Multibed Needle Trap Devices for on Site Sampling and

Anal. Chem. 2009, 81, 5851–5857

Multibed Needle Trap Devices for on Site Sampling and Preconcentration of Volatile Breath Biomarkers Maren Mieth,*,† Sabine Kischkel,† Jochen K. Schubert,† Dietmar Hein,‡ and Wolfram Miekisch† Department of Anesthesiology and Intensive Care Medicine, University Rostock, Schillingallee 35, 18057 Rostock, Germany, and PAS Technology Deutschland GmbH, Richard-Wagner-Strasse 10, 99441 Magdala, Germany To facilitate their use in trace gas analysis, the adsorption capacity of needle trap devices (NTDs) was increased by combining three adsorbent materials and increasing total adsorbent amount. The use of 22 gauge needles, application of internally expanding desorptive flow technique without cryofocusation and a new on site alveolar sampling method for NTDs provided sensitivity in the parts per trillion range of VOC concentrations without loosing precision or linearity. LODs were 0.4 ng/L for isoprene, 0.5 ng/L for dimethyl sulphide, 0.9 ng/L for 2-butenal, 1.0 ng/L for hexane, 1.2 ng/L for pentane, 2.3 ng/L for hexanal, 5.3 ng/L for pentanal, and 8.3 ng/L for acetone. R of calibration curves were consistently >0.98. Loss of volatile aldehydes during storage for 7 days was less than 10%. Needle trap devices packed with more than one adsorbent material represent a promising alternative to SPE and SPME for analysis of volatile organic compounds in the low parts per billion/parts per trillion range. Crucial problems of clinical breath analysis concerning sensitivity of analytical methods, limited stability, and decomposition of breath compounds during sampling and storage could be solved. Micro-packed needles represent a novel and robust means of sample preparation for trace analysis in gaseous matrices. Needle traps can easily be transported and stored. Because of the low thermal mass, sample volumes are small and desorption is fast. A new desorption method for needle trap devices (NTDs) was introduced recently by Eom1 that works without special narrow neck liner2 or extra gas supplies. Nor is there a need for desorption by water vapor,3 which may affect separation. Recent applications of micro-packed needles have mainly been described for use in the environmental field, for example, for preconcentration of BTEX and higher alkanes (C6-C15).4-7 Important VOCs in breath gas are C2-C6 alkanes, alkenes, * To whom correspondence should be addressed. E-mail: maren.mieth@ med.uni-rostock.de. Phone: +49-381-494-5955. Fax: +49-381-494-5942. † University Rostock. ‡ PAS Technology Deutschland GmbH. (1) Eom, I. Y.; Pawliszyn, J. J. Sep. Sci. 2008, 31, 2283–2287. (2) Wang, A.; Fang, F.; Pawliszyn, J. J. Chromatogr. A 2005, 1072, 127–135. (3) Prikryl, P.; Kubinec, R.; Jurdakova, H.; Sevcik, J.; Ostrovsky, I.; Sojak, L.; Berezkin, V. Chromatographia 2006, 64, 65–70. (4) Eom, I. Y.; Tugulea, A. M.; Pawliszyn, J. J. Chromatogr. A 2008, 11961197, 3–9. 10.1021/ac9009269 CCC: $40.75  2009 American Chemical Society Published on Web 06/23/2009

