific compounds the run time could still be decreased considerably by, e.g., backflushing the first column after the heart cut. Heart-cut GC/MS proved to be a valuable tool when biomarkers had to be determined from a breath matrix containing abundant concentrations of contaminants. As this occurs regularly in the hospital environment heart-cut GC/MS can help to solve crucial problems of clinical breath analysis. Patient Study. Substances in breath gas may originate from different sources. Exhaled blood-borne substances may be generated in the body by metabolism, may come from intravenous infusions like drugs, or may represent contaminations from different materials used in the clinical environment.39 Other substances in breath gas come from ambient air or are generated in the airways.8,10,40 Figure 3 shows the profile of three bloodborne substances normalized to the value determined 30 min after surgery. Normalization of peak areas was done to eliminate interindividual variations. The concentration profile of acetone is (37) Luong, J.; Gras, R.; Yang, G.; Cortes, H.; Mustacich, R. J. Sep. Sci. 2008, 31, 3385–3394. (38) Gunnar, T.; Engblom, C.; Ariniemi, K. J. Chromatogr., A 2007, 1166, 171– 180. (39) Wahl, H. G.; Hoffmann, A.; Haring, H. U.; Liebich, H. M. J. Chromatogr., A 1999, 847, 1–7. (40) Miekisch, W.; Schubert, J. K. TrAC, Trends Anal. Chem. 2006, 25, 665– 673.

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similar to profiles measured by means of SPME-GC/MS by Pabst et al.41 The intravenous anesthetic Propofol was found in seven patients, who had total intravenous anesthesia based on this drug. The concentration profile shows the expected increase at the beginning of surgery and development of a steady state during the stay in the intensive care unit (ICU). In four patients, we found 1,2-dichloroethane, which was identified by retention time and mass spectrum. 1,2-Dichloroethane showed highest concentrations directly after stopping the heart-lung machine (HLM) and then exhibited a decrease over time. Subsequent calibration of 1,2dichloroethane showed concentrations in breath gas up to 7.6 nmol/L. The true origin of 1,2-dichloroethane is unknown. Since the substance was only found in 4 of 11 patients we assumed that nonexhaustive outgassing of the HLM tubing after sterilization by means of ethylenoxide was the reason for exhalation of 1,2dichloroethane, which is known as carcinogenic. Figure 4 shows the concentration profiles of two contaminants. 2,2,4,6,6-Pentamethylheptane was found mainly in breath samples which were taken in the ICU. By testing fresh tubing sets in the laboratory we proved that the substance came off the respiratory circuits. The ventilatory tubing was changed when patients were transferred from the operation theater (OR) to the ICU and consisted of different materials. Subsequent calibration of 2,2,4,6,6pentamethylheptane showed concentrations in breath gas up to 31.7 nmol/L in the ICU. 2-Propanol is a good example for contamination through disinfectants. In the OR ambient concentrations of 2-propanol were high due to disinfection of large skin areas of patients. On the (41) Pabst, F.; Miekisch, W.; Fuchs, P.; Kischkel, S.; Schubert, J. K. J. Cardiothorac. Surg. 2007, 2, 37.

Figure 3. Exhaled and inspired concentrations of blood-borne substances from patients undergoing cardiac surgery. Acetone (n ) 11) is an endogenous metabolite, Propofol (n ) 7) is an anesthetic drug, and 1,2-dichloroethane (n ) 4) is an exogenous contaminant: t1 ) after induction of anesthesia; t2 ) after sternotomy; t3 ) 5 min after end of ECC; t4 ) 30 min, t5 ) 60 min, t6 ) 90 min, t7 ) 120 min, and t8 ) 150 min after end of surgery; Ins1 ) inspiration during surgery; Ins2 ) inspiration in ICU. Analytical Chemistry, Vol. 82, No. 6, March 15, 2010

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Figure 4. Exhaled and inspired concentrations of exogenous substances from patients undergoing cardiac surgery. 2,2,4,6,6-Pentamethylheptane (n ) 11) comes off the tubing material used for mechanical ventilation in the ICU, 2-propanol (n ) 11) originates from skin disinfectants used in the OR. t1 ) after induction of anesthesia; t2 ) after sternotomy; t3 ) 5 min after end of ECC; t4 ) 30 min, t5 ) 60 min, t6 ) 90 min, t7 ) 120 min, and t8 ) 150 min after end of surgery; Ins1 ) inspiration during surgery; Ins2 ) inspiration in ICU.

