Application of Two-Dimensional 1H−31P Inverse NMR

Anal. Chem. 1997, 69, 2694-2700

Application of Two-Dimensional 1H-31P Inverse NMR Spectroscopy to the Detection of Trace Amounts of Organophosphorus Compounds Related to the Chemical Weapons Convention Christine Albaret, Daniel Lœillet, Patrick Auge´, and Pierre-Louis Fortier*

Centre d’EÄ tudes du Bouchet, DGA, 91710 Vert le Petit, France

Two-dimensional (2D) 1H-31P NMR techniques are applied to samples containing trace amounts of organophosphorus (OP) compounds related to the Chemical Weapons Convention. Because these techniques allow the recording of 31P-filtered spectra with the sensitivity of proton NMR, the problem of overlapping in 1H NMR spectra is nearly canceled, while simultaneous 31P and 1H NMR information is obtained in a fast way. How these experiments are carried out is described. The advantages of 2D techniques are compared with respect to their 1D counterparts, and new developments are proposed for rapid and reliable assignment without the need for reference data of the spiked OP chemicals. Since the signing of the Convention for Prohibition of Chemical Weapons in 1993,1 interlaboratory tests (Proficiency Tests)2,3 have been organized in order to designate laboratories accredited for verifying the application of the Convention. In these tests, participating laboratories have to identify, within a time limit of 15 days, compounds spiked at the trace level in different matrices (water, soil, rubber, paint, etc.). Since it is a very sensitive method and can be coupled to a separation technique, e.g., gas or liquid chromatography (GC or LC), mass spectrometry (MS) has been mainly used during the first Proficiency Tests. Though NMR spectroscopy is considered to be less sensitive, this technique has also been applied by some laboratories during the first Proficiency Tests.3,4 Criteria for identification by NMR rely upon the comparison of spectra with reference data.5 These data can be obtained either from libraries or by recording spectra of the polluted (or blank) sample spiked with the presumed chemicals. (1) Convention on the Prohibition of the Development, Production, Stockpilling, and Use of Chemical Weapons and their Destruction, signed in Paris in January 1993. Printed and distributed by the Provisional Technical Secretariat for the Preparatory Commission for the Organisation for the Prohibition of Chemical Weapons. Depositary of the Convention is the Secretary-General of the United Nations. (2) International Interlaboratory Comparison (Round-Robin) Test for 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, 1994; Vol. F. (3) 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; Vol. H. (4) Mesilaakso, M.; Tolppa, E.-L. Anal. Chem. 1996, 68, 2313-2318. (5) Recommended Operating Procedures for Sampling and Analysis in the Verification of Chemical Disarmament; The Ministry for Foreign Affairs of Finland: Helsinki, 1994.

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This requires that library spectra have been recorded under the same conditions of solvent, temperature, concentration, etc., or that the laboratory would have been able to synthesize all presumed compounds within the time limit. These criteria might be very difficult to meet in most cases, and NMR has been used essentially in order to confirm results obtained by other analytical methods.3 In the tests, since most of the compounds related to the Convention are organophosphorus (OP) molecules,1 31P NMR spectroscopy is primarily used to check for the presence of such OP chemicals. Although the sensitivity of 31P NMR is low, this method is interesting since the corresponding spectra usually exhibit no overlapping signals. However, the different families of OPs related to the Convention resonate in relatively narrow spectral regions, so characterization of this type of chemicals using only 31P NMR spectroscopy is considered to be insufficient.2 For this reason, in order to confirm the structure of the spiked chemicals through assignment of their protons, NMR spectroscopy of the more sensitive nucleus 1H is also performed. The problem with 1H NMR is that spectra often present strong overlapping signals. Indeed, in the absence of a separation technique, the extraction procedures used to prepare NMR samples (see the Experimental Section for more details) are ineffective in eliminating the background molecules. Thus, in the case where strong signals overlap peaks of interest, it is impossible to assign the corresponding protons. The advantages of each nucleus (sensitivity for proton, and spectra free of background noise for phosphorus) were combined. This was achieved by recording inverse two-dimensional 1H-31P NMR spectra.6 Indeed, such techniques obtain proton and phosphorus chemical shift information in a single experiment, with the sensitivity of 1H. In the first part of this paper, we outline how two-dimensional (2D) heteronuclear single quantum coherence (HSQC) experiments7,8 can be applied to a Proficiency Test sample and the advantages of these experiments with respect to their one-dimensional (1D) counterparts. The second part of the article describes new developments in the field of 2D NMR that could help in the assignment of spiked OPs without the need for reference data. EXPERIMENTAL SECTION Samples. Two Proficiency Test samples have been selected for this study, each of them being provided with a blank one in (6) Morris, G. A.; Freeman, R. J. Magn. Reson. 1978, 29, 433-435. (7) Bodenhausen, G.; Ruben, D. J. Chem. Phys. Lett. 1980, 69, 185-189. (8) Norwood, T. J.; Boyd, J.; Campbell, I. D. FEBS Lett. 1989, 255, 369-371. S0003-2700(97)00063-2 CCC: $14.00