aldehydes, ketones, and alcohols. In addition, exhaled air is saturated with water vapor. Therefore, NTDs set up for BTEX analysis did not work well for trace analysis in exhaled air. For that reason, breath analyses have mainly been carried out by means of SPE8 or SPME.9,10 Although reliable results could be obtained with these methods large sample volumes and complex sample procedures were required in the case of SPE. SPME does not work for all compounds occurring in human breath and sensitivity of SPME can not be enhanced by modification of sampling procedures. In principle, these problems could be solved by adaptation of the needle trap technique to breath gas analysis, as NTDs combine the advantages and avoid the disadvantages of SPE and SPME. Clinical application of breath gas analysis could be facilitated11,12 if direct sampling and on site sample preparation could be realized by means of customized NTDs. This study was intended to modify the needle trap technique for application in breath analysis. For that purpose, adsorption capacity was increased especially for small molecules by combining different adsorbent materials and increasing total adsorbent amount. For a fast and easy desorption, the internally expanding desorptive flow technique was adapted to a 22 gauge needle without cryofocusing and a new reliable direct alveolar sampling method for needle trap devices was developed. EXPERIMENTAL SECTION Chemicals and Materials. Reference substances n-pentane, n-hexane, n-heptane, C1-C6 standard mixture, 2,2-dimethylbutane, 2-methylpentane, 3-methylpentane, 2-methylbutane, dimethyl sulphide, isopropanol, isoprene, and acetone were obtained from Sigma-Aldrich (Steinheim, Germany). Aldehyde standard mixtures were purchased from Ionimed Analytik (5) Jurdakova, H.; Kubinec, R.; Jurcisinova, M.; Krkosova, Z.; Blasko, J.; Ostrovsky, I.; Sojak, L.; Berezkin, V. G. J. Chromatogr. A 2008, 1194, 161– 164. (6) Koziel, J. A.; Odziemkowski, M.; Pawliszyn, J. Anal. Chem. 2001, 73, 47– 54. (7) Saito, Y.; Ueta, I.; Kotera, K.; Ogawa, M.; Wada, H.; Jinno, K. J. Chromatogr. A 2006, 1106, 190–195. (8) Sanchez, J. M.; Sacks, R. D. Anal. Chem. 2003, 75, 2231–2236. (9) Grote, C.; Pawliszyn, J. Anal. Chem. 1997, 69, 587–596. (10) Miekisch, W.; Schubert, J. K. Trends Anal. Chem. 2006, 25, 665–673. (11) Schubert, J. K.; Miekisch, W.; Geiger, K.; Noldge-Schomburg, G. F. Expert Rev. Mol. Diagn. 2004, 4, 619–629. (12) Miekisch, W.; Kischkel, S.; Sawacki, A.; Liebau, T.; Mieth, M.; Schubert, J. K. J. Breath Res. 2008, 2, 026007.

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(Innsbruck, Germany). Adsorbent materials for the needle trap package were purchased from Supelco (Bellefonte, PA). Gas tight syringes were purchased from Hamilton (Bonaduz, Switzerland), and 0.1 L gas bulbs were from Supelco (Bellefonte CA); 20 mL headspace vials, Teflon coated rubber septa, and crimp caps were purchased from Gerstel (Muelheim an der Ruhr, Germany). Needles and autosampler syringes were obtained from PAS Technology (Magdala, Germany). Helium and nitrogen of purity 5.0 (i.e., 99.999%) were purchased from Linde (Vienna, Austria), and Tedlar bags were purchased from SKC (Eighty Four, PA). Five minute epoxy glue was purchased from UHU (Buehl, Germany); 1 mL single use sampling Luer syringes were purchased from Transcoject (Neumuenster, Germany). The aspirating pump AP-20 was purchased from Komyo Rikagaku Kogyo K.K. (Kanagawa, Japan). Preparation of Standard Gas Mixtures. Specific amounts of reference substances were transferred into a 100 mL evacuated gas sampling bulb by means of a 10 µL syringe. The gas sampling bulb was equilibrated with nitrogen. A specific volume of this gas mixture was transferred into a Tedlar bag filled with nitrogen by means of a 1 mL syringe. This gas mixture was further diluted either with nitrogen, or with breath gas to obtain the desired concentration levels. Humidified nitrogen was prepared by piping nitrogen through a gas sampling bottle filled with distilled water at a temperature of 45 °C. Instrumentation. A Varian Star 3400 CX gas chromatograph equipped with a Varian Saturn 2000 mass spectrometer was used for separation and detection of the volatile organic compounds desorbed from the needle trap devices. A RTX 502.2 capillary column (60 m; 0.32 mm; 1.8 µm film thickness) from Restek (Bad Soden, Germany) was used for the investigation of the dry purging times, conditioning times, the influence of different numbers of sampling cycles, and the in vivo experiments. For the investigation of different desorption temperatures, carry over, the influence of sampling volume, and the method validation a CP PoraBond Q (25 m; 0.32 mm; 5 µm film thickness) capillary column from Varian was used. The column temperature program for the RTX 502.2 worked as follows: 40 °C for 2 min, 10 °C/min to 60 °C for 4 min, 8 °C/min to 90 °C, 10 °C/min to 170 °C for 2 min, and 15 °C/min to 260 °C. The oven temperature for the PoraBond Q column was programmed 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, and 15 °C/min to 260 °C for 6 min. The front line pressure was 15 psi for both columns. For thermal desorption, the needle trap devices were plugged by a Teflon cap at the Luer lock end. The whole length of the needle (60 mm) was then inserted into the GC injector (Varian 1078 Injector) through a septum inlay. The injector was equipped with a 0.8 mm i.d. SPME inlet liner (Supelco, Bellefonte, PA). A GC-autosampler (Concept, PAS Technology, Germany) with a purge flow configuration before desorption was used for automated sampling. Needle Trap Devices. The NTD consisted of a 22-gauge stainless steel needle (60 mm × 0.41 mm i.d., 0.72 mm o.d.). A spiral plug was positioned inside the needle and the adsorbent material was aspirated into the needle by means of a pump in the way that the first 10 mm of adsorption surface consisted of Tenax (mesh size 35/60), followed by 10 mm Carbopack X (mesh size 60/80) and 10 mm Carboxen 1000 (mesh size 60/80). The 30 mm 5852


