Screening and Identification of Organophosphorus Compounds

Proficiency Test are the low spiking level (1-10 μg/mL or μg/g) and the tight timetable for the analysis. Identification of spiked chemicals, includ...
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Anal. Chem. 2006, 78, 3715-3722

Screening and Identification of Organophosphorus Compounds Related to the Chemical Weapons Convention with 1D and 2D NMR Spectroscopy Harri Koskela,*,† Nicoleta Grigoriu,‡ and Paula Vanninen†

VERIFIN, University of Helsinki, P.O. Box 55, FIN-00014 Helsinki, Finland, and Ministry of National Defence, NBC&Ecology Defence Scientific Research Center, Soseaua Oltenitei 225, Sector 4, Bucharest, Romania

Two-dimensional 1H-31P Fast-HMQC was tested for determination of the presence in low concentrations of organophosphorus compounds related to the Chemical Weapons Convention. This method, based on inverse detection, demonstrated high sensitivity and selectivity. Background signals, such as solvent peaks, are suppressed with good efficiency, and organophosphorus compounds present at a concentration level 1-10 µg/mL can be detected within a few hours. In addition, phosphorus-selective one-dimensional 1H-31P HSQC-TOCSY was applied to produce a complete proton spectrum of selected organophosphorus compound from a sample containing intense background resonances. Application of the methods presented in this paper resulted in considerably improved performance of NMR spectroscopy as a complementary technique for screening as well as identification of chemical warfare agents in environmental samples. The joint concern of nations to prevent the production and use of chemical warfare agents (CWA) led in 1993 to an agreement, the Chemical Weapons Convention (CWC),1 for the prohibition of the development, production, stockpiling, and use of chemical weapons and on their destruction. The goal of the Convention is that existing stockpiles should be destroyed and new production prevented. The Technical Secretariat of the Organisation for Prohibition of Chemical Weapons (OPCW)2 is the governing body that implements the CWC internationally. The Director General of the OPCW nominates designated laboratories where authentic, usually environmental samples can be sent for verification analysis. The designated laboratories must report all scheduled chemicals1 in these samples. Identification requires confirmation by two * To whom correspondence should be addressed. E-mail: Harri.T.Koskela@ helsinki.fi. Fax: +358-9-191 50437. † University of Helsinki. ‡ NBC&Ecology Defence Scientific Research Center. (1) 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. (2) The Organisation for Prohibition of Chemical Weapons Headquarters, Johan de Wittlaan 32, 2517 JR, The Hague, The Netherlands, http://www.opcw.org. 10.1021/ac052148c CCC: $33.50 Published on Web 04/22/2006

© 2006 American Chemical Society

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

spectrometric methods; no quantitative results are given. Each year, the OPCW organizes Proficiency Tests in which participating laboratories analyze three samples in different matrixes (aqueous or organic liquid, soil, sand, concrete, paint or rubber) and report any scheduled chemicals found. The challenging features of the Proficiency Test are the low spiking level (1-10 µg/mL or µg/g) and the tight timetable for the analysis. Identification of spiked chemicals, including sample preparation, analysis of the chemicals, and reporting of results must take place within 15 days of the arrival of the samples. So far, analytical techniques characterized by high sensitivity, such as gas and liquid chromatography combined with mass spectrometry (LC/MS, GC/MS), have been favored for the analysis.3,4 Nuclear magnetic resonance (NMR) spectroscopy has played a minor role in CWA analysis owing to the modest sensitivity and relatively high cost of the instrument. Nevertheless, the most of the scheduled chemicals are organophosphorus (OP) compounds (Chart 1), which are conveniently screened for in a liquid sample with a simple 31P{1H} NMR experiment.5,6 General environmental chemistry has also appreciated 31P{1H} NMR in this context.7 Complete structure elucidation is not possible on the basis of a 31P{1H} NMR spectrum alone due to the low information content. (3) Rautio, M., Ed. Methodology and Instrumentation for Sampling and Analysis in the Verification of Chemical Disarmament; 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; The Ministry for Foreign Affairs of Finland: Helsinki, 1994. (4) Hooijschuur, E. W. J.; Kientz, C. E.; Brinkman, U. A. T. J. Chromatogr., A 2002, 982, 177-200. (5) Enqvist, J.; Hesso, A.; Rahkamaa, E.; Bjo ¨rk, H.; Piispanen, H.; Siivinen, K.; Kentta¨maa, H.; Sivonen, A.; Ali-Mattila, E. Identification of Potential Organophosphorus Warfare Agents: An Approach for the Standardization of Techniques and Reference Data; The Ministry for Foreign Affairs of Finland: Helsinki, 1979. (6) Mesilaakso, M.; Tolppa, E.-L. Anal. Chem. 1996, 68, 2313-2318. (7) Cade-Menun, B. J. Talanta 2005, 66, 359-371.