© 1997 American Chemical Society

Figure 1. Spiked OP compounds. Protons are identified by letters.

the tests. Samples were prepared according to standard Recommended Operating Procedures:5 (1) Soil. The spiked and blank samples were provided by the Republic of Korea as part of the Second Official Proficiency Test. The spiked sample contained three OP compounds (see Figure 1): (2-methoxyethyl)isopropyl methylphosphonate (I), diethyl isopropylphosphonate (II), and (2-methoxyethyl)ethyl isopropylphosphonate (III).9 Samples were extracted with 2 × 10 mL of CH2Cl2, filtered, and concentrated to dryness under a nitrogen gas flow at 25 °C. The material was resuspended in 500 µL of CDCl3. (2) Liquid. These samples were provided by Switzerland as part of the First Official Proficiency Test. The spiked sample contained three compounds related to the chemical warfare agent tabun (see Figure 1): ethyl N,N-dimethylphosphoramidocyanidate or tabun (IV), diethyl N,N-dimethylphosphoramide (V), and (dimethylamino)phosphoryl dichloride (VI). Spiking levels were 50 ppm for each chemical.10 For the preparation, a volume of 50 µL of CDCl3 was simply added to 450 µL of the solution. NMR Spectroscopy. One-dimensional 31P and {1H}31P NMR spectra were recorded at 25 °C on a Bruker Avance DPX360 MHz instrument equipped with a quadrupole probe. Proton decoupling in the {1H}31P NMR spectra was achieved using a WALTZ-16 composite pulse decoupling (CPD) sequence.11 All proton and two-dimensional experiments were recorded at 25 °C on a Bruker Avance DRX500 spectrometer equipped with a quadrupole Xinverse probe. Decoupling of phosphorus in the 1D 1H and 2D 1H-31P correlation experiments was achieved using a GARP CPD sequence.12 Heteronuclear correlation spectra were recorded using the “echo-antiecho” scheme.13 In this experiment, the positive and negative orders of coherence are selected in two successive transients by applying suitable pulsed field gradients.14-16 This leads to a pure absorption spectrum. Due to the two-step (9) Laudares, M. Preliminary Evaluation of Results: Second Official OPCW Interlaboratory Proficiency Test; Preparatory Commission for the Organisation for the Prohibition of Chemical Weapons, 1997. (10) Bruce, P.; Laudares, M. “Preliminary Evaluation of Results: First Official OPCW/PTS Inter-laboratory Comparison Test: Proficiency Test; Preparatory Commission for the Organisation for the Prohibition of Chemical Weapons, 1996. (11) Shaka, A. J.; Keeler, J.; Freeman, R. J. Magn. Reson. 1983, 53, 313-340. (12) Shaka, A. J.; Barker, P. B.; Freeman, R. J. Magn. Reson. 1985, 64, 547552. (13) Davis, A. L.; Keeler, J.; Laue, E. D.; Moskau, D. J. Magn. Reson. 1992, 98, 207-216. (14) Bax, A.; DeJong, P. G.; Mehlkopf, A. F.; Smidt, J. Chem. Phys. Lett. 1980, 68, 567-580. (15) Davis, A. L.; Laue, E. D.; Keeler, J. J. Magn. Reson. 1991, 94, 637-644.

