Solid-Phase Microextraction for the Analysis of Human Breath

normal human breath by gas chromatography and mass spec- trometry.1. These voliatile organic compounds (VOCs) are pro- duced by metabolic processes ...
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Anal. Chem. 1997, 69, 587-596

Solid-Phase Microextraction for the Analysis of Human Breath Christoph Grote† and Janusz Pawliszyn*

The Guelph-Waterloo Center for Graduate Work in Chemistry, Department of Chemistry, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1

Solid-phase microextraction (SPME) has been applied to the quantitative determination of ethanol, acetone, and isoprene in human breath. The method involves extraction and preconcentration with a fused silica fiber coated with a polymeric stationary phase, desorption at 200 °C, and assay by gas chromatography/mass spectrometry. Three different fiber coatings have been evaluated with regard to sensitivity, linear range, precision, and detection limits. Typical RSD values in the range 2%-6% could be obtained, depending on the fiber coating and the compound investigated. The calibration curves for the compounds are reproducible and linear over the concentration ranges found in human breath samples. The method is capable of detecting concentrations of acetone and isoprene reported for healthy subjects. The influence of temperature and humidity on the extraction process has been studied in detail. A linear relationship between log K versus 1/T allows the calibration of the method for any given temperature. The device is portable, economical, and easy to use in patient sampling. In recent years there has been increased interest in the determination of breath compounds in medicine and clinical toxicology. More than 100 compounds have been identified in normal human breath by gas chromatography and mass spectrometry.1 These voliatile organic compounds (VOCs) are produced by metabolic processes and partition from the blood stream via the alveolar pulmonary membrane into the alveolar air. This implies that the concentration measured in breath is related to the concentration in blood. Breath analysis can be used as a diagnostic tool because increased or decreased concentrations of some compounds have been associated with various diseases or altered metabolism.2 The major advantage of breath analysis is that it is a noninvasive procedure. It is well accepted by patients because it does not incur the inconvenience of the collection of blood or urine samples. Although many diseases have not yet been examined in the context of using breath analysis as a diagnostic tool, the usefulness has already been demonstrated by the investigation of various clinical conditions. Volatile hydrocarbons, especially ethane and pentane, have been used as markers of lipid peroxidation, the oxidative degradation of † Present address: Department of Analytical Chemistry, Fraunhofer Institute of Toxicology and Aerosol Research, Nikolai-Fuchs-Strasse 1, D-30625 Hannover, Germany. (1) Krotoszynski, B. K.; Gabriel, G.; O’Neill, H. J. Chromatogr. Sci. 1977, 15, 239-244. (2) Manolis, A. Clin Chem. 1983, 29 (1), 5-15.

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© 1997 American Chemical Society

polyunsaturated fatty acids.3 Several organic compounds can be associated, for example, with lung cancer,4 liver disease,5,6 and myocardial infarction.7 Increased concentrations (>50 nmol/L) of acetone have been detected in the breath of patients suffering from diabetes, a chronic disorder in which either the pancreas is unable to produce insulin or the body is unable to effectively use insulin.8-10 Moreover, breath acetone can be used to monitor patients on diets. A weight reduction of about 225 g/week is indicated by an acetone concentration of 500 nmol/L. Eating meals with a high carbohydrate content significantly decreases this concentration.10 Breath acetone analysis can, therefore, be used as a motivational tool during weight-loss programs. Inhalation toxicology is another area of interest. Exposure of workers to environmental pollutants can be monitored by breath analysis.11 Routine breath analysis is applied for the determination of ethanol because its concentration in breath is directly related to the concentration in blood. A breath-to-blood ratio of 1:2100 is used when breath alcohol measurement devices are calibrated to reflect blood alcohol concentrations.12 Values displayed by breathalyzers show the blood concentration of alcohol that would be assumed by the breath alcohol concentration. Breath compounds are present in nanomolar or even lower quantities.1 To improve the sensitivity and precision of the determination of these compounds, the sample has to be concentrated before assay with gas chromatography/mass spectrometry (GC/MS). The three main methods currently utilized for preconcentration are chemical interaction, adsorptive binding, and cold trapping. For example, cold trapping utilizes solid adsorbents cooled to temperatures of liquid nitrogen or solid carbon dioxide.13-15 Trapping systems operated at ambient temperatures (3) Van Gossum, A.; Decuyper, J. Eur. Respir. J. 1989, 2, 287-291. (4) Gordon, S. M.; Szidon, J. P.; Krotoszynski, B. K.; Gibbons, R. D.; O’Neill, H. J. Clin. Chem. 1985, 31 (8), 1278-1282. (5) Kaji, H.; Hisamura, M.; Saito, N.; Murao, M. Clin. Chim. Acta 1978, 85, 279-284. (6) Chen, S.; Mahadevan, V.; Zieve, L. J. Lab. Clin. Med. 1970, 75, 622-627. (7) Weitz, Z. W.; Birnbaum, A. J.; Sobotka, P. A.; Zarling, E. J.; Skosey, J. L. Lancet 1991, 337, 933-935. (8) Levey, S.; Balchum, O. J.; Medrano, V.; Jung, R. J. Lab. Clin. Med. 1964, 63, 574-584. (9) Rooth, G.; Ostenson, S. The Lancet 1966, ii, 1102-1105. (10) Crofford, O. B.; Mallard, R. E.; Winton, R. E. Trans. Am. Clin. Climatol. Assoc. 1977, 88, 128-139. (11) Raymer, J. H.; Pellizzari, E. D.; Thomas, K. W.; Cooper, S. D. J. Exposure Anal. Environ. Epidemiol. 1991a, 1 (4), 439-451. (12) Van Berkom, L. C. Alcohol Drugs Driving 1991, 7, 229-234. (13) Van Gossum, A.; Shariff, R.; Lemoyne, M.; Kurian, R.; Jeejeebhoy, K. N. Am. J. Clin. Nutr. 1988, 48, 1394-1399. (14) Roberts, R. J.; Rendak, J.; Butcher, J. Dev. Pharmacol. Ther. 1983, 6, 170178.

