Detection of Trace Amounts of Chemical Warfare Agents and Related

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Anal. Chem. 1996, 68, 2313-2318

Detection of Trace Amounts of Chemical Warfare Agents and Related Compounds in Rubber, Paint, and Soil Samples by 1H and 31P{1H} NMR Spectroscopy Markku Mesilaakso* and Eeva-Liisa Tolppa

Finnish Institute for Verification of the Chemical Weapons Convention, P.O. Box 55, FIN-00014 University of Helsinki, Helsinki, Finland

1H

and 31P{1H} NMR spectroscopy was used in the identification of chemical warfare agents and related compounds present in trace amounts in rubber, paint, and soil samples. The work was done as part of an international Trial Proficiency Test of laboratory performance. NMR spectroscopy provided identifications of bis(2-chloroethyl) sulfide (mustard), bis(2-chloroethylthio)ethane (sesquimustard), dibenz[b,f][1,4]oxazepine (CR), bis(2-hydroxyethyl) sulfide (thiodiglycol), and cyclohexyl ethylphosphonate, and the presence of cyclohexyl methyl ethylphosphonate, 2-methoxyethyl pinacolyl methylphosphonate, and ethylphosphonic acid was supported. One of the spiking compounds, dithiane 1,4-cyclohexane-1oxide, was not detected. This report describes sample preparations, experiments, spectral analyses, and the results obtained by NMR spectroscopy. The most frequently used analytical method in the international interlaboratory comparison (round robin) tests for verification of chemical disarmament has been gas chromatography/mass spectrometry in electron ionization (GC/MS-EI) and chemical ionization (GC/MS-CI) modes.1,2 Gas chromatography/Fourier transform infrared spectroscopy (GC/FT-IR) and NMR spectroscopy3,4 have been used considerably less. Test samples, spiked with low quantities of analytes, have included air, soil, water, rubber, paint, and concrete and, in addition, wipe, liquid, and solid samples collected from reactors, pipelines, storage containers, and aqueous and organic wastes of a chemical facility.1,2 For unambiguous identification of a compound, two different spectrometric methods giving a consistent result are considered more reliable than two mass spectrometric techniques (e.g., GC/ (1) (a) F.1 Testing of Existing Procedures. (b) F.2 Testing of Procedures on Simulated Industry Samples. (c) F.3 Testing of Procedures on Simulated Military Facility Samples. (d) F.4 Validating of Procedures for Water and Soil Samples. F. International Interlaboratory Comparison (Round Robin) Test for the Verification of Chemical Disarmament. In Methodology and Instrumentation for Sampling and Analysis in the Verification of Chemical Disarmament; Rautio, M., Ed.; The Ministry for Foreign Affairs of Finland: Helsinki, 1990-1993. (2) H.1 First Interlaboratory Comparison Test. H. Interlaboratory Comparison Test Coordinated by the Provisional Technical Secretariat for the Preparatory Commission for the Organisation for the Prohibition of Chemical Weapons. In Methodology and Instrumentation for Sampling and Analysis in the Verification of Chemical Disarmament; Rautio, M., Ed.; The Ministry for Foreign Affairs of Finland: Helsinki, 1994. (3) Reference 1a, pp 72-78, 215-246; ref 1b, pp 83-106, 301-334; ref 1c, pp 71-82, 277-296; ref 1d, pp 73-84, 201-210. (4) Reference 2, pp 85-102, 203-218.