one hand, the NTDs got contaminated even within the short time when the tip end had to be opened for connection to the respiratory circuit; on the other hand transdermal uptake of 2-propanol may have played a role. Ambient 2-propanol concentrations found in the ICU were much lower and consequently yielded much lower concentrations on the needles. The results of the patient study prove that NTDs combined with heart-cut GC/MS represent a method of choice when highthroughput breath analysis is to be performed reliably under clinical conditions. Comprehensive GC × GC/TOF-MS. We used heart-cut GC/ MS to enhance the selectivity of the separation system and to quantify a number of predefined target compounds. To further enhance the selectivity of the chromatographic system for a nontargeted screening and identification of still unknown bio2548

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markers comprehensive GC × GC was applied to the analysis of breath samples. Polarity and film thickness of the columns used in this study were chosen according to what has already been known concerning application of GC × GC systems to breath gas.3,4 Although preconcentration volumes were much lower for NTDs (20 mL) than for conventional SPE (>250 mL), columns and detector got overloaded when samples from patients were analyzed who had sevoflurane for anesthesia. A reasonable interpretation of data from these samples was not possible. For the patient study only samples which were taken before or a sufficiently long time after application of sevoflurane could be used. As these problems will regularly be encountered in clinical breath analysis the choice of the GC columns has to be optimized.

Figure 5. Two-dimensional chromatogram showing exhaled substances (n-pentane to n-decane) from a patient after induction of anesthesia. For peak assignment refer to Table 2. Peaks were identified according to mass spectra, relative retention times, and retention index (alkane).

Table 2. Compound List and Retention Times (RT) for the Comprehensive GC × GC/TOF-MS Chromatogram no.

compd name

RT (s) 1st column

RT (s) 2nd column

no.

compd name

RT (s) 1st column

RT (s) 2nd column

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

acetaldehyde 1,1,2,2-tetrafluoroethane sevolflurane methanthiol ethanol 1-pentene n-pentane acrolein furane acetone 2-propanol isoprene 2,2-dimethylbutane 2-methyl-propan-2-ol carbondisulfide 2-methylpentane 2-methylpropanal 3-methylpentane methacrolein crotonaldehyde 1-hexene n-hexane methyl vinyl ketone butane-2,3-dione propanol butanal 3-methylfurane 2-methylfurane butan-2-one benzene

215 215 230 240 240 260 260 260 265 265 265 275 290 295 300 325 335 350 355 345 365 375 380 385 340 385 390 405 395 510

1.05 1.00 1.59 1.00 1.98 1.00 0.98 1.39 1.24 1.31 1.94 1.04 1.02 1.74 1.10 1.40 1.26 1.05 1.37 1.46 1.08 1.07 1.74 1.99 2.94 1.41 1.36 1.42 1.47 1.45

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

n-butan-1-ol 1-heptene n-heptane pentan-2-one pentan-2,3-dione pentanal 1,4-dioxan ethylfurane 2,4-dimethylfurane 2,5-dimethylfurane toluene n-pentan-1-ol 1-octene n-octane hexan-2-one hexanal ethylbenzene m/p-xylene 1-nonene heptane-2-one n-nonane o-xylene heptanal styrene benzaldehyde 1-decene n-decane octan-2-one octanal limonene

545 570 590 585 610 610 645 600 615 565 770 785 820 840 835 860 1000 1020 1060 1070 1075 1075 1100 1075 1250 1285 1295 1285 1315 1370

3.17 1.13 1.11 1.60 2.10 1.61 1.96 1.47 1.48 1.55 1.59 3.52 1.17 1.13 1.66 1.62 1.56 1.56 1.18 1.65 1.15 1.63 1.60 1.92 3.07 1.19 1.15 1.60 1.57 1.34