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Although complete structural identification of pure chemicals can be concluded with 1H NMR, interpretation of the spectrum of an unpurified sample may be all but impossible due to the intense background level of the interfering sample matrix and impurities. A number of sample preparation methods8 involving enrichment, purification, and solvent exchange significantly assist the application of 1H NMR for identification. However, part of the analyte is always lost during these procedures, and sample contamination may occur. Except, for matrixes such as soil where sample pretreatment is mandatory, it is preferable, therefore, that samples are analyzed as such. Recent method development in NMR spectroscopy has provided sophisticated polarization transfer experiments, which allow selective observation of proton nuclei with scalar coupling to a selected heteronucleus, while effectively suppressing the background signals. Among these, inverse-detected two-dimensional (2D) 1H-31P correlation experiments offer a powerful technique for the screening of OP compounds relevant to the CWC. 2D heteronuclear single quantum coherence (HSQC)9-based methods have good information content; unambiguous identification of compounds is possible in some cases, and identification of the type of compound and the structure of the phosphorus-bound alkyl moiety is possible for all OP chemicals. Identification of the ester side chain is often hampered by the low intensity of resonances caused by strong splitting of JHH coupling and weak or absent JHP coupling. Acquisition of 2D 1H-31P HSQC spectra within reasonable time, however, requires spiking levels of 50-100 µg/mL analyte.9 Thus, sample enrichment is mandatory for Proficiency Test samples. With one-dimensional (1D) analogues of HSQC,10 a spectrum can be acquired within 1 h even from samples with spiking levels of 5-10 µg/mL. 1D HSQC methods are thus highly useful for screening. Chemical shift information on phosphorus coupled to protons is of course lost, and all OP compounds, relevant and irrelevant, appear in the spectrum. The essential requirement of a method for screening of very dilute samples is sensitivity. Inverse detection can be exploited with benefit; theoretically, inverse detection of phosphorus compounds offers over nine times the sensitivity of direct detection.11 While the use of pulsed field gradients for coherence selection reduces the sensitivity by factor x2, the efficiency of pulsed field gradients in rejection of unwanted signals outweighs the loss in sensitivity.12 However, the efficiency of polarization transfer and the effects of relaxation must also be taken into account when the goal is optimum signal-to-noise ratio within a certain time unit. With 1D NMR experiments, optimization of repetition time and excitation pulse angle with respect to T1 times according to the Ernst angle equation13 has proved a good way to obtain considerable savings in the acquisition time for insensitive nuclei. Ross (8) Rautio, M., Ed. Recommended Operating Procedures for Sampling and Analysis in the Verification of Chemical Disarmament; The Ministry for Foreign Affairs of Finland: Helsinki, 1994. (9) Albaret, C.; Loeillet, D.; Auge´, P.; Fortier, P.-L. Anal. Chem. 1997, 69, 26942700. (10) Meier, U. C. Anal. Chem. 2004, 76, 392-398. (11) Ernst, R. R.; Bodenhausen, G.; Wokaun, A. Principles of Nuclear Magnetic Resonance in One and Two Dimensions; Oxford University Press: Oxford, 1990. (12) Croasmun, W. R., Carlson, R. M. K., Eds. Two-Dimensional NMR Spectroscopy: Applications for Chemists and Biochemists, 2nd ed.; VCH Publishers: New York, 1994. (13) Ernst, R. R. Adv. Magn. Reson. 1966, 2, 1-135.