S0003-2700(96)00085-6 CCC: $12.00

© 1996 American Chemical Society

MS-EI and GC/MS-CI).5 In NMR, compounds are identified through reference to spectra included in a database or spectrum library,6 through comparison with a spectrum of the authentic compound, or through spiking the test sample with an authentic compound.7 Even when the data are inadequate for unambiguous identification, they may still provide support to an identification made with other analytical methods.4,8 Recording of one-dimensional 1H, 13C{1H}, 19F, 31P{1H}, and/ 31 or P NMR spectra forms the basis of the NMR method.7 If needed, two-dimensional correlation techniques may be applied as well.9,10 By way of example, the detection limit of a compound when observing 1H at 400 MHz may be as low as 1 µg/mL (1 ppm). The first Trial Proficiency Test11 coordinated by the Preparatory Commission for the Organisation for the Prohibition of Chemical Weapons/Provisional Technical Secretariat (OPCW/ PTS) took place in January 1995. The participating laboratories were asked to identify all analytes deemed relevant to the Chemical Weapons Convention:12 chemical warfare agents, their precursors and degradation products, and decontamination products. Samples (matrices) included a piece of rubber, a piece of painted metal, and two portions of soil. Corresponding blank samples were provided for all test samples so that the total number of samples, with an additional paint sample, was nine. The spiking level was 8-15 ppm. Detailed spiking information, as supplied after the test, is given in Table 1. Fifteen days were allowed for sample preparation, analysis, and reporting. Our laboratory applied 1H and 31P{1H} NMR, GC, GC/MS-EI, GC/MS-CI, and GC/FT-IR to the identification task. Sample (5) Reference 1d, pp 117-118. (6) An in-house 1H, 13C{1H}, 19F, 31P{1H}, 31P, and 2D NMR Spectrum Library of Chemical Warfare Agents and Related Compounds has been developed. (7) Recommended Operating Procedures for Sampling and Analysis in the Verification of Chemical Disarmament; Rautio, M., Ed.; The Ministry for Foreign Affairs of Finland: Helsinki, 1994. (8) Wils, E. R. J.; Hulst, A. G.; Verwiel, P. E. J.; van Krimpen, S. H.; Niederhauser, A. Fresenius J. Anal. Chem. 1992, 343, 297-303. (9) Martin, G. E.; Zektzer, A. S. Two-Dimensional NMR Methods for Establishing Molecular Connectivity: A Chemist’s Guide to Experiment Selection, Performance, and Interpretation; VCH Publishers, Inc.: New York, 1988. (10) Reference 1d, pp 73-84, 201-210. (11) For test nomenclature, see: Horwitz, W. Pure Appl. Chem. 1994, 66, 19031911. (12) Convention on the Prohibition of the Development, Production, Stockpiling and Use of Chemical Weapons and on their Destruction; Signed in January 1993; Printed and distributed by the Provisional Technical Secretariat of the Preparatory Commission for the Organisation for the Prohibition of Chemical Weapons. The Depositary of this Convention is the Secretary-General of the United Nations, from whom a certified true copy can be obtained.

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Table 1. Sample Matricesa and Structures, Names, and Amounts of Spiking Compounds in the First Trial Proficiency Test Coordinated by the Preparatory Commission for the Organisation for the Prohibition of Chemical Weapons/Provisional Technical Secretariat

a Sample matrices were as follows. Rubber: 6 g of material made of polyolefins containing high boiling hydrocarbons; 2000 µg of diesel fuel was added. Paint: 20 mm × 40 mm plate of aluminum covered with a 60 µm layer of alkyd paint; 200 mg of diesel fuel was added. Soil-1: 50 g of soil with low silica (sand) but high organic matter, carbonate, and clay content. Soil-2: 50 g of soil with very low carbonate and organic matter, moderate clay, and high silica (sand) content.

preparations, experiments, analyses performed, and results in NMR spectroscopy are described here, and the GC/MS and FTIR results will be published elsewhere.13 EXPERIMENTAL SECTION General Sample Preparation, Chemical Shift References. General sample preparation consisted of extraction of the test material (rubber, paint, soil) and of the further preparation required for a particular analytical method. Recommended Operating Procedures (ROPs) for the sample preparation were followed.7 Many of the extracts were colored and contained nondissolved particles. In NMR sample preparation, three rules were followed: non-deuterated solvent was replaced by the corresponding deuterated solvent, nondissolved material (dust, humus) was carefully filtered out, and external chemical shift references were applied. The NMR tubes (Wilmad 507-PP, 5 mm o.d., routine quality) and the other glassware were new. A total of 20 NMR samples were prepared. (13) So¨derstro¨m, M. T.; Bjo¨rk, H.; Ha¨kkinen, V. M. A.; Kostiainen, O.; Kuitunen, M.-L.; Rautio, M. Identification of Compounds Relevant to the Chemical Weapons Convention Using Selective GC Detectors, GC-MS and GC/FTIR in an International Trial Proficiency Test. J. Chromatogr., in press.

