Application of Low-Temperature Glassy Carbon-Coated Macrofibers

Anal. Chem. 2003, 75, 1604-1614

Application of Low-Temperature Glassy Carbon-Coated Macrofibers for Solid-Phase Microextraction Analysis of Simulated Breath Volatiles Matthew Giardina and Susan V. Olesik*

Department of Chemistry, The Ohio State University, 100 West 18th Avenue, Columbus, Ohio 43210

With increasing interest in the detection of disease-related volatile organic compounds (VOCs) found in human breath, breath analysis could prove to be a very useful diagnostic tool, especially for the early detection of lung cancer. Solid-phase microextraction (SPME) is a technique well suited for breath analysis and has been applied to studying VOCs in the nanomolar concentration range. However, many compounds of interest in human breath are excreted at picomolar concentrations and may be unsuitable for analysis using conventional SPME sorbent phases. To extend the concentration range of conventional SPME, a novel 4-cm-long, low-temperature glassy carbon (LTGC) macrofiber was developed. The LTGC SPME macrofibers were used to extract five lung cancer-related VOCs (2-methylheptane, styrene, propylbenzene, decane, undecane) at conditions simulating human breath, and they were analyzed via gas chromatography/mass spectrometry. Results show that detection limits are lower using the SPME macrofibers compared to a conventional SPME fiber, in the low- to sub-picomolar range for the compounds of interest, which should be adequate for the analysis of these compounds in human breath. Also, the LTGC SPME macrofibers demonstrate significantly greater extraction efficiencies, sensitivity, and peak identification accuracy compared to that of commercial PDMS/DVB fibers without excessive chromatographic peak broadening. The use of SPME macrofibers broadens the potential range of application of SPME where the rapid extraction of very low levels of volatile compounds is required.

No mechanical instrumentation or pumps are needed for sampling, thereby reducing the complexity of collection and limiting the possibility of cross-contamination due to sample carryover from one individual to another, which is more likely with complex breath-sampling devices.2-4 Collection of breath volatiles using SPME may be applied either passively or actively. Active sampling involves the collection of the breath volatiles while the individual expels breath over a fiber attached to a simple mouthpiece. Passive sampling requires the collection of breath in a sample bag or container for extraction at a later time. The interest in studying the volatile organic compounds (VOCs) found in human breath is a result of the identification and correlation of certain compounds (i.e., markers) with a variety of diseases. On average, human breath contains several thousand endogenous VOCs.5 Many of these compounds can be attributed to normal metabolic processes in which VOCs passively diffuse into the breath stream from the blood via the alveoli.4,5 For example, the presence of straight-chain hydrocarbons is a result of free radical-initiated lipid peroxidation of polyunsaturated fatty acids found in cellular membranes.6 Accordingly, any changes in the amount of endogenous hydrocarbons present in breath can be linked to a number of disorders that affect this particular metabolic pathway. Increased levels of hydrocarbons in breath have been associated with individuals suffering from pulmonary, liver, autoimmune, bowel, and neurologic diseases to name a few.6 Other VOCs in breath are found to be markers of more specific pathological conditions such as isoprene for hypercholesterolemia and acetone for diabetes.1-3 In light of this information, a number of researchers have attempted to identify particular VOCs that are associated with lung cancer.7-11

Solid-phase microextraction (SPME) has demonstrated a great deal of potential in the study of breath volatiles. In recent publications, it was successfully used to extract and quantify levels of isoprene, acetone, and ethanol in simulated and actual breath samples.1,2 SPME is uniquely suited for the study of breath volatiles because of its relative simplicity, consisting of an extracting material coated on a fused-silica or stainless steel fiber.

(3) Manolis, A. Clin. Chem. 1983, 29, 5-15. (4) Phillips, M. Anal. Biochem. 1997, 247, 272-278. (5) Phillips, M.; Herrera, J.; Krishnan, S.; Zain, M.; Greenberg, J.; Cataneo, R. N. J. Chromatogr., B 1999, 729, 75-88. (6) Kneepkens, C. M. F.; Lepage, G.; Roy, C. C. Free Radical Biol. Med. 1994, 17, 127-160. (7) Gordon, S. M.; Szidon, J. P.; Krotoszynski, B. K.; Gibbons, R. D.; O’Neill, H. J. Clin. Chem. 1985, 31, 1278-1282. (8) O’Neill, H. J.; Gordon, S. M.; O’Neill, M. H.; Gibbons, R. D.; Szidon, J. P. Clin. Chem. 1988, 34, 1613-1618. (9) Preti, C.; Labows, J. N.; Kostelc, J. G.; Aldinger, S.; Daniele, R. J. Chromatogr. 1988, 432, 1-8. (10) Phillips, M.; Gleeson, K.; Hughes, J. M. B.; Greenberg, J.; Cataneo, R. N.; Baker, L.; McVay, W. P. Lancet 1999, 333, 1930-1933. (11) Rizvi, N.; Hayes, D. F. Lancet 1999, 353, 1897-1898.

* To whom correspondence should be addressed. Phone: (614) 292-0733. Fax: (614) 292-1685. E-mail: [email protected]. (1) Hyspler, R.; Crhova, S.; Gasparic, J.; Zadak, Z.; Cizkova, M.; Balasova, V. J. Chromatogr.. B 2000, 739, 183-190. (2) Grote, C.; Pawliszyn, J. Anal. Chem. 1997, 69, 587-596.

