Structural Characterization of Chemical Warfare Agent Degradation

May 27, 2010 - ... Weapons Convention (CWC),(2) but there is still a possibility for the existence of nonstate actors with intentions to perform terro...
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Anal. Chem. 2010, 82, 5331–5340

Structural Characterization of Chemical Warfare Agent Degradation Products in Decontamination Solutions with Proton Band-Selective 1H-31P NMR Spectroscopy Harri Koskela,* Ullastiina Hakala, and Paula Vanninen VERIFIN, University of Helsinki, P.O. Box 55, FIN-00014 Helsinki, Finland Decontamination solutions, which are usually composed of strong alkaline chemicals, are used for efficient detoxification of chemical warfare agents (CWAs). The analysis of CWA degradation products directly in decontamination solutions is challenging due to the nature of the matrix. Furthermore, occasionally an unforeseen degradation pathway can result in degradation products which could be eluded to in standard analyses. Here, we present the results of the application of proton band-selective 1H-31P NMR spectroscopy, i.e., band-selective 1D 1H-31P heteronuclear single quantum coherence (HSQC) and bandselective 2D 1H-31P HSQC-total correlation spectroscopy (TOCSY), for ester side chain characterization of organophosphorus nerve agent degradation products in decontamination solutions. The viability of the approach is demonstrated with a test mixture of typical degradation products of nerve agents sarin, soman, and VX. The proton band-selective 1H-31P NMR spectroscopy is also applied in characterization of unusual degradation products of VX in GDS 2000 solution. The devastating consequences of modern chemical warfare became evident during the First World War,1 and raised a strong antipathy to chemical weapons among nations. Currently most of the nations have signed the Chemical Weapons Convention (CWC),2 but there is still a possibility for the existence of nonstate actors with intentions to perform terror attacks (e.g., the Tokyo subway attack in 1995 by members of Aum Shinrikyo).3 In order to improve the preparedness, considerable resources in military research have been assigned to develop suitable protection against chemical warfare agent (CWA) attack. One of the focuses in this kind of research has been on the efficient decontamination of * To whom correspondence should be addressed. E-mail: Harri.T.Koskela@ helsinki.fi. Fax: +358-9-191 50437. (1) Ivarsson, U., Nilsson, H., Santesson, J., Eds. A FOA Briefing Book on Chemical Weapons; FOA S-172 90; Swedish Defence Research Agency: Stockholm, Sweden, 1992. (2) 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 (http://www.opcw.org/docs/cwc_eng.pdf). The Depositary of this Convention is the Secretary-General of the United Nations, from whom a certified true copy can be obtained. (3) Okumura, T.; Hisaoka, T.; Yamada, A.; Naito, T.; Isonuma, H.; Okumura, S.; Miura, K.; Sakurada, M.; Maekawa, H.; Ishimatsu, S.; Takasu, N.; Suzuki, K. Toxicol. Appl. Pharmacol. 2005, 207, S471-S476. 10.1021/ac100867x  2010 American Chemical Society Published on Web 05/27/2010

contaminated skin, equipment, vehicles, land areas, and other surfaces. The main purpose of decontamination of CWAs is to avoid secondary casualties and to maintain the operational capacity. The other use of decontamination solutions is to safely dispose of large quantities of CWAs. Nowadays several different decontamination solutions that are composed of strong alkaline chemicals or oxidizing agents are being used for effective decontamination of CWAs, but the research in development of new decontamination systems is still active. The CWC has emphasized the importance of destruction of existing chemical weapons and munitions and, in addition, prevention of the production of CWA. The Organisation for the Prohibition of Chemical Weapons (OPCW),4 which implements the CWC internationally, can conduct a challenge inspection to a site alleged to have nondeclared chemical production. The international network of analytical laboratories designated by the OPCW support the CWC by maintaining the capability of verifying the presence of chemicals related to the CWC in samples taken by the OPCW inspection teams. It is a plausible scenario that certain decontamination solution containers can be found during the challenge inspection and samples from the containers are sent to the designated laboratories for the verification analysis. The detoxification with decontamination solutions can in some cases cause unprecedented degradation pathways to occur, resulting in unknown degradation products. Therefore, it is crucial for the verification purposes to understand the reactions of the CWAs in decontamination solutions and what types of compound are formed. Majority of the chemicals scheduled by the CWC are organophosphorus (OP) compounds, which the most notorious are nerve agents sarin, soman, and VX. A structural feature of the CWCrelated OP compounds (Scheme 1) is that they typically bear alkyl group bonded directly to phosphorus (alkyl side chain) and indirectly by means of another element, e.g. oxygen, sulfur, or nitrogen (ester side chain; this naming is adopted for the rest of the text). While the cleavage of P-C bond can occur in certain conditions,5 the bond is usually quite stable and the alkyl side chain remains intact in decontamination solutions. The ester side chains, on the other hand, are more susceptible for chemical reactions, and the detoxification of OP nerve agents is typically a result of changes in the ester side chains.6 (4) Organisation for the Prohibition of Chemical Weapons Headquarters. Johan de Wittlaan 32, 2517 JR, The Hague, The Netherlands (http://www.opcw. org). (5) Schowanek, D.; Verstraete, W. J. Environ. Qual. 1991, 20, 769–776.