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A sample to serve as an external chemical shift reference was prepared for each solvent. For 1H NMR, 3-(trimethylsilyl)-1propanesulfonic acid (TSPSA, Fluka, >99%, δH ) 0.015 ppm) was dissolved in D2O (Merck Uvasol, 99.8% D), and tetramethylsilane (TMS, Aldrich, 99.9%, δH ) 0.00 ppm) was dissolved in acetoned6 (Merck Uvasol, >99.8% D) and CD2Cl2 (Aldrich, >99.6% D). For 31P NMR, 85 wt % phosphoric acid (Aldrich, 99.999%, δP ) 0.00 ppm) was placed into a coaxial insert tube (Wilmad WGS5BL), which in turn was placed into an outer tube (Wilmad 507PP) containing the same solvent as that used for the sample. The chemical shift origin in a spectrum was determined by using the same spectrum reference frequency as was used for the reference spectrum. Rubber Sample Extracted with Acetone. The rubber sample was extracted by two 10 min sonications with 10 mL of acetone (Riedel-de Hae¨n, pa.) each time. After combination of the extracts, the solution was filtered through filter paper (Whatman 4), and 10 mL of the extract was concentrated to 1-2 mL under a gentle flow of nitrogen gas at room temperature. The solution was colorless but contained an oily phase which was separated off. The sample vial and the oily phase were rinsed successively with 2 × 300 µL and 200 µL of acetone-d6, and the

rinsing and sample solutions were combined. The resulting solution was concentrated to 0.1-0.2 mL and filtered into an NMR tube through a cotton plug placed in the neck of a Pasteur pipet. The vial was rinsed successively with 2 × 200 µL and 150 µL of acetone-d6, and the rinsing solutions were filtered through cotton into the NMR tube. Rubber Sample Extracted with Water. The same piece of rubber sample was extracted by two 10 min sonications with 10 mL of distilled deionized water each time. The extracts were combined, and 10 mL of the solution was concentrated on a rotary evaporator to 0.7 mL and further by a gentle flow of nitrogen at 35-45 °C to about 0.2 mL. Next, 600 µL of D2O was added, and the solution was reevaporated to about 0.3 mL. This was repeated. Then, 400 µL of D2O was added, and the solution was concentrated to about 0.3 mL. The solution was filtered into an NMR tube through a cotton plug placed in the neck of a Pasteur pipet. The sample vessel was rinsed with 2 × 200 µL of D2O, and the rinsing solutions were filtered through cotton into the NMR tube. Sample pH was 9.2. Paint Sample Extracted with Acetone. The paint sample was extracted and filtered in the same way as the rubber sample to obtain 1-2 mL of solution for NMR sample preparation. Filtration through paper (Whatman 4) did not remove all of the nondissolved particles. The sample was evaporated by nitrogen gas nearly to dryness, 1 mL of acetone-d6 was added, and the solution was filtered into a vial through an HPLC filter unit (0.45 µm, Millipore Millex FH13). The sample vessel was rinsed with 2 × 300 µL and 200 µL of acetone-d6, and the rinsing solutions were added to the vial. The resulting solution was evaporated to 0.2 mL and filtered into an NMR tube through a cotton plug placed in the neck of a Pasteur pipet. The vial was rinsed with 2 × 200 µL and 150 µL of acetone-d6, and the rinsing solutions were filtered through cotton into the NMR tube. Paint Sample Extracted with Water. The paint sample was extracted with distilled water in the manner described above for the rubber sample. Sample pH was 8.9. Soil Samples Extracted with Water. A 10 g portion of soil was extracted with distilled water as described above for the rubber sample, and 20 mL of brownish solution was obtained. After centrifugation and paper filtration, 10 mL of sample was taken for NMR and concentrated on a rotary evaporator to about 2 mL. The remaining solution was filtered with an HPLC filter unit (0.45 µm, Millipore Millex-HV). The sample vessel and the filter unit were rinsed with 2 × 300 µL of D2O, and the portions were combined in a vial with the previous solution. The resulting solution was concentrated in a gentle flow of nitrogen at 25-40 °C to a volume of 300 µL. Then, 600 µL of D2O was added, and the solution was again concentrated to about 300 µL. The solution was filtered into an NMR tube through a cotton plug placed in the neck of a Pasteur pipet. The vial was rinsed with 2 × 200 µL of D2O, and the rinsing solutions were filtered into the NMR tube. The pH was 8.7 in the sample of soil-1 and 9.1 in the sample of soil-2. Soil Samples Extracted with Dichloromethane. A dichloromethane sample of soil was prepared in the same way as the water sample of soil. A 15 mL portion of the extract was concentrated under a gentle flow of nitrogen to about 1.5 mL, and about 1 mL was taken for the NMR sample. The solution was dried for 15 min with Na2SO4 and then filtered into another vessel. The Na2SO4 was rinsed with 2 × 300 µL of CD2Cl2, and