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10.1021/ac025984k CCC: $25.00

© 2003 American Chemical Society Published on Web 02/27/2003

Lung cancer is a devastating disease. In the United States, more deaths are attributed to lung cancer than breast, prostate, and colon cancers combined. It is estimated that 169 400 new cases will be diagnosed and 154 900 deaths will be attributed to lung cancer this year.12 Needless to say, the overall prognosis for individuals suffering from this disease is disappointing. The 5-year survival rate is less than 15% for all types of lung cancer.12 It is believed this outcome is partially a result of inadequate screening techniques. In hopes that by diagnosing the cancer during very early changes in lung tissue, before masses are large enough to be imaged by CT, the survivability rate for this virulent disease will improve. An early screening technique has a great deal of potential in combating lung cancer especially when used in combination with new cutting-edge therapies utilizing antiangiogenesis agents, monoclonal antibodies, and vaccines. In an effort to achieve this goal, new screening techniques including breath analysis are being investigated.7-11 Phillips et al. correlated the presence of 22 VOCs as markers of lung cancer.10 Using discriminant analysis, the researchers correctly predicted 71.7% of patients with lung cancer and 66.7% of those without in a cross-sectional study consisting of 108 high-risk participants. In light of these encouraging results, the authors suggested that larger studies encompassing individuals from the general population should be included to improve the specificity of this diagnostic tool. However, the breath collection device used by the researchers was complex, consisting of tubing, valves, and an adsorbent trap, in which sampling was controlled by a microprocessor.4,5 Due to the complexity of the sampling device, the efficacy of conducting large-scale studies is dubious. Also, it must be noted that any new technique for detecting lung cancer must lead to a significant improvement in cure rates to offset the cost of the screening method. In general, a screening method should translate into a cost per life-year saved of no more than $50 000.13 With this in mind, a simpler breath collection strategy may be warranted. SPME has the desired characteristics as one such technique that should be investigated due to its relative simplicity and cost-effectiveness. The concentrations of the compounds identified in Phillips’ work were not explicitly determined; however, it was suggested that many of these compounds are present at extremely low concentrations in the nanomolar to picomolar range. The published data concerning SPME breath analysis focused mainly on studying breath VOCs toward the higher end of the concentration spectrum, in the nanomolar range.1,2 For SPME to successfully be applied in the extraction of compounds at lower concentration range, the overall extraction efficiency must be increased. One approach to extending the useful range of SPME for compounds in the subnanomolar range is to increase the amount of extracting material per fiber. From fundamental thermodynamic principles, increasing the coating thickness of the SPME fibers does increase extraction efficiencies.14 However, the ability to increase the amount of extracting material by increasing the fiber coating thickness and diameter is limited due to the configuration of an (12) In Cancer Facts and Figures; American Cancer Society: Atlanta, GA, 2002. (13) Henschke, C. L.; Naidich, D. P.; Yankelevitz, D. F.; McGuinness, G.; McCauley, D. I.; Smith, J. P.; Libby, D.; Pasmantier, M.; Vazquez, M.; Koizumi, J.; Flieder, D.; Altorki, N.; Miettinen, O. S. Cancer 2001, 92, 153159. (14) Pawliszyn, J. Solid-Phase Microextraction: Theory and Practice; Wiley-VCH: New York, 1997.

unmodified GC injection port. On the other hand, current SPME fibers are produced on the order of 1 cm in length and only occupy a minor portion (∼1/8) of a typical GC injection port. Therefore, increasing the length of the extracting fiber is a logical approach to extending the lower concentration limits of SPME. In an earlier work, low-temperature glassy carbon (LTGC)SPME fibers prepared with conventional dimensions exhibited unique retention characteristics.15 The dominant mechanism of extraction was based upon the ability of a solute molecule to engage in dispersive interactions with the surface of the LTGC. The amount of solute extracted was shown to be dependent upon the cross-sectional surface area and the polarizability of the molecule. The LTGC phase also showed a range of selectivity that was a function of the final processing temperature of the oligomer. For certain compounds, the LTGC processed at low temperatures (300-400 °C) displayed extraction selectivity similar to PDMS and for higher processing temperature (600-800 °C), the selectivity was closer to PDMS/DVB. In this study, the use of a novel SPME format was explored in the form of LTGC macrofibers for the extraction of five out of the top six compounds identified in Phillip’s work. The LTGC SPME macrofibers were prepared with a length of 4 cm and a coating thickness of ∼11 µm. Extractions were performed on vapor-phase mixtures of 2-methylheptane, styrene, propylbenzene, decane, and undecane under simulated breath analysis conditions to model the effects of humidity and other interfering compounds. The performance of the macrofibers was evaluated by comparing the extraction efficiencies, sensitivity, limit of detection, chromatographic band broadening, and peak identification accuracy to a commercial PDMS/DVB fiber. This investigation into the use of macrofibers is an initial attempt to aid in the development of a simple screening technique for the wide-scale evaluation of breath analysis as a diagnostic tool for lung cancer. EXPERIMENTAL SECTION Instrumentation. A Hewlett-Packard 5890 Series II Plus gas chromatograph equipped with an electronic pressure-controlled split/splitless injector port and a 5972 mass-selective detector (MSD) was used for the analysis of all the compounds. The instrument was calibrated daily by injecting replicate samples of a 100 ppb standard solution diluted in dichloromethane. A smallvolume injection sleeve with an inner diameter of 0.75 mm was used in conjunction with an SPB-5 capillary column (1.0-µm film thickness, 30 m long, 0.25-mm inner diameter), both purchased from Supelco (Bellefonte, PA). A 65-µm poly(dimethylsiloxane)/ divinylbenzene (PDMS/DVB) fiber was used for comparative extractions and was also purchased from Supleco. This fiber was chosen because it was shown to have extraction characteristics similar to that of the LTGC processed at intermediate temperatures15 and because it is recommended by the manufacturer for the extraction of semivolatile compounds over a mass range of 90-150 amu. For analysis of the macrofibers, the injection port temperature was held at a constant 320 °C. The oven temperature was held at 50 °C for 1 min and then ramped 20 °C/min to 200 °C followed by a second ramp of 40 °C/min to a final temperature of 280 °C. The mass spectrometer transfer line was held at 280 °C. The mass (15) Giardina, M.; Olesik, S. V. Anal. Chem. 2001, 73, 5841-5851.