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Figure 1. SPME device modified for breath analysis.

have also been used.16 Several published reviews discuss the currently used sampling techniques.2,17 These methods require complex devices, and each method has its particular problems. The methods based on adsorptive binding or cold trapping are susceptible to contamination and require an additional water trap to remove the large amount of water vapor present in human breath. Otherwise this water would saturate the traps, and after thermal desorption it could damage the GC column. Gas sampling bags, often used to transport a definite volume of breath to a place where the concentration and analysis are carried out, can be the source of contamination and may lead to selective adsorption at the inner wall. Current sampling devices used to preconcentrate breath compounds at the time of sample collection normally consist of a complex arrangement of tubing and valves which often lead to a buildup of contaminants and loss of analytes by leakage. The difficulty in quantitative delivery of the sample to a gas chromatograph is another problem of the collection methods used currently. A technique is needed that does not concentrate H2O or CO2 and that can be applied directly to the mouth to preconcentrate the compounds so that possible contamination due to tubing material is avoided. Solid-phase microextraction (SPME), developed in recent years by Pawliszyn et al.,18-20 is a rapid, inexpensive, and efficient technique for sampling solid, liquid, or gaseous samples. It requires no solvents or complicated apparatus and provides linear results over a wide range of analyte concentrations. Analysis of the extracts is performed using GC, GC/MS, or HPLC.18-20 In the SPME technique, a fused silica fiber coated with a polymeric stationary phase is contained in a specially designed syringe whose needle protects the fiber when septa are pierced. The fiber is directly exposed to a liquid or gaseous sample to extract and concentrate the analytes. After the absorption equilibration is attained, the fiber is withdrawn into the needle and introduced into an injector of a gas chromatograph, where the extracted compounds are thermally desorbed and analyzed. SPME can be performed manually or by an autosampler. The method is economical, because one single fiber can be used repeatedly. Unlike in conventional methods for analysis of gaseous samples, modified equipment like a complex valve injection system, a thermal desorption device, or a cooling trap is not required by SPME. The SPME fiber is easily cleaned by desorbing any (15) Snider, M. T.; Balke, P. O.; Oerter, K. E.; Francalancia, N. A.; Pasko, K. A.; Robbins, M. E.; Gerhard, G. S.; Richard, R. B. Life Chem. Rep. 1985, 3, 168-173. (16) Lawrence, G. D.; Cohen, G. Anal. Biochem. 1982, 122, 283-290. (17) Schaeffer, H. J. J. High Resolut. Chromatogr. 1989, 12, 69-81. (18) Chen, J.; Pawliszyn, J. Anal. Chem. 1995, 76, 2530-2533. (19) Zhang, Z.; Yang, M. J.; Pawliszyn, J. Anal. Chem. 1994, 66, 844A-853A. (20) Boyd-Boland, A. A.; Pawliszyn, J. Anal. Chem. 1996, 68, 1521-1529.