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Scheme 1. General Structure of Scheduled OP Compounds (Excluding Pyrophosphonates and Phosphonites)

Separation techniques like gas and liquid chromatography combined with mass spectrometry (GC/MS, LC/MS) are powerful techniques in analysis of chemicals related to CWC.7-9 Analysis of CWA degradation products in decontamination solutions is, however, challenging due to interfering chemicals in the sample matrix. Before GC/MS techniques are applicable, extensive sample pretreatment steps like purification and derivatization of the nonvolatile degradation products are needed.10 LC/MS, although a sensitive method suitable for detection of polar degradation productions,10–12 requires sample pretreatment to minimize the level of interfering ions in order to avoid ion suppression. Nuclear magnetic resonance (NMR) spectroscopy has been an important technique in the studies of toxic OP compounds.13 Because NMR analysis sets only a few limitations for the sample matrix, it has facilitated in situ monitoring of the OP nerve agent degradation. Recently, 31P{1H} NMR spectroscopy was used in determination of the effectiveness of the new system derived from Decon Green to decontaminate soman and VX.14 Similarly, the decontamination reaction and formed degradation products of sarin, soman, and VX were analyzed by 31P HRMAS NMR spectroscopy in a polymer-based CWA decontamination system.15 The information derived from the phosphorus chemical shift gives a general class of the OP compound but is insufficient to describe the side chain structures. In recent years, several proton-phosphorus (1H-31P) correlation experiments like one- and two-dimensional 1 H-31P heteronuclear single quantum coherence (1D/2D 1H-31P HSQC),16,17 and two-dimensional 1H-31P fast heteronuclear multiple quantum coherence (2D 1H-31P fast-HMQC)18 have been proposed that are of great help in determination of the side chain structures of the OP compounds related to the CWC. The protons of the alkyl side chain (Table 1) have usually strong JHP couplings, and their characterization is quite straightforward with the 1H-31P correlation methods. Complete assignment of protons of the ester (6) Yang, Y.-C.; Berg, F. J.; Szafraniec, L. L.; Beaudry, W. T.; Bunton, C. A.; Kumar, A. J. Chem. Soc., Perkin Trans. 2 1997, 607–613. (7) Rautio, M., Ed. Methodology and Instrumentation for Sampling and Analysis in the Verification of Chemical Disarmament; H. Interlaboratory Comparison Test Coordinated by the Provisional Technical Secretariat for the Preparatory Commission for the Organisation for the Prohibition of Chemical Weapons. H.1 First Interlaboratory Comparison Test; The Ministry for Foreign Affairs of Finland: Helsinki, Finland, 1994. (8) Kientz, Ch. E. J. Chromatogr., A 1998, 814, 1–23. (9) Mesilaakso, M., Ed. Chemical Weapons Convention Chemical Analysis. Sample Collection, Preparation and Analytical Methods; John Wiley & Sons Ltd.: Chichester, U.K., 2005. (10) Black, R. M.; Muir, B. J. Chromatogr., A 2003, 1000, 253–281. (11) Black, R. M.; Read, R. W. J. Chromatogr., A 1997, 759, 79–92. (12) Black, R. M.; Read, R. W. J. Chromatogr., A 1998, 794, 233–244. (13) Koskela, H. J. Chromatogr., B 2010, 878, 1365–1381. (14) Waysbort, D.; McGarvey, D. J.; Creasy, W. R.; Morrissey, K. M.; Hendrickson, D. M.; Durst, H. D. J. Hazar. Mater. 2009, 161, 1114–1121. (15) Bromberg, L.; Schreuder-Gibson, H.; Creasy, W. R.; McGarvey, D. J.; Fry, R. A.; Hatton, T. A. Ind. Eng. Chem. Res. 2009, 48, 1650–1659. (16) Meier, U. C. Anal. Chem. 2004, 76, 392–398. (17) Albaret, C.; Loeillet, D.; Auge´, P.; Fortier, P.-L. Anal. Chem. 1997, 69, 2694– 2700. (18) Koskela, H.; Grigoriu, N.; Vanninen, P. Anal. Chem. 2006, 78, 3715–3722.

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Table 1. Typical Proton Chemical Shift and JHP Coupling Ranges of Selected Alkyl Side Chains Directly Bonded to Phosphorus in the OP Compounds Related to the CWCa

a

R

δH (ppm)

-CH3 -CH2-CH3 -CH2-CH3 -CH-(CH3)2 -CH-(CH3)2 -CH2-CH2-CH3 -CH2-CH2-CH3

1.0-2.0 1.6-2.0 1.1-1.3 1.6-2.7 1.0-1.4 1.3-2.1 1.3-1.8

2,3

JHP (Hz)

15-19 15-19 19-22 15-20 16-22 18-20 13-17

The source is the OPCW central analytical database, OCAD.

