Anal. Chem. 1998, 70, 3015-3020
Trace Analysis of Explosives in Seawater Using Solid-Phase Microextraction and Gas Chromatography/Ion Trap Mass Spectrometry Stacy-Ann Barshick* and Wayne H. Griest
Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831
Complex matrixes typically cannot be analyzed directly to obtain the selectivity and sensitivity required for most trace analysis applications. To circumvent this problem, solid-phase microextraction (SPME) techniques were used to preconcentrate analytes selectively prior to gas chromatographic/ion trap mass spectrometric analysis. This approach was applied to the trace analysis of explosives and their metabolites in seawater. The choice of SPME sorbent phase was shown to be important especially for the amino metabolites of trinitrotoluene (TNT) and RDX, which were extracted better on polar phases. Although equilibration times were quite lengthy, on the order of 30 min or greater, a sampling time of only 10 min was shown to be sufficient for achieving low part-perbillion (ppb) to part-per-trillion (ppt) detection limits for TNT and the amino metabolites in real seawater samples. While SPME was ideal for rapid screening of explosives in seawater samples, methods for improving the reproducibility and accuracy of quantification are still being investigated. The release of explosives from unexploded ordnance or from contaminated property into various bodies of water, and the subsequent identification of the biotransformation products of these explosives, is an environmental concern because of the toxicity of most explosives.1-4 Studies have shown that the detection of unexploded ordnance becomes more complex when exposure to environmental or biological processes, which can lead to the formation of transformation products or metabolites, is involved.5-8 Biotransformation complicates the detection process by not only reducing the level of parent compound available for detection but also affecting the extraction process by introducing metabolites of often greater polarity and water solubility. Trini(1) Roberts, W. C.; Hartley, W. R. Drinking Water Health Advisory: MUNITIONS; U.S. Environmental Protection Agency. Office of Drinking Water Health Advisories, Lewis Publishers: Boca Raton, FL, 1992. (2) Hartley, W. R.; Roberts, W. C.; Commons, B. J. Drinking Water Health Advisory: MUNITIONS; U.S. Environmental Protection Agency. Office of Drinking Water Health Advisories, Lewis Publishers: Boca Raton, FL, 1994. (3) Tan, E. L.; Ho, C.-h.; Griest, W. H.; Tyndall, R. L. J. Toxicol. Environ. Health 1992, 36, 165-175. (4) Caton, J. E.; Griest, W. H. J. Liq. Chromatogr. Relat. Technol. 1996, 19, 661-677. (5) Walsh, M. E. Environmental Transformation Products of Nitroaromatics and Nitramines. Literature Review and Recommendations for Analytical Development, Special Report 90-2, U.S. Army Cold Regions Research and Engineering Laboratory, Hanover, NH, 1990. S0003-2700(98)00060-2 CCC: $15.00 Published on Web 05/29/1998
© 1998 American Chemical Society
trotoluene (TNT), for example, is reduced at the nitro function to form aminodinitrotoluene and other metabolites that are more soluble than the parent compound.9,10 The objective of this project was to investigate sampling and analysis methods for trace detection of explosives in seawater as part of an effort for the remediation and cleanup of areas contaminated by unexploded ordnance. To detect explosives and their metabolites in seawater, a method was investigated based on gas chromatography/ion trap mass spectrometry (GC/ITMS). Complex matrixes, however, typically cannot be analyzed directly without sacrificing some degree of selectivity and sensitivity. To circumvent this problem, liquid-liquid or solid-phase extraction techniques can be applied as a means of preconcentrating analytes selectively prior to gas chromatographic/ion trap mass spectrometric analysis. Alternatively, a relatively new technique, solid-phase microextraction (SPME), can be used to extract, preconcentrate, and introduce analytes for GC/ITMS analysis. SPME is a fast, sensitive, and solvent-free technique for extracting organic compounds from headspace or aqueous samples.11-13 This technique involves exposing a fused-silica fiber that has been coated with a stationary phase to a sample where the analytes partition from the sample to the coating until equilibrium is achieved. The fiber is then removed from the sample, and the extracted analytes are thermally eluted in the injector of a gas chromatograph. SPME has been used