the rinsing solutions and the main solution were combined. The resulting solution was concentrated to about 100 µL. After addition of 300 µL of CD2Cl2, the solution was evaporated to about 50 µL and filtered through cotton into an NMR tube. The sample vessel was rinsed with 4 × 200 µL of CD2Cl2, and the rinsing solutions were filtered into the NMR tube. Alkaline Water Samples of Soils. After 1H NMR experiments, 40 µL of 5 M NaOD (prepared from 40% NaOD in D2O, Fluka) was added to the previously prepared water NMR sample of soil. The pH was 13.4 in the sample prepared from soil-1 and 13.5 in the sample prepared from soil-2. Phosphorus-31 NMR spectra were obtained only from alkaline water solutions or organic solutions. Blank Samples and Authentic Reference Samples.14 Blank samples for NMR were prepared in a way similar to that for spiked samples. Reference samples of authentic compounds were prepared to correspond to the test samples in solvent and pH, but the concentrations were 2 or 3 orders higher (1-50 mg/mL). Spectra. ROPs were followed in testing of the spectrometer performance, in selection of chemical shift references, and in experiments.7 The Bruker AMX-400 NMR spectrometer was equipped with quattro nucleus (H, C, F, P) and dual (F/P) probeheads and with a variable-temperature control unit. Prior to the test period, the proton line shape and sensitivity, phosphorus sensitivity, and pulse lengths were checked for both probeheads. Sample temperatures were controlled to +23.5 °C, except for one experiment on a paint sample, where the temperature was controlled to 39.5 °C. The observed nuclei were 1H and 31P. Proton was detected using a sequence that allows simultaneous solvent suppression with presaturation,15 and phosphorus was detected with proton broadband (composite pulse) decoupling.16 Proton-coupled phosphorus spectra were observed with gated proton decoupling.17 The experimental conditions were selected to allow effective pulsing: 90° pulses were used, and the pulse interval was selected to be approximately 6 s in proton and 4.2 s in phosphorus observation. The number of scans varied from 64 to 10 770, but typically the proton spectra were accumulated with about 1800 and the phosphorus spectra with several thousand scans. The probehead was separately tuned for each sample. The magnetic field homogeneity adjustment was performed carefully, and, always before final accumulation, trial spectra with a few scans were acquired to check the line shape and line width. During the test period, 30 1H spectra, one HH-COSY spectrum, nine 31P{1H} spectra, and one 31P spectrum were recorded from test samples. Just before and during the test period, a total of 25 spectra were recorded for testing of spectrometer performance, for external chemical shift referencing, or for analysis of solvent blanks. Fourteen 1H and six 31P{1H} spectra of authentic reference compounds were recorded. RESULTS AND DISCUSSION Spectra of Rubber Samples. Acetone extraction of the rubber produced a strong background in both the sample and (14) Warning: In view of their toxicity, mustard and sesquimustard should be handled only in specialized laboratories. Dibenz[b,f][1,4]oxazepine is a strongly irritating chemical. (15) Derome, A. E. Modern NMR Techniques for Chemistry Research; Pergamon Press: Oxford, 1987; pp 172-173. (16) Reference 15, pp 162-164. (17) Martin, M. L.; Martin, G. J.; Delpuech, J.-J. Practical NMR Spectroscopy; Heyden and Son Ltd.: London, 1980; pp 235-236.