Figure 5 shows a two-dimensional chromatogram from a sample taken immediately after induction of anesthesia (without sevoflurane). The indicated analytes (Table 2) were identified according to their spectra and retention indexes (if available). Some of the peaks with low intensities are only visible on single m/z traces and, therefore, are not visible on the total ion chromatogram shown in the figure. About 3 times more substance

peaks than listed in Table 2 could be observed from the low preconcentration volumes used for the NTDs, but not clearly identified. As there are a large number of unidentified substances which may originate from the ambient air or from the patient, recognition of new biomarkers by means of comprehensive GC × GC is difficult. The huge number of targets can cause problems in data Analytical Chemistry, Vol. 82, No. 6, March 15, 2010

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Figure 6. Panel a shows a pattern of normalized peak areas from compounds in Table 2. Each line represents a compound which is normalized to each other compound (column) in Table 2. Panel b shows the comparison of two patterns from different clinical states of the same patient. Results from samples gathered immediately after sternotomy were compared to those from samples taken 60 min after sternotomy. 2550

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analysis and visualization. Especially in the field of metabolomics much effort has been made to handle this kind of data.17,23,42-46 Since any IS is specific for a restricted number of substances, internal standardization of unknown substances is hardly possible. To overcome this problem patterns of deconvoluted areas normalized to every other compound could be used. In this way, different conditions of the same patient or identical conditions of different patients can be compared to each other. Figure 6a shows a pattern of normalized peak areas from compounds listed in Table 2. Each line represents a compound which is normalized to every other compound (columns) in Table 2. Figure 6b shows the comparison of patterns from different clinical states of the same patient. Results from samples gathered immediately after sternotomy were compared to those from samples taken 60 min after sternotomy. The “hot” colors in row 11 and the “cold” colors in row 10 indicate higher concentrations of 2-propanol and lower concentrations of acetone in the samples taken immediately after sternotomy. This is consistent with the data shown in Figures 3 and 4. Green colors at the crossing of rows and columns indicate that the corresponding compounds identified by the column number show a similar behavior. For example, the green color at the crossing of row 11 (2-propanol) and column 28 indicates that 2-methylfurane behaves in a similar way as 2-propanol. The cold colors in row 11 indicate that the corresponding compounds (e.g., sevoflurane) show a relative increase in concentration that is higher than that for 2-propanol. (42) Pierce, K. M.; Hoggard, J. C.; Mohler, R. E.; Synovec, R. E. J. Chromatogr., A 2008, 1184, 341–352. (43) Pierce, K. M.; Hope, J. L.; Hoggard, J. C.; Synovec, R. E. Talanta 2006, 70, 797–804. (44) Almstetter, M. F.; Appel, I. J.; Gruber, M. A.; Lottaz, C.; Timischl, B.; Spang, R.; Dettmer, K.; Oefnert, P. J. Anal. Chem. 2009, 81, 5731–5739. (45) Groger, T.; Welthagen, W.; Mitschke, S.; Schaffer, M.; Zimmermann, R. J. Sep. Sci. 2008, 31, 3366–3374. (46) Groger, T.; Schaffer, M.; Putz, M.; Ahrens, B.; Drew, K.; Eschner, M.; Zimmermann, R. J. Chromatogr., A 2008, 1200, 8–16.

This method of data processing can serve as a filter to identify unknown substances which exhibit characteristic changes relative to other compounds. In a second step, these substances can be characterized in more detail, identified, and quantified, e.g., by means of heart-cut GC/MS. CONCLUSION Needle trap devices optimally meet the requirements for reliable and reproducible sampling and preconcentration in the clinical environment due to small sample volumes, full compatibility with alveolar sampling, and stability of substance adsorption. Due to enhanced selectivity and fast detection comprehensive GC × GC/TOF-MS is a powerful tool for screening purposes. Needle trap sampling in combination with comprehensive GC × GC/TOF-MS holds promise for identification of unknown substances and for recognition of changes in substance concentrations under varying clinical conditions. High concentrations of contaminants limit the applicability of this technique in the ICU or OR. Heart-cut GC/MS provides optimal solutions for this problem, but due to its mode of action the total number of compounds that can be characterized is limited. Automated needle trap processing in combination with heart-cut GC/MS represents a valuable tool for quantitative high-throughput analysis of targeted compounds and works well even in the presence of high concentrations of contaminants. Needle trap sampling combined with hyphenated chromatographic techniques can thus be used to provide well-tailored solutions for complex problems occurring in clinical breath analysis. Received for review January 8, 2010. Accepted February 10, 2010. AC100061K

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