An Improved Approach for Accurate Quantitation of Benzene, Toluene

Anal. Chem. 2006, 78, 5375-5383

An Improved Approach for Accurate Quantitation of Benzene, Toluene, Ethylbenzene, Xylene, and Styrene in Blood David M. Chambers,* David O. McElprang, Michael G. Waterhouse, and Benjamin C. Blount

Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, Georgia 30341

Widespread exposure to benzene, toluene, ethylbenzene, xylene, and styrene (BTEXS) and the potential for this exposure to cause health effects drives the need to develop improved methods for measuring exposure. In this work, we demonstrate our latest assay for quantifying BTEXS in blood and characterize sources of both positive and negative biases. This method involves blood sample collection using common techniques followed by static headspace sampling using solid-phase microextraction and gas chromatography/mass spectrometry analysis. We found that the greatest and unexpected source of positive bias was from contamination of butyl rubber materials used in sample preparation consumables such as Vacutainer stoppers, syringe plungers, and sample vial septa. Conversely, the primary cause of negative bias observed was from the diffusion loss of BTEXS from blood during transfer into sample vials. By minimizing or eliminating these and other sources of bias, we improved method accuracy and precision to within 10% while maintaining low-picogram per milliliter detection. Furthermore, upon comparison of these results with those from other laboratories, we observe substantially lower blood BTEXS levels reported to date for nonoccupationally exposed nonsmokers. A relatively unbiased method, as such, will help elucidate any potential associations between adverse health effects and human exposure to low levels of BTEXS.

more, toluene, ethylbenzene, and xylene have been demonstrated to cause cancer in laboratory animals and have been called “slow carcinogens”.7 Studies of occupational exposure to toluene, ethylbenzene, and xylene also indicate increased cancer risk.8-10 The available data on chronic BTEXS exposure and adverse health effects in humans are mainly limited to occupational exposure. In the case of benzene, some studies of chronic exposure demonstrate an etiological relationship between exposure and cancer.11-13 Although the exposure levels described in these studies are considered low for occupational exposures, they far exceed what is typically encountered in the nonoccupational environment. Nonoccupational exposure to BTEXS is ubiquitous (e.g., fuel, emissions); however, the greatest nonoccupational exposure levels occur in tobacco smokers. In fact, it has been reported that the increased leukemia risk associated with smoking may be caused by benzene in tobacco smoke.14-16 These findings are important because they relate health effects associated with high-level occupational exposure to low-level chronic exposure, which is presumed for cigarette smokers. As with much of this epidemiologic work where sources and environmental concentrations have been measured, body burden is only presumed. This burden may not correlate with environmental levels because potential exposure pathways are numerous and complex. Thus, several laboratories have developed different biomonitoring approaches using blood, breath, and urine to assess trace BTEXS exposure.

Occupational exposure to benzene, toluene, ethylbenzene, xylene, and styrene (BTEXS) is associated with numerous adverse health effects and raises concerns about nonoccupational exposure levels in the general population.1-3 In particular, benzene, which is classified as a human carcinogen by the Environmental Protection Agency (EPA),4 is well documented to increase the risk of acute myeloid leukemia upon long-term exposure.5,6 Further-

(7) Maltoni, C.; Conti, B.; Cotti, G.; Belpoggi, F. Am. J. Ind. Med. 1985, 7, 415-46. (8) Yin, S. N.; Linet, M. S.; Hayes, R. B.; Li, G. L.; Dosemeci, M.; Wang, Y. Z.; Chow, W. H.; Jiang, Z. L.; Wacholder, S.; Zhang, W. U.; Dai, T. R.; Chao, X. J.; Zhang, X. C.; Ye, P. Z.; Kou, Q. R.; Meng, J. F.; Zho, J. S.; Lin, X. F.; Ding, C. Y.; Kneller, R.; Blot, W. J. Am. J. Ind. Med. 1994, 26, 383-400. (9) Steineck, G.; Plato, N.; Gerhardsson, M.; Norell, S. E.; Hogstedt, C. Int. J. Cancer 1990, 45, 1012-17. (10) Silverman, D. T.; Levin, L. I.; Hoover, R. N. J. Natl. Cancer Inst. 1989, 81, 1480-83. (11) Lindquist, R.; Nilsson, B.; Eklund, G.; Gahrton, G. Cancer 1987, 60, 137884. (12) Egeghy, P. P.; Nylander-French, L.; Gwin, K. K.; Hertz-Picciotto, I.; Rappaport, S. M. Ann. Occup. Hyg. 2002, 46, 489-500. (13) Glass, D. C.; Gray, C. N.; Jolley, D. J.; Gibbons, C.; Sim, M. R.; Fritschi, L.; Adams, G. G.; Bisby, J. A.; Manuell, R. Epidemiology 2003, 14, 569-77. (14) Korte, J. E.; Hertz-Picciotto, I.; Schulz, M. R.; Ball, L. M.; Duell, E. J. Environ. Health Perspect. 2000, 108, 333-39. (15) Mitacek, E. J.; Brunnemann, K. D.; Polednak, A. P.; Limsila, T.; Bothisuwan, K.; Hummel, C. F. Oncol. Rep. 2002, 9, 1399-403. (16) Thomas, X.; Chelghoum, Y. Leuk. Lymphoma 2004, 45, 1103-09.

