Determination of Trace Amounts of Chemical Warfare Agent

Nov 1, 2007 - of fluorine by hydroxyl group,5 and O-ethyl S-2-(diisopropylamino)- ... www.opcw.org/docs/cwc_eng.pdf (the depositary of this Convention...
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Anal. Chem. 2007, 79, 9098-9106

Determination of Trace Amounts of Chemical Warfare Agent Degradation Products in Decontamination Solutions with NMR Spectroscopy Harri Koskela,* Marja-Leena Rapinoja, Marja-Leena Kuitunen, and Paula Vanninen

VERIFIN, University of Helsinki, P.O. Box 55, FIN-00014 Helsinki, Finland

Chemical warfare agents (CWAs) were first used on a large scale in World War I. Because of the devastating effects of this form of warfare considerable resources in military research have been assigned to develop suitable protection against CWA attack. The development has been concentrated on the physical protection of a body and respiration, medical protection involving pretreatment and therapy, and detection for alarming, monitoring, identification, and verification of CWA1 but also on the efficient decontamination of contaminated skin, equipment, vehicles, land areas, and other surfaces.2,3 The Chemical Weapons Convention (CWC),4 which came into force in 1997, has emphasized the importance of destruction of existing chemical weapons and munitions.

Most of the CWAs can be detoxified through hydrolysis. Phosphonofluoridates such as isopropyl methylphosphonofluoridate (sarin) and pinacolyl methylphosphonofluoridate (soman) are hydrolyzed in aqueous solutions through nucleophilic substitution of fluorine by hydroxyl group,5 and O-ethyl S-2-(diisopropylamino)ethyl methylphosphonothiolate (VX) undergoes autocatalytic hydrolysis.6 In order to increase hydrolysis rate, specific decontamination solutions have been developed. The most used approaches in the optimization of CWA detoxification are the pH control of the solution with strong alkali and the addition of hydrolyzing or oxidizing reagents.7-9 Four decontamination solutions, sodium hypochlorite (NaOCl), sodium hydroxide-polyethylene glycol (PEG), potassium hydroxide-ethanol (KOH/EtOH), and decontamination solution 2 (DS2), were selected for this study (Table 1). These solutions are inexpensive, easy to prepare, and in worldwide use. The NaOCl solution, which represents early bleach-based decontamination solutions,2 is highly corrosive. Its effectiveness is decreased in a cold weather and by loss of an active chlorine content which gradually decreases with time. Although these days more sophisticated solutions for the destruction of nerve agents are available, the strong oxidizing properties of the NaOCl solution10 make it also applicable for the sanitation of biohazard substances. The PEG and KOH/EtOH solutions are examples of aqueous solutions with strong alkali salts and additional organic solvents and are commonly used in designated laboratories of the Organisation for Prohibition of Chemical Weapons (OPCW)11 for the decontamination of analytical samples. These solutions are effective in the decontamination of phosphonofluoridates like sarin and soman, while the degradation rate of VX is slower. DS2 was developed by the U.S. Army in 1960 for an efficient decontamina-

* Corresponding author. E-mail: [email protected]. Fax: +358-9191 50437. (1) A FOA Briefing Book on Chemical Weapons; FOA S-172 90; Swedish Defence Research Agency: Stockholm, Sweden, 1992. (2) Yang, Y.-C.; Baker, J. A.; Ward, J. R. Chem. Rev. 1992, 92, 1729-1743. (3) Talmage, S. S.; Watson, A. P.; Hauschild, V.; Munro, N. B.; King, J. Curr. Org. Chem. 2007, 11, 285-298. (4) Convention on the Prohibition of the Development, Production, Stockpiling and Use of Chemical Weapons and on their Destruction, signed in January 1993. 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). Accessed June 21, 2007.