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contaminants in a hot GC injector. The SPME technique coupled with GC has been used to analyze volatile and semivolatile air compounds.21 The fiber can be exposed directly to the air or to a sample collected in a gas sampling bulb. This paper reports on the first application of solid-phase microextraction for the analysis of human breath. The validation of the method was based on ethanol, acetone, and the nonpolar compound isoprene, which was found to be the main endogenous hydrocarbon of human breath.1 EXPERIMENTAL SECTION Chemicals and Materials. Chemicals. Acetone (99.9%+, HPLC grade) was purchased from Sigma-Aldrich (Mississauga, ON, Canada) and isoprene (99%, inhibited with 100 ppm p-tertbutylcatechol) from Aldrich (Milwaukee, WI). Undernatured ethanol (99.9%+, H2O < 0.1%) was obtained from a local pharmacy. Carbon disulfide was purchased from BDH Inc. (Toronto, ON, Canada). Dimethyldichlorosilane (Supelco, Canada) was used to deactivate the glassware. For the Varian GC/MS system, helium was purchased from Praxair (Waterloo, ON, Canada). Materials. Gas sampling bulbs (0.5 and 1.0 L) were used for standard gas preparation (Supelco). Prior to the first use, all bulbs were silanized to deactivate the interior glass surface using a 10% (v/v) solution of dimethyldichlorosilane in toluene. The solution was kept in the bulbs for 12 h. After the bulbs were rinsed with toluene and methanol several times, they were dried at 100 °C. After use, the gas bulbs were cleaned with deionized water, followed by rinsing with methanol and drying at 100 °C. The bulbs were then purged with purified nitrogen for 30 min to remove any contaminants. The SPME fibers and the holder were purchased from Supelco. Prior to their first use, new fibers have been conditioned under a helium stream to clean the fiber from any contaminants present in the coating. Conditioning was performed in a normal GC injection port following the instructions of the manufacturer. SPME Device Modified for Breath Analysis. The SPME fiber can be directly exposed into the mouth of a subject. To avoid the subject’s tongue coming into contact with the fiber during sampling, the fiber has to be protected. This is achieved simply by using a small piece of inert tubing which fits onto the depth guide of the SPME device. The tubing also serves as a mouthpiece, which can be easily replaced for hygienic reasons. One little aperture in the tubing allows the subject to replace the dead volume in the tubing and to exhale during the sampling procedure. Figure 1 shows the modified SPME device. The breath sampling procedure is described below. Preparation of Standards. Preparation of Standards for the Instrument Calibration. The standards used to calibrate the mass (21) Chai, M.; Pawliszyn, J. Environ. Sci. Technol. 1995, 29, 693-701.

spectrometer were prepared in carbon disulfide. Three stock solutions containing 100 µL of acetone, 200 µL of ethanol, and 20 µL of isoprene, respectively, were prepared in 50.0 mL volumetric flasks. Eight standards were then prepared by taking appropriate aliquots of the stock solutions and diluting them with carbon disulfide in 25.0 mL volumetric flasks. The solutions were mixed in an ultrasonic bath for 15 min and refrigerated at 4 °C. For analysis, 0.5 µL aliquots of the solutions were injected into the instrument. Manual injections were performed using a 10 µL syringe (Hamilton, Reno, NV). Preparation of Gaseous Standards. The stock solutions of acetone and ethanol were prepared in 50.0 mL volumetric flasks using deionized water as a solvent (NANOpure, ultrapure water system, Barnstead/Thermolyne, Dubuque, IA). The solutions were mixed in an ultrasonic bath for 15 min. The temperature of human breath is about 35 °C. Moreover, it is almost saturated with water vapor. It has been shown that both temperature and humidity affect the extraction of analytes on the fiber.21 These results require the setup of a calibration curve with gas standards kept at 35 °C and spiked with the appropriate amount of water to give a saturated atmosphere. A water bath big enough to accommodate four gas sampling bulbs at the same time was used, which made the procedure quite practical. A thermostat with a circulator was installed to keep the temperature at 35 °C. A second thermostat was used to heat water flushing through a copper coil at the bottom of the water bath to ensure a more accurate temperature control. The temperature of this additional thermostat was controlled by a contact thermometer. Manual adjustment was not necessary, and the temperature could be kept constant in a small range of approximately (0.2 °C. The temperature was measured using a commercial digital oral thermometer. Its accuracy is reported to be (0.1 °C in the range between 33 and 43 °C. The amount of water which is necessary to give a saturated atmosphere was calculated by the ideal gas law using the vapor pressure of water at 35 °C. The 1.0 L bulbs had to be spiked with 39.7 µL of water to give a saturated atmosphere. This water evaporates to about 55 mL of vapor, which causes a significant pressure increase in the gas bulb. Therefore, the bulb was first spiked with 34 µL of pure water. After the water had been evaporated at 35 °C, the pressure was decreased to atmospheric pressure by quickly turning the stopcock of the bulb. Then 5 µL of water containing the appropriate amount of ethanol and acetone was spiked into the gas sampling bulb through a half-hole septum using a microsyringe (Hamilton). The total amount of water injected gave a relative humidity of approximately 100%. Standards with higher concentrations of ethanol were prepared by spiking neat ethanol into the bulb using a 1.0 µL syringe with no dead volume (SGE, Australia). Since isoprene is not miscible with water, the procedure described above could not be used for this analyte. Therefore, isoprene was spiked into the bulb using a gas-tight syringe (Hamilton). The appropriate aliquots were taken from gaseous stock solutions obtained by spiking a 500 mL gas sampling bulb with 10 and 20 µL of pure isoprene, respectively. The gas sampling bulbs were kept at 35 °C for at least 30 min prior to analysis to guarantee a homogeneous mixture. The isoprene stock solutions were kept in the dark at room temperature. Using this procedure, no visible condensation occurred on the inner wall of the sampling bulb. Condensation must be