Table 2. Typical Proton Chemical Shift and JHP Coupling Ranges of Selected Ester Side Chains in OP Compounds Related to the CWCa

a The source is the OPCW central analytical database, OCAD. *, only one entry found. n.d., no data.

side chains (Table 2) could not be made with the 1H-31P correlation spectroscopy alone, because the proton-phosphorus coupling normally extends only to protons of the first carbon beyond the heteronucleus. The protons of the remaining ester side chain can be, however, detected using total correlation spectroscopy (TOCSY)-based19,20 approaches. Consequently, suitable 1D and 2D 1 H-31P HSQC-TOCSY17,18 experiments have been proposed to alleviate the identification of the ester side chain structure. However, these experiments suffer from the homonuclear coupling evolution during the HSQC part, which causes distorted resonance lineshapes and loss of the peak intensities. In addition, the intense resonances of the alkyl chain directly bonded to phosphorus can mask the ester side chain resonances, making the structure characterization difficult. These problems can be, however, addressed using a bandselective approach. Band-selective pulses21,22 are useful when only a part of the spectral region needs either excitation, inversion, or refocusing. Band selectivity is mostly used to reduce the dimensionality of multidimensional experiment, like transforming a 2D NMR experiment into the 1D analogue, but the other commonly used approach is to use band-selective excitation in the selected dimension of a multidimensional experiment in order to study only the resonances of interest.23 Here, we present the results of the application of band-selective proton pulses in the 1H-31P correlation experiments in selective detection of the ester side chain resonances. With the proposed approach, the relevant ester resonances with JHP were detected with clean line shape and good sensitivity in the band-selective 1D 1H-31P HSQC spectrum. In (19) (20) (21) (22) (23)

Braunschweiler, L.; Ernst, R. R. J. Magn. Reson. 1983, 53, 521–528. Davis, D. G.; Bax, A. J. Am. Chem. Soc. 1985, 107, 2820–2821. Geen, H.; Freeman, R. J. Magn. Reson. 1991, 93, 93–141. Freeman, R. Prog. Nucl. Magn. Reson. Spectrosc. 1998, 32, 59–106. Kessler, H.; Mronga, S.; Gemmecker, G. Magn. Reson. Chem. 1991, 29, 527–557.

addition, the approach improved the performance of the 2D 1 H-31P HSQC-TOCSY analysis of ester side chain structures by increasing the sensitivity of the TOCSY correlation peaks and eliminating the overlapping resonances of the alkyl side chain. EXPERIMENTAL SECTION Sample Preparation. Model degradation products isopropyl methylphosphonic acid (IPMPA, purity >99%), pinacolyl methylphosphonic acid (PMPA, >95%), and ethyl methylphosphonic acid (EMPA, >95%) were synthesized by the Spiez laboratory, Switzerland, and methylphosphonic acid (MPA, 98%) was purchased from Fluka, Switzerland. O-ethyl S-(2-diisopropylaminoethyl) methylphosphonothiolate (VX, >80%) was synthesized by the Finnish Defense Forces Technical Research Centre. Deuterated methanol (MeOH-d4, 99.8 d-%), 3-(trimethylsilyl) propionic2,2,3,3-d4 acid sodium salt, (TSP-d4, 98 d-%), and deuterium oxide (D2O, 99.9 d-%), were purchased from Aldrich. Tetramethylsilane (TMS, >99.8%) was purchased from Fluka, Switzerland. Potassium hydroxide (KOH, p.a.) was purchased from J.T. Baker, U.K. Ethanol (EtOH, technical grade) was purchased from Alko, Finland. The GDS 2000 solution, provided by the Finnish Defense Forces Technical Research Centre, was purchased from Ka¨rcher, Germany. The KOH/EtOH solution (pH ∼14.8) was prepared by mixing 1 part (of volume) 20% w/v potassium hydroxide solution with 1 part ethanol. Preliminary tests were performed with a sample containing 100 µg/mL of IPMPA, PMPA, EMPA, and MPA in KOH/EtOH solution. The degradation of VX in GDS 2000 solution was studied by adding 1 mL of GDS 2000 solution on 10 mg of VX. The solution was stirred for 24 h before analysis. Additional samples were prepared for external proton chemical shift calibration by adding 10 mg of TSP-d4 in 1 mL of KOH/EtOH solution and 10 µL of TMS in 1 mL of GDS 2000 solution. NMR samples were prepared from the above samples by taking 0.5 mL of the sample solution and adding 0.1 mL of deuterated solvent, D2O for the KOH/EtOH solutions and MeOH-d4 for the GDS 2000 solutions and transferring the mixture in an NMR tube. Additionally, the external proton chemical shift calibration NMR samples were equipped with a glass insert containing 85% phosphoric acid in order to acquire external phosphorus chemical shift calibration. The studied solutions were corrosive to glass; therefore, disposable 5 mm Wilmad HIP-7 NMR tubes were selected for this study and discarded after use. NMR Experiments. The experiments were carried out using a Bruker DRX 500 NMR spectrometer (Bruker BioSpin, Germany) equipped with a 5 mm inverse z-gradient broadband probe head at 290 K. The pulse sequences used in the NMR analyses are shown in Figure 1. The difference in the proposed experiments to the conventional 1D HSQC and 2D HSQC-TOCSY experiments is the application of proton-band selectivity in the insensitive nuclei enhanced by polarization transfer (INEPT)24 periods. The concept of proton band selectivity has been used earlier in the insensitive nuclei assigned by polarization transfer (INAPT)25 experiment for the assignment of carbons with 3JCH coupling to the selected proton. In our approach, instead of all proton pulses, only the 180° rectangle pulse in the INEPT periods is replaced with a band-selective refocusing pulse. There are two commonly used (24) Morris, G. A.; Freeman, R. J. Am. Chem. Soc. 1979, 101, 760–762. (25) Bax, A. J. Magn. Reson. 1984, 57, 314–318.