for sampling a diverse number of analytes in both headspace and aqueous phases for a variety of applications including environmental,14,15 forensic,16,17 toxicological,18-20 and food.21-24 (6) Checkai, R. T.; Major, M. A.; Nwanguma, R. O.; Amos, C. T.; Phillips, C. T.; Wnetsel, R. S.; Sadusky, M. C. Transport and Fate of Nitroaromatic and Nitramine Explosives in Soils from Open Burning Detonation Operations at Anniston Army Depot. ERDEC Technical Report 135, U.S. Army Edgewood Research, Development, and Engineering Center, Aberdeen Proving Ground, MD, 1993. (7) Kaplan, D. L.; Kaplan, A. M. Reactivity of TNT and TNT-Microbial Reduction Products with Soil Components. Natick/TR-83/041 Technical Report, U.S. Army Natick Research and Development Laboratories, Natick, MA, 1983. (8) Greene, B.; Kaplan, D. A.; Kaplan, A. M. Degradation of Pink Water Compounds in Soil-TNT, RDX, HMX. Technical Report 85/036, AD-A157954, U.S. Army Natick Research and Development Laboratories, Natick, MA, 1985. (9) Kaplan, D. L.; Kaplan, A. M. Appl. Environ. Microbiol. 1982, 44, 757-760. (10) Griest, W. H.; Tyndall, R. L.; Stewart, A. J.; Caton, J. E.; Vass, A. A.; Ho, C.-H.; Caldwell, W. M. Environ. Toxicol. Chem. 1995, 14, 51-59. (11) Zhang, Z.; Pawliszyn, J. Anal. Chem. 1993, 65, 1844-1852. (12) Arthur, C. L.; Potter, D. W.; Buchholz, K. D.; Motlagh, S.; Pawliszyn, J. LCGC 1992, 10, 656-661. (13) Louch, D.; Motlagh, S.; Pawliszyn, J. Anal. Chem. 1992, 64, 1187-1199. (14) James, K. J.; Stack, M. A. J. High Resolut. Chromatogr. 1996, 19, 515-519.
Analytical Chemistry, Vol. 70, No. 14, July 15, 1998 3015
This paper will address the application of SPME to the trace analysis of explosives in seawater.
removed from the fiber by desorbing for 3 min in the injection port at 250 °C.
EXPERIMENTAL SECTION Chemicals. Recrystallized TNT and hexahydro-1,3,5-trinitro1,3,5-triazine (RDX) were supplied by the Naval Explosive Ordnance Disposal Technical Division of the Naval Surface Warfare Center (Indian Head, MD). 2-Amino-4,6-dinitrotoluene (2ADNT), 4-amino-2,6-dinitrotoluene (4ADNT), and 2,4-dinitrotoluene (DNT) were purchased from Aldrich Chemical Co. (Milwaukee, WI). DNT was used as an internal standard. All standards were prepared in HPLC-grade acetonitrile (J. T. Baker, Phillipsburg, NJ). Simulated ocean water was prepared using 38.3 g of Instant Ocean salt (Aquarium Systems, Mentor, OH) dissolved in 1 L of Milli-Q water. GC/ITMS System. All analyses were carried out on a Finnigan MAT GCQ gas chromatograph/ion trap mass spectrometer (Finnigan MAT, San Jose, CA). Negative chemical ionization was used with methane as the reagent gas at a source pressure of 100 mTorr. The ion source temperature was maintained at 150 °C. Ionization times were set using automatic gain control.25 Data were acquired in the full-scan detection mode from 60 to 450 amu at a rate of 0.5 s/scan. A filament/multiplier delay time of 3 min was used. Sample introduction was performed using a standard split/ splitless-type injector operated in the splitless injection mode. Splitless time was 3 min for SPME fiber desorptions and 1 min for liquid injections. The injection port was maintained at a temperature of 250 °C for thermal desorption and 180 °C for injection. Separation was performed on a 10 m × 0.25 mm × 0.25 µm DB-5MS capillary column (J&W Scientific, Folsom, CA). Transfer line temperature was 225 °C. The column oven was initially held at 80 °C for 3 min, programmed to 150 °C at a rate of 20 °C/min and then to 200 °C at 15 °C/min. Electronic pressure control was used to maintain a constant linear velocity of 90 cm/s. Helium was used as the carrier gas. Sample Extraction. The solid-phase microextraction device consisted of a fused-silica fiber coated with a sorbent phase (Supelco, Bellefonte, PA). The operating procedure for SPME has previously been described.12 In this study, 5-mL sample volumes were extracted by immersing the fiber in a sample that was stirred rapidly and consistently with a magnetic stir bar. Choice of fiber sorbent phase and sampling time were optimized, and the results will be discussed below. Analytes were completely