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spectrometer was tuned using PFTBA mass fragments 50, 131, and 219. The MSD was operated in scan mode monitoring masses between 40 and 200 m/z; however, only selected ions (the base peaks) were used for quantification: 2-methylheptane (43 m/z), styrene (104 m/z), propylbenzene (91 m/z), decane (57 m/z), and undecane (57 m/z). For the analysis of the PDMS/DVB fibers, the same GC/MS method was used with the exception of using an injection port temperature of 250 °C. Chemicals. All the chemicals used for model extractions, 2-methylheptane, styrene, propylbenzene, decane, undecane, and isoprene, were purchased from Sigma-Aldrich (St. Louis, MO) with a purity of 99% or greater. The LTGC oligomer 1,3-diethynyl(5-trimethylsilyl)benzene was prepared in-house using a previously documented procedure.16 LTGC SPME Macrofiber Preparation. The LTGC SPME macrofibers were prepared using a four-step process. First, porous silica beads were encapsulated in the LTGC oligomer precursor and then thermally treated to form the glassy carbon. The porous silica is needed to act as a solid support to provide a large surface area to the glassy carbon. Next, the porous LTGC-encapsulated silica was immobilized on stainless steel fibers using a sol-gel process.17 After the films were dried and washed, a second encapsulation and thermally treatment procedure was carried out to ensure that any silica surface formed during the sol-gel process was encapsulated in LTGC. In the final step, the SPME LTGC macrofibers were mounted into a modified 7000 series syringe for evaluation (Hamilton Co., Reno, NV). The LTGC-encapsulated silica was prepared using a method previously published with a few modifications.15 The porous silica with mean particle diameter of 5 µm and a surface area of 401 m2/g (Phenomonex, Torrance, CA) was encapsulated with the LTGC oligomer using an evaporative coating process. To form the LTGC, the oligomer-encapsulated silica was thermally processed in a quartz tube furnace to a final temperature of 350 °C using a linear temperature ramp of 1 °C/min, starting from ambient room temperature. The final temperature was maintained for a minimum of 10 h. All thermal processing was carried in an atmosphere of 5% hydrogen/95% nitrogen. The macrofiber substrates were prepared by cutting 12.2-cm ((0.1 cm) lengths of 316 stainless steel wire with a diameter of 200 µm (Small Parts, Miami, Lakes, FL). To enhance the thermal stability of the steel, a thin film of elemental silicon was applied to the steel using the Silcosteel process (Restek, Bellefonte, PA). Before coating the Silcosteel substrates with the LTGC-encapsulated silica, the silicon films were oxidized by heating them to 350 °C in air for ∼2 h and then hydrolyzing in a (10:1) solution of concentrated H2SO4/HNO3 heated to 95 °C for ∼30 min. The LTGC-encapsulated silica was coated on the hydrolyzed Silcosteel macrofibers using a sol-gel process. Approximately 0.8 g of formamide (Fisher Scientific, Pittsburgh, PA) was added to 3.2 g of Kasil No. 1, a potassium silicate solution, (PQ Corp., Valley Forge, PA). The sol-gel solution was mixed and then centrifuged. The supernatant liquid was removed and placed into a clean 10 × 75 mm culture tube. A 4-cm section of the modified stainless steel fiber was first dipped into the sol-gel solution and then into to LTGC-encapsulated porous silica. The dipping process was (16) Callstrom, M. R.; Neenan, T. X.; McCreery, R. L.; Alsmeyer, D. C. J. Am. Chem. Soc. 1990, 112, 4954-4956. (17) Shoup, R. D. Colloid and Interface Science; Academic Press: New York, 1976.

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Figure 1. Image of LTGC macrofiber (top) and commercial PDMS/ DVB fiber (bottom).

repeated 4 times. The fibers were dried at room temperature and ambient air for a minimum of 24 h. The coated fibers were rinsed with water and methanol and then conditioned in a quartz tube furnace to 350 °C for at least 1 h in an atmosphere of 5% hydrogen/ 95% nitrogen. The final encapsulation of the LTCG SPME macrofibers with the LTGC oligomer was performed by suspending the macrofiber in a solution containing 7 mg of the LTGC oligomer dissolved in 30 mL of heptane and 30 mL of methylene chloride. The solution was gradually heated to evaporate the methylene chloride, allowing to LTGC oligomer to precipitate onto the coated macrofiber. To keep the volume of solvent constant during the coating procedure, neat heptane was added to the coating solution as the methylene chloride evaporated. After this final encapsulation step, the macrofibers were thermally processed to 350 °C in a quartz tube furnace under an atmosphere of 5% hydrogen/95% nitrogen. Heating was carried out using a linear temperature ramp of 1 °C/min, starting from ambient room temperature. The final temperature was maintained for a minimum of 10 h and under an atmosphere of 5% hydrogen/95% nitrogen. The finished macrofibers (Figure 1) were mounted into modified Hamilton syringes in a manner similar to those previously published.14 Extraction Procedure. A stock solution of vapor standard was prepared by injecting 1 µL of each of the five neat compounds into a 1-L Septa-Jar vial (Fisher Scientific). The vial was equilibrated in a water bath set to 35 °C for 30 min. To dilute the VOCs to the desired concentrations before extraction, aliquots of the vapor stock solution were withdrawn into a gastight syringe and then diluted by injecting the vapor into clean 1-L Septa-Jar vials equilibrated at 35 °C. The water bath was large enough to accommodate 12 1-L containers at once. All diluted samples were allowed to equilibrate for a minimum of 30 min before extractions. New stock solutions and dilutions were prepared on the same day extractions were preformed. Between trials, the vials and caps were rinsed with small portions of hexane, dichloromethane, and methanol and were dried overnight in an oven set to 200 °C for the glass vials and 70 °C for the Septa-Jars. RESULTS AND DISCUSSION This section is divided into five parts. The first subsection discusses the coating technique and process of preparing the LTGC macrofibers. The second subsection presents data concern-

Figure 2. Extraction efficiencies of PDMS/DVB and LTGC macrofibers. Sampling was conducted under static conditions for 2 min at 35 °C. The concentrations of vapor standards were 6.2, 8.8, 7.2, 5.2, and 4.7 pM for 2-methylheptane, styrene, propylbenzene, decane, and undecane, respectively.