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Chart 2. Structures and Proton Numbering of Alkylphosphonic Acids Used in the Detection Limit Studiesa

a

Schedule codes are from the CWC1

and co-workers14 have described an interesting way to apply the Ernst angle optimization in inverse-detected 2D NMR spectroscopy. Their modified heteronuclear multiple quantum coherence (HMQC) experiment, Fast-HMQC, made possible the measurement of a 2D 1H-15N correlation spectrum from a 1 mM protein sample within 1 min, thus enabling real-time monitoring of protein-ligand interactions. In this paper, we demonstrate the application of 2D 1H-31P Fast-HMQC for screening of Proficiency Test samples for organophosphorus compounds present in concentrations of 1-10 µg/ mL (∼10-100 µM). The screening is done within a reasonable time, and no extensive sample preparation (purification, enrichment, solvent exchange) is required for aqueous or organic liquid samples. We also propose a modified, phosphorus-selective 1D 1H-31P HSQC-TOCSY experiment that can be used for the identification of OP compounds found with Fast-HMQC or conventional 31P{1H} NMR. EXPERIMENTAL SECTION Sample Preparation. The detection limit for Fast-HMQC was established with a dilution series of water samples (D1, D2, D3) containing different amounts of alkylphosphonic acids (Chart 2): methylphosphonic acid (MPA) ethylphosphonic acid (EPA), propylphosphonic acid (PPA), and isopropylphosphonic acid (IPPA). The chemicals in the dilution series contain four types of alkyl side chain directly bonded to phosphorus, which are relevant to the CWC and must be identified in Proficiency Test report. The chosen chemicals have no ester side chain(s) since the scalar coupling between phosphorus and the protons of the ester side chain is typically very weak or absent. NMR samples for the studies of detection limit were prepared as follows. A stock solution of MPA, EPA, and PPA with targeted concentrations of 1000 µg/mL was prepared in 2 mL of distilled water containing 20 mM ammonium acetate buffer. PPA contained IPPA as 3.1% impurity, and IPPA thus appears in much lower concentration than the other chemicals. A 10-fold dilution was made from the original stock solution to prepare 2 mL of working solution with concentrations of roughly 100 µg/mL. The dilution was repeated for the preparation of 2 mL of working solution with concentrations of roughly 10 µg/mL, which is the typical spiking level in Proficiency Test samples. NMR samples were prepared in 5-mm NMR tubes by adding 0.1 mL of D2O to 0.5 mL of solution. Exact concentrations of the chemicals in the NMR samples are presented in Table 1. The 18th Proficiency Test samples, consisting of organic (O), water (W), and soil (S) samples, and the corresponding blank (14) Ross, A.; Salzmann, M.; Senn, H. J. Biomol. NMR 1997, 10, 389-396.

Chart 3. Scheduled Chemicals Spiked in Samples of the 18th Proficiency Testa

a The spiking level of the compounds was 10 µg/mL in the organic and water samples and 10 µg/g in the soil sample. Molar concentrations for this spiking level are 72.5 (1), 72.5, (2), 42.2 (3), 37.9 (4), and 50.0 µM (5).

Table 1. Concentrations of MPA, EPA, PPA, and IPPA in NMR Samples of the Dilution Seriesa sample code D1 D2 D3 a

MPA

concentration level (µg/mL) in D2O EPA PPA IPPA

673.5 67.3 6.7 (70.2 µM)

1001.3 100.1 10.0 (91.0 µM)

893.2 89.3 8.9 (72.0 µM)

30.9 3.1 0.3 (2.5 µM)

Molarity of chemicals in D3 sample is also shown.

samples (OB, WB, SB) were prepared in Belgium.15 The scheduled OP compounds spiked at level 10 µg/mL (10 µg/g for the soil sample) are presented in Chart 3; the samples also contained nonrelevant phosphates with spiking level of 10-100 µg/mL. The NMR sample of the water sample was prepared simply by adding 0.1 mL of D2O and 0.5 mL of water sample to a 5-mm NMR tube. The NMR sample of the organic sample was prepared in the same manner by adding 0.1 mL of acetone-d6 and 0.5 mL of organic sample to a 5-mm NMR tube. Five grams of soil sample was extracted with 5 mL of dichloromethane. The extract was evaporated almost to dryness with nitrogen gas flow, then dissolved in 0.6 mL of CD2Cl2, and transferred to a 5-mm NMR tube. The recovery of the extraction was determined with a Hewlett-Packard (HP) 5890 Series II GC equipped with an HP autosampler and an HP 5972 mass-selective detector. Capillary column was HP5MS (30 m × 0.25 mm i.d., 0.25-µm film). The column temperature was programmed from 40 (1 min) to 280 °C at 10 °C/min and held at final temperature for 10 min. Splitless mode with time of 1 min and injector temperature 250 °C was used. The carrier gas was helium at a flow of 35 cm/s at 40 °C. The transfer line temperature was 280 °C. The ionization mode was electron impact (70 eV), (15) Preliminary Evaluation of the Results: Eighteenth Official Proficiency Test; Technical Secretariat of the Organisation for the Prohibition of Chemical Weapons, 2006; Vols. 1, 2.