avoided because the analytes would partition into the aqueous phase. Selection of SPME Coating. Four different types of coatings, a 100 µm polydimethylsiloxane (PDMS) fiber, an 85 µm polyacrylate fiber, a 65 µm polydimethylsiloxane/divinylbenzene fiber, and a 65 µm Carbowax/divinylbenzene fiber, were assessed by comparing the extraction time profile and sensitivity of SPME analysis with these fibers. The PDMS fibers with thinner films were not investigated because, although they equilibrate faster, they have a lower capacity which is not useful for analyzing highly volatile trace level compounds. Analytical Instrument Parameters. Instrument Calibration. A Varian 3400 GC coupled with a Varian Saturn ion trap mass spectrometer (Varian Chromatography Systems, Walnut Creek, CA) was used to carry out the experimental work. Data acquisition was achieved using the Varian Saturn software. A 30 m SPB-5 column with 1.0 µm film thickness was connected to a 30 m SPB-5 column with 0.25 µm film thickness using a glass seal connector (Supelco). Removing this thick film column was easily possible without shutting down the mass spectrometer. Carbon disulfide was chosen as a solvent because it elutes after the target compounds. The best chromatographic results were obtained by using a precolumn and starting the column temperature at 45 °C, slightly below the boiling point of the solvent. During injection, the septum-equipped programmable injector (SPI) was kept at the same low temperature of 45 °C and then ramped to 200 °C with a rate of 200 °C/min. The acquisition was stopped before the large amount of solvent entered the ion trap to protect the electron multiplier. The column temperature was then ramped to 120 °C with a rate of 30 °C/min to remove all the solvent. Since the peaks were narrow, the highest possible scan rate was applied (0.14 s/scan). Different characteristic masses for the three analytes have been checked for the quantification. At higher concentrations of ethanol, the mass spectrum changes due to secondary reactions occurring in the ion trap. A steady decrease in detector response factor with increasing concentration was observed when only m/z ) 45 was used for the quantification of ethanol. In contrast, the response increased when m/z ) 47 was used. Due to reactions in the trap, the fragment M - 1 (m/z ) 45) turned into M + 1 (m/z ) 47). Therefore, the sum of m/z ) 45 and m/z ) 47 was used to quantify ethanol. As for acetone, both m/z ) 43 (base peak) and m/z ) 58 (molecular ion) can be used for quantification. Although the fragment m/z ) 58 is more significant for acetone than the common fragment m/z ) 43, the latter one was used during the study, because of the much higher intensity of the peak. The quantitation of isoprene gave good results when m/z ) 53, m/z ) 67, or the sum of both characteristic masses was taken. Instrument Parameters for the Analysis of Gaseous Standards and Breath Samples. Several column temperatures have been investigated. Since the time required for the thermal desorption of the volatile analytes in the hot injector port is relatively short ( 0.99) 0-34 µmol/L 0-25 µmol/L 0-545 nmol/L 0-136 nmol/L 0-100 nmol/L 0-100 nmol/L

ethanol acetone isoprene

5.7 2.2 3.9

Precision, %RSD (n ) 8) 8.3 4.2 12.8

1.7 4.8 3.2

ethanol acetone isoprene

20 5.0 2.1

Limits of Detection (nmol/L) 4.6 5.2 1.1

5.8 1.8 0.3

0-8 µmol/L 0-545 nmol/L 0-100 nmol/L

the fiber at equilibrium, Vf is the volume of the fiber coating, and c, is the concentration remaining in the sample at equilibrium.23 The problem in determining the K value is that cs is the concentration at equilibrium (after the extraction took place) and not the initial concentration of the sample, cs°. In certain cases, cs in eq 1 can be replaced by the initial concentration cs°. This simplification is possible when the sample volume Vs is at least 2 orders of magnitude larger than the product KVf.24 This assumption is true when 1.0 L gas sampling bulbs are used, considering a constant fiber volume of the PDMS fiber of Vf ) 6.91 × 10-4 cm3 and relatively small K values (less than 1000) for the highly volatile compounds of interest. The determination of K values is then possible by using the equation

K ) nf/cs°Vf

Conditions: relative humidity, 98%; extraction temperature, 35 °C; extraction time, 1 min; desorption time, 15 s; desorption temperature, 200 °C.

(2)

a

be used, the fiber length has to be taken into account as well, because a variability in the length of (1 mm would affect the results by (10%. Effect of Temperature on the Extraction. The temperature effect on the extraction process was studied by analyzing a standard with 99% relative humidity. Since the effect of temperature on the extraction of volatile compounds has already been investigated,21,24 only three temperatures (26, 35, and 45 °C) have been studied. At each temperature, four measurements were performed with each type of fiber. Since the instrument was calibrated prior to the analysis, this study was also used to determine the partition coefficients (K values) at different temperatures. The partition coefficient K between the fiber coating and the gaseous sample matrix can be expressed as

K ) nf/csVf

(1)

where nf is the amount of analyte extracted from a gas sample by (24) Martos, P. A.; Pawliszyn, J. Anal. Chem., in press.