ing the general extraction performance of the LTGC macrofibers compared to a commercial PDMS/DVB fiber with respect to efficiency, sensitivity, chromatographic performance, and extraction mechanism. The third section analyzes the effect of potentially interfering compounds that are present in human breath. The fourth subsection presents a statistical evaluation of the extraction performance and forms the basis of comparison between the LTGC SPME macrofiber and PDMS/DVB fiber. In addition, the fourth subsection includes the calculation of detection limits. The final subsection discusses the issues of chromatographic peak identification related to mass spectrometric detection. LTGC SPME Macrofiber Preparation. The 4-cm macrofibers prepared in this study followed the same general procedure previously documented for the preparation of 1-cm LTGC SPME fibers with the addition of a few improvements.15 The most notable addition is the secondary encapsulation procedure of the macrofibers in which a final oligomer coating step is included to cover the silica surface introduced during the sol-gel bonding step. Because of the secondary encapsulation, it was necessary to modify the stainless steel using a pretreatment step to improve its thermal stability. Initial experiments indicated that the untreated stainless steel fibers degraded upon thermal treatment. Bare stainless steel fibers turned a bluish-pink after exposure to temperatures as low as 500 °C in the reducing atmosphere. Leaching of some of the steel components into the porous silica/ sol-gel coating was also apparent after processing blank fibers containing no LTGC oligomer. In this case, the characteristic white appearance of the porous/sol-gel silica turned a dark brown indicating the possible diffusion of steel components into the coating. To make the stainless steel fibers inert, they were treated with a thin film of elemental silicon using the Silcosteel processes (Restek). However, introduction of the silicon film reduced the wettability of the stainless steel fibers; therefore, it was necessary to hydrolyze the silicon film to restore adequate coating characteristics to the substrates. Although it was not explicitly studied, it is possible that the silanol groups produced on the surface of the silicon film after hydrolysis were available to participate in bonding to the sol-gel/porous silica structure similar to the manufacturing of MTX (Restek) capillary gas chromatographic

columns. If this is indeed the case, this method of preparing a general support for SPME by bonding the porous silica to a chemically and thermally inert substrate should be amenable to applying a variety of coating phases and could potentially prove to be a great improvement over the current technology of SPME fabrication. Extraction Performance. Initial experiments were performed to evaluate the extraction characteristics of the macrofibers. It should be noted that all extractions were carried out before thermodynamic equilibrium was achieved between the fiber coating and vapor phase, in the linear portion of the Langmuir isotherm. This was necessary to reduce any potential effects of competitive adsorption between solute molecules.18 Figure 2 shows the extraction efficiency of the fibers as measured by the percent of analyte extracted during a 2-min static sampling period. As a point of reference, the extraction efficiencies of a commercial PDMS/DVB 65-µm fiber were compared. As illustrated by the graph, there is an overall improvement in extraction efficiency of a factor of ∼5 relative to the commercial fiber. The next set of experiments was conducted to measure the extraction sensitivity. In this case, five-point calibration curves were constructed for the gaseous compounds during 2-min static extractions. Each concentration standard was measured in duplicate. The extraction sensitivity was determined by the slope of the calibration curves after fitting the lines using a standard method of least squares. All calibration curves were linear under the concentration ranges studied giving R2 values of 0.99 or greater. Figure 3 compares the extraction sensitivities of the LTGC macrofiber with that of the PDMS/DVB fiber under the same extraction condtions. The overall improvement in extraction sensitivity using the macrofibers over the commercial fiber is a factor of 4 on average. The 4-5-fold improvement in the extraction efficiencies and sensitivities with the macrofiber compared to the commercial fiber is to be expected as indicated by our previous research where the extraction recoveries of 1-cm LTGC fibers were of a magnitude similar to that for the 1-cm PDMS/DVB fibers.15 Accordingly, (18) Semenov, S. N.; Koziel, J. A.; Pawliszyn, J. J. Chromatogr., A 2000, 873, 39-51.


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Figure 3. Extraction sensitivity of PDMS/DVB and LTGC macrofibers. Sampling was conducted under static conditions for 2 min at 35 °C. The concentration ranges of vapor standards were 6-92, 9-131, 7-108, 5-77, and 5-71 pM for 2-methylheptane, styrene, propylbenzene, decane, and undecane, respectively.

Figure 4. Half-height peak widths for chromatographic separations for PDMS/DVB and LTGC macrofiber extractions. The concentrations of vapor standards were 31, 45, 36, 26, and 24 pM for 2-methylheptane, styrene, propylbenzene, decane, and undecane, respectively.

increasing the length from 1 to 4 cm correspondingly increases the extraction efficiency and sensitivity by approximately the same ratio. To ensure that using the macrofibers did not lead to excessive band broadening during the gas chromatographic separation, the half-height peak widths for each of the chromatographic peaks were studied. A significant increase in the peak widths would indicate a reduction in separation performance during analysis. This could potentially be a significant problem, especially considering the overall number of VOCs contained in breath. However, no significant increase in chromatographic peak widths was noted as compared to the PDMS/DVB fiber (Figure 4). The reason for this is due to the relatively high injection port temperature of 320 °C, which is the maximum recommended operating temperature of the column. Effects of Interfering Compounds. Because breath contains a multitude of components, the potential effect of interfering compounds is great, especially when using solid-phase extraction media. Perhaps the greatest interfering compound in breath is 1608


water vapor because it is present in such high concentration. To model the effects of water vapor on the extraction of the breath volatiles, extractions were carried out at 100% relative humidity (RH). To produce environments of 100% RH at 35 °C, 39 µL of purified water was injected into the 1-L vials before the analytes were injected into the vials. The 100% RH systems were allowed to equilibrate for 30 min before extractions were performed. On average, the amount extracted with the LTGC macrofibers is reduced by a factor of 1.5 (Figure 5). Interestingly, the impact of the water vapor on the PDMS/DVB fiber is negligible, which is in contrast to studies that indicated that the extraction efficiencies of these phases are highly dependent upon water vapor concentration.2,19 Nonetheless, it can be seen that high concentrations of water in the system reduce the overall extraction efficiency of the macrofibers by competitively interfering. Therefore, any extraction procedure must carefully control the amount of water vapor present during the extraction. This is easily accomplished (19) Gorecki, T.; Yu, X.; Pawliszyn, J. Analyst 1999, 124, 643-649.

Figure 5. Effect of humidity on extraction recoveries for PDMS/DVB and LTGC macrofibers. Sampling was conducted under static conditions for 2 min at 35 °C. Vapor concentrations for 2-methylheptane, styrene, propylbenezene, decane, and undecane were 31, 44, 36, 26, and 24 pM, respectively.