with an electron multiplier voltage of 200 V above the “maximum sensitivity autotune” value. The concentration of chemicals in soil sample extract was estimated by comparing the total ion chromatogram peak area of 5-chloro-2-methylaniline (5 µg/mL, 35.5 µM) of a separate quality control (QC) sample to the peak area of the target chemical. The estimated recovery of analytes was ∼50%, resulting in the NMR sample concentration of ∼40 µg/ mL. Blank samples were prepared analogously. An authentic reference sample corresponding to compound 5 was prepared in a 5-mm NMR tube by adding 0.5 mL of acetone solution containing S,S-diethyl methylphosphonodithiolothionate at a level of 240 µg/mL (1.2 mM) and then 0.1 mL of acetone-d6. NMR Experiments. All proton-detected experiments were carried out on a Bruker DRX 500 equipped with 5-mm broadband inverse probe head with z-gradient at 290 K. 31P{1H} NMR spectra were measured with a Bruker AMX 400 equipped with a 5-mm quattro-nucleus probe head (31P, 13C, 19F, 1H) at 290 K. All spectra were processed with Bruker XWIN-NMR 3.0 software. T1 relaxation times of OP compounds in Proficiency Test samples are needed for optimization of NMR experiments. Direct determination of relaxation times is, however, not possible due to low concentrations of the compounds. The D1 sample was used to determinate the typical range of proton and phosphorus T1 values in organophosphorus compounds. The spin-lattice relaxation times were determined in inversion-recovery experiments (Table 2); proton decoupling was used during acquisition in phosphorus T1 experiments. Acquired maximum T1 times were applied to optimize the repetition times of NMR experiments used in this study. 1H NMR experiments were done with 5.4-s acquisition time and 4-s relaxation delay. The 1H NMR spectrum of the authentic reference sample of 5 was acquired with 16 scans (4 dummy scans) and the 1H NMR spectrum of the soil sample with 64 scans (no dummy scans). Total acquisition times were 3 and 10 min, respectively. The spectra were weighted with exponential window function (LB ) 1.0 Hz) prior to Fourier transform. Analytical Chemistry, Vol. 78, No. 11, June 1, 2006

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Figure 1. Pulse sequences for 1H-31P Fast-HMQC (A) and phosphorus-selective 1D 1H-31P HSQC-TOCSY (B). Narrow and wide bars denote 90° and 180° pulses, respectively, and unfilled bars denote pulsed field gradients. Pulses are along the x axis if not stated otherwise. Phase cycles: φ1 ) {x, -x}, φ2 ) {x, x, -x, -x}, φR ) {x, -x, -x, x}. The unfilled half-ellipse denotes a selective excitation pulse. The rectangle with diagonal line represents an adiabatic sweep pulse. The GARP decoupling is applied on phosphorus during the TOCSY and zero-quantum filter steps. Delay ∆ is the polarization-transfer delay, and δ corresponds to the total duration of the gradient pulse and gradient recovery delay.

31P{1H} NMR spectra were acquired using 45° excitation pulse angle. The repetition time (the sum of acquisition time and relaxation delay) was calculated according to the Ernst angle13 with respect to the T1(max) of phosphorus (3.90 s; see Table 2). The 31P{1H} NMR spectrum from the D1 sample was measured with 128 scans (32 dummy scans), resulting in the total acquisition time of 3 min. 31P{1H} NMR spectra from the D3 sample and Proficiency Test samples were measured with 16 384 scans (32 dummy scans), resulting in a total acquisition time of 5 h 30 min for each spectrum. The 31P{1H} NMR spectra were weighted with exponential window function (LB ) 1 Hz) prior to Fourier transform. Some changes were made to the original Fast-HMQC14 sequence (Figure 1A). The refocusing period was omitted to minimize relaxation losses, and the spectra were acquired without composite pulse decoupling. The Fast-HMQC used an excitation pulse Rernst, which is optimized according to the Ernst angle condition:14

cos(Rernst) ) -exp(-TPS/T1) exp(-Tr/T1)