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This equation can only be applied for the 100 µm PDMS fiber because this coating can be treated as a “normal” liquid. We believe that, due to presence of solid polymer particules (DVB) in the cross-linked structure of the Carbowax/DVB and PDMS/ DVB, these fibers extract the analytes primarily by adsorption rather than by absorption. Therefore, the equilibrium theory on which eq 2 is based cannot be applied. For these fibers, a different K value can be defined, taking into account the surface area of the fibers. Since this surface area is not known, the K values have been determined only for the 100 µm PDMS fiber. To determine the K values, a standard containing 136 nmol/L ethanol, 109 nmol/L acetone, and 100 nmol/L isoprene was prepared. Three different temperatures were investigated. The relative humidity was set at 99% in all cases. Four replicate extractions were performed at each temperature. The average detector responses were converted into the absolute amount of analytes extracted by the fiber, nf, using a calibration curve generated prior to the study. The K values for ethanol, acetone, and isoprene were 88, 101, and 59 at 26 °C, 53, 72, and 45 at 35 °C, and 35, 43, and 40 at 45 °C, respectively, for the 100 µm PDMS fiber. The relatively low K values are due to the low boiling points and high vapor pressures of the analytes. Several extractions can be performed using one bulb without a significant depletion in

Table 3. Effect of Temperature on the Extraction Process of the 100 µm PDMS Fibera log K ) a(1/T) + b compound

a

b

r2

ethanol acetone isoprene

2014 1866 852

-4.8 -4.2 -1.1

0.9935 0.9887 0.9425

a Concentrations of analytes in the standard: ethanol, 136 nmol/L; acetone, 109 nmol/L; and isoprene, 100 nmol/L. Relative humidity, 99%; extraction temperatures investigated, 26, 35, and 44 °C; extraction time, 1 min; desorption time, 15 s.

the concentration because of the small amount of analyte extracted during one sampling process. As can be seen, the K values strongly depend on the temperature. Once the relationship between K and T is determined, the plot of log K vs 1/T gives a straight line.24 Using the equation of that line allows the determination of K at any given temperature. It has to be mentioned that the K values are calculated for a relative humidity of 99%. Table 3 shows the data obtained when log K is plotted versus 1/T for the 100 µm PDMS fiber. As predicted by the theory, a straight line was obtained. Since Carbowax/DVB and PDMS/DVB coatings cannot be treated as “normal” liquids, a partition coefficient has to be defined which takes into account the adsorption process. It is assumed for the linear portion of the adsorption isotherm that the amount extracted by the fiber is proportional to the concentration in the sample. Therefore, a similar relationship between K or the mass of analyte extracted and the temperature should be obtained as well. Since the volume and the surface of these fibers are not known, a new K value was not defined. The log of the mass versus 1/T was plotted instead, giving straight lines similar to those shown in Table 3. Changing the temperature had a significant effect on the amount of analyte extracted by the fiber. The positive slope in the figures illustrates that the amount of analyte extracted by the fiber increases with decreasing temperature, indicating that the extraction is an exothermic process. The study demonstrates that the temperature had to be carefully monitored. However, the temperature of the standards does not necessarily have to be monitored at the same temperature as human breath, as long as the saturated humidity is carefully adjusted. The equations of the straight lines obtained as described above allow the calculation of K at any given temperature. Knowing K and the amount of analyte extracted by the fiber, the concentration of the sample can be calculated. Effect of Relative Humidity on the Extraction. The effect of the relative humidity on the amount of analyte extracted by each fiber type has been investigated for three different humidities (12.6%, 90.7%, and 100%). Each standard was prepared freshly. The 5 µL of water containing the appropriate amount of acetone and ethanol which was always spiked into the gas sampling bulbs determined the lower limit of the humidity range. Not more than three extractions were performed out of the same bulb. Since the extraction efficiency largely depends on the temperature, the water bath was carefully kept at 35 °C. To exclude an instrumental drift in response, the analysis of the standard with a relative humidity of 90.7% was repeated after the analysis of the standard with 100% relative humidity. No difference was observed. Error

Figure 4. Effect of relative humidity on the extraction. Relative humidities investigated: 12.6%, 90.7%, 100%. Extraction time, 1 min; extraction temperature, 35 °C; desorption time, 15 s; desorption temperature, 200 °C; ethanol, 136 nmol/L, quantitation ions m/z ) 45 + m/z ) 47; acetone, 109 nmol/L, quantitation ion m/z ) 43; isoprene, 100 nmol/L, quantitation ions m/z ) 53 + m/z ) 67.