Figure 6. Effect of isoprene on extraction recoveries for LTGC macrofibers. Sampling was conducted under static conditions for 2 min at 35 °C and 100% RH. Vapor concentrations for 2-methylheptane, styrene, propylbenezene, decane, and undecane were 31, 44, 36, 26, and 24 pM, respectively.

by spiking the sample containers with a sufficient amount of water to ensure vapor saturation.2 Another potential interference present in human breath at relatively high concentrations is isoprene, which is a byproduct of cholesterol synthesis. It is estimated the average concentration of isoprene in breath is ∼5 nM.1 This compound contains two conjugated π-bonds, allowing the molecule to adopt a planar geometrical configuration. Therefore, it is expected that the glassy carbon surface would have a high affinity for this compound and therefore cause potential interfering adsorption. To gauge the effect of adding the isoprene to the system, the 1-L vials were injected with the appropriate amount of isoprene to achieve a concentration level of 5 nM. Extractions were then performed at 100% RH with and without 5 nM isoprene and then compared. Figure 6 shows the result. Again, the overall amount extracted was reduced by a factor of 1.5.

One way to mitigate the impact of interfering compounds might be to reduce the sampling time. By reducing the sampling time, the competitive adsorption can be eliminated by returning the extraction profile to the linear region of the isotherm where the number of adsorption sites is greater than the number of adsorbed molecules. Figure 7 illustrates this principle. Two sets of extractions were carried out, one in which the five analytes were extracted in the presence of 100% RH and 5 nM isoprene and the other containing the five analytes in the presence of 100% RH alone. By subtracting the peak areas of the compounds extracted at 100% RH by the peak areas of the compounds in the 100% RH and isoprene at different time intervals, the effect of reducing the sampling time can be discerned. As expected, the interference of the isoprene is lessened as the sampling time interval decreases from 2 min to 1 min to 30 s. Ideally, it would be expected that at the point at which the interfering effect is totally eliminated, the Analytical Chemistry, Vol. 75, No. 7, April 1, 2003

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Figure 7. Effect of sampling time on extraction efficiencies for LTGC macrofibers with and without the addition of isoprene. The y-axis is the peak area for each compound extracted in the presence of isoprene and 100% RH subtracted from the peak area for extractions carried out in 100% RH alone. Vapor concentrations for 2-methylheptane, styrene, propylbenezene, decane, and undecane were 62, 88, 72, 52, and 47 pM, respectively.

difference in the peak areas would be zero. However, the bars on the graph indicate that a negative effect is occurring. In other words, at small sampling intervals, the isoprene in the system is enhancing the extraction of the five analytes. The cause of this behavior is currently under investigation. However, the practical effect of controlling sampling time to reduce interference is noted. Statistical Methods. The Student’s t- and F-tests were used as a basis to compare the performance of LTGC macrofibers with respect to the PDMS/DVB fibers to ensure unbiased evaluation.20 All significance testing and confidence limits were determined at the 95% confidence level (P ) 0.05). For the first series of extractions carried out under ambient humidity at 35 °C with no interfering compounds present (Figure 1), the average extraction recovery, standard deviation, relative standard deviation, and confidence limits were calculated for both the LTGC macrofiber and PDMS/DVB fiber and are given in Table 1. The second column in Table 1 lists the vapor concentration (picomolar) for each compound in the 1-L vial on which the extractions were performed. The fiber recovery data are given in terms of amount extracted (picograms) and are shown in the third column in Table 1. These units were chosen because the concentration of these analytes in human breath are thought to be in the low-picomolar range and are usually discussed in terms of these units. The extraction recoveries are listed in the absolute amount extracted (picograms) to provide a clear comparison between the fibers instead of the more ambiguous units of concentration, which would require a precise knowledge of the fiber surface area or volume. The variability in the extraction performance for each fiber was compared with respect to each analyte using the F-test. According to a two-tailed F-test, the variance in the extraction data showed significant differences between the LTGC and PDMS/ DVB fibers for each compound extracted except decane. The calculated F-values for 2-methylheptane, styrene, propylbenzene, decane, and undecane were 10.6, 9.2, 4.6, 1.9, and 4.1, respectively. The critical F-value used to compare variances for 2-methylheptane, styrene, propylbenzene, and decane was 3.7 for 10 degrees 1610 Analytical Chemistry, Vol. 75, No. 7, April 1, 2003

Table 1. Absolute Recoveries and Statistics for Replicate Extractions (High Concentration)a

compound LTGC 2-methylheptane styrene propylbenzene decane undecane PDMS/DVB 2-methylheptane styrene propylbenzene decane undecane

std vapor concn in 1-L vial (pM)

av extn recov (pg)

SD (pg)

RSD

95% confid interval ((pg)

6.2 8.8 7.2 5.2 4.7

237 240 165 256 260

59 25 9 18 26

0.25 0.11 0.06 0.07 0.10

40 17 6 12 20

6.2 8.8 7.2 5.2 4.7

52 42 57 70 30

14 6 4 11 13

0.28 0.15 0.07 0.16 0.42

10 4 3 8 9

a N ) 11 observations for each compound except for decane, which was N ) 9 for LTGC and N ) 10 for PDMS/DVB.

of freedom (df ) 10) for both fibers. The F-critical value for the decane determination was 4.9 with 8 and 9 degrees of freedom for the LTGC and PDMS/DVB, respectively. Therefore, the precision in extractions was significantly different for the majority of compounds. (This phenomenon will be considered in greater detail following the comparison of the mean extraction recoveries.) The mean extraction recoveries for the PDMS/DVB fiber and LTGC macrofiber were compared using a one-tailed t-test20 under the assumption that the larger fiber did indeed extract more analyte. For the compounds 2-methylheptane, styrene, propylbenzene, and undecane; the t-values were calculated by taking into account that the population standard deviations were found to be significantly different based on the results of the F-test above. On the other hand, for decane, a pooled standard deviation was used to calculate the t-value. The calculated t-values were found to be (20) Miller, J. C.; Miller, J. N. Statistics for Analytical Chemistry; Wiley & Sons: New York, 1984.