Figure 1. Schematic drawing of a 22 gauge stainless steel needle trap device with a multilayer sorbent bed consisting of Carboxen 1000 (A), Carbopack X (B), and Tenax (C) particles. The sorbent particles were positioned less than 1 mm from the opening of the NTD and were held in the needle by means of a stainless steel spiral plug.

packing was positioned less than 1 mm away from the opening of the NTD (Figure 1) and kept in place by means of a small amount of epoxy glue. NTD manufacturing procedures have already been described in more detail in recent literature.1,13 Needles had no side orifice. Prior to first use NTDs were conditioned in a GC injector at 300 °C for 20 h with a permanent helium flow to eliminate contaminations. Afterward, the Luer lock end was sealed with a Teflon cap, and the NTDs were stored in a needle tray. Mode of Sampling. Three different methods of loading the NTDs were used. Automated Sampling. Standards or humidified nitrogen were transferred into a 20 mL evacuated sealed headspace glass vial by means of a gastight syringe. The needle traps were connected to an autosampler syringe (1 mL) and loaded through the septa of the glass vials by moving the plunger of the syringe up and down automatically. Manual Sampling. Standards were prepared in Tedlar bags. The needle traps were connected to a 1 mL single use sterile syringe. The needles were then pierced through the septum of the Tedlar bag and loaded by moving the plunger of the syringe manually up and down outside the bag. Moving the plunger up within 1 s and down within 1 s once was defined as one sampling cycle. In Vivo Sampling. A single use Luer lock plug with septum was connected to the respiratory circuit by means of a stainless steel T-piece (Figure 2). For manual sampling the needle was pierced through the septum until the tip of the needle 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. For pump sampling the needle was positioned in the same way and connected to a 100 mL aspirating pump. One hundred milliliters of mixed expiratory breath gas were sampled through the action of the pump. Dry Purging Time. A breath sample was spiked with 270 ng/L 2,3-dimethylbutane, 230 ng/L pentane, 270 ng/L hexane, and 312 ng/L heptane. The NTDs were automatically loaded during 15 sampling cycles. The loaded needles were connected to a customized syringe having an additional gas inlet below the plunger. Prior to desorption helium at a flow of 100 mL/min was purged through the gas inlet of the syringe for different lengths of time (0, 5, 7, 10, 12, 15, 20, 25, and 30 s) at 22 °C to remove the remaining water. Desorption Temperature. The effect of three different injector temperatures (250, 285, and 300 °C) onto substance desorption from the NTDs was investigated. The humidified gas (13) Gong, Y.; Eom, I. Y.; Lou, D. W.; Hein, D.; Pawliszyn, J. Anal. Chem. 2008, 80, 7275–7282.