coherence pathway selection, the sensitivity of this experiment is reduced by a theoretical factor of 21/2. This loss of sensitivity was compensated by refocusing and detecting the two orthogonal in-phase magnetization components,17 as described in sequence A (Figure 2). Delay τ was set to 1/4 J, while τ1 was set to 1/6 J in order to optimize the INEPT transfer for all proton multiplicities.18-20 In a modified HSQC experiment, in order to decouple selectively the methyls of the compounds, the refocusing 1H 180° pulse in the middle of the 31P evolution period was replaced by a selective one (see sequence B in Figure 2). This soft pulse was generated by using an amplitude modulated spin-inversion-shaped pulse of the BURP type (e.g., I-BURP).21 Since this pulse was considerably longer than the corresponding hard one (5 ms instead of 20 µs), the enhanced scheme of sequence A was not retained in order to prevent loss of signal due to relaxation of the nuclei. HSQC-TOCSY22 was recorded using the scheme described in C (Figure 2). The isotropic mixing was achieved using a MLEV-17 magnetization transfer sequence applied along the x axis.23 In all spectra, chemical shifts were referenced to internal trimethylsilyl (δ ) 0 ppm) in the case of 1H and external phosphoric acid (δ ) 0 ppm) in the case of 31P. RESULTS AND DISCUSSION Use of a HSQC Experiment in a Proficiency Test. The advantages of 2D HSQC can be illustrated in the case of the soil sample. This sample had been polluted with three organophosphonates (see Figure 1). Among these chemicals, only II had been previously synthesized in our laboratory. Therefore, NMR was essentially used to support identification made by MS. The chemicals were easily detected by 31P NMR, but the 1H spectra of both extracts exhibited strong signals in the aliphatic region (Figure 3). Only five additional signals could be observed in the spectrum of the spiked extract that did not appear in the blank one (see Figure 4). As it can be judged, signals from protons Ie, (16) Keeler, J.; Clowes, R. T.; Davis, A. L.; Laue, E. D. Methods Enzymol. 1994, 239, 145-207. (17) Cavanagh, J.; Palmer, A. G., III; Wright, P. E.; Rance, M. J. Magn. Reson. 1991, 91, 429-436. (18) Mc Lafferty, F. W. Tandem Mass Spectrometry; Wiley Interscience: New York, 1983. (19) Pegg, D. T.; Doddrell, D. M.; Brooks, W. M.; Bendall, M. R. J. Magn. Reson. 1981, 44, 32-40. (20) Palmer, A. G., III; Cavanagh, J.; Wright, P. E.; Rance, M. J. Magn. Reson. 1991, 93, 151-170. (21) Geen, H.; Freeman, R. J. Magn. Reson. 1991, 93, 93-141. (22) Norwood, T. J.; Boyd, J.; Heritage, J. E.; Soffe, N.; Campbell, I. D. J. Magn. Reson. 1990, 87, 488-501. (23) Bax, A.; Davis, D. G. J. Magn. Reson. 1985, 65, 355-360.

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Figure 2. Sequences used in the NMR experiments. Phase cycling is as follows: φ1 ) x,-x; φ2 ) x,x,-x,-x; φ3 ) y,y,-y,-y; receptor ) x,-x,-x,x; (B and C) φ1 ) x,-x; φ2 ) x,x,-x,-x; φ4 ) x,x,x,x,-x,-x,-x,-x; receptor ) x,-x,-x,x. SLx denotes a 1 ms spin-lock pulse along x to suppress the residual solvent resonance,28 and ∆ is the gradient pulse length (here 1 ms). Quadrature in the f1 dimension was obtained by recording the odd increments with G1/G2 ) γ(1H)/γ(31P) and the even increments with G1/G2 ) -γ(1H)/γ(31P) and φ3 phase-inverted in the case of the enhanced experiment (sequence A). Fourier transform was applied using the “echo-antiecho” treatment in the Bruker software UXNMR.

Figure 3. Superimposition of the 1D 1H spectra of the soil extracts (top, blank sample; bottom, spiked sample).