Figure 1. Pulse sequence for band-selective 1D 1H-31P HSQC (A) and band-selective 2D 1H-31P HSQC-TOCSY (B). Narrow and wide bars represent 90° and 180° pulses, respectively, and the unfilled half-ellipses denote selective refocusing pulses; pulses are along the x axis if not stated otherwise. Phase cycles are φ1 ) {x,-x}, φ2 ) {x,x,-x,-x}, φR ) {x,-x,-x,x}. The optional phosphorus decoupling is applied during the acquisition. The 90° phosphorus pulse prior to acquisition is used for to transform any antiphase proton magnetization with respect to phosphorus to multiple-quantum coherence, thus assuring a clean line shape in the spectrum when the phosphorus decoupling is omitted during the acquisition. TOCSY mixing is performed with the MLEV-17 pulse train with spin locks on the beginning and the end of the train. The polarization transfer delay ∆ is optimized for the studied JHP coupling. Open rectangles represent gradient pulses. Gradient strengths are 80%, 32%, 70%, and 58% for g1, g2, sg1, and sg2, respectively, from the maximum gradient field strength.

band-selective refocusing pulses, Gaussian cascade pulse (Q3)26,27 and REBURP.28 We chose Q3 for this task, because contrary to the REBURP pulse, the JHP coupling will not evolve during the Q3 pulse, as discussed by Lescop and co-workers.29 This means that the duration, and thus the selectivity of the refocusing pulse and the INEPT polarization transfer delay can be set independently. As a trade-off, relaxation can attenuate the signals slightly more during the long INEPT periods. The pulse shapes, durations, and rf powers were calculated with ShapeTool, a pulse calculation tool in the spectrometer’s operating software (TopSpin 1.3, Bruker BioSpin). For the KOH/EtOH sample analysis, the band-selective 1D 1 H-31P HSQC experiment shown in Figure 1A was optimized for the average of 3JHP couplings of the P-O-CHn substructure (Table 2) by setting the polarization transfer delay ∆ according to Jopt ) 10 Hz (∆ ) 1/(2Jopt)). The repetition time was set for 10 s. The transmitter frequencies for proton and phosphorus were 4.7 and 30 ppm, respectively. The band-selective Q3 pulses were set at δH 4.2 ppm, and the bandwidth was set to 600 Hz, resulting in the pulse duration of 5746.67 µs. The spectrum width was set for 12 ppm. The spectra were acquired with 128 scans (8 dummy scans), giving the total measurement time of 23 min. The conventional 1D 1H-31P HSQC experiment16,30 from the KOH/EtOH sample was measured with the 1D pulse (26) (27) (28) (29) (30)

Emsley, L.; Bodenhausen, G. Chem. Phys. Lett. 1990, 165, 469–476. Emsley, L.; Bodenhausen, G. J. Magn. Reson. 1992, 97, 135–148. Geen, H.; Freeman, R. J. Magn. Reson. 1991, 93, 93–141. Lescop, E.; Kern, T.; Brutscher, B. J. Magn. Reson. 2010, 203, 190–198. Koskela, H.; Rapinoja, M.-L.; Kuitunen, M.-L.; Vanninen, P. Anal. Chem. 2007, 79, 9098–9106.

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Table 3. Characteristic Degradation Products of Sarin, Soman, and VXa

a The CAS numbers are shown in square brackets. Schedule codes are from the CWC.2 The extractable NMR parameters are evaluated from the KOH/EtOH sample analysis. n.d., not detected.

sequence shown in Figure 1A using rectangular 180° pulses instead of band-selective pulses; otherwise the acquisition parameters were the same. The band-selective 2D 1H-31P HSQC-TOCSY experiment from the KOH/EtOH sample was measured with the 2D pulse sequence shown in Figure 1B. Number of scans was 16 per increment; the number of dummy scans was 64. The TOCSY step was executed using the MLEV-17 pulse train31 with duration of 60 ms, rf field strength of 7812.5 Hz, and bracketed by 2.5 ms spin-lock pulses. Spectral widths in the proton and phosphorus dimensions were 10 and 15 ppm, respectively. The transmitter frequencies for proton and phosphorus were 4.7 and 24 ppm. The band-selective Q3 pulses were set at δH 4.2 ppm, and the bandwidth was set to 600 Hz, resulting in the pulse duration of 5746.67 µs. The repetition time was set for 3.0 s. Polarization transfer delay ∆ was optimized according to Jopt ) 10 Hz (Table 2). The optional phosphorus decoupling during acquisition was performed with low-power adiabatic CHIRP-95 pulses32 using the M4P5 supercycle33 in order to minimize the sample heating. Quadrature detection in the F1 dimension was accomplished with a gradient-based echo-antiecho selection by changing the polarity of the g2 pulse, resulting in a phase-sensitive spectrum. The 2D spectrum was acquired with 4k × 256 points. Total measurement time was 3 h, 48 min. The 2D 1H-31P HSQC-TOCSY experiment from the KOH/EtOH sample was measured with the 2D pulse sequence shown in Figure 1B using rectangular 180° pulses instead of band-selective pulses; otherwise the acquisition parameters were the same. The (31) Bax, A.; Davis, D. G. J. Magn. Reson. 1985, 65, 355–360. (32) Fu, R.; Bodenhausen, G. J. Magn. Reson. A 1996, 119, 129–133. (33) Fujiwara, T.; Nagayama, K. J. Magn. Reson. 1988, 77, 53–63.