Table 2. Absolute Recoveries and Statistics for Replicate Extractions (Low Concentration)a


std vapor concn in 1-L vial (pM)

av extn recov (pg)

SD (pg)

RSD

95% confid interval ((pg)

1.5 2.2 1.8 1.3 1.2

nd 34 10 24 30

4.9 1.4 7.5 10

0.15 0.14 0.31 0.34

6 2 9 13

1.5 2.2 1.8 1.3 1.2

nd 9 3 nd nd

4.5 0.9

0.49 0.26

7 2

a N ) 5 for all extractions performed with the LTGC; N ) 4 for all extractions performed with the PDMS/DVB. ND, no chromatographic peaks were detected.

9.4 (df ) 12), 22.9 (df ) 12), 31.3 (df ) 14), 26.5 (df ) 20), and 23.8 (df ) 11) for 2-methylheptane, styrene, propylbenzene, decane, and undecane, respectively. The one-sided critical t-values used for comparison of all the compounds except decane was 1.8. For decane, the t-value was 1.7. In all cases, the null hypothesis can be rejected and all extractions performed with the LTGC macrofiber lead to a significantly greater amount of analyte extracted. The finding that extraction precisions are different between the two fibers is a result of heterocedastic behavior in which the standard deviations of the measurements are not constant and vary with concentration.21 Heterocedasticity has been noted in the literature for a number of analytical methods including gas chromatography,22-27 and several statistical techniques have been developed to compensate for such occurrences including the use of weighted least-squares regression. The nonuniform variance in the extraction data was thought to be a function of the amount of extracted analyte and not of the fiber configuration. This was confirmed by comparing the PDMS/DVB data in the first series of extractions to the lower level extractions performed in the third series of experiments with the LTGC (Table 2). The concentrations in the third series of extractions are reduced by a factor of 4, so the effect of the larger extraction efficiency of the LTGC macrofiber compared to the PDMS/DVB fiber was mitigated. Table 2 lists the extraction recoveries, standard deviation, relative standard deviation, and confidence intervals for these extractions. For the LTGC macrofiber, all analyte concentrations in the 1-L vials were sufficiently high enough to be extracted with the (21) Miller, J. N. Analyst 1991, 116, 3-14. (22) Schwartz, L. M. Anal. Chem. 1979, 51, 723-727. (23) Zorn, M. E.; Gibbons, R. D.; Sonzogni, W. C. Anal. Chem. 1997, 69, 30693075. (24) Garden, J. S.; Mitchell, D. G.; Mills, W. N. Anal. Chem. 1980, 52, 23102315. (25) Claeys, M.; Markey, S. P.; Maenhaut, W. Biomed. Mass Spectrom. 1977, 4, 122-128. (26) Rodriguez, L. C.; Casado, A. G.; Campana, A. M. G.; Vilchez, J. L. Chromatographia 1998, 47, 550-556. (27) Kurtz, D. A., Ed. Trace Residue Analysis: Chemometric Estimations of Sampling, Amount, and Error; ACS Symposium Series 284; American Chemical Society: Washington, DC, 1985.

exception of 2-methylheptane. For PDMS/DVB, only styrene and propylbenzene could be extracted at the lower concentration level. To determine whether the amount extracted correlated with the variability, the variance of the high-concentration extractions using the PDMS/DVB fiber (Table 1) was compared to that of the lower concentration extractions using the LTGC macrofiber (Table 2) for all the compounds except 2-methylheptane. This comparison was chosen on the basis of the fact that the absolute recoveries of the both fibers were on the same order of magnitude and therefore would be less likely to exhibit differences in variance based on heterocedastic behavior. A two-tailed F-test was performed to determine whether there was any significant difference in population standard deviations. The calculated F-values for styrene, propylbenzene, decane, and undecane were 1.4, 3.9, 1.8, and 3.5, respectively. The critical F-value used to compare variances for styrene, propylbenzene, and decane was 4.5 (df ) 4 for LTGC and df ) 10 for PDMS/DVB). The F-critical value for the undecane determination was 4.7 for 4 and 9 degrees of freedom for the LTGC and PDMS/DVB, respectively. Therefore, the variances were not statistically different, indicating that the system does indeed exhibit heterocedastic behavior based on the amount extracted and not the fiber configuration. In the second set of experiments, the extraction sensitivities were compared using the slope of the calibration curves generated from a series of vapor standards (Figure 3). Statistical comparisons of analytical method sensitivity have been documented in the chemical literature for linear regressions under assumed uniform variance using the t-test.28,29 The comparison of sensitivities under nonuniform variance is more complex due to the nonlinear dependence of variance on concentration as opposed to uniform variance where the standard deviation in the slope and intercept can be easily calculated.23 However in most circumstances, weighted least-squares (WLS) regressions compared to ordinary least-squares (OLS) regression produce minimal difference in determining the slope.23 The greatest differences in using a WLS regression usually occurs in determination of the intercept and in the back calculation of unknown concentrations predicted by the WLS parameters.20 Table 3 shows the calculated slope using both a WLS regression and OLS regression. For the WLS, the weighting factor was defined as the inverse of the variance (1/s2) for each data point to account for the higher weight given to the concentration values with smaller variance. The procedure followed for the WLS regression is outlined in greater detail in Miller and Miller.20 The standard deviation, confidence interval, and relative standard deviation associated with the OLS regression are also given in Table 3. To test whether the differences in extraction sensitivities between the two fibers are significant, first the OLS variances were compared using a two-tailed F-test. For the compounds 2-methylheptane, styrene, propylbenzene, decane, and undecane, the calculated F-values are 16.3, 5.9, 6.3, 5.7, and 9.0, respectively, and the F-critical value is 9.6 (df ) 4 for both fibers). Therefore, no differences in precision could be established at the 95% confidence level for the compounds except for 2-methylhepatane. To determine whether the extraction sensitivities performed with the LTGC macrofiber are indeed great than the PDMS/DVB fiber, (28) Sorrell-Raschi, L. A.; Tomasic, M. Am. J. Vet. Res. 1998, 59, 1519-1522. (29) Chen, S. T.; Thompson, R. C.; Poust, R. I. J. Pharm. Sci. 1981, 70, 12881289.