bars were defined as mean (2SD and calculated individually for triplicate measurements of each point. The results are illustrated in Figure 4. It can be seen from Figure 4a that the extraction efficiency of the 100 µm PDMS fiber is not significantly affected by the amount of water present in the bulb. Considering the error in triplicate measurements, there is no significant difference in the amount of ethanol and acetone extracted with varying relative humidity. The amount of isoprene extracted increases with increasing humidity. About 21% less is extracted at a relative humidity of 12.6% compared to a relative humidity of 100%. The effect observed for isoprene follows a trend opposite to what would be expected. One reason for this phenomenon might be a slightly higher concentration of isoprene in the gas phase at saturated Analytical Chemistry, Vol. 69, No. 4, February 15, 1997

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humidity. Although no visible condensation of water was observed on the inner glass wall of the sampling bulbs, there is a certain amount of water adsorbed at the active sites of the glass. Due to its polarity, water has a higher affinity to these sites than isoprene, which might also be adsorbed to some degree. At higher relative humidity, more water is present, which can replace some of the adsorbed isoprene, resulting in a higher concentration in the gas phase. It is not the purpose of this study to carefully investigate the mechanism by which humidity affects the extraction process. It should rather demonstrate that the standards have to be prepared at 100% relative humidity because this also affects the accuracy of this analytical method. The importance of humidity is even more obvious when the Carbowax/DVB and PDMS/DVB fibers were investigated. The hypothesis that these fibers extract the analytes by adsorption rather than by absorption is consistent with the observations. The affinity to the coating material now plays a major role, and replacement of analytes by compounds with higher affinity is very likely to occur. Whereas for all three compounds the difference in extraction efficiency between a relative humidity of 90.7% and 100% is not significant, the response differs significantly at a relative humidity of 12.6%. When the relative humidity is increased from approximately 12% to 100%, the amount of acetone extracted by the fiber drops by 13% and that of isoprene by 33%. This can be explained by replacement of these compounds by water. Ethanol shows the opposite trend. When the relative humidity increases from about 12% to 100%, the response increases by a factor of 2. Hence, the more water that is present causes more ethanol to be extracted. This is likely caused by a change of the properties of the sorbent material, depending on the amount of sorbed water at different humidities. A hypothesis that adequately explains the data may be that water will be included in the porous structure of the polymeric Carbowax/DVB coating. This water, now available as a kind of stationary phase, will lead to a higher extraction efficiency for polar ethanol molecules. These molecules have high affinity to water due to possible formation of hydrogen bonds. Whereas the nonpolar isoprene molecule has no affinity to water, the polar acetone provides less hydrogen bonding interaction. Thus, acetone and isoprene do not show the same effect. The extraction efficiency for the 65 µm PDMS/DVB fiber strongly depends on the relative humidity as well (Figure 4c). The amount of analytes extracted by the fiber coating decreases by about 50% for all the analytes of interest when the relative humidity increases from 12% to 100%. This result indicates that water replaces some of the analytes from the fiber due to the higher affinity to the coating. All findings obtained for the three types of fibers investigated in this study clearly show that it is crucial to set up the standards at 100% relative humidity to obtain accurate results with the SPME method. Effect of Other Matrix Compounds on the Extraction. Extraction efficiency for trace level compounds might be affected by other compounds present in much higher concentrations in the sample. In breath this can occur when the subject has consumed alcoholic beverages. Even after one drink, the concentration of ethanol is much higher than the concentrations of the other compounds of interest. To investigate the effect of high ethanol concentrations on the extraction of acetone and isoprene, the following experiment has been performed. Standards with the same concentra594

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tions as used for the calibration curves were prepared. The relative humidity was set at 98%. Thirty minutes after acetone and isoprene had been spiked into the bulb, two extractions were performed using the 100 µm PDMS fiber. Ethanol was then spiked into the same bulb, and 30 min later the bulbs were extracted again in duplicate. Using this procedure, six different concentrations were analyzed. It was found that high ethanol concentrations (i.e., 17.0 µmol/L ethanol compared to 136 nmol/L acetone and 20.0 nmol/L isoprene) do not significantly affect the extraction of acetone and isoprene when the 100 µm PDMS fiber is used. This allows reliable quantification of acetone and isoprene, even if large amounts of ethanol are present in the breath. A short study has also been performed to check the effect of high ethanol concentrations on the extraction of acetone and isoprene when the Carbowax/DVB and the PDMS/DVB fibers were used. Two standards containing 136 nmol/L ethanol, 109 nmol/L acetone, and 100 nmol/L isoprene were prepared with a relative humidity of 98%. The extraction was performed at a temperature of 35 °C. Each standard was analyzed three times with the same type of fiber. Then, 0.2 µL of pure ethanol was spiked into the bulb, giving a new ethanol concentration of 3.55 µmol/L, which is approximately 26 times greater than before. This standard which still has the same acetone and isoprene concentration was then analyzed again three times. All six extractions in total were performed out of the same bulb. It was found that the effect of high ethanol concentrations on the extraction of acetone and isoprene depends on the type of fiber used for extraction. The Carbowax/DVB fiber indicates no difference in sensitivity to acetone and isoprene when the ethanol concentration is significantly increased. In this case, the concentrations of the compounds are probably too small, so that the active sites of the fiber are not completely occupied. The results obtained for the 65 µm PDMS/DVB fiber indicate that ethanol replaces acetone and isoprene from the fiber coating. A decrease in sensitivity by about 10% is observed, demonstrating that, whenever high ethanol concentrations are present in human breath, the uptake of acetone and isoprene by the PDMS/DVB coating will be affected. Limit of Detection. The sensitivity of the SPME technique was considered in terms of limit of detection (LOD). The limits were estimated on the basis of the signal-to-noise ratios obtained with standards containing the compounds of interest at low concentration levels. Since the sensitivity of the PDMS/DVB fiber is much higher compared to those of the PDMS and Carbowax/ DVB fibers, two standards with different concentrations were prepared in such a way that the signal-to-noise ratios for the analytes were less than 20 when the characteristic masses were monitored. The LOD was defined as that concentration of an analyte which produced a signal 3 times greater than the baseline noise with GC/MS detection. The average signal-to-noise ratio of four measurements was used to calculate the LOD. The fiber blanks always contained coeluting impurities at the same position as acetone with the same characteristic fragment m/z ) 43. Therefore, eight blank samples at a relative humidity of 98% were analyzed, and the average peak are of these measurements was considered as noise. The signal-to-noise was then calculated manually, and the limit of detection was determined in the same way as described above. The results are shown in Table 2. The results indicate that all three types of fiber coatings are suitable for detecting low-level concentrations of ethanol, acetone, and