IIIc,f, and IIc are difficult to distinguish. No signals could be detected for the P-C side chains of I, II, and III, which would allow an unambiguous assignment of these compounds. It must be emphasized that assignment of the isopropyls of II and III is 2696 Analytical Chemistry, Vol. 69, No. 14, July 15, 1997

of great interest. Indeed, it is not possible to distinguish between a n-propyl and an isopropyl by analysis of the mass spectra obtained in either standard mode (electronic impact and chemical ionization). Therefore, in this test, a MS-MS study18 was

Figure 4. Expansions of the spectra of Figure 3 (top, blank sample; bottom, spiked sample), showing the signals that could be detected by comparison of the spectra.

necessary to determine the nature of the propyls, and NMR was of considerable help in confirming the mass results. The 2D HSQC experiment7,8 was recorded using pulsed field gradients in order to eliminate the unwanted signals arising from protons not coupled to the heteronucleus of interest (see the Experimental Section). For organophosphonates, the 1H-31P coupling constants are known to range from 0 to 22 ppm.24 Therefore, in order to screen for all coupling nuclei and minimize loss of signal due to relaxation during the INEPT sequences, J was set to an intermediate value of 12 Hz. The 2D map is presented on Figure 5, with the 1D {1H}31P and {31P}1H spectra plotted along the f1 and f2 axes. As can be seen, the 2D map is very clean, and each cross peak can be easily connected to a phosphorus signal. Thus, by extracting 1D rows at the frequency of each phosphorus, it is possible to link the coupled proton to its respective molecule (Figure 5). The negative sign of some cross peaks is due to a different refocusing of the orthogonal magnetization components during the two consecutive reverse INEPT6 sequences of the experiment.17 Compound II could be completely assigned using the HSQC experiment. In particular, the isopropyl was clearly detected, (24) Crutchfield, M. M.; Dungan, C. H.; Letcher, J. H.; Mark, V.; Van Wazer, J. R. P31 Nuclear Magnetic Resonance; Wiley Interscience: New York, 1967; Vol. 5.

demonstrating the power of the method. For this compound, the chemical shifts extracted from the 2D map were able to be compared with those of the pure chemical, both spectra being recorded under the same conditions of solvent and temperature. The values were found to fit very well (Table 1). Note that, among the four proton resonances, only one (e.g., IIc) could be extracted by comparison of the 1D 1H spectra (see Figure 4). For I and III, there were no reference data. Therefore, the 2D experiment was used to support the identification made by mass spectrometry. In the case of chemical I, methyl Ia was easily assigned to the most intense signal. Cross peaks corresponding to CH Ie (at 4.71 ppm) and CH2 Ic (at 3.60 ppm) were also observed. No cross peak was detected for CH2 Ib, although the corresponding J3 coupling constant with 31P is expected to be greater than the J4 of CH2 Ic. However, the multiplet signal of CH2 Ib probably spreads in the 1H dimension due to the nonequivalence of the methylene protons. Hence, the cross peak is not observed. This spreading also explains why the peak could not be detected by comparison of the spiked and blank extract 1D spectra (see Figure 3). In the next section, how to get information on such protons will be explained. Finally, the O-CH3 protons of I (Id) were assigned by 1D to the signal at 3.40 ppm exhibiting no multiplicity (Figure 3). Concerning III, all protons could be correlated to the phosphorus except CH3 IIIg and O-CH3 Analytical Chemistry, Vol. 69, No. 14, July 15, 1997

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Figure 5. 2D HSQC 1H-31P map of the spiked soil extracted with CH2Cl2. {31P}1H and {1H}31P 1D spectra are plotted along the axis. The HSQC correlation spectrum was recorded using 32 increments in the f1 dimension; 256 scans were accumulated per increment. J was set to 12 Hz. Table 1. Summary of the Results Obtained for Compound II (Soil)a 1Ha

reference spectra (CDCl3) sample spectra (CDCl3) 1D 1H/31P 2D 1H-31P a

1.95 1.94

1Hb

1Hcc′

1.18

4.09, 4.13

1.18

4.10 4.10

1Hd

Table 2. Summary of the Results Obtained for Compounds I and III (Soil)a 31P

1.32

35.95

1.32

35.97 36.0

Chemical shifts are given in ppm.

sample spectra (CDCl3)

Compound I 1Ha 1Hb 1Hc

assigned from 1D 1H/31P assigned from 2D 1H-31P

1.49

sample spectra (CDCl3)