Figure 2. In vivo sampling from a mechanically ventilated animal (pig). Breath samples were taken through a septum port (yellow) in the respiratory circuit. Two different sampling modes were applied: (a) alveolar breath gas was drawn through the NTD under control of expired CO2 during 10 manual sampling cycles (10 mL). For that purpose, the plunger of a 1 mL single use syringe was moved up during expiration and moved down during inspiration. (b) 100 mL of mixed expiratory breath gas were drawn through the NTD by means of an aspiration pump through the same port as in a (not shown in the figure).

mixture used for that purpose contained volatile substances in concentrations from 80 ng/L (propane) to 520 ng/L (acetone). Substance concentrations were chosen in the way to mimic concentrations in breath gas. Samples were taken automatically by means of the autosampler. Conditioning Time. NTDs were automatically loaded (10 mL) with a humidified standard containing volatile substances in concentrations from 2.5-4.5 µg/L. Needles were conditioned in the hot injector for 0, 900, or 1680 s and then loaded, desorbed, and analyzed. Carry Over. NTDs were loaded manually through 15 sampling cycles (15 mL). Gas mixtures prepared for that purpose contained isoprene 1.4 µg/L, acetone 7.9 µg/L, isopropanol 6.3 µg/L, aldehydes 0.8 µg/L, and n-alkanes 0.6 µg/L. Substance concentrations were chosen in the way to mimic concentrations in the upper range of naturally occurring breath gas. The manually loaded NTDs were then inserted into a GC injector (at 300 °C), removed from there 0.5 min later, and stored in a needle tray. During storage the Luer lock end of the NTD was sealed with a Teflon cap. After 30 min a second injection of the NTD was performed. Influence of the Number of Sampling Cycles. A breath sample was spiked with 270 ng/L 2,3-dimethylbutane and 2,2dimethylbutane, 230 ng/L pentane, 270 ng/L hexane, and 312 ng/L heptane. The needles were loaded in the way that different numbers of sampling cycles were performed in the autosampler. To demonstrate that reliability and linearity of adsorption was not affected by the number of sampling cycles applied, NTDs were exposed to gas mixtures containing different concentrations of volatile substances during a varying number of sampling cycles. Gas mixtures prepared for this experiment contained volatile substances in concentrations mimicking low, middle, and high ranges of concentrations in breath gas. In a series of three experiments, concentrations and number of sampling cycles had been chosen in the way that the product of concentrations and sampling cycles should yield identical amounts of volatile substances adsorbed onto the NTDs, that is, 5 sampling cycles from

the mixture with highest substance concentrations, 15 cycles from the mixture with moderate concentrations, and 25 cycles from the mixture with lowest concentrations should result in identical substance amounts adsorbed onto the NTDs. Method Validation. For method validation, two different numbers of sampling cycles were used. Fifteen sampling cycles were used for determination of the normal calibration curve. Ten concentration levels in a concentration range of 47.4-7900.0 ng/L for acetone, 32.6-3264.0 ng/L for isoprene, 3.4-339.6 ng/L for dimethyl sulphide, 2.4-768.9 ng/L for n-alkanes, and 3.5-1061.7 ng/L for n-aldehydes were analyzed with four needle trap devices. In addition, 4 NTDs were exposed to 6 blank samples from a Tedlar bag filled with nitrogen during 15 sampling cycles. LOD was directly determined by repeated analysis of the blank.14 To enhance sensitivity, three needle trap devices were exposed to gas mixtures during 25 sampling cycles. Substance concentrations in the mixtures ranged from 0.8 to 22.1 ng/L for acetone, from 0.8 to 22.8 ng/L for isoprene, from 0.2 to 4.8 ng/L for dimethyl sulphide, from 0.1 to 5.4 ng/L for n-alkanes, and from 0.1 to 6.2 ng/L for n-aldehydes. In total, 6 discrete concentration levels were used for these experiments. All samples were taken manually. Effects of Storage. Immediately prior to analysis, a Tedlar bag was filled with mixed expiratory breath gas and spiked with an alkane standard mixture (100-192 ng/L) and an aldehyde standard mixture (122-221 ng/L). A needle trap device was loaded through 15 sampling cycles by manual sampling. The needle was desorbed immediately afterward. After desorption, the needle was stored for 5 min outside the injector at room temperature for cooling. The same needle was loaded in the same way as before and sealed with Teflon caps on both ends. The needle was stored for 10 h, 3 days, or 7 days before the second desorption. RESULTS AND DISCUSSION Mode of Sampling. In contrast to blood or tissue analysis, sampling of breath is completely noninvasive, and testing can be done repeatedly and frequently without any burden to the patient and without any risk for the staff collecting the samples. Potential applications of breath analysis include the detection of lung diseases, recognition of inflammatory and malignant processes in the body, as well as detection of special diseased states such as allograft rejection and renal failure. For our experiments, we developed an on site sampling technique optimized for the needs of clinical in vivo sampling. It can be applied (at the bedside) without need of sophisticated equipment by means of a T-piece and any gastight syringe with standard Luer connection. By moving the plunger of the syringe up and down under control of expired CO2, one can achieve reliable alveolar sampling.12 Only alveolar substance concentrations reflect concentrations of endogenous compounds in blood.11,15 Regardless of the analytical method applied afterward, controlled alveolar sampling is a basic requirement for biomarker recognition in breath analysis. Most breath biomarkers occur in the part per trillion volume (pptV) range and often hundreds of other endogenous or exog(14) Huber, W. Accredit. Qual. Assur. 2003, 8, 213–217. (15) van den Velde, S.; Quirynen, M.; van Hee, P.; van Steenberghe, D. Anal. Chem. 2007, 79, 3425–3429.