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band-selective 2D 1H-31P HSQC-TOCSY spectra from the GDS 2000 sample were measured using a phosphorus spectral width of 5 ppm and transmitter frequency of 23.9 ppm, and the number of scans was increased to 32 in order to facilitate detection of minor components. The polarization transfer delay ∆ was set separately for phosphonic acids and phosphonamidic acids by optimizing the delay with the band-selective 1D 1H-31P HSQC experiments for the best signal-to-noise; Jopt ) 10 Hz and Jopt ) 14 Hz was used for phosphonic acids and phosphonamidic acids, respectively. The band-selective Q3 pulses were set at δH 3.85 ppm and δH 2.95 ppm for phosphonic acids and phosphonamidic acids, respectively, and the bandwidth was set to 250 Hz for both cases, resulting in the pulse duration of 13792 µs. Otherwise the acquisition parameters were the same. Total measurement time was 7 h, 38 min per spectrum. The 2D 1H-31P HSQC experiment from the GDS 2000 sample was optimized for the typical 2JHP couplings by setting the polarization transfer delay ∆ according to Jopt ) 17 Hz (Table 1). Acquisition was performed using 8 scans per increment; the number of dummy scans was 32. Spectral widths in the proton and phosphorus dimensions were 10 and 5 ppm, respectively. Otherwise the acquisition parameters were similar as with the conventional/band-selective 2D 1H-31P HSQC-TOCSY. Total measurement time was 4 h, 43 min. 31 P{1H} NMR spectra from the KOH/EtOH and GDS 2000 samples were measured using a 45° excitation pulse angle. The repetition time was set for 1.2 s according to the Ernst angle equation.34 The transmitter frequency for phosphorus was 80 (34) Ernst, R. R. Adv. Magn. Reson. 1966, 2, 1–135.

Figure 2. 1D 1H-31P HSQC spectrum, Jopt ) 17 Hz (A), 1D 1H-31P HSQC spectrum, Jopt ) 10 Hz (B), band-selective 1D 1H-31P HSQC spectrum, Jopt ) 10 Hz (C), and band-selective 1D 1H-31P HSQC spectrum with phosphorus decoupling, Jopt ) 10 Hz (D) from the sample containing 100 µg/mL MPA, EMPA, IPMPA, and PMPA in the KOH/EtOH solution. The overall spectra are plotted at the same intensity levels. The Hb resonances of EMPA, IPMPA, and PMPA are shown in the expansions. The Ha resonances of the OP compounds are shown at δH 1.0-1.4 ppm in parts A and B. Residual water (at δH 5.1 ppm) and ethanol (at δH 1.2 and 3.7 ppm) resonances are more pronounced in the band-selective spectra (C, D).

ppm, and the spectrum width was set for 200 ppm. The number of scans was 4096 (64 dummy scans). The total measurement time was 1 h, 24 min. Processing of the spectra was conducted using spectrometer operating software (TopSpin). Line broadenings (LB) of 1.0 and 3.0 Hz were used in apodization of the 1D 1H-31P HSQC (conventional and band-selective) and the 31P{1H} NMR spectra, respectively. The phase-sensitive 2D spectra were processed with squared cosine apodization on both dimensions (i.e., using QSINE window function with SSB ) 2). The number of real points after Fourier transform for the 1D 1H-31P HSQC (conventional and band-selective), 31P{1H} NMR, band-selective 2D 1H-31P HSQC-TOCSY, and 2D 1H-31P HSQC spectra were 32k, 128k, 8k × 512, and 8k × 512, respectively.

External chemical shift calibration for decontamination solutions spectra was obtained by correcting the spectral reference () 0 ppm frequency of the spectrum in MHz) with values taken from the 1H NMR spectra and 31P{1H} NMR spectra of the external chemical shift calibration samples. RESULTS AND DISCUSSION The preliminary tests of the concept were conducted on the decontamination solution KOH/EtOH sample, which was spiked with a mixture of OP compounds (100 µg/mL MPA, EMPA, PMPA, and IPMPA). As discussed earlier,30 the intense level of background signals in this decontamination solution makes the analysis with 1H NMR futile. With the conventional 1D 1H-31P HSQC experiment optimized for the 17 Hz JHP couplings Analytical Chemistry, Vol. 82, No. 12, June 15, 2010

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(corresponds to typical 2JHP, see Tables 1 and 3), the background signals can be eliminated and the proton resonances of the OP compounds with JHP couplings are easily spotted. While the methyl groups directly bonded to phosphorus give intense resonances at δH 1.0-1.4 ppm, the ester side chain resonances with JHP at δH 3.8-4.6 ppm appear weak and distorted (Figure 2A). In an attempt to improve the ester resonance intensities by setting the Jopt closer to the 3JHP of the studied OP compounds (10 Hz, see Tables 2 and 3), the situation, in fact, comes much worse (Figure 2B). Theoretically, this can be explained as follows: consider a HH′P spin system of one proton (H) coupled to a remote proton (H′) and a phosphorus (P), with δH * δH′ * δP, JHP * 0, JHH′ * 0, and JH′P ) 0. According to product operator calculations,35 the density function of the observable spin H magnetization after the 1D 1H-31P HSQC experiment equals