* 3.60

Compound III 1Hb 1Hc 1Hd

1Ha

1Hd

1He

3.40

* 4.71

1He

1Hf

1H/31P

assigned from 1D * * 3.40 * 4.17 assigned from 2D 1H-31P 1.99 1.19 4.17 3.60

IIIe. As in the case of I, the P-C isopropyl side chain was easily detected from the map. Methyl IIIg could be assigned using 1H 2D TOCSY (not shown), while the O-CH3 protons were assigned to the same peak as the O-CH3 protons of I. Table 2 summarizes the results obtained by each technique (1D and 2D) for I and III. It is clear from this table that the 2D experiment contains more information than the combined 1D 1H and 31P spectra and appears particularly interesting in the case where the sample has been spiked with more than one OP compound. Finally, the measuring time for this 2D experiment was a bit longer than the one required to get the {1H}31P spectrum (4 h 25 min instead of 3 h 15 min). However, the information contained in the map, as described, is essentially richer. Design of New 2D Experiments for the Characterization of OP Compounds. As mentionned in the introduction, Proficiency Tests have to be completed within a time limit of 15 days. Regarding the difficulty of the tests, it is not certain that a participating laboratory would be able to synthesize all presumed 2698 Analytical Chemistry, Vol. 69, No. 14, July 15, 1997

1Hf

31P

30.81 30.8

1Hg

31P

36.59 36.6

a Chemical shifts are given in ppm. Stars refer to resonances which were detected by comparison of the 1D spectra but which could not be definitively assigned to one of the chemicals.

chemicals within this period. It has also been shown that shifty results are expected to occur in NMR round-robins due to the dependence of chemical shifts upon sample conditions (e.g., solvent, concentration of the chemicals, pH (if relevant), or temperature).25 Therefore, an unambiguous assignment might be very difficult to achieve in many circumstances. In the last section, it was shown that it was possible to get partial assignment from 2D HSQC experiments. In these experiments, the intensity of a 2D cross peak depends upon two factors: the coupling of proton(s) with phosphorus, which governs the magnitude of the magnetization transfer, and the multiplicity of the proton(s), which determines the spreading of the cross peak (25) Grzonka, M.; Davies, A. N. Spectrosc. Eur. 1994, 6, 32-33.

Figure 6. {1H}31P 1D spectrum of the spiked liquid sample. Expansions of the coupled

31P

spectrum are shown.

Figure 7. Expanded plots of the selectively decoupled HSQC experiment. The corresponding extracted columns are shown on the right of each expansion. The spectrum was recorded using 256 increments in the f1 dimension; eight scans were accumulated per increment. J was set to 12 Hz.

in the f2 dimension. For instance, in the case of P-C side chains of OPs I, II, and III, cross peaks were easily detected because the coupling constants are relatively strong (>10 Hz), and the methyl protons are equivalent. The problem is more intricate when the coupling constants are weaker and when protons are inequivalent, as is the case for the -O-CH2- protons of I (Ic). Since several OP compounds belonging to the scheduled lists are organophosphonates,1 it appears important to find a method allowing characterization of such protons. The easiest way to obtain this J3 coupling information is to extract it from the coupled 1D 31P spectrum. Considering the poor sensitivity of this nucleus and the low concentration of the species in test samples, a long acquisition time is necessary to record a spectrum with adequate signal/noise ratio. This technique was applied to the spiked liquid sample for which the concentrations of the polluting OPs were relatively high (e.g., 50-100 ppm).10 As described in the Experimental Section, this sample contained three OP compounds related to tabun (IV), among which two were esters (tabun and V). The 1D {1H}31P spectrum is shown in Figure 6, with expansions of the coupled spectrum. Three days was necessary to record the coupled spectrum, a time which is quite long in comparison to the 15 days given for the tests. Hence, in order to get the same information faster, the HSQC sequence was modified. The idea was to take advantage of the signals arising from the coupling of the -N-(CH3)2 methyls with the phosphorus. These nuclei are, indeed, relatively strongly coupled (J ≈ 11 Hz),24 and the proton signal of the methyls is not split. Hence, the corresponding cross peaks are expected to be intense and should be used to read the J3 passive constants information in the 31P dimension. For this purpose, the methyl protons were selectively decoupled during the 31P evolution period of the HSQC experiment. This was achieved by applying a 180° shaped pulse centered on the methyl region instead of the usual hard pulse in the sequence (see the Experimental Section). As a result, cross peaks of methyl IVa and Va presented multiplicities in the f1 dimension consistent with the structure of each compounds (Figure 7): a triplet for IV (passive coupling with one CH2) and