(1) Gist, G. L.; Burg, J. R. Toxicol. Ind. Health 1997, 13, 661-714. (2) Fay, M.; Eisenmann, C.; Diwan, S.; De Rosa, C. Toxicol. Ind. Health 1998, 14, 571-776. (3) Low, L. K.; Meeks, J. R.; Mackerer, C. R. Toxicol. Ind. Health 1988, 4, 49-75. (4) Sonawane, B.; Bayliss, D.; Valcovic, L.; Chen, C.; Rodan, B.; Farland, W. J. Toxicol. Environ. Health A 2000, 61, 471-72. (5) Schnatter, A. R.; Rosamilia, K.; Wojcik, N. C. Chem. Biol. Interact. 2005, 153, 9-21. (6) Mehlman, M. A. Ann. N.Y. Acad. Sci. 2002, 982, 137-48. 10.1021/ac060341g Not subject to U.S. Copyright. Publ. 2006 Am. Chem. Soc.

Published on Web 06/22/2006

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Measurement of BTEXS in blood is useful because it provides concentration levels in the blood that is circulating directly to target tissue(s).17,18 Although blood sample collection is more invasive than collection of breath or urine, we find sample integrity of BTEXS throughout collection, transportation, and storage can be more easily maintained.19 This sample handling advantage is mainly attributed to the relatively nonpolar characteristic of blood, which results in less diffusion loss than with water or urine, ease in handling a liquid as opposed to a gas, and the off-the-shelf ability for hermetic collection and storage of blood provided by Vacutainers (i.e., vacuum blood vials). Thus, we choose to assess human exposure to volatile organic compounds (VOCs) by measuring these compounds in blood rather than in urine20 or breath.21-23 Purge and trap (P&T) and headspace injection are among the extraction approaches most often used for VOC blood analyses;24,25 however, headspace solid-phase microextraction (SPME) is emerging as an alternative that targets nonpolar species.26 One of the primary obstacles that we have encountered in implementing these methods has been from contamination interference. Until recently, the sources of contamination have not been well understood, making them difficult to control. Interestingly, the greatest sources were found to be certain materials that come in to close contact with the samples. For example, our work has revealed that many butyl rubber materials contain substantial quantities of BTEXS as a residue. The presence of BTEXS in this material may be attributed to its use as a processing aid during compounding or as cleaning solvents for forming equipment. This finding is important because butyl rubber is used in Vacutainers, disposable syringes, and as headspace vial septa. Therefore, an important objective of this work is to identify relatively high sources of BTEXS contamination and loss that can bias biomonitoring analyses and to describe methods that are effective in minimizing these biases. Although certain aspects of method execution are not typically emphasized in published scientific methods, identifying and minimizing biases are the most crucial steps in ensuring method accuracy. Implementing these error minimization procedures can improve detection limits, accuracy, and precision. Here we describe improvements to methods for analyzing BTEXS in human blood by identifying and minimizing contamination sources as well as loss mechanisms relevant to these compounds. EXPERIMENTAL SECTION Reagents and Materials. Benzene, toluene, ethylbenzene, m-/ p-xylene, o-xylene, and styrene of g99% purity were purchased from Sigma-Aldrich Corp. (Milwaukee, WI); 13C6-benzene, (17) Ramsey, J. D.; Flanagan, R. J. J. Chromatogr. 1982, 240, 423-44. (18) Angerer, J.; Scand. J. Work Environ. Health 1985, 11, suppl 1, 49-52. (19) Ashley, D. L.; Bonin, M. A.; Cardinali, F. L.; McCraw, J. M.; Wooten, J. V. Environ. Health Perspect. 1996, 104, 871-77. (20) Imbriani, M.; Ghittori, S. Int. Arch. Occup. Environ. Health 2005, 78, 1-19. (21) Wallace, L. A.; Buckley, T. J.; Pellizzari, E. D.; Gordon, S. M. Environ. Health Perspect. 1996, 104, 861-69. (22) Fenske, J. D.; Paulson, S. E. J. Air Waste Manage. 1999, 49, 594-98. (23) Heinrich-Ramm, R.; Jakubowski, M.; Heinzow, B.; Christensen, J. M.; Olsen, E.; Hertel, O. Pure Appl. Chem. 2000, 72, 385-436. (24) Ashley, D. L.; Bonin, M. A.; Cardinali, F. L.; McCraw, J. M.; Holler, J. S.; Needham, L. L.; Patterson, D. G., Jr. Anal. Chem. 1992, 64, 1021-29. (25) Seto, Y. J. Chromatogr., A 1994, 674, 25-62. (26) Mills, G. A.; Walker, V. J. Chromatogr., A 2000, 902, 267-87.