( ) ( )

σ ∝ Hx cos2

π JHH′ π JHP sin2 + 2 Jopt 2 Jopt

( ) ( )

JHH′ π JHP sin2 0.5HyHz′ sin π Jopt 2 Jopt

(1)

where the term Hx represents the in-phase magnetization, the term HyHz′ represents the antiphase magnetization with respect to the remote H′ spin, and Jopt is the coupling constant used for optimization of the INEPT polarization transfer delays (∆ ) 1/(2Jopt)). If JHP is equal to Jopt, then the second trigonometric part of the both Hx and HyHz′ terms are equal to 1 and can be disregarded from the analysis. Then we can consider the remaining trigonometric parts of the spin terms. When Jopt approaches JHH′, then the in-phase H magnetization attenuates to zero due to the cosine part. The remaining sine part of the HyHz′ spin term will also attenuate, but with reduced rate, meaning that when 0.5 < JHH′/Jopt < 1.0, the most of the spin H magnetization is in antiphase (Figure 3), giving the distorted lineshapes shown in parts A and B of Figure 2. This problem caused by homonuclear coupling evolution is well-know when the INEPT polarization transfer delay is optimized for heteronuclear coupling that is close to the homonuclear couplings, and it can hinder the application of the HSQC experiment in proton-carbon long-range correlation spectroscopy.36 The lineshapeproblemcanbealleviatedwithCPMG-basedapproaches,37,38 but a better solution would be achievable if the homonuclear coupling could be refocused. This is possible when the coupled protons appear in the spectrum with individual spectral regions. Consider the 1D 1H-31P HSQC experiment where the proton 180° pulses excite exclusively the H spin. In this case the density function of the observable spin H magnetization for the HH′P spin system is

( )

σ ∝ Hx sin2

π JHP 2 Jopt

(2)

(35) Sørensen, O. W.; Eich, G. W.; Levitt, M. H.; Bodenhausen, G.; Ernst, R. R. Prog. Nucl. Magn. Reson. Spectrosc. 1983, 16, 163–192. (36) Croasmun, W. R.; Carlson, R. M. K., Eds. Two-Dimensional NMR Spectroscopy: Applications for Chemists and Biochemists, 2nd ed.; VCH Publishers: New York, 1994; pp 482-483. (37) Luy, B.; Marino, J. P. J. Am. Chem. Soc. 2001, 123, 11306–11307. (38) Koskela, H.; Kilpela¨inen, I.; Heikkinen, S. J. Magn. Reson. 2003, 164, 228– 232.

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Figure 3. 1D 1H-31P HSQC experiment polarization transfer profiles of in-phase (Hx) and antiphase (HyHz′) terms for HH′P spin system, when Jopt ) JHP (see eq 1).

As a result of the selective 180° proton pulses, the homonuclear coupling evolution has refocused and the antiphase spin term HyHz′ will no longer appear in the equation. This means, that we should get the in-phase peaks in the spectrum, and no antiphase distortions should be visible. This approach is quite feasible in studies of OP compound ester side chains because the protons of the carbon next to the heteronucleus usually appear in distinctly different chemical shift range than the rest of the ester side chain protons (cf. Tables 2 and 3). The effect to the studied OP compound resonances is quite nicely shown in Figure 2C, where band-selective Q3 refocusing pulses are applied on the Hb resonances at δH 3.8-4.6 ppm during the INEPT periods of the 1D 1H-31P HSQC experiment. These selected Hb resonances will appear in pure phase, as shown in Figure 2C. In addition, because no loss of polarization will occur due to homonuclear coupling evolution, the peaks appear with much higher intensity; the signal-to-noise-ratio of Hb(EMPA) was roughly 6-fold higher in Figure 2C compared to parts A and B of Figure 2. Phosphorus decoupling can be applied to simplify the appearance of the resonances, and then it is easier to distinguish the multiplicities in Figure 2D, i.e., septet, quartet, and quartet for Hb(IPMPA), Hb(PMPA), and Hb(EMPA), respectively, and evaluate the JHH couplings (cf. Table 3). The multiplicity information can be used to indicate the number of protons three bonds further in the ester side chains (i.e., Hc protons, see Table 3) and help to derive the structural information. The advances of the proposed approach can be further demonstrated in the 2D 1H-31P HSQC-TOCSY analysis of the KOH/ EtOH sample. When the magnetization of the Hb protons of the studied OP compounds (Table 3) is not in pure phase after the HSQC step, the following TOCSY step will not be efficient in the Hartmann-Hahn transfer of the magnetization along the proton spin system of the ester side chains. The resonances will appear weak and with a twisted shape or they may be even lost (Figure 4A). Furthermore, the Ha resonances of the OP compounds will appear with high intensity and can overlap the TOCSY transfer peaks (the Hc resonances) in the same chemical shift range (Figure 4A). With the band-selective approach, the resonances of the ester side chains will appear with higher intensity and cleaner line shape, and since the Ha resonances of the OP compounds will not overlap these peaks, the interpretation is easier (Figure 4B). Interpretation of the multiplicities and the structure of the side chains can be further simplified with application of phosphorus decoupling during the