(1)

where Tr is the repetition time (the sum of acquisition time and relaxation delay), which was set for 1.0 s. TPS equals the pulse sequence duration (TPS ) ∆ + 3δ + t1). The T1 was set for T1(max) of protons (2.16 s; see Table 2). Polarization transfer delay ∆ equal to (2 × 24 Hz)-1 was used to give a good compromise between polarization-transfer efficiency and minimization of relaxation losses. Delay δ was 1.1 ms, corresponding to total duration of gradient pulse (1 ms) and gradient recovery delay (100 µs). If 3718

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Table 2. Proton and Phosphorus T1 Relaxation Times of Alkylphosphonic Acids in D1 Sample Measured in Inversion-Recovery Experiments T1 (s) nucleus

MPA

EPA

PPA

IPPA

Ha Hb Hc P

1.88

1.59 2.16

n/aa n/aa

3.61

3.90

1.30 1.30 1.73 3.61

a

n/a

Overlapped by stronger signals.

the t1 is assumed zero, then the result for Rernst is 128.5°. Gradient strengths for coherence selection (g1, g2) were 28.0 and 22.7 G/cm for Fast-HMQC. Quadrature detection for the Fast-HMQC in the F1 dimension was accomplished with a gradient-based echo spectrum selection, and the spectrum was processed in magnitude mode. The axial peak displacement was achieved with the StatesTPPI method by inverting the phases φ1 and φR on every second increment of t1. Spectral width for phosphorus dimension was set to correspond to the typical chemical shift range for OP compounds, from -10 to 90 ppm. Spectral width in the proton dimension was from 0 to 10 ppm. Fast-HMQC spectra from D1 and D3 were acquired with 8k × 128 points, and spectra from Proficiency Test samples were acquired with 8k × 200 points. The number of dummy scans was 32, and the number of scans per increment was 16 for the D1 sample and 100 for the D3 sample and Proficiency Test samples. Total acquisition time was 34 min for the D1 sample, 3 h 40 min for the D3 sample, and 5 h 46 min

Figure 2. Expansions from the 500-MHz 1H-31P Fast-HMQC spectra of the D1 sample (A) and D3 sample (B). The F2-skyline projections of Fast-HMQC and 31P{1H} NMR spectra are plotted along the F2 and F1 axes, respectively. The cross-peak of Hb protons of IPPA, which is present in trace amount in sample D3 (0.3 µg/mL), can be detected, but the 31P{1H} NMR spectrum shows no signal. The cross-peak of Hc(PPA) with a small JHP coupling constant (1.28 Hz) is also distinguishable.

for each Proficiency Test samples. Linear prediction was conducted for the indirect dimension, and squared cosine weighting was used for both dimensions. Spectral size was 8k × 512 after Fourier transform. Phosphorus-selective 1D 1H-31P HSQC-TOCSY (Figure 1B) uses shaped 31P-pulses with offset and bandwidth matched to the phosphorus resonance of interest for selective excitation. The 270° Gaussian pulse was found best suited for this task. Proper pulse shape, duration, and power were calculated with Bruker Shape Tool software. INEPT steps were optimized for JHP ) 24 Hz (∆ ) (2 × 24 Hz)-1) to obtain a compromise between polarizationtransfer efficiency and minimization of relaxation losses during the rather long polarization-transfer delay. Delay δ was 1.1 ms, corresponding to the total duration of the gradient pulse (1 ms) and gradient recovery delay (100 µs). A TOCSY step (MLEV17)16-18 with 2.5-ms spin-lock pulses at the beginning and end and duration of 64 ms (∼(2 × 3JHH)-1) was employed in order also to detect those protons of the selected OP compound that have no JHP coupling. GARP decoupling19 was applied on phosphorus (16) (17) (18) (19)