(5) Larsson, L. Acta Chem. Scand. 1957, 11, 1131-1142. (6) Yang, Y.-C.; Szafraniec, L. L.; Beaudry, W. T.; Rohrbaugh, D. K.; Procell, L. R.; Samuel, J. B. J. Org. Chem. 1996, 61, 8407-8413. (7) Epstein, J.; Callahan, J. J.; Bauer, V. E. Phosphorus 1974, 4, 157-163. (8) Yang, Y.-C.; Szafraniec, L. L.; Beaudry, W. T. J. Org. Chem. 1993, 58, 69646965. (9) Yang, Y.-C.; Szafraniec, L. L.; Beaudry, W. T.; Rohrbaugh, D. K. J. Am. Chem. Soc. 1990, 112, 6621-6627. (10) Epstein, J.; Bauer, V. E.; Saxe, M.; Demek, M. M. J. Am. Chem. Soc. 1956, 78, 4068-4071. (11) The Organisation for Prohibition of Chemical Weapons Headquarters, Johan de Wittlaan 32, 2517 JR, The Hague, The Netherlands. http://www.opcw.org.

Decontamination solutions are used for an efficient detoxification of chemical warfare agents (CWAs). As these solutions can be composed of strong alkaline chemicals with hydrolyzing and oxidizing properties, the analysis of CWA degradation products in trace levels from these solutions imposes a challenge for any analytical technique. Here, we present results of application of nuclear magnetic resonance spectroscopy for analysis of trace amounts of CWA degradation products in several untreated decontamination solutions. Degradation products of the nerve agents sarin, soman, and VX were selectively monitored with substantially reduced interference of background signals by 1D 1H-31P heteronuclear single quantum coherence (HSQC) spectrometry. The detection limit of the chemicals was at the low part-per-million level (210 µg/mL) in all studied solutions. In addition, the concentration of the degradation products was obtained with sufficient confidence with external standards.

9098 Analytical Chemistry, Vol. 79, No. 23, December 1, 2007

10.1021/ac0713196 CCC: $37.00

© 2007 American Chemical Society Published on Web 11/01/2007

Table 1. Decontamination Solutions and Their Constituents decontamination solution

constituent

sodium hypochlorite (NaOCl)

10% w/v (1.4 M) NaOCl in water

sodium hydroxidepolyethylene glycol (PEG)

9.7% w/v (2.4 M) NaOH 21.7% w/v (ca. 1.1 M) PEG 300 in water

potassium hydroxideethanol (KOH/EtOH)

10% w/v (1.8 M) KOH in 50%/50% (v/v) ethanol-water solution

decontamination solution 2 2% w/w (0.5 M) NaOH (DS2) 28% w/w (3.7 M) 2-methoxyethanol 70% w/w (6.8 M) diethylenetriamine

Table 2. Characteristic Degradation Products of Sarin, Soman, and VXa

a Schedule codes are from the CWC (ref 4). The CAS numbers are shown in brackets.

tion solution usable also in cold conditions.12 It is corrosive, and therefore certain precautions must be followed when it is used on rubber, paint, and leather surfaces.2 The primary hydrolysis products of sarin, soman, and VX in aqueous solutions are isopropyl methylphosphonic acid (IPMPA), pinacolyl methylphosphonic acid (PMPA), and ethyl methylphosphonic acid (EMPA), respectively (Table 2). In a slower reaction, the ester side chains are hydrolyzed, producing the secondary degradation product, methylphosphonic acid (MPA). Contrary to the other solutions in this study, DS2 is nonaqueous, and the degradation reactions follow different pathways. The conjugate base of 2-methoxyethanol is the reactive component which rapidly transforms the nerve agents to diesters, which are further decomposed via slower reactions to IPMPA (sarin), PMPA (soman), and EMPA (VX).13 As these compounds are common degradation products both in aqueous and nonaqueous solutions, they are used as marker chemicals in determination of nerve agent use. (12) Jackson, J. B. Development of Decontamination Solution DS2; CWLR, 2368; 1960. (13) 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.