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13C 9toluene, 13C -ethylbenzene, 13C -m-/p-xylene, 2H 9o-xylene, 7 6 6 6 and 13C6-styrene were from Cambridge Isotope Laboratories, Inc.

(Andover, MA); performance test standards were made using EPA 524 VOC mix A from Sigma-Aldrich Corp.; high-performance liquid chromatography (HPLC) grade water was from Mallinckrodt Baker, Inc. (Phillipsburg, NJ); P&T grade methanol was from Honeywell Burdick & Jackson (Muskegon, MI), and HPLC grade methanol was from J. T. Baker (Phillipsburgh, NJ). Commercially available water often contains unacceptable levels of volatile organic contaminants. To remove these contaminants, HPLC grade water was helium purged and distilled in-house. Equipment for the distillation and helium purge system consisted of a Fuchs continuous distiller fitted with a bubbler to allow helium stripping during the distillation. The equipment and procedure, which are described in previous work,27 involved purging the water for 17 h with ultra-high-purity helium (i.e., 99.9999%) followed by 4 h of reflux before the water was collected by distillation. The water was then immediately transferred while hot with a 10-mL serological pipet to either 5-, 10-, or 25-mL glass ampules and sealed with an oxygen/natural gas flame. Standard crimp-top 10-mL headspace vials were purchased from MicroLiter Analytical Supplies, Inc. (Suwanee, GA). The 20mm headspace vial septa used are from MicroLiter (PN 20-0055 Level 4 produced by Integrated Liner Technologies, Albany, NY). These septa were nominally 20-mm diameter and 3 mm thick and were composed of silicone with a poly(tetrafluoroethylene) (PTFE) barrier layer between 0.10 and 0.15 mm thick. The headspace vial septa were cleaned by the producer to meet our minimal specifications equivalent to 17 h at 110 ( 10 °C and either vacuum below 1.3 kPa or nitrogen purging. Before use, the septa were then reprocessed in-house for 17 h at 100 °C under vacuum below 1.3 kPa to remove any residue or contaminants from packaging, shipping, and storage. The headspace vials were sealed with a standard 20-mm-diameter steel-insert crimp cap obtained from Sun-Sri (Duluth, GA). Sample Preparation. An intermediate internal standard mixture was prepared from neat labeled isotopes, which were serially diluted with P&T grade methanol to achieve concentrations of 0.422 µg/mL for 13C6-benzene, 6.935 µg/mL for 13C7toluene, 4.850 µg/mL for 13C6-ethylbenzene, 8.558 µg/mL for 13C -m-/p-xylene, 5.211 µg/mL for 2H -o-xylene, and 9.059 µg/ 6 6 mL for 13C6-styrene. This stock solution was flame-sealed in glass ampules and stored at -70 °C. A working internal standard solution was prepared weekly by diluting the intermediate stock solution with methanol by 200:1 and then storing at -20 °C in the 25-mL volumetric flask in which it was prepared. Before analysis, 40 µL of the internal standard working solution was added to all unknowns, blanks, standards, and quality control specimens. All glassware was rinsed three times with methanol and baked at 150 °C for at least 17 h prior to use. A BTEXS standard solution set consisting of seven calibration concentrations was prepared in a manner similar to the internal standard by formulating intermediate solutions with methanol (P&T grade) that were stored at -70 °C in flame-sealed glass ampules. Aqueous calibration standards were prepared fresh weekly by transferring 40 µL of each intermediate standard (27) Cardinali, F. L.; McCraw, J. M.; Ashley, D. L.; Bonin, M. A. J. Chromatogr. Sci. 1994, 32, 41-45.