RESULTS AND DISCUSSION For SPME sampling, there are four parameters to control for optimum performance: fiber polarity, fiber coating thickness, sample medium (pH and ionic strength), and rate of sample agitation. Fiber polarity must be either strongly polar or nonpolar in nature because only 1 cm of fiber is exposed to the sample matrix. Fiber coating thickness determines the rate of diffusion of an analyte from the sample matrix into the coating and the subsequent rate of desorption. The effects of salt and pH relative to the sample medium can decrease the solubility of certain analytes and enhance extraction. Last, properly applied sample agitation, through mechanical stirring or sonication, can enhance extraction efficiency and reduce equilibration times by increasing rates of diffusion. This is especially helpful for analytes with higher molecular weights or smaller diffusion coefficients.13 Stirring was maintained at a rapid and consistent setting using a magnetic stirrer, and no further optimization was performed for this parameter. The remaining parameters were taken into consideration in the development of the analytical method as discussed below. Selection of SPME Fiber. Four commercially available SPME fibers differing in sorbent-phase coating were evaluated for the extraction of explosives and metabolites from seawater. The phases evaluated ranged from the nonpolar poly(dimethylsiloxane) (pdms, 100-µm film) to the polar polyacrylate (pa, 85µm film). Nonpolar pdms (65 µm) and polar Carbowax (cw, 65 µm) coatings adsorbed onto a porous polymer material (divinylbenzene, dvb) were also evaluated. The addition of the polymer adsorbent acts to increase the available surface area and may enhance the extraction of analytes compared to the phases without the divinylbenzene. Fiber selectivity was evaluated for the extraction of a 5-mL sample containing 50 ppb of DNT, TNT, RDX, 2ADNT, and 4ADNT in simulated ocean water. The solution was sampled for 10 min and desorbed for 3 min at 250 °C. The desorption temperature used was within the recommended operating temperature range of each of the fibers. No carry-over on second desorptions was found for any of the fibers, indicating complete removal of analytes from the fibers on initial desorption using these conditions. The results of the fiber comparison study are shown in Figure 1. The extraction efficiency of the fibers was based on the average peak area counts of the analyte for three replicate analyses. The results show that no one fiber was suitable for all of the analytes. The pdms fiber was not suitable for any of the analytes, while the pa fiber was moderately efficient only for the ADNTs. The pdmsdvb combination was highly efficient for extracting the nitrotoluenes, but was less efficient for the more polar ADNTs and RDX. The most suitable fiber for general use was the polar cw-dvb coating which extracted all of the analytes with relatively good efficiency. This can be attributed to both the polar Carbowax phase and the increased surface area provided by the divinylbenzene polymer. Other factors that can effect extraction efficiency include ionic strength and sample pH. Addition of salt to the sample prior to extraction can increase the ionic strength of the solution and affect the solubility of the analytes. Depending on the analyte, this can
(15) Chai, M.; Pawliszyn, J. Environ. Sci. Technol. 1995, 29, 693-701. (16) Steffen, A.; Pawliszyn, J. Anal. Commun. 1996, 33, 129-131. (17) Furton, K. G.; Bruna, J.; Almirall, J. J. High Resolut. Chromatogr. 1995, 18, 625-629. (18) Degel, F. Clin. Biochem. 1996, 29, 529-540. (19) Ishi, A.; Seno, H.; Kumazawa, T.; Watanabe, K.; Hatori, H.; Suzuki, O. Chromatographia 1996, 43, 331-333. (20) Chiarotti, M.; Marsili, R. J. Microcolumn Sep. 1994, 6, 577-580. (21) Chin, H. W.; Bernhard, R. A.; Rosenberg, M. J. Food Sci. 1996, 61, 11181122. (22) Matich, A. J.; Rowan, D. D.; Banks, N. H. Anal. Chem. 1996, 68, 41144118. (23) Garcia, D. D.; Magnaghi, S.; Reichenbacher, M.; Danzer, K. J. High Resolut. Chromatogr. 1996, 19, 257-262. (24) Ng, L. K.; Hupe, M.; Harnois, J.; Moccia, D. J. Sci. Food Agric. 1996, 70, 380-388. (25) March, R. E.; Hughes, R. J. Quadrupole Storage Mass Spectrometry; John Wiley: New York, 1989; Chapter 6.
3016 Analytical Chemistry, Vol. 70, No. 14, July 15, 1998
Table 1. Analysis of Calibration Standards
LODa
(ppt) LOQb (ppt) corr coeffc % rsd at LOQd
TNT
RDX
4ADNT
2ADNT
10 15 0.985 6
325 790 0.947 16
10 15 0.982 20
5 10 0.986 15
a Limit of detection was calculated using the equation LOD ) X + 3σ, where X is the mean blank signal and σ is the standard deviation of the blank signal. b Limit of quantification was calculated using the equation, LOQ ) X + 10σ. c Correlation coefficient was determined from the linear regression analysis of four standards in the range of 50 ppt to 2 ppb. d Percent relative standard deviation was based on three replicate analyses (n ) 3).