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Figure 1. (A) 400 MHz 1H NMR spectrum obtained from a sample prepared from the acetone extract of the rubber sample in acetoned6. Number of scans was 1430, and the experiment time was 2 h. The resonances of spiking chemicals are shown by arrows. (B) An expanded region of the spectrum of the blank rubber sample. (C) The corresponding region of spectrum A. (D) The spectrum of the authentic bis(2-chloroethyl) sulfide (1).

the blank, especially in the region from 0.8 to 2 ppm (Figure 1A), and owing to severe overlapping, no signals of suspected chemicals were found. Almost the whole area of the resonances in Figure 1A is from the background and the solvent. In addition, the backgrounds of the blank and the sample did not match exactly. Three resonances of spiking chemicals were nevertheless detected in the region 2.9-3.9 ppm (Figure 1C). At 3.754 ppm the AA′ part and at 2.982 ppm the XX′ part of an AA′XX′ spin system were found. The XX′ part partially overlapped with a resonance from the background. The resonances matched well in chemical shifts and multiplicities (Figure 1C) with those of bis(2-chloroethyl) sulfide (1, Figure 1D). Corresponding chemical shifts of authentic 1 were δ(H-2) ) 3.755 and δ(H-1) ) 2.983 ppm. On the basis of these spectra, 1 was considered identified also by NMR. The doublet (J ) 10.7 Hz) at 3.644 ppm was very likely due to protons H-7 of cyclohexyl methyl ethylphosphonate (2), identified by GC/MS and GC/FT-IR. Because of overlapping with background, however, no other resonances of 2 were revealed in the proton spectrum. The phosphorus resonance of 2 was found at δ(P) ) 34.36 ppm. NMR spectral parameters of authentic cyclohexyl methyl ethylphosphonate in acetone-d6 were δ(H-R) ) 1.71 (J ) 18.2, 7.6 Hz), δ(H-β) ) 1.09 (J ) 19.7, 7.6 Hz), δ(H-1) 2316 Analytical Chemistry, Vol. 68, No. 14, July 15, 1996

Figure 2. (A) 400 MHz 1H NMR spectrum obtained at 39.5 °C from a sample prepared from the acetone extract of paint in acetone-d6. Number of scans was 1952, and the experiment time was 3.5 h. The resonances of spiking chemicals are shown by arrows. (B) An expanded region of spectrum A. (C) The corresponding region of the spectrum of the authentic bis(2-chloroethylthio)ethane (3). (D) Highfrequency region of spectrum A. (E) The corresponding region of the spectrum of the authentic dibenz[b,f][1,4]oxazepine, 4.