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Figure 3. Effects of dry purging. Breath gas spiked with standard was adsorbed onto NTDs and the effect of dry purging with a constant helium flow (100 mL/min) was investigated. The purging time varied from 2 to 30 s. After purging, NTDs were desorbed at 300 °C for 0.5 s. The figure shows selected analytes standardized to 0 s dry purging time (no purging) as a function of purge time: gray triangle, isoprene; black diamond, acetone; gray box with plus sign, 2,3dimethylbutane; gray box, heptane; gray box with asterisk, hexane; gray circle, pentane.

enous compounds having orders of magnitude higher concentrations are found simultaneously in exhaled air. Hence, adequate sampling preparation, proper separation, identification, and quantification are basic requirements for reliable recognition of endogenous biomarkers. GC/MS coupled with an adequate preconcentration technique is the method of choice for that task.15,16 In Vivo Sampling. Figure 2 shows in vivo sampling from a mechanically ventilated animal. The amount of acetone adsorbed into the needles during manual alveolar sampling of 10 mL was seventy times higher than during mixed expiratory sampling of 100 mL of breath gas by means of an aspiration pump. The reasons for the seventy times lower efficacy of mixed expired sampling are elution of already adsorbed substances by inspired air and dilution of the whole sample by dead space air. Additional improvement of adsorption with the alveolar method may have been achieved through bidirectional sampling which was a consequence of the up and down movement of the plunger. This hypothesis was affirmed by the fact that we found excellent linearity and sensitivity for the manual (alveolar) sampling method despite using considerably higher sampling flows than those

described by Eom et al. for BTEX analysis.4 They recommended sampling flows e1.9 mL/min, whereas flows used in this study were as high as 60 mL/min. Effect of Dry Purging Time. Removal of water prior to analysis is a common method when solid phase extraction is used with humid matrices. Since breath samples are saturated with water vapor we investigated different purging times to evaluate the effect of (dry)purging onto the NTDs. Figure 3 shows peak areas of selected analytes standardized to 0 s dry purging time (no purging) as a function of purge time. For all substances dry purging led to a decrease of measured concentrations indicating loss of compounds during the purging step. Depending on the physicochemical properties of the substances the loss after 2 s (purge time) was up to 45%. Therefore, dry-purging prior to analysis cannot be recommended. In addition, remaining water content in the needle traps did not decrease substance concentrations or hamper analysis. On the contrary, some water in the NTDs may help to improve desorption of compounds.3 Effect of Sampling Cycles (Total Volume of Samples). Increasing the volume drawn through the NTD through additional ups and downs of the syringe plunger results in a linear increase of adsorbed substance amounts. No saturation occurred up to 40 sampling cycles (40 mL). Figure 4 shows peak areas as a function of the number of sampling cycles. In contrast to SPME, the sensitivity of the method can be enhanced considerably in this way. SPME is a fast and reliable method for the detection of volatile substances in breath gas and is based on distribution effects between gaseous and solid (liquid) phases. Sensitivity is defined by the physicochemical properties of the coatings and cannot be enhanced by modification of the sampling procedures. Especially low boiling substances may have a very small distribution coefficient with the SPME material. In principle, sensitivity of SPE can be improved when total sample volumes are increased. But sample volumes necessary to capture substances in the parts per trillion range may be as high as 3 L. Therefore, reliable alveolar sampling becomes more difficult with SPE, long sampling times and additional steps prior to analysis such as water removal or cryofocusation are required and the patient may be stressed by the sampling procedures.