Table 2. Accuracy of SPME Method Figure 1. Relative extraction efficiency for a 10-min sampling time using SPME fibers containing polyacrylate (pa), Carbowax-divinylbenzene (cw-dvb), poly(dimethylsiloxane)-divinylbenzene (pdmsdvb), and poly(dimethylsiloxane) (pdms) stationary phases. Extraction efficiencies are shown for DNT, TNT, ADNTs (sum of 2ADNT and 4ADNT), and RDX (signal multiplied by 100).
TNT
RDX
4ADNT
2ADNT
actual conca
716
560
784
836
day 1 conc foundb % rsdc % errord
682 ( 95 14 5
598 ( 111 18 7
679 ( 44 6 13
747 ( 42 6 11
day 2 conc found % rsd % error
808 ( 154 19 13
868 ( 670 76 55
681 ( 60 9 13
862 ( 195 22 3
day 3 conc found % rsd % error
625 ( 122 20 13
311 ( 322 104 44
554 ( 128 23 29
642 ( 65 10 23
ave conc found % error
705 2
592 6
638 18
750 10
a Actual concentrations of analytes in the fortified simulated ocean water sample given in pg/mL (ppt). b Reported concentrations (in ppt) are given for the mean ( std dev based on triplicate analyses (n ) 3). c Percent relative standard deviations are used as a measure of precision. d Percent errors are used as a measure of accuracy. e Average concentration over the 3-day period.
Figure 2. Partitioning equilibration (adsorption) time profiles using the cw-dvb fiber for determination of optimum sampling times. Sampling profiles are shown for DNT, TNT, ADNTs (sum of 2ADNT and 4ADNT), and RDX (signal multiplied by 100).
either enhance or limit extraction efficiency. Likewise, changing pH can minimize solubility where acidic and basic compounds are less soluble at low and high pH, respectively. Of the analytes used in this study, only the monoamino metabolites of TNT would be affected by these factors. The salt content of the simulated ocean water was approximately 4% and the pH was 8.0. It has been reported that salt concentrations up to 1% do not have a significant effect on extraction efficiency.12,26 Sodium chloride concentrations greater than 1%, however, have been reported to increase adsorption and improve detection limits by an order of magnitude, while also increasing equilibration times.26 Literature reports also state that sample pH in the range of 4-10 does not have a significant impact on extraction efficiency; however, exposure to more acidic or basic samples can cause deterioration of the fiber coating.12,26 For this method, at a sample pH of 8.0 and assuming a pKa of ∼1-2.5 for the nitroaniline compounds, (26) Arthur, C. L.; Killam, L. M.; Buchholz, K. D.; Pawliszyn, J. Anal. Chem. 1992, 64, 1960-1966.
the monoamino metabolites would be uncharged and already in a form that would be most efficiently extracted. Determination of Sampling Time. The amount of analyte adsorbed by the fiber depends on the analyte’s distribution constant and the thickness of the stationary phase. Maximum adsorption occurs when equilibrium is achieved between the solution and stationary phase. The time required to reach equilibrium also depends on the analyte’s diffusion coefficient, where higher molecular weight analytes have longer equilibration times than lower molecular weight analytes.13 Because analytes have different distribution constants and diffusion coefficients, the partitioning equilibration time between each analyte and the stationary phase should be established. An adsorption versus time profile was determined for each of the explosives by sampling a solution containing 5 ppb of each analyte with the 65-µm cw-dvb fiber. The results are shown in Figure 2. Equilibration times for the explosives in water ranged from 15 min for RDX to greater than 60 min for the ADNTs. Based on the curves shown in Figure 2, a sampling time of 60 min compared to 10 min only offers an improvement in sensitivity of about 3-4 times for most of the analytes. This improvement in detection capability was considered Analytical Chemistry, Vol. 70, No. 14, July 15, 1998
3017
Table 3. Comparison of SPME and SPE Results for TNT and RDX concentration, ng/L (ppt) TNT SPE
a
RDX SPE
sample
HPLC
GC/ITMS
SPME GC/ITMS
HPLC
GC/ITMS
SPME GC/ITMS
A B C D E F G H I J
595 1910 185 280 165 {85}a {40} {100} 200 150
621 957 313 213 157 117 57 92 179 105
1685 3357 482 647 507 256 207 266 585 225
620 1027 185 285 nd nd 660 660 445 240
304 407 173 74 74 38 268 338 261 121
1283 3425 ndb nd nd {386} 1892 nd {653} nd
Numbers in { } indicate reported concentrations are semiquantitative only (> LOD but < LOQ). b nd signifies analyte was not detected (