a quintuplet for V (passive coupling with two CH2). The cross peak of methyl VIa with phosphorus was unaffected by the pulse, the unusual shape of the peak being due to an isotopic effect of the chlorine nuclei. The J3 coupling constants could also be easily obtained using this scheme (8.9 and 7.5 Hz for IV and V, respectively). It is emphasized that the recording of this experiment is expected to be longer than that for the classical HSQC. Indeed, resolution in the f1 dimension and suitable signal/noise ratio are required for the analysis of the spectrum. In this case, the total acquisition time could be reduced to 1 h 30 min using appropriate folding in the f1 dimension. This kind of experiment could be particularly attractive in the case where the solvent peak falls into the O-CH2 (or O-CH) region. Indeed, pulsed field gradients are not completely efficient in eliminating the solvent.16 This results in the presence of a residual signal in the 2D map that could prevent the observation of the cross peaks at the chemical shift of interest. In that case, the selective decoupling technique will provide useful information. Any of the HSQC experiments previously described can give information on weakly coupled or noncoupled nuclei, for example, the case of the methyls of the O-R moieties of IV and V for which the coupling constant is less than 0.7 Hz. In order to transfer the in-phase magnetization throughout the 1H spin system, a TOCSY mixing sequence26 was applied just after the reverse INEPT sequence of the HSQC experiment.22,27 Using this scheme, two cross peaks were detected corresponding to the methyl protons c of IV and V whose signals are overlapped in the 1D 1H spectrum (see Figure 8). This scheme is applicable to longer O-R chains, providing that the 1H-1H coupling would allow the magnetization transfer to occur. It is emphasized that this experiment was far less sensitive than the HSQC one, and 4 h 30 min were necessary to record the 2D map presented here. Therefore, in the case where the spiking level is above 50 ppm (26) Braunschweiler, L.; Ernst, R. R. J. Magn. Reson. 1983, 53, 521-528. (27) Otting, G.; Wu ¨ thrich, K. J. Magn. Reson. 1988, 76, 569. (28) Messerle, B. A.; Wider, G.; Otting, G.; Weber, C.; Wu ¨ thrich, K. J. Magn. Reson. 1989, 85, 608-613.

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clean maps in a very easy way, demonstrating the power of 2D techniques. New experiments were also developed to provide full assignment of spiked compounds without the need for reference spectra. These techniques are not restricted to the detection of chemicals related to the Chemical Weapons Convention but could be widely used in environmental studies for the screening of OP polluting substances.

Figure 8. HSQC-TOCSY spectrum of the spiked liquid sample. {31P}1H and {1H}31P 1D spectra are plotted along the axis. The spectrum was recorded using 32 increments in the f1 dimension; 256 scans were accumulated per increment. J was set to 12 Hz. The mixing period was 40 ms long.

or if the sample can be concentrated prior to NMR analysis, this type of experiment is appropriate. CONCLUSION Two-dimensional 1H-31P HSQC was successfully applied to the characterization of OP compounds in complex mixtures. This allowed the simultaneous extraction of 1H and 31P information from

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ACKNOWLEDGMENT We thank NC-Laboratory (Switzerland) and Agency for Defense Development (Republic of Korea) for providing the test samples, and the Provisional Technical Secretariat of the Organisation for the Prohibition of Chemical Weapons for coordinating the First and Second Official Proficiency Tests. We are grateful to C. Thenault for the preparation of the samples. We thank Dr. Stoven, Ecole Polytechnique (France), for helpful discussions, and W. P. Ashman, U.S. Army Edgewood Research Development and Engineering Center (MD), for proofreading the manuscript. Received for review January 21, 1997. Accepted April 23, 1997.X AC9700639 X

Abstract published in Advance ACS Abstracts, June 15, 1997.