Figure 4. Amounts of adsorbed substance increased linearly when additional ups and downs of the syringe plunger (sampling cycles) were performed. Standard spiked breath gas was sampled with 1 to 40 sampling cycles. The figure shows selected VOC peak areas as a function of theoretical total substance amount adsorbed onto the needle: black box, 2,3-dimethylbutane (R 2 ) 0.9880); black triangle, pentane (R 2 ) 0.9917); medium gray box, hexane (R 2 ) 0.9957); light gray box, heptane (R 2 ) 0.9849); light gray diamond, 2,2-dimethylbutane (R 2 ) 0.9895). 5854


Figure 5. NTDs were exposed to gas mixtures containing different concentrations of volatile substances during a varying number of sampling cycles. Gas mixtures prepared for this experiment contained volatile substances in concentrations mimicking low, middle, and high ranges of concentrations in breath gas. Five sampling cycles from the mixture with highest substance concentrations, 15 cycles from the mixture with moderate concentrations, and 25 cycles from the mixture with lowest concentrations should result in identical substance amounts adsorbed onto the NTDs. NTDs were desorbed in a 300 °C GC injector for 30 s. The figure shows mean counts and standard deviations for different numbers of sampling cycles (4 NTDs): light gray, 5 sampling cycles; medium gray, 15 sampling cycles; dark gray, 25 sampling cycles.

In NTD loading, moving up the plunger of the syringe during expiration and down during inspiration provides an easy method for direct breath gas sampling as the up or down movement can easily be accomplished in less than one second. In this way, sampling can also be done during alveolar phases of multiple breathing cycles. Taking samples with more than 40 cycles is not feasible in clinical practice because sampling times become too long. For other applications, such as headspace analysis, it may be useful to increase the number of sampling cycles even beyond 40. To confirm this hypothesis and to show that the effects were independent of concentration, NTDs were exposed to different substance concentrations and varying numbers of sampling cycles. Figure 5 shows that the experiments with 5, 15, or 25 manual sampling cycles yielded almost identical responses when corresponding concentrations and number of sample cycles were chosen in the way that the total amount of substance adsorbed should be identical. Desorption and Reconditioning. Figure 6 shows mean peak areas for selected VOCs at different desorption temperatures between 250 and 300 °C. For most compounds desorbed amounts only slightly increased at 300 °C when compared to lower temperatures. This suggests that for the NTD used in this study the internally expanding flow enables fast and complete desorption in the way that temperature (Eom1) and temperature gradients (Wang2) in the GC inlet did not have a decisive effect on desorption. This can be explained by the fact that Wang et al. used conventional flow through desorption and Eom worked with NTDs containing only small amounts of one adsorbent. Eom suggested to use a desorption temperature of 300 °C and a desorption time of 1.5 min combined with cryogenic refocusing for his NTDs filled with 10 mm of Carboxen to overcome the problem of peak broadening. Probably because of the different desorption geometry cryofocusation after desorption was not

Figure 6. Effects of different desorption temperatures. A humidified standard (mimicking substance concentration in breath gas) was adsorbed during 10 sampling cycles onto NTDs and desorbed in a hot injector at 250, 285, and 300 °C for 30 s. The figure shows mean peak areas of selected VOCs at each desorption temperature. All needle trap devices had the same amount of substances adsorbed and were desorbed for 30 s in an injector with different temperatures: light gray, 250 °C; medium gray, 285 °C; black, 300 °C.