) 4.36, δ(H-2) ) δ(H-6) ) 1.91 and 1.50, δ(H-3) ) δ(H-5) ) 1.73 and 1.36, δ(H-4) ) 1.52 and 1.27, δ(H-7) ) 3.66 (J ) 10.8 Hz), and δ(P) ) 34.64 ppm. Compound 2 was not identified by NMR owing to inadequate data, but its presence was supported. The 1H NMR spectrum of a sample in D2O (pH 9.2) showed a triplet at 2.772 ppm (J ) 6.3 Hz) due to protons H-1 of bis(2hydroxyethyl) sulfide (6), a hydrolysis product of 1. H-2 was found at 3.771 ppm (J ) 6.3 Hz), overlapped with two narrow lines of the background. The library data of authentic 6 in D2O were δ(H-1) ) 2.757 (J ) 6.3 Hz) and δ(H-2) ) 3.755 ppm (J ) 6.3 Hz). Spectra of Paint Samples. The background was also strong in the sample prepared from the acetone extract of paint (Figure 2A). Backgrounds in the sample and the blank were almost identical. The spectral range from 0.8 to 3 ppm was very crowded by strong resonances of aliphatic hydrocarbons, water, and the solvent. A broad resonance (probably due to water) appeared at 2.88 ppm at 23.5 °C. No resonances were found in the 31P{1H} spectrum. Because of overlapping of the broad resonance with two resonances of interest, the temperature of the sample was increased to 39.5 °C to move the broad resonance upfield to 2.77

Figure 3. (A) 400 MHz 1H NMR spectrum obtained from the sample (pH 8.7) prepared from the water extract of soil-1 in D2O. Number of scans was 6410, and the experiment time was 11 h. The resonances of spiking chemicals are shown by arrows. (B) An expanded region of spectrum A. (C) The corresponding region of the spectrum of the authentic bis(2-hydroxyethyl) sulfide (6).

ppm. At 39.5 °C, the 1H spectrum revealed the AA′ part of an AA′XX′ spin system at 3.731 ppm and the XX′ part at 2.947 ppm (Figure 2B). A singlet was found at 2.861 ppm. The resonances matched well with those of bis(2-chloroethylthio)ethane (3, Figure 2C). Corresponding chemical shifts of authentic 3 were δ(H-2) ) 3.730, δ(H-1) ) 2.947, and δ(H-3) ) 2.862 ppm. In the region of aromatic protons, several multiplets were revealed in the range 7.19-7.57 ppm, and a broad singlet was found at 8.579 ppm (Figure 2D). The resonance patterns matched well with those of dibenz[b,f][1,4]oxazepine (4, Figure 2E). The proton chemical shifts of authentic 4 in acetone-d6 at 39.5 °C were δ(H-1) ) 7.52, δ(H-2) ) 7.30, δ(H-3) ) 7.55, δ(H-4) ) 7.22, δ(H6) ) 7.18, δ(H-7) ) 7.27, δ(H-8) ) 7.21, δ(H-9) ) 7.33, and δ(H11) ) 8.58 ppm. Similarity of resonance patterns with those of authentic compounds, including almost identical chemical shifts, provided an identification of 3 and 4. Neither compound was found in the water extract of paint. The lists of chemicals in the Chemical Weapons Convention do not include 4, but it is a wellknown riot control agent whose use is forbidden as a method of warfare.12 Spectra of Soil-1 Samples. The 1H spectrum of the sample prepared from the water extract of soil-1 (Figure 3A) revealed a background that was weak from 1 to 3 ppm but strong from 3.2 to 4 ppm (cf. Figures 1A and 2A). In this case, the sample prepared from the blank did not well represent the background found in the sample.