Figure 4. Expansions of the 2D 1H-31P HSQC-TOCSY spectrum (A), band-selective 2D 1H-31P HSQC-TOCSY spectrum (B), and bandselective 2D 1H-31P HSQC-TOCSY spectrum with phosphorus decoupling (C) from the sample containing 100 µg/mL MPA, EMPA, IPMPA, and PMPA in the KOH/EtOH solution. The expansions are plotted at the same intensity levels, negative contour levels are marked with gray. The 1D 1H-31P HSQC and 31P{1H} NMR spectra are plotted as projections along the F2 and F1 axes, respectively. The Ha resonances of EMPA, PMPA, and IPMPA are shown at δH 1.25-1.35 ppm in part A, partially overlapping the Hc resonances. In the band-selective 2D 1H-31P HSQCTOCSY spectra (B and C), the Ha resonances are not shown and the assignment of TOCSY transfer peaks (Hc peaks) is straightforward. Furthermore, the peaks are with quite clean line shape, facilitating the determination of the peak multiplicities. Multiplicity determination and ester side chain characterization can be further assisted with phosphorus decoupling (C). The isolated Hd (PMPA) protons are not shown due to lack of JHH. Residual methyl resonance from ethanol gives some t1 noise at δH 1.2 ppm.

acquisition (Figure 4C). Naturally, TOCSY transfer will only show the protons belonging to the same spin system, and therefore isolated parts of the ester side chain like isobutyl group of PMPA will not be shown. Since some antiphase magnetization will be generated during the TOCSY step,19 evaluation of accurate JHH couplings is not possible. The proposed approach was tested in the study of VX degradation in GDS 2000 solution. This decontamination solution is described as ready-to-use, nonaqueous decontamination agent for detoxification of CWAs on material surfaces.39 GDS 2000 is a highly alkaline organic decontaminant containing diethylenetri-

amine, several aminoalcohols, and sodium alcoholates.40 The GDS 2000 sample was spiked with VX and analyzed after 24 h. The first analysis was performed with 31P{1H} NMR, which revealed several peaks at the chemical shift range of δP 24.1-25.3 ppm (Figure 5). This chemical shift range is typical for phosphonic acids; OP compounds with sulfur like phosphonothiolates and (39) Toepfer, H.-J.; Kostron, M. Symposium Proceedings on NBC 2009, 7th Symposium on CBRNe Threats; Defense Forces Technical Research Centre: Ylo ¨ja¨rvi, Finland, 2009; pp 190-194. (40) Richardt, A.; Blum, M.-M., Eds. Decontamination of Warfare Agents. Enzymatic Methods for the Removal of B/C Weapons; Wiley-VCH: Weinheim, Germany, 2008; p 88.

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Figure 5. Expansions of the 2D 1H-31P HSQC spectrum (A), band-selective (δH 3.8-3.9 ppm) 2D 1H-31P HSQC-TOCSY spectrum with phosphorus decoupling (B), and band-selective (δH 2.8-3.0 ppm) 2D 1H-31P HSQC-TOCSY spectrum with phosphorus decoupling (C) from a GDS 2000 sample spiked with 10 mg/mL of VX. The 1D 1H-31P HSQC and 31P{1H} NMR spectra are plotted as projections along the F2 and F1 axes, respectively. The 2D 1H-31P HSQC spectrum (A) shows several OP compounds with methyl bonded directly to phosphorus and ester proton correlations at chemical shifts typical for P-O-CHn substructures (δH 3.8-3.9 ppm) and at chemical shifts typical for P-N-CHn substructures (δH 2.8 -3.0 ppm). The band-selective (δH 3.8-3.9 ppm) 2D 1H-31P HSQC-TOCSY spectrum (B) shows the ester side chain resonances of EMPA and compounds 3 and 4, and the band-selective (δH 2.8-3.0 ppm) 2D 1H-31P HSQC-TOCSY spectrum (C) shows the ester side chain resonances of compounds 1 and 2.

phosphonothionates appear usually at chemical shift ranges δP 35-65 ppm and δP 75-100 ppm, respectively (source: the OPCW central analytical database, OCAD). The 2D 1H-31P HSQC spectrum showed that all the degradation products preserved methyl bonded to phosphorus (Figure 5A, Ha cross peaks at δH 1.15-1.30 ppm), and the two main products, based 5338

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on the ester side chain correlations (δH 2.8-3.0 ppm), had ester side chains containing other heteronucleus than oxygen between the phosphorus and alkyl group (cf. Table 2). Only the minor degradation products indicated the presence of P-O-CHn substructures by giving ester correlations at chemical shift range δH 3.8-3.9 ppm (Table 2). On the basis of the

Table 4. Unusual OP Degradation Products of VX Found in GDS 2000a

a

The CAS numbers are shown in square brackets. Schedule codes are from the CWC.2 The extractable NMR parameters are shown.

constituent of GDS 2000, it was assumed that these major degradation products may contain P-N-CHn substructures. Therefore, two band-selective 2D 1H-31P HSQC-TOCSY measurements were conducted using the aforementioned proton chemical shift bands in order to reveal the ester side chain structures. With the band-selective 2D 1H-31P HSQC-TOCSY spectra, the characterization of the ester side chains of the minor phosphonic acids (Figure 5B) and the major phosphonamidic acids (Figure 5C) was straightforward (Table 4). In the spectrum optimized for band δH 3.8-3.9 ppm of the minor compounds, we can see that the correlations at δP 24.26 ppm are from the ethyl ester side chain of EMPA (Figure 5B). Typically phosphorus of VX reacts with nucleophiles so that one or both of the ester side chains are displaced, but it seems that, under the described reaction conditions, this most typical degradation pathway is less common in GDS 2000. The dominant reactions are formation of substitution products with the compounds of the decontamination solution (Table 4). Formation of this kind of products is not rare in decontamination solutions, e.g., some of the components of DS241 will form substitution products with sarin, soman, and VX producing characteristic phosphonates.42,43 However, to the best of our knowledge, the phospho(41) Jackson, J. B. Development of Decontamination Solution DS2; CWLR, 2368; 1960.