Figure 7. Carry over. NTDs were loaded manually through 15 sampling cycles (15 mL). Gas mixtures prepared for that purpose contained isoprene 1.4 µg/L, acetone 7.9 µg/L, isopropanol 6.3 µg/ L, aldehydes 0.8 µg/L and n-alkanes 0.6 µg/L. Substances concentrations were chosen in the way to mimic concentrations in the upper range of naturally occurring breath gas. The manually loaded NTDs were then inserted into a GC injector (at 300 °C), removed from there 0.5 min later and stored in a needle tray. During storage the Luer lock end of the NTD was sealed with a Teflon cap. After 30 min a second injection of the NTD was performed. The figure shows a detail from a total ion chromatogram: Upper trace 1st desorption (offset ) 45 kCounts), lower trace 2nd injection after storage.

necessary with the multibed needles used in this study. The expandable volume between the Teflon seal and the 30 mm adsorbent bed was about 20% higher than in a 23 gauge needle with 10 mm packing. Because of the combination of different materials, having different adsorbent strengths, instantaneous desorption of substances was facilitated. A desorption time of 0.5 min was sufficient for a good repeatability without additional reconditioning. After exposure of the NTDs to standard mixtures containing high concentrations of alkanes, aldehydes, acetone, isoprene, and isopropanol, which typically occurs as a contaminant in the clinical environment carry over was e1% after desorption without additional reconditioning (Figure 7). Analytical Chemistry, Vol. 81, No. 14, July 15, 2009

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Table 1. Reproducibility (RSD %, n ) 5) of Measurements with Different Times of Conditioning at 300 °C before Analysisa condition time (s)

isoprene

acetone

butane, 2.2-dimethyl-

butane, 2-methyl-

heptane

hexane

pentane

pentane, 2-methyl-

0 900 1680

6.8 7.4 6.4

11 9.9 3.8

5.4 8.8 7.9

8.6 9.8 10

0.9 4.0 3.5

4.8 3.8 3.6

2.8 7.9 5.7

6.9 5.5 5.4

a A humidified standard (2.5-4.5 µg/L) was sampled with 10 sampling cycles. After desorption (30 s), the needle was conditioned in the hot injector for 0, 900, or 1680 s. Afterward the needle was used again without further preparation.

Table 2. Interneedle Variationa substances isoprene acetone dimethyl sulphide pentane hexane pentanal hexanal 2-butenal

% relative standard deviation 19.7 8.9 13.0 7.5 2.2 15.2 10.4 13.7

(32.6 ng/L) (47.4 ng/L) (3.4 ng/L) (4.6 ng/L) (5.4 ng/L) (5.2 ng/L) (7.1 ng/L) (4.9 ng/L)

16.6 5.9 4.7 12.8 17.1 11.1 11.2 14.5

(914 ng/L) (664 ng/L) (95.1 ng/L) (110 ng/L) (129 ng/L) (213 ng/L) (255 ng/L) (177 ng/L)

12.8 4.7 1.5 10.8 10.6 10.6 9.4 14.5

(1958 ng/L) (4740 ng/L) (340 ng/L) (657 ng/L) (769 ng/L) (885 ng/L) (1062 ng/L) (490 ng/L)

rb

LOD [ng/L]

0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.98

14.8 13.5 5.3 1.9 5.4 19.5 27.8 33.9

a Relative standard deviations of analyses at three concentration levels with 4 different NTDs and 15 sampling cycles. b Correlation coefficient of the calibration curve.

Figure 8. Recoveries (%) of spiked breath gas samples after storage for 10 h, 3 days, and 7 days. Breath gas collected in a Tedlar bag and spiked with standard, was adsorbed through 15 sampling cylces onto NTDs and desorbed at 300 °C for 30 s. Directly after 1st desorption the needles were cooled down to room temperature, and the adsorption process was repeated with the same standard mixture. The needles were capped with Teflon seals at both ends and stored at room temperature for 10 h, 3 days, and 7 days before 2nd desorption at 300 °C for 30 s: light gray, 10 h; medium gray, 3 days; black, 7 days.