The triplet at 2.750 ppm (J ) 6.3 Hz) and another overlapping triplet at 3.749 ppm (J ≈ 6.3 Hz) (Figure 3B) could be assigned by comparing the spectrum to that of the blank (this part of the blank spectrum was better). On the basis of these spectra, bis(2-hydroxyethyl) sulfide (6, Figure 3C) was identified. Some close homologues of 6 (e.g., the corresponding disulfide) may produce a very similar spectrum, and it is then recommended to spike the sample with an authentic compound for verification. The amount of 6 in the NMR sample may have been approximately 30 µg, if 50% extraction efficiency of soil is assumed. The proton spectrum (Figure 3A) revealed doublets (J ) 17.5 Hz) at 1.627 and 1.624 ppm in the P-CH3 proton region of diesters, doublets (J ) 6.4 Hz) at 1.295 and 1.290 ppm, and singlets at 0.923 and 0.918 ppm. This pointed to pinacolyl methylphosphonate homologues, likely diesters. If one of the O-alkyls were different from pinacolyl, the molecule would be present as diastereomers, providing an explanation for the doubled resonances. The rest of the resonances overlapped with the background. In the 31P{1H} spectrum (pH 13.4), resonances were found at 35.54 and 35.05 ppm (region of alkylphosphonate diesters) and at 26.04 ppm (an alkylphosphonate monoester). The molecular weights of the unknown compounds were determined by GC/MS-CI. Two peaks with close retention times in the total ion chromatogram revealed molecules of the same mass, m/z 238. MS-EI spectra were interpreted, and 2-methoxyethyl pinacolyl methylphosphonate 5 was deduced as the best candidate. Synthesis of 5 resulted in the target compounds but also in a variety of byproducts. Because of limited time and amount of the mixture, no further purification was carried out. Identification of 5 was based on GC/MS-EI, GC/MS-CI, and GC/ FT-IR data.13 The following NMR spectral parameters were determined for authentic 5 from a sample in D2O (pH 8.1) prepared from the synthesized mixture: δ(H-R) ) 1.633 and 1.629 (J ) 17.5 Hz), δ(H-3) ) 3.42, δ(H-4) ) 1.304 and 1.298 (J ) 6.4 Hz), δ(H-6) ) 0.931 and 0.925 ppm; and at pH 13.5, δ(P) ) 35.50 and 35.01 ppm. The resonance at δ(P) ) 26.04 ppm (pH 13.4) in the test sample matched well with that of pinacolyl methylphosphonate (25.99 ppm), identified as a minor component by GC/MS-EI and CI-MS/MS.13 According to the 31P{1H} spectrum, the amount was comparable to the amount of 5, which may be explained by the extremely alkaline conditions in the NMR sample, which would slowly degrade 5 to pinacolyl methylphosphonate. Resonances of dithiane 1,4-cyclohexane-1-oxide (7) overlapped with the background. No spiking compounds were found by NMR in the dichloromethane extract of soil-1. Spectra of Soil-2 Samples. Background in the proton spectrum of the sample (pH 9.1) prepared from the water extract of soil-2 was very similar to that shown in Figure 3A. Several resonances were found (Figure 4A) in the region from 1 to 2 ppm: a doublet (J ) 17.0 Hz) of triplets (J ) 7.7 Hz) at 1.047 ppm, a doublet (J ) 18.8 Hz) of quartets (J ) 7.7 Hz) at 1.555 ppm, and broad multiplets at 1.93, 1.73, 1.35, and 1.18 ppm. In addition, a broad multiplet was found at 4.08 ppm. The doublet of triplets and the doublet of quartets clearly indicated the presence of a CH3CH2-P moiety, and the broad multiplets indicated the presence of a cyclic group. A lower intensity triplet was found at about 1.0 ppm, partially overlapping with the doublet of triplets at 1.047 ppm. The 31P{1H} NMR spectrum (pH 14) revealed Analytical Chemistry, Vol. 68, No. 14, July 15, 1996

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No spiking compounds were found by NMR in the dichloromethane extract of soil-2.

Figure 4. Expanded regions of the 400 MHz 1H NMR spectra recorded from a sample (pH 9.1) prepared from the water extract of soil-2 (A) and from a mixture of cyclohexyl ethylphosphonate (8) and ethylphosphonic acid (9) (B) in D2O. Number of scans in spectrum A was 5960, and the experiment time was 10 h. (C) An expanded region of the 162 MHz 31P{1H} NMR spectrum recorded from a sample (pH 14) prepared from the water extract of soil-2. (D) The corresponding region of the spectrum from the mixture of 8 and 9. Number of scans in spectrum C was 1686, and the experiment time was 2 h.