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Table 3. Comparison of Extraction Sensitivitiesa


vapor calibration range (pM)

WLS slope (ng/ppt)

OLS slope (ng/ppt)

OLS SD (ng/ppt)

OLS 95% CI ((ng/ppt)

OLS RSD

6-92 9-131 7-108 5-77 5-71

152 198 176 187 214

145 204 184 200 233

4 5 4 8 13

13 17 14 25 40

0.03 0.03 0.02 0.04 0.05

6-92 9-131 7-108 5-77 5-71

44 46 41 49 43

44 50 44 54 47

1 2 2 3 4

3 7 5 11 13

0.02 0.04 0.04 0.06 0.09

a Five standards with duplicate measurements for each calibration. OLS, ordinary least squares; WLS, weighted least squares; CI, confidence interval. Units of slope are in nanogram of analyte extracted per ppt sample concentration.

a one-tailed t-test was used. For styrene, propylbenzene, decane, and undecane; a pooled standard deviation was needed to calculate the t-value since no difference in standard deviations could be established based upon the F-test. For 2-methylheptane, the t-value was calculated using separate standard deviations based upon the results of the F-test as well. The calculated t-values for 2-methylheptane, styrene, propylbenzene, decane, and undecane were 53.9 (df ) 7), 59.6 (df ) 8), 66.7 (df ) 8), 37.9 (df ) 8), and 31.2 (df ) 7), respectively. The t-critical value in all cases was 1.9; therefore, the extraction sensitivities are significantly greater for the LTGC macrofiber. In the third set of extractions, the concentration of the vapor standards was 4 times lower than in the first series of standards and interfering agents (water vapor and isoprene) present in the system during the extraction. The reason for reducing the concentration of the analytes was based on estimations of the detection limits using the higher concentration standards and the perceived nonuniformity in variance. Interfering compounds were included to better model actual extraction conditions. A number of factors need to be considered before choosing a method to calculate the limit of detection (LOD) because of the general importance given to this figure of merit. The main difficulty involves the lack of a single accepted method that is used by all researchers. Two important factors must be considered before choosing a method. First, a LOD method should be chosen that is consistent with the basic principal of defining a concentration at which a signal is produced that can be reliable distinguished from background. Second, the LOD should serve as a valid figure of merit to compare the performance of similar techniques. The body of previous work in a particular technique dominates this later concern. With these considerations in mind, the method of LOD that was used to evaluate the performance of the LTGC macrofibers was chosen based on the repetitive measurement of a single low-concentration standard. This method is theoretically sound,30 and it has been used to evaluate the performance of SPME fibers in the extraction of other breath volatiles.1,2 The calculation of the LODs was based on the following formula: (30) Armbruster, D. A.; Tillman, M. D.; Hubbs, L. M. Clin. Chem. 1994, 40, 1233-1238.

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(Cstd) s LOD ) 3 ) 3(Cstd) jx (S/N)

()

(1)

where Cstd is the concentration of the standard, jx is the average peak area, and s is the standard deviation corresponding to the average peak area. This formula is applied by using the inverse of the relative standard deviation (RSD) as a measure of the signalto-noise ratio (S/N); therefore, LOD is found by back-calculating the concentration at which the S/N is equal to a defined value of 3. One of the basic assumptions involved in the application of eq 1 is that the variance in the measurement of the low-concentration standard used to calculate the S/N is uniform and is not different from the actual LOD.31 Depending on how close the standard concentration is to the actual LOD, this may or may not be true. Therefore, it is important that the standards used to calculate the LOD based on eq 1 are sufficiently close to the actual LOD. It is for this reason that an initial determination of the LOD was carried out using the first series of extraction data. The estimates for the first set of LOD calculations are given in left-hand side in Table 4. For all of the compounds extracted, the estimated LODs are lower than the actual vapor concentrations by a factor of 3.7 on average, with a range of 1.3 for 2-methylheptane to 5.8 for propylbenzene. Therefore, a second set of vapor standards was prepared with lower concentrations by a factor of 4 (Table 2) to produce a better estimate of the LOD. The right-hand side in Table 4 lists the results of the LOD estimates at lower concentrations. All analytes could be extracted at this lower range except for 2-methylheptane using the LTGC fiber and only styrene and propylbenzene could be extracted using the PDMS/DVB fiber. The difference in the standard concentrations and the estimated LTGC LODs in this case is only a factor of 1.7 on average, with a range of 1.0 for decane to 2.4 for propylbenzene. This was deemed to be sufficiently close as not to exhibit significant difference in variance between the standard concentrations and the estimated LODs. This is based on the fact that, for decane and undecane, the LODs are close to the actual extraction concentrations and that the confidence intervals estimated for the LODs encompass the extraction concentration as well (the calculation of the confidence intervals for the LOD will be (31) Boumans, P. W. J. M. Spetrochim. Acta 1978, 33B, 625-634.

Table 4. Initial Limit of Detection Estimationa highconcentration level


Table 5. Peak Identification Accuracy

lowconcentration level

std vapor std vapor concn in concn in 1-L vial LOD 95% CI 1-L vial LOD 95% CI (pM) (pM) ((pM) (pM) (pM) ((pM) 6.2 8.8 7.2 5.2 4.7

4.6 2.8 1.2 1.1 1.4

1.0 0.6 0.3 0.2 0.4

1.5 2.2 1.8 1.3 1.2

nd 0.95 0.75 1.2 1.2

6.2 8.8 7.2 5.2 4.7

5.2 4.0 1.4 2.5 6.0

1.2 0.9 0.3 0.6 1.4

1.5 2.2 1.8 1.3 1.2

nd 3.2 1.4 nd nd

0.4 0.3 0.5 0.5 1.3 0.6

a N ) 11 observations for each compound except for decane, which was N ) 9 for LTGC and N ) 10 for PDMS/DVB.