Table 1 shows relative standard deviations for the analysis of selected compounds after NTD conditioning times between 0 and 1680 s (30 s desorption time). Conditioning time did not affect repeatability and sensitivity of the measurements significantly. Thus, NTDs can provide significantly shorter intervals for multiple analyses when compared to SPE and SPME. Linearity and Sensitivity (Limits of Detection and Quantification). Table 2 shows relative standard deviations for analyses of volatile substances in three different concentrations when 15 sampling cycles (15 mL) were applied to four NTDs. The correlation coefficients were in the range of 0.98 for 2-butenal and 0.99 for all other compounds. All calibration curves showed good linearity. The standard deviations were acceptable if one takes into account that needle trap devices and standard mixtures were 5856


handmade and sampling was done manually. In addition, Table 2 shows limits of detection determined from analysis of 6 blanks from Tedlar bags filled with nitrogen. Sample preparation was identical and consisted of 15 sampling cycles with 4 NTDs. Pentane showed a LOD of 1.9 ng/L; acetone showed a LOD of 13.5 ng/L, and 2-butenal showed one of 33.9 ng/L. To improve detection limits more sampling cycles (25) were performed with three NTDs. The LODs were found at 0.4 ng/L for isoprene, 0.5 ng/L for dimethyl sulphide, 0.9 ng/L for 2-butenal, 1.0 ng/L for hexane, 1.2 ng/L for pentane, 2.3 ng/L for hexanal, 5.3 ng/L for pentanal, and 8.3 ng/L for acetone. This demonstrates that the sensitivity of the method can be increased and extended into the ppt range of VOC concentrations by simply performing more ups and downs of the syringe plunger, that is, by increasing the total sample volume without losing precision or linearity. Storage. In breath analysis, two principally different approaches are applied for on site sampling. Sampling in gastight bags (e.g., Tedlar) or direct preconcentration on sorbent traps. Stability of breath constituents in bags is limited.17,18 Within 10 h concentrations of aldehydes, such as hexanal fell to 65% of the initial concentrations when samples were stored in Tedlar bags.19 Neither are reactive compounds stable in these recipients20 and even short storage times may cause considerable losses. Hence, the number of samples that can be collected and processed consecutively during clinical studies is limited. For the same reasons, duration of storage and transport of breath samples is restricted to a few hours. Figure 8 shows mean recoveries in percent after storage of loaded NTDs for 10 h, 3 days, and 7 days (16) Sanchez, J. M.; Sacks, R. D. Anal. Chem. 2006, 78, 3046–3054. (17) McGarvey, L. J.; Shorten, C. V. Aihaj 2000, 61, 375–380. (18) Steeghs, M. M.; Cristescu, S. M.; Harren, F. J. Physiol. Meas. 2007, 28, 73–84. (19) Beauchamp, J.; Herbig, J.; Gutmann, R.; Hansel, A. J. Breath Res. 2008, 2, 046001. (20) Mochalski, P.; Wzorek, B.; Sliwka, I.; Amann, A. J. Chromatogr. B 2009, 877, 189–196.

at room temperature. Aldehydes had excellent recoveries >90% after 7 days of storage. Recoveries of alkanes depended on the size of the molecules. After 3 days, there was a loss of butane of 56%, but only 15% for pentane. After 7 days the recovery of pentane fell below 65%. Aldehydes were described as potential markers of oxidative stress, therefore a reliable screening method with direct sampling and good stability for aldehydes would be desirable. Regarding the recovery of just 65% for hexanal after storage for 10 h in Tedlar bags,19 NTDs represent a good alternative for the storage of breath gas aldehydes. Alkanes larger than butane should be measured within 3 days.

CONCLUSION Needle trap devices packed with more than one adsorbent material represent a promising alternative to SPE and SPME for analysis of volatile organic compounds in the low part per billion/ part per trillion range. Crucial problems of clinical breath analysis concerning sensitivity of analytical methods, limited stability, and decomposition of breath compounds during sampling and storage can be solved though the smart combination of sampling and sample preparation described in this study. Received for review April 30, 2009. Accepted June 5, 2009. AC9009269


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Multibed Needle Trap Devices for on Site Sampling and

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