Table 1. Standard Concentrations in Water concn level

benzene (pg/mL)

toluene (pg/mL)

ethylbenzene (pg/mL)

m-/p-xylene (pg/mL)

o-xylene (pg/mL)

styrene (pg/mL)

1 2 3 4 5 6 7

12.0 38.1 120 381 1200 3810 12000

12.1 38.2 121 382 1210 3820 12100

12.0 37.9 120 379 1200 3790 12000

16.5 52.1 165 521 1650 5210 16500

6.13 19.4 61.3 194 613 1940 6130

25.9 81.9 259 819 2590 8190 25900

solution into 25 mL of water. Final BTEXS concentrations differed by analyte and are given in Table 1. A standard blank was prepared along with calibration standards to assess contamination during standard preparation. Following addition of internal standard, standard solutions were delivered into headspace vials, crimpsealed, and stored at 4 °C until analysis. Quality control (QC) samples were used to verify performance of the analytical assay. These samples were prepared by adding concentrated standards of BTEXS into fetal bovine serum (Hyclone Laboratories, Logan, UT) that first underwent a cleaning process to reduce background BTEXS levels. This process involved adding 200 cleaned headspace vial septa to 2 L of serum in a 4-L glass bottle with a PTFE-lined cap. The purpose of this treatment is to adsorb a broad range of VOCs, including BTEXS, without contaminating or foaming the serum. This procedure reduces but does not completely eliminate all BTEXS compounds in the serum. The mixture was allowed to equilibrate for 15 h at 4 °C in a refrigerator that had recently been outgassed at room temperature. After equilibration of the bovine serum, 1 L was decanted into a cleaned 2-L round-bottom flask, to separate the serum from the headspace vial septa. QC serum samples were prepared at two different concentrations by spiking intermediate stock solutions on separate occasions into serum in the 2-L flask. The low-level QC samples were prepared at a targeted concentration that was 6.5 times the lowest standard, and the high-level QC samples were prepared at a targeted concentration that was 30 times the lowest standard. After thorough mixing to allow BTEXS to equilibrate into the sera, aliquots were flame sealed into glass ampules and stored at -70 °C. On the day of use, an aliquot of QC serum was thawed, mixed, and sampled as though it were an unknown. Mean concentrations of BTEXS in each QC pool were measured by 20 separate determinations. Blind quality control samples were evaluated by an independent QC officer using Westgard QC rules.28 If the results from analysis of a quality control sample did not meet Westgard rules for any BTEXS analyte, then all results for the analyte in that run were rejected. Blood samples were collected in 10-mL Vacutainers that were reprocessed to minimize residue contamination.29 This procedure included removing and processing the Vacutainer stopper for 21 days under 1.3 kPa vacuum at 80 °C. Vacutainers that underwent this procedure delivered no level of BTEXS above our method detection limit in water stored horizontally (in contact with the stopper) for 6 days at 4 °C. However, unprocessed stoppers were (28) Westgard, J. O.; Barry, P. L.; Hunt, M. R.; Groth, T. Clin. Chem. 1981, 27, 493-501. (29) Cardinali, F. L.; McCraw, J. M.; Ashley, D. L.; Bonin, M.; Wooten, J. J. Chromatogr. Sci. 1995, 33, 557-60.