isoprene in human breath. Jones et al. reported a range of isoprene concentrations found in human breath of 1.60-10.33 nmol/L.25 A mean acetone concentration in normal subjects of 23.2 nmol/L was reported by Phillips and Greenberg.26 This indicates that all fibers are capable of detecting concentrations of acetone and isoprene found in the breath of healthy subjects. Stability of Standards. To ensure that the developed method will be practical for everyday use, the gaseous standards should be stable for at least 1 work day. Once prepared in the morning, the standards could then be used during the day to quantify the compounds of interest. To study the stability of the standards, four gas sampling bulbs were spiked with the analytes in the same way as described earlier (98% relative humidity). The temperature of the water bath was carefully kept at 35 °C. The standards were extracted using the 100 µm PDMS fiber. The extraction time was 1 min and the desorption time 15 s. The first extraction was performed 30 min after the analytes were spiked. The other times investigated were 1, 2, 4, 8, 24, and 48 h. During the study, seven extractions in total were performed out of each bulb. After the end of the study (48 h), a control sample with the same concentration was prepared and analyzed 30 min after the analytes had been spiked to check for any trend in instrument response during the study. The mean value for four replicate measurements was calculated for each time period investigated. The relative standard deviation was then calculated for these mean values (n ) 7) to evaluate the stability of the standards during 48 h. The RSD values were 4.4% for ethanol, 3.7% for acetone, and 3.8% for isoprene. Assuming an inherent random error and comparing the results with those for the control sample, it was found that the standards are stable for at least 2 days. Thus, several calibration standards could be prepared at one time and stored in the water bath for use throughout the working day. Storage Time. An analysis normally cannot be carried out immediately after the collection of the sample. Therefore, it is essential to know how long a sample can be stored on the fiber. The storage time for selected VOCs in air has previously been investigated.21 The best results were obtained by sealing the needle of the SPME device with a septum and keeping the sampled fiber in a box filled with dry ice. Martos improved the storage by retracting the fiber approximately 1.5 cm back into the needle.24 Therefore, the fiber was removed from the holder, retracted into the needle, and embedded in a Thermogreen LB-2 septum (Supelco) immediately after extraction. The fiber was kept in a box with dry ice for 8 h. It has been shown that the standards are stable during 1 working day. Before and after analyzing the fibers which were stored on dry ice for 8 h, two control samples were run to monitor any instrumental drift. Since one single fiber of eacy type was used to study the storage conditions, the whole procedure was repeated on consecutive days to ge three values for each fiber type. The standards were prepared fresh daily. The results have been statistically evaluated by comparing the mean values of the control samples which had been analyzed prior to the stored sample (n ) 6) with the mean values of the stored samples (n ) 3) by applying Student’s t-test at the 95% confidence level.27 Prior to the t-test, an F-test was applied to verify that the standard deviations did not differ significantly. (25) Jones, A. W.; Lagesson, V.; Tagesson, C. J. Chromatogr. B 1995, 672, 1-6. (26) Phillips, M.; Greenberg, J. J. Chromatogr. B 1987, 422, 235-238. (27) Miller, J. C.; Miller, J. N. Statistics for Analytical Chemistry, 2nd ed.; Ellis Horwood Limited: Chichester, England, 1988.