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. Bax, A.; Davis, D. G. J. Magn. Reson. 1985, 65, 355-360. Shaka, A. J.; Barker, P. B.; Freeman. R. J. Magn. Reson. 1985, 64, 547552.

during the TOCSY step and the zero-quantum filter to prevent line shape distortions due to JHP coupling. The zero-quantum filter20 was exploited to clean the zero-quantum artifacts from the resonance line shapes; adiabatic sweep pulse (smoothed chirp) was used with duration of 20 ms and sweep width of 40 kHz. Pulse shape, duration, and power was calculated with Bruker ShapeTool software, and proper gradient strength was optimized using an experiment described in the supporting material of ref 20. Gradient strengths for coherence selection (g1, g2) were 44.8 and 17.9 G/cm for 1D HSQC-TOCSY. Spoiler gradient strengths (sg1-4) were 39.2, 32.5, 39.2, and 5.6 G/cm, respectively. Acquisition time and relaxation delay were 1.3 and 3.0 s, respectively. An 1D HSQCTOCSY spectrum of the authentic reference sample of 5 was acquired with 32 scans (8 dummy scans) and the corresponding spectrum of the soil sample with 1538 scans (32 dummy scans), giving total acquisition times of 3 min and 1 h 54 min, respectively. The spectra were weighted with exponential multiplication (LB ) 1.0 Hz) prior to Fourier transform. RESULTS AND DISCUSSION The Fast-HMQC spectrum acquired from the D1 sample (Figure 2) shows almost all the cross-peaks of alkyl protons from (20) Thrippleton, M. J.; Keeler, J. Angew. Chem., Int. Ed. 2003, 42, 3938-3941.

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Figure 3. 500-MHz 1H-31P Fast-HMQC spectra of organic (A), water (B), and soil (C) samples. The F2-skyline projection and 31P{1H} NMR spectrum are plotted along the F2 and F1 axes, respectively. The phosphate background is visible in the δP range from -5 to 2 ppm. The F2 traces along the cross-peaks corresponding to the spiked 1 (δP 31.13 ppm) and 2 (δP 7.37 ppm) are shown below the spectrum of the organic sample (A). The F2 traces along the cross-peaks corresponding to the spiked 3 (δP 10.64 ppm) and 4 (δP 34.08 and 33.67 ppm for two diastereoisomers) are shown below the spectrum of the water sample (B). The F2 traces along the cross-peaks corresponding to the spiked 3 (δP 8.71 ppm), 4 (δP 29.96 and 29.07 ppm for two diastereoisomers), and 5 (δP 79.77 ppm) are shown below the spectrum of the soil sample (C).

alkylphosphonic acids. Although the Ha cross-peak of IPPA is missing because of its low intensity, caused by the strong JHH splitting, the Hb cross-peak of IPPA is clearly visible, confirming the presence of IPPA in the sample. The same cross-peaks can be seen in the spectrum of the D3 sample, where concentrations are 100-fold lower, within total acquisition time of 4 hours. The 31P{1H} NMR spectrum of the D3 sample after almost 45% longer acquisition showed no evidence of IPPA. A weak correlation for the Hc protons of PPA was observable even though JHP constant is very small (1.28 Hz) compared to the coupling constant used for optimization of the polarization-transfer delay (24 Hz). Although some OP chemicals with level below 1 µg/mL can be detected without difficulty by Fast-HMQC, others may appear with low intensity due to the variation of JHP’s and homonuclear splitting. Detection will then require exceedingly long acquisition. The practical detection limit of the method within reasonable acquisition time is 1-10 µg/mL. Fast-HMQC was tested during the official 18th Proficiency Test. Figure 3A shows the spectrum of the organic sample measured in 5 h 46 min. A first look on the spectrum shows phosphate background in the δP range from -5 to 2 ppm.7 These 3720 Analytical Chemistry, Vol. 78, No. 11, June 1, 2006