found to deliver up to 60 ng/mL total BTEXS in water stored under these conditions. To prepare the blood samples for analysis, we placed the samples on a rotating mixer in a level 1 (type II class A/B3) biological safety cabinet at room temperature for a minimum of 15 min. Blood was removed from the Vacutainers with a 5-mL glass barrel gastight syringe (Hamilton). Once 3 mL of blood was drawn into the syringe, the blood was dispensed into a tared 10mL headspace vial. The internal standard working solution (40 µL) was added to the vial, which was then capped and weighed. Sample weights were used to correct the final concentrations. Quality control fetal bovine serum samples and water blanks were prepared using the same technique as for the blood samples. Method reagents and materials were measured for BTEXS using the same sample preparation and analysis approach as for QC and blood samples. Reagents were analyzed in a 10-mL headspace vial as a neat solution or diluted with redistilled HPLC water to yield 3 mL and then spiked with internal standard. BTEXS concentrations in the Vacutainer stoppers and headspace vial septa were measured by performing consecutive 5-mL methanol extractions of the material. Each extraction was performed in a sealed 10-mL headspace vial that was placed for 15 min in an ultrasonic bath. For analysis, 40 µL of the methanol extract and 40 µL of the internal standard working solution were added to 3 mL of water. Instrumentation. The headspace SPME/GC/MS instrumentation and methods used for these analyses have been described previously.30 Electron impact ionization MS response was measured in selected ion-monitoring mode using a primary quantification ion, a confirmation ion, and an internal standard ion using m/z 78 (40 ms), 82 (40 ms), and 84 (40 ms) for benzene; m/z 91 (100 ms), 92 (100 ms), and 98 (80 ms) for toluene; 91 (50 ms), 106 (40 ms), and 97 (30 ms) for ethylbenzene and the xylenes; and 104 (30 ms), 103 (20 ms), and 110 (20 ms) for styrene, where the dwell time is stated in parentheses. Quantification. The quantification approach used for this work was the same as previously described.30 Blood samples with response ratios above that of the highest calibrator were decreased in volume and reanalyzed. Water-based calibration standards were used instead of serum or blood because of the broad variation of VOC levels found in biological samples. A weighted, 1/x, leastsquares model was fit to the calibration data, where x is the standard concentration. Instrument response was calibrated with every sample batch analyzed within a maximum 24-h period. Samples were analyzed (30) Chambers, D. M.; McElprang, D. O.; Mauldin, J. P.; Hughes, T. M.; Blount, B. C. Anal. Chem. 2005, 77, 2912-19.

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Table 2. Levels of Benzene, Toluene, Ethylbenzene, Xylene, and Styrene in Water after Storage in Vacutainers from Two Manufacturers before and after Cleaninga

BD (gray) uncleaned (lot 0103528) BD (gray) cleaned (lot 0103528) SM (gray) uncleaned (lot 419815) SM (gray) cleaned (lot 419815) a

benzene (pg/mL)

toluene (pg/mL)

ethylbenzene (pg/mL)

15

259

43

124

50

6

5

5

3

8

7

>1

17

111

10100

28200

24500

99

2

3

30

111

111

8

m-/p-xylene (pg/mL)

o-xylene (pg/mL)

styrene (pg/mL)

Values outside the standard set range are linearly extrapolated.

beginning with the four lowest concentration standards in order of increasing concentration, then a low-concentration QC, a highconcentration QC, the unknowns, a second low-concentration QC, a second high-concentration QC, and ending with the remaining standards in order of increasing concentration. RESULTS AND DISCUSSION Biomonitoring gives a snapshot of the internal concentration of a toxicant and provides an effective proof of exposure. However, accurate assessment can be difficult to achieve especially for ubiquitous volatile chemicals. Not only do researchers encounter positive and negative biases (i.e., systematic error) during sample collection, processing, and analysis, but the challenge also exists of describing the population because exposure routes can vary among individuals. Likely causes for positive bias resulting from method execution stem from contamination by the surrounding environment. Although potential contamination sources can include entrainment from the atmosphere, the largest sources we have encountered are from materials, matrixes, and reagents involved in sample collection and preparation. Conversely, the most likely source of negative bias is diffusion loss to the surrounding environment or into materials that come in contact with standard and blood samples. Substantial contamination sources that have previously been identified include (1) the Vacutainer into which blood is collected, transported, and stored, (2) the gastight syringe that is used to hermetically transfer blood from the Vacutainer, (3) environmental absorption that occurs during the transfer of blood from the gastight syringe and internal standard into the headspace vial, and (4) diffusion through the headspace vial septum that passes through either the PTFE/glass interface or the PTFE barrier. Loss occurs through routes similar to those described above for contamination; however, the internal standard will compensate for this once it has been added and equilibrated. In addition, positive and negative biases in results can occur if the standard set is inaccurate or the concentration of the internal standard stock solution changes. These errors can result if a formulation error or contamination of reagents used to prepare standards and internal standards occurs. Material and Reagent Characterization. Because of the ubiquitous nature of BTEXS, its presence in materials and reagents used to collect and prepare samples should be characterized. Once sources are identified and minimized, accuracy, precision, and 5378