At the 95% confidence level, the data revealed no significant loss of analytes, even after a storage time of 8 h for the Carbowax/ DVB and the PDMS/DVB fibers. The mean values obtained for the PDMS fiber differed significantly for acetone (increase by 10% after storage of 8 h), whereas for ethanol and isoprene no significant difference was observed. This phenomenon was not further investigated. Breath Analysis. Breath Sampling. As mentioned before, the amount of analyte extracted by the fiber is independent of the sample volume if the sample volume is large compared to the fiber volume (Vs . KVf). That is true when the fiber is exposed directly into the mouth. Moreover, the analyte uptake of the fiber does not depend on the flow rate for analytes with K values lower than 5000, which has been shown by Martos and Pawliszyn.24 The whole breath normally contains about two-thirds alveolar breath. The other third is dead space air of the respiratory tract (mouth, nose, pharynx, trachea, and bronchi), with lower concentrations of breath compounds.28 To obtain reproducible samples, it is important to use alveolar air for breath analysis. The following procedure should gaurantee that the fiber is exposed mainly to the end exhaled breath with the highest concentration of compounds of interest. Using the modified SPME device described above, the subject has to inhale through the nose and hold the breath for about 5 s to attain equilibration between the blood stream and the alveolar air. Then the subject exhales approximately the first third of the breath quickly through the mouth into the specially designed mouthpiece. The end part of the breath should be exhaled as slowly as possible until the total extraction time of 30 s is reached. Following this procedure was quite convenient and easy as stated by the subject involved in this study. Quantitation of Acetone and Isoprene. Three breath samples from a healthy subject were obtained by the method described above (65 µm PDMS/DVB fiber) and assayed for acetone and isoprene immediately after the extraction (RSD ) 1.2% for acetone, 5.7% for isoprene). Background samples from the room air and fiber blanks assayed prior to the collection of the breath samples did not show any peaks eluting at the same time as acetone and isoprene. The concentrations of acetone and isoprene in the breath were determined to be 38 ( 1.1 nmol/L for acetone and 10 ( 1.4 nmol/L for isoprene at the 95% confidence level. These values are within the range found for healthy people.25,26 Figure 5 shows the total ion chromatogram of a breath sample taken 1 h after the consumption of approximately 0.5 L of beer and stored for 3 h on dry ice prior to analysis. The peaks, which were identified by comparing the retention times and mass spectra with reference materials, correspond to air, CO2 (1), acetaldehyde (2), ethanol (3), acetone (4), isoprene (5), and carbon disulfide (6). CONCLUSIONS The present work has shown that solid-phase microextraction is applicable for the collection of breath samples. SPME provides an effective method for the quantitative analysis of ethanol, acetone, and isoprene. Comparing the SPME method to currently used sampling techniques which are usually cumbersome and costly shows numerous advantages. SPME is a rapid method because the sampling process takes less than 1 min. Hence, donating a breath sample does not require much effort from the (28) Periago, J. F.; Prado, C.; Ibarra, I. J. Chromatogr. A 1993, 657, 147-153.

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Figure 5. GC/MS chromatogram of a breath sample. Breath sample taken after the consumption of alcohol, 65 µm Carbowax/ DVB fiber. Peak assignment: air, CO2 (1), acetaldehyde (2), ethanol (3), acetone (4), isoprene (5), and carbon disulfide (6). Extraction time, 30 s; desorption time, 15 s; desorption temperature, 200 °C.

patient. A complete analysis is performed in less than 10 min, which allows a much higher sample throughput compared to other methods described so far. The technique is sufficiently sensitive to detect 5.8 nmol/L ethanol, 1.8 nmol/L acetone, and 0.3 nmol/L isoprene, respectively. The response was linear over the concentration ranges of interest, and the data obtained for accuracy and precision are satisfactory. However, further evaluation of this new sampling approach against well-understood blood measurements is necessary before it can be relied upon for clinical measurements. The device is easy to handle, and because of its simplicity, the buildup of contaminants is unlikely. The mouthpiece can be

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further improved by adding two one-way valves. This would not only simplify the sampling procedure, but it could also be used to provide the subject with a pure air supply for inhalation to reduce atmospheric contamination. The technique is economical, and since the device is portable, it is suitable for patient sampling. Samples can be stored on the fiber for 8 h without any significant loss of analytes. This storage time can certainly be increased and should be investigated in further studies. Breath samples can, therefore, be easily obtained at the patient’s room without the requirement to carry large equipment. The SPME device is simple enough for the potential use in a physician’s consulting room. To date, the method is limited to compounds with relatively high concentrations in human breath, but as more coatings become available, the sensitivity and selectivity of the method could be improved. The initial results of this study justify further research in this new use of SPME for application to other compounds of interest in human breath. ACKNOWLEDGMENT Financial support from the Natural Sciences and Engineering Research Council of Canada, Supelco, and Varian is greatly appreciated. This work was presented in part at the 21st International Symposium on Chromatography, September 1520, 1996, Stuttgart, Germany.

Received for review July 25, 1996. Accepted November 18, 1996.X AC960749L X

Abstract published in Advance ACS Abstracts, January 1, 1997.