chemicals are not relevant to the CWC and can be disregarded from analysis, as they appear in a different region from the scheduled chemicals. Examination of the proton and phosphorus chemical shifts and multiplicity of the cross-peak of Ha(1) provides a clear indication of the presence of methylphosphonate (Chart 3) in the sample. The cross-peak of Hb(1) shows that the methylphosphonate has a methyl ester side chain. For the ethyl ester side chain, only a small cross-peak from Hc(1) can be seen, while Hd(1) cannot be detected owing to the absence of JHP coupling between P(1) and Hd(1). The interesting part of the FastHMQC spectrum is the evidence for Ha of 2. While 1JHP of Ha(2) is 685 Hz, exceeding the optimization of the polarization-transfer delay, sufficient polarization is still transferred during the experiment for a clear correlation. This correlation is also a clear demonstration of the presence of phosphite (Chart 3) in the sample. The cross-peak for Hb(2) can be seen as well, indicating the presence of ester side chain(s) with alkyl chains longer than one carbon. Again the absence of JHP coupling prevents the observation of Hc(2) correlation. The Fast-HMQC spectrum of the water sample revealed two scheduled chemicals (Figure 3B). The cross-peaks at δP 10.64 ppm

Figure 4. 500-MHz 1H NMR spectrum of the authentic reference sample (200 µg/mL of S,S-diethyl methylphosphonodithiolothionate in 5:1 acetone/acetone-d6) corresponding to 5 (A), phosphorus-selective 1D 1H-31P HSQC-TOCSY spectrum of the authentic reference sample corresponding to 5 (B), 1H NMR spectrum of the soil sample (C), and phosphorus-selective 1D 1H-31P HSQC-TOCSY spectrum of the soil sample (D).

suggest the presence of phosphoramidates (Chart 3). A closer look at the cross-peak shape of Ha(3) shows a characteristic pattern for the methine protons of N,N-diisopropyl phosphoramidates, and the appearance of weak resonance at δH 1.1 ppm (Hb(3)) confirms the assignment. The resonance of Hc(3) again indicates the presence of alkyl ester side chain(s) in the structure. Hd(3) is not seen. Compound 4 (dipinacolyl methylphosphonate) is interesting for its diastereoisomerism. The pinacolyl ester side chains have a chiral center, which means that the chemical has three diastereoisomers. Two of them were more prominent in the water sample, giving rise to two sets of cross-peaks at δP 34.08 and 33.67 ppm. Ha(4) produces two doublets of different diaster-

eomers at δH 1.4 ppm, and the resonances of Hb(4) are also visible at δH 4.0 ppm. Three relevant chemicals were found in the Fast-HMQC spectrum of the soil sample (Figure 3C). Two of them, 3 and 4, were also spiked in the water sample. The δP of the third compound, 5, is in the typical range of phosphonothionates (79.77 ppm). However, the chemical shift of one of the proton resonances of 5 is unorthodox. The chemical shift of Ha of this sulfur analogue of diethyl methylphosphonate is very high and easily suggests assignment as a resonance of methylthioester side chain, instead of a methyl directly bonded to phosphorus. The chemical shift of Hb(5), on the other hand, is very typical for methylene protons Analytical Chemistry, Vol. 78, No. 11, June 1, 2006