resonances at 30.33 and 25.24 ppm (Figure 4C), where the intensity of the latter was one-third that of the former. Cyclohexyl ethylphosphonate (8) and ethylphosphonic acid (9) were identified by GC/MS and GC/FT-IR as their methyl and trimethylsilyl derivatives.13 For NMR, a mixture of authentic 8 and 9 (the latter as minor component) was prepared. With sample pH adjusted to match the spectrum with that of the test sample, well-matched resonances of 8 were obtained (cf. parts A and B of Figure 4). Also, a doublet of triplets of H-β of 9 was found at 1.03 ppm. However, the resonance of R-protons of 9 overlapped with a multiplet at about 1.38 ppm. The 31P{1H} NMR spectra of the sample (pH 14) and the authentic mixture were identical (Figure 4C,D). NMR spectral parameters (pH 9.1) of authentic 8 were δ(HR) ) 1.562 (J ) 17.0, 7.7 Hz), δ(H-β) ) 1.054 (J ) 18.8, 7.7 Hz), δ(H-1) ) 4.087, δ(H-2) ) δ(H-6) ) 1.93 and 1.35, δ(H-3) ) δ(H5) ) 1.73 and 1.31, δ(H-4) ) 1.54 and 1.16 ppm, and δ(P) ) 30.34 ppm (pH 14). NMR parameters (pH 8.3) of authentic 9 were δ(H-R) ) 1.430 ppm (J ) 16.7, 7.6 Hz), δ(H-β) ) 1.026 (J ) 17.7, 7.6 Hz), and δ(P) ) 25.54 ppm (pH 14). Compound 8 was identified in soil-2 but not 9, although NMR results provided strong evidence of the presence of the latter. 2318

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DISCUSSION At spiking levels used in this test (see Table 1), no sensitivity problems occurred where the background did not interfere. The problems in identifying the compounds by NMR were all due to background, leading to serious overlapping in many cases (e.g., with 7). However, backgrounds in phosphorus spectra were clean, and, as a result, five (1, 3, 4, 6, and 8) of the nine spiking compounds were identified by NMR. In addition, a contribution to the identification of three (2, 5, and 9) of the four remaining compounds was obtained. For analyses like those discussed here, the sample preparation and the NMR experiments need to be performed with special care. Preferably a non-deuterated solvent should be replaced by the corresponding deuterated solvent, and the sample should always be filtered into the NMR tube. Filtration of the sample guarantees the best sample condition for homogeneity adjustment, the narrowest line, and hence the best resolution and sensitivity, all of which are needed to resolve signals of interest from the background. The blank samples play an important role, although representative blanks may be difficult to obtain from real target environments. The duration of experiments needs to be selected so as to obtain sufficient S/N, and experiments lasting hours are common. The samples prepared from authentic compounds, needed for identification, should be similar to the test samples in solvent, pH, temperature, and chemical shift reference. For unambiguous identification, the blank or the test sample should be spiked with the suspected chemical. All chemicals shown in Table 1 were identified by at least two of the GC/MS-EI, GC/MS-CI, GC/FT-IR, and NMR methods, and no false identifications occurred. CONCLUSIONS NMR spectroscopy in 1H and/or 31P observation deserves consideration as a method for the identification of chemical warfare agents and related compounds, even in trace amounts. Sensitivity on a high-field (e.g., 400 MHz) spectrometer will generally be sufficient if the NMR sample is of good quality and enough instrument time is available. The single disadvantage of the NMR method appears to be the unpredictable amount and spectral range of the background always present in environmental samples. The analytical methodology described here should also be applicable to other types of compounds and solid sample matrices. ACKNOWLEDGMENT The authors thank Dr. M. A. Tambute of the Centre d’Etudes du Bouchet (CEB, France) for the test samples and the Preparatory Commission for the Organisation for the Prohibition of Chemical Weapons/Provisional Technical Secretariat for organizing the first Trial Proficiency Test. Thanks also to M.-L. Kuitunen, S. Lemettinen, and K. Rosendahl of the Institute for their expert help in sample preparation. Received for review January 30, 1996. Accepted April 18, 1996.X AC960085F X

Abstract published in Advance ACS Abstracts, June 1, 1996.