namidic acids have not been reported in the degradation studies of OP nerve agents, nor is there any NMR data about phosphonamidic acids in the OCAD. One related study by Ga¨b et al.44 describes the formation of aminoalcohol products when OP nerve agents were dissolved in commonly used aminoalcohol buffer solutions, but only O-esters (phosphonates) were detected. According to a study by Palomer et al.,45 the stability of N-phosphonyl-R-aminoalcohols is highly pH-dependent; these phosphonamidates are stable in alkaline conditions, but in acidic conditions they undergo phosphonyl group migration to phosphonates. The reversal of this migration in the pH changes has not been observed. This can indicate that in the buffer solutions with close to neutral conditions, N-esters (phosphonamidic acids) do not exist, but the formation of phosphonamidic acids, based on our results, can happen in highly alkaline decontamination solutions containing aminoalcohols. Therefore, one must be careful with analytical techniques like GC/MS and LC/MS, which require (42) Beaudry, W. T.; Szafraniec, L. L.; Leslie, D. R. Reactions of Chemical Warfare Agents with DS2. Product Identification by NMR. I. Organophosphorus Compounds; CRDEC-TR-364; 1992. (43) Yang, Y.-C.; Baker, J. A.; Ward, J. R. Chem. Rev. 1992, 92, 1729–1743. (44) Ga¨b, J.; John, H.; Melzer, M.; Blum, M.-M. J. Chromatogr., B 2010, 878, 1382–1390. (45) Palomer, A.; Cabre´, M.; Mauleo´n, D.; Carganico, G. Synth. Commun. 1992, 22, 2373–2380.

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extensive sample pretreatment, that the composition of the degradation products is not irreversibly altered before the analysis. Nevertheless, further studies are needed to determine the stability of these phosphonamidic acids in the commonly applied sample preparation procedures46 and the formation of these compounds in other decontamination solutions that contain aminoalcohols (e.g., GD 5 and GD 640). CONCLUSIONS As shown, the proton band-selective 1H-31P correlation experiments are excellent methods in the characterization of ester side chains of OP nerve agent degradation products. The best use of these methods would be in stepwise progression: The presence of OP compounds and possibly their quantity could be screened with the conventional 1D 1H-31P HSQC30 and band-selective 1D 1 H-31P HSQC analyses, further characterization of the OP compounds would be facilitated with 2D 1H-31P HSQC17 and 2D 1 H-31P fast-HMQC18 analyses by establishing the correlation between the phosphorus and the alkyl/ester side chains, and the final evaluation of the ester side chain structures could be conducted with the band-selective 2D 1H-31P HSQC-TOCSY analysis. These techniques would be quite useful in studies of (46) Rautio, M., Ed. Recommended Operating Procedures for Sampling and Analysis in the Verification of Chemical Disarmament; The Ministry for Foreign Affairs of Finland: Helsinki, Finland, 1994. (47) Cullinan, D. B.; Hondrogiannis, G.; Henderson, T. J. Anal. Chem. 2008, 80, 3000–3006. (48) Koskela, H.; Vanninen, P. Anal. Chem. 2008, 80, 5556–5564. (49) Ga¨b, J.; Melzer, M.; Kehe, K.; Wellert, S.; Hellweg, T.; Blum, M.-M. Anal. Bioanal. Chem. 2010, 396, 1213–1221.

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degradation reactions of OP nerve agents and give information for the development of new decontamination systems. Because of the TOCSY transfer, the limit of detection in the band-selective 2D 1H-31P HSQC-TOCSY is not as good as with, e.g., 2D 1H-31P fast-HMQC experiment,18 but as shown above, levels of 100 µg/ mL of typical OP nerve agent degradation products were detected within reasonable total measurement times (under 4 h). With the use of suitable sample enrichment procedures and application of new probe head techniques,47,48 the band-selective 2D 1H-31P HSQC-TOCSY experiment or the band-selective modification of the phosphorus-selective 1D 1H-31P HSQC-TOCSY experiment reported earlier18 will also be applicable in the OPCW Proficiency Test analyses. The band-selective 1D 1H-31P HSQC would also be a valuable technique in kinetic studies49 for the measurement of degradation rates of OP compounds. The techniques shown above are not limited to the CWC-related OP compounds; their application in, e.g., environmental analysis of OP pesticides should be feasible. ACKNOWLEDGMENT The supply of chemicals from Swiss NBC Defense Establishment, Spiez Laboratory, 3700 Spiez, Switzerland, and the Finnish Defense Forces Technical Research Centre are gratefully acknowledged. Our colleague Martin So¨derstro¨m is acknowledged for his comments on the manuscript.

Received for review April 2, 2010. Accepted May 10, 2010. AC100867X