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detection limit of the analyses should be improved. The material supplies used in this work that might contaminate samples include materials used in field blood collection (i.e., Luer adapter or butterfly set), glass vials, headspace vial septa, gastight syringes, and Vacutainer stoppers. As part of our protocol, these materials are routinely screened for residue and contamination. In our experience, the BTEXS residue in the butyl rubber (e.g., Vacutainer stoppers) has been the most persistent, whereas relatively low levels of BTEXS are typically found in the other materials. For this reason, we focus this discussion on the butyl rubber Vacutainer stopper materials. Furthermore, we do not intend to compare contamination and residue concentrations seen among lots and manufacturers, but rather will call attention to the magnitudes that we typically encounter and the degree to which these levels can bias the analytical results. In our experience, the largest BTEXS residue differences occur between manufacturers rather than among lots from the same manufacturer. Results shown in Table 2 exemplify the substantial contamination difference that occurs between Vacutainers labeled as Becton-Dickinson (BD) and Sherwood Medical (SM) in water stored horizontally for 6 days at 4 °C. In stoppers from both these manufacturers, toluene, ethylbenzene, and xylene contamination is relatively high, requiring cleaning or at least considerable background correction. The ethylbenzene and xylenes were particularly high for the vials labeled SM and resulted in contamination that exceeded 10 ng/mL. Similarly, high BTEXS residue levels have been found in the butyl rubber used in other laboratory supplies including headspace vial septa and syringe plungers. The high BTEXS levels in these materials are indicative of a synthesis byproduct rather than contamination. Because of the difficulties encountered in diffusing VOCs out of butyl rubber, we avoid this material if possible. Unfortunately, all Vacutainers use clay-filled butyl rubber as the stopper material. Thus, careful cleaning of this material is necessary before use. We have tried to remove contaminating residue from the SM stoppers as with the BD stoppers. Although this process removes >99% of the ethylbenzene and xylenes, enough residue remains to contaminate samples drawn into these vials. This cleaning procedure involves heating under vacuum as described elsewhere.29 We used water in our evaluation of Vacutainer contamination instead of blood because blood tends to have relatively high levels of VOCs complicating the measurement at low levels, and VOCfree water is easier to obtain and handle.

Figure 1. Blood benzene, toluene, ethylbenzene, xylene, and styrene contamination immediately upon collection and after storage in a recleaned Sherwood Medical labeled vial stored 6 days vertically at 5 °C. DL, detection limit.

To evaluate whether the SM vials were not suitable for blood collection and would cause approximately the same level of contamination as seen with the water, we sampled and stored blood in the recleaned SM vials. In this evaluation, seven 10-mL vials of blood were collected from the same individual. An initial blood sample was collected through a butterfly set directly into a gastight syringe and then analyzed immediately. In addition, one sample was collected into a Vacutainer and analyzed immediately. For samples analyzed 2, 16, and 24 days after collection, two samples were taken from each Vacutainer. As shown in Figure 1, contamination from the Vacutainer stopper was greatest for o-xylene (110 pg/mL) and m-/p-xylene (200 pg/mL). Contamination levels for the other compounds were lower and near or below the method detection limits. Evaluation for adsorption loss of 100 pg/mL levels of BTEXS from blood stored at 4 °C for up to 10 weeks have been reported elsewhere.31 Loss under these conditions was found to be less than 10% for all the analytes except for benzene (13%) and ethylbenzene (20%). Even though diffusion rates are relatively slow at 4 °C, absolute biases can be large if handling high concentrations. These data emphasize the importance of screening and cleanup of butyl materials as well as minimizing sample storage times. In addition to materials, the reagent water and methanol were also evaluated for potential BTEXS contamination. Table 3 shows a comparison of BTEXS levels in reagents used to formulate the standards and internal standard. Typically, BTEXS levels in HPLC grade water fall below our lowest standard concentrations and are only slightly above our detection limits for ethylbenzene and the xylene. In our experience, BTEXS levels can vary from lot to lot, and thus, we have found it necessary to prescreen the HPLC water lots. In addition, the possibility of a poor container seal can create variability within a lot. As a result, HPLC water is redistilled and stored in ampules in small quantities. A possibility of contamination exists in preparing this water for future use; however, samples from these lots are screened to verify that no relatively large VOC contamination exists; the resulting materials contain measurably lower levels as seen in Table 3. (31) Blount, B. C.; Kobelski, R. J.; McElprang, D. O.; Ashley, D. L.; Morrow, J. C.; Chambers, D. M.; Cardinali, F. L. J. Chromatogr., B In press.