discussed later on). For styrene and propylbenzene, there was no difference in variance between the extractions performed with both fibers over a factor of 3 in the quantity of analyte extracted. Therefore, it can be assumed that LODs are sufficiently close to the actual extraction concentrations, which only differ ny a factor of 2.4 at most, to be within uniform variance range. Since the variance in LOD measurements is constant (discussed above), confidence intervals can be generated for the LOD estimates based on confidence intervals associated with the analyte extraction recoveries (xj) alone. By this reasoning, the average peak area (xj) given in eq 1 is the only independent variable and the other quantities remain fixed. To generate a confidence interval for a particular LOD, first the upper and lower confidence interval limits associated with an average peak area (xj) value are calculated. Since the variance is assumed to be constant, the S/N for each limit can be calculated by dividing each limit in jx by the standard deviation. Then, these S/N limits are scaled linearly by dividing each by the factor (xj/s)/(Cstd) to transform them into confidence interval limits associated with the LOD. These values are listed in Table 4. To determine whether there were any significant differences in the extraction recoveries of the LTGC and PDMS/DVB fibers for styrene and propylbenzene at the lower concentration level (the only compounds extracted by both fibers), an F- and t-test were used. The calculated F-values for styrene and propylbenzene are 1.2 and 2.2, respectively, with df ) 4 for the LTGC fiber and df ) 3 for the PDMS/DVB fiber. The two-tailed F-critical value is 15.1; therefore, there is no significant difference in precision between the two fibers for these compounds. Next, a one-tailed t-test was used to see whether the amount extracted by the LTGC fiber is significantly greater than the PDMS/DVB fiber. Since there was no significant difference in the precision of the analyses, a pooled standard deviation was used to calculate the t-values. For styrene and propylbenzene, the t-values were found to be 7.9 and 8.0 with 7 degrees of freedom for both measurements. The critical t-value was 1.9 for both extractions; therefore, the amount extracted using the LTGC fiber was significantly greater. In summary, the extraction efficiencies using the LTGC macrofiber were found to be significantly greater than conven-


ion ratio (no. of peak ions/ions in NIST library)

direct match (out of 1000)

reverse search (out of 1000)

probability (%)

13/51 16/29 19/64 18/50 21/43

781 893 890 841 888

781 926 890 898 888

64.6 36.4 71.0 54.6 49.9

7/51 6/29 6/64 9/50 6/43

754 826 831 651 659

754 826 831 651 659

58.3 43.7 50.6 0.00 1.53

tional PDMS/DVB fibers for all the compounds studied. The variance in the extraction measurements is nonuniform and depends on the quantity of analyte extracted, independent of the type of extracting phase or configuration of the fiber. The extraction sensitivities of the macrofibers with respect to these compounds were also significantly greater. The LOD measured for each compound was the low-picomolar to subpicomolar concentration range. For vapor standards prepared at the lower concentration level (1.2-2.2 pM), the LTGC macrofiber showed significantly greater extraction efficiency and lower detection limits for styrene, propylbenzene, decane, and undecane compared to the commercial fiber. In fact, only two of the five compounds could be extracted with the commercial fiber compared to the four out of five analytes extracted with the LTGC macrofiber for the lower concentration standards. Compound Identification Accuracy. The final aspect of macrofiber extraction evaluation was the accuracy of peak identification. This is an important factor in breath analysis considering the abundance of VOCs. Four measures were used to gauge effectiveness of peak identification. The first was ratio of the number of ions present in a given total ion chromatogram (TIC) for a particular compound to the number of ions listed in a NIST mass spectral database library. The second is based on the direct matching results between the compound of interest and the library spectrum. The third criterion is based on the results of a reverse search in which any ions found in the sample that are not listed in the library for the compound are ignored. The fourth criterion is the probability that the compound can be matched correctly by the database. This is somewhat dependent upon the uniqueness of the spectrum compared to other compounds present in the library. For the direct matching and reverse search, the perfect match results in a value of 1000. According to the literature, values less than 600 represent a poor match, 700-800 a fair match, 800-900 a good match, and greater than 900 is an excellent match. The probability factor is listed as a percent.32,33 The results of the search of the peak identification study are given in Table 5. The ratio of ions present in a TIC to those in the library is just a rough indication of how many parameters are available for matching spectra. On average, the LTGC macrofiber (32) Stein, S. E. J. Am. Soc. Mass Spectrom. 1994, 5, 316-323. (33) NIST Mass Spectrometry Spectral Search Program, Version 1.6; Gaithersburg, MD, 1997.


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extractions provide ∼2.5 times the number of ions per TIC peak than PDMS/DVB fiber. For the direct matching and reverse search, the match factors for all the compounds are greater using the LTGC macrofibers compared to PDMS/DVB. The improvement in match factors for the first three compounds ranged from 27 to 100 points. For the last two compounds, the improvement in match factors range from 190 to 247, indicating the detrimental effect of lower extraction efficiencies for the higher molecular weight compounds using the PDMS/DVB. These low extraction efficiencies with the commercial fiber translate into very low probabilities for identifying decane and undecane 0.00 and 1.53%, respectively. For the first three compounds, LTGC also provides higher probability for identification except for styrene, 36.4% compared to 43.7% for PDMS/DVB. The lower overall probability for styrene is a result of the disparity in the direct and reverse matching procedures. Since the reverse search shows a better match than the direct match for styrene (893 versus 926), the presence of extraneous ions in the spectrum reduces the overall probability of identification. CONCLUSION The data presented in this study show that LTGC can be successfully applied in a large-scale format to prepare macrofibers. The ability to produce macrofibers using LTGC is by virtue of the high thermal stability of the glassy carbon polymer itself. The

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LTGC macrofibers display improved extraction characteristics over the PDMS/DVB fibers with respect to extraction efficiency, limits of detection, and peak identification accuracy. The general improvement in performance of the macrofibers indicates the efficacy of using the fibers for the analysis of cancer-related breath volatiles in the low- to subpicomolar concentration range and warrants further investigation and development as a screening technique. The development of macrofibers extends the realm of applications for SPME and may show potential applications for the rapid extraction and analysis of ultratrace volatiles. ACKNOWLEDGMENT The authors thank Jasmy Methapara for her contribution to the research during the initial phase of this study. We also thank the National Science Foundation (NSF) for support as well as funding from Procter and Gamble and the Grants in Areas of National Needs (GAANN) program. We are grateful to the following companies for providing their services and products making this work possible: Restek, PQ, and Phenomenex Corporations.

Received for review July 25, 2002. Accepted January 10, 2003. AC025984K

Application of Low-Temperature Glassy Carbon-Coated Macrofibers

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