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of ethylthioester side chain(s). Hc(5) is not shown due to the absence of JHP coupling. The parameters applied for calculation of Rernst were tested with the theoretical sensitivity equations given in ref 14 and were found to be good all-around values for optimizing the sensitivity for the detection of OP chemicals with proton T1 values within the range 1-3 s. This range covers well the T1 values of alkyl moiety bonded to phosphorus of OP compounds in water and some organic solvents (e.g., dichloromethane). The parameters need to be recalculated, if the T1 range of analytes differs from the above due to solvent or other effects. Theoretical sensitivity gain with the Ernst angle excitation for the T1 range 1-3 s with the above parameters was 8-11% higher compared to a normal 90° excitation, which means 15-19% saving in total acquisition time. The saving is significant when the low concentrations of analytes require very long experiment times. Complete assignment of protons of the ester side chains could not be made because the proton-phosphorus coupling normally extends only to protons of the first carbon beyond the heteronucleus (oxygen, sulfur, or nitrogen). The polarization can be transferred between the protons of the ester side chain under isotropic mixing conditions using a TOCSY transfer step, though the transfer causes some loss in sensitivity. The efficiency of background signal suppression with pulsed-field gradient coherence selection is also jeopardized. If complete proton assignment is necessary, the application of TOCSY transfer can help if the following conditions are met: (i) the sample should not contain intense background signals (like solvent peaks) that overlap the resonances of interest, and (ii) the observed chemical should not have a complex JHH coupling network. With these conditions in mind, we chose 5 from the soil sample for the test. The magnetization of interest must be chosen before the TOCSY transfer step. As we intended to detect a selective proton spectrum of a single OP compound, 1D 1H-31P HSQC was a good starting element for this experiment (Figure 1B). The selection of the magnetization of one phosphorus compound is conducted with selective pulses set to the frequency of the phosphorus resonance. The actual chemical shift of the phosphorus of interest can be obtained from a 31P{1H} NMR or a Fast-HMQC spectrum. The bandwidth of the selective pulses must be set to correspond to the total width of the phosphorus resonance including the coupling splitting; normally a bandwidth of 200 Hz suffices. However, a bandwidth larger than 1000 Hz is required if the phosphorus has one-bond coupling to fluorine (as in phosphonofluoridates; see Chart 1, R2 ) F, R3 ) OR) or proton (as in phosphites, Chart 1, R1 ) H, R2, R3 ) OR). In the case of phosphonic difluorides (Chart 1, R2, R3 ) F) the required bandwidth is larger than 2000 Hz. There could be a selectivity problem if additional phosphorus resonances appear close to this region of the phosphorus spectrum. The isotropic mixing time should be set as small as possible, since the relaxation during the mixing decreases the sensitivity. In the case of 5, a duration corresponding to (2 × 3JHH)-1 gave sufficient polarization transfer. The isotropic mixing also produces zero-quantum coherence, which distorts the line shape. Thrippleton and Keller20 have demonstrated that a simultaneous adiabatic inversion pulse and

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Analytical Chemistry, Vol. 78, No. 11, June 1, 2006

pulsed-field gradient offer an effective method for zero-quantum coherence filtering. We applied this approach in our experiment to minimize distortions in the spectrum. Phosphorus-selective 1D 1H-31P HSQC-TOCSY was tested with the authentic reference sample containing 200 µg/mL (1 mM) of 5 (Figure 4A). The phosphorus-selective 1D 1H-31P HSQC-TOCSY spectrum of the authentic sample (Figure 4B) shows all the proton resonances of the analyte. Due to incomplete suppression of the solvent resonance, a small artifact appears at 2.1 ppm. The intensity ratios of lines in multiplets are slightly biased, but the multiplicity and coupling magnitudes are easily extracted. The integrals of resonances reflect the amount of polarization that has been transferred, not the number of protons, which must be taken into account in the interpretation of the spectrum. A normal 1H NMR spectrum of the soil sample is shown in Figure 4C, revealing high background of the interfering impurities (aromatics, sulfides, amines, diesel, and solvent signals). The phosphorus-selective 1D 1H-31P HSQC-TOCSY spectrum of the soil sample (Figure 4D), acquired under 2 hours, is a clean spectrum showing only the proton resonances of 5. CONCLUSIONS We have demonstrated that 2D 1H-31P Fast-HMQC is an excellent method for screening of organophosphorus compounds related to the CWC. The method accurately revealed organophosphorus compounds in very dilute samples within the same time as the traditional 31P{1H} NMR experiment. The analysis can be conducted from a sample with spiking level as low as 1-10 µg/ mL, which means that the aqueous and organic samples of the OPCW Proficiency Test can be analyzed as such without the need of concentration, solvent exchange, or other sample preparation. The Fast-HMQC spectrum also gave structural information useful for identification of the chemicals. Phosphorus-selective 1D 1H-31P HSQC-TOCSY demonstrated its potential for the identification of OP compounds. The experiment gives a selective proton spectrum of a single OP compound in the sample, which can be used for unambiguous identification of that compound. The methods presented here represent a valuable addition to the methods presently available for the verification of chemical warfare agents in environmental samples. They should also be useful for the detection of pesticides and naturally occurring organophosphorus compounds in environmental samples. ACKNOWLEDGMENT The Internship Programme of the International Cooperation and Assistance Division, OPCW (Project L/ICA/ICB/100597/05) has sponsored this work. The authors thank Mr. Matti Kuula and Mrs. Terhi Taure-Gesterberg for their assistance in the sample preparation and the NMR experiments during the 18th Proficiency Test and Mr. Olli Kostiainen for his assistance in the GC/MS quantization. Received for review December 6, 2005. Accepted February 20, 2006. AC052148C