Potential contamination of reagents from the laboratory environment was evaluated not only where the samples are prepared but also where they are stored. For this evaluation, we studied the amount of contamination that can be adsorbed by the HPLC water stored open in the laboratory for 6 h, compared with that stored open in a refrigerator at 4 °C for the same period of time. As shown in Table 3, we observed relatively high levels of BTEXS contamination from inside a refrigerator whereas that from the laboratory air was relatively small. Despite the fact that the vapor pressure drops with temperature, this finding suggests that VOCs can concentrate in the refrigerator air from storage of samples and standards or condense from the laboratory air. Because storing headspace vial samples in a refrigerator can increase BTEXS levels for blank and low-concentration samples that are not hermitically sealed, we isolate samples in separate jars and store them in a refrigerator that is cleaned periodically. Table 3 shows BTEXS levels found in both HPLC and P&T grade methanol as measured in 40 µL of materials that was spiked in 3 mL of water. This small quantity of methanol was used to be consistent with the amount added to the unknown, QC, blank, and standard samples. Nevertheless, the HPLC grade methanol was found to have ethylbenzene, m-/p-xylene, and o-xylene levels that exceed that which can be tolerated for producing the lowest concentration standards. The P&T grade methanol, whose BTEXS levels fall below the detection limits as shown in Table 3, is used in the standard and internal standard formulations and does not require reprocessing or hermetic storage if shelf life and handling are minimized. To minimize BTEXS adsorption, we limit the shelf life of unopened bottles of methanol to ∼6 months and a bottle once opened is only used in one application. Method Characterization. By effectively reducing the BTEXS contamination as previously discussed, we are able to achieve method detection limits (MDL) in the low-picogram per milliliter range. Despite our efforts to reduce contamination as much as possible, interferences still occur in the analysis and raise the MDL higher than if they were only a function of instrument response. The effect of interferences on the MDL among analytical runs is characterized in this work using detection limit criteria established by the standard deviation at zero concentration (i.e., 3S0). The 3S0 detection limit is defined as three times the standard deviation at an extrapolated zero concentration and is the concentration at which the measured value exceeds the uncertainty in the measurement. The standard deviation at zero concentration is estimated as the y-intercept of a linear regression analysis of the standard deviation (in units of concentration) of the calibrators analyzed throughout the study versus their concentrations for the four lowest standards. These detection limits and corresponding concentration ranges for the standard set are given in Table 4. We also extrapolated linearly below the lowest MDL to approximate certain low-level contamination. These extrapolated detection limit values were calculated from the lowest standard to a concentration at which an instrumental signal-to-noise ratio (S/N) of 3 is achieved. This value is independent of the origin of that signal, including background contamination. The relatively high 3S0 detection limit values, which are a factor of 2 above the lowest standard, likely result from low-level transient contamination or loss during preparation and analysis of the standard set. Although this variation is difficult to characterize in Analytical Chemistry, Vol. 78, No. 15, August 1, 2006

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Table 3. Levels of Benzene, Toluene, Ethylbenzene, Xylene, and Styrene in Standard Water and Methanol Matrixes

HPLC water redistilled ampulized HPLC water HPLC water equilibrated in laboratory HPLC water equilibrated in sample refrigerator 13 µL/mL HPLC grade methanol 13 µL/mL P&T grade methanol

benzene (pg/mL)

toluene (pg/mL)

ethylbenzene (pg/mL)

m-/p-xylene (pg/mL)

o-xylene (pg/mL)

styrene (pg/mL)