In implementation of the CWC, designated analytical laboratories of the OPCW must be capable of verifying the presence of scheduled chemicals in samples taken by OPCW inspection teams from a site alleged to have nondeclared chemical production. Separation techniques like gas and liquid chromatography combined with mass spectrometry (GC/MS, LC/MS) are powerful techniques in this verification analysis.14-16 However, analysis of trace amounts of CWA degradation products in decontamination solutions sets a challenge due to interfering chemicals in the sample matrix. Extensive sample pretreatment steps including purification and derivatization of the nonvolatile degradation products are needed before GC/MS techniques are applicable.17 LC/MS, although a sensitive method suitable for detection of water-soluble degradation productions,17-19 still requires sample pretreatment in order to minimize the level of interfering ions that cause ion suppression and thereby decreased signal intensities. Nuclear magnetic resonance (NMR) spectroscopy sets only a few limitations for the sample matrix. Therefore, it would be an ideal technique for the direct analysis of the degradation products in untreated decontamination solutions at trace levels. However, traditional NMR methods20,21 are not adequate for this task. The intense signals from the main components of the solution matrix cause problems in dynamic range in 1H NMR analysis and can mask resonances of the chemicals of interest. 31P{1H} NMR offers a selective way to detect relevant organophosphorus compounds, but the low sensitivity of phosphorus hinders its application for the analysis of these compounds at trace level. The common structural feature in the studied degradation products (Table 2) is that they bear a methyl group bonded to phosphorus. This methyl gives a doublet at a characteristic proton chemical shift with a distinct JHP splitting (Table 3) and would offer a way to identify these degradation products, e.g., against library spectra of the OPCW central analytical database.11 Recently, benefits of the proton-phosphorus correlation experiments have been demonstrated in the OPCW proficiency test analyses.22-24 This approach offers a selective and sensitive way to detect proton resonances of organophosphorus compounds, while the background signals can be eliminated. In this study, we have applied the one-dimensional proton-phosphorus heteronuclear single quantum coherence (1D 1H-31P HSQC) experiment for analysis (14) 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; H.1 The Ministry for Foreign Affairs of Finland: Helsinki, 1994. (15) Kientz, Ch. E. J. Chromatogr., A 1998, 814, 1-23. (16) Mesilaakso, M., Ed. Chemical Weapons Convention Chemical Analysis. Sample Collection, Preparation and Analytical Methods; John Wiley & Sons Ltd.: Chichester, U.K., 2005. (17) Black, R. M.; Muir, B. J. Chromatogr., A 2003, 1000, 253-281. (18) Black, R. M.; Read, R. W. J. Chromatogr., A 1997, 759, 79-92. (19) Black, R. M.; Read, R. W. J. Chromatogr., A 1998, 794, 233-244. (20) 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. (21) Mesilaakso, M.; Tolppa, E.-L. Anal. Chem. 1996, 68, 2313-2318. (22) Albaret, C.; Loeillet, D.; Auge´, P.; Fortier, P.-L. Anal. Chem. 1997, 69, 26942700. (23) Meier, U. C. Anal. Chem. 2004, 76, 392-398. (24) Koskela, H.; Grigoriu, N.; Vanninen, P. Anal. Chem. 2006, 78, 3715-3722.

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Table 3. Spectral Parameters for Ha Resonance of the Chemicals in the Solutionsa spectral chemical parameter IPMPA PMPA

KOH/EtOH

DS2

1.30 ( 0.01 1.27 ( 0.01 1.29 ( 0.03 1.10 ( 0.01 16.2 ( 0.1 16.2 ( 0.1 16.1 ( 0.1 16.0 ( 0.2

δ(Ha)

1.29 ( 0.01 1.26 ( 0.01 1.27 ( 0.03 1.10 ( 0.01 16.4 ( 0.3 16.0 ( 0.3 15.8 ( 0.2 16.2 ( 0.2

HP

δ(Ha) 2J

MPA

PEG

δ(Ha) 2J HP 2J

EMPA

NaOCl

HP

δ(Ha) 2J

HP

1.30 ( 0.01 1.27 ( 0.01 1.30 ( 0.03 1.10 ( 0.01 16.3 ( 0.2 16.1 ( 0.1 15.9 ( 0.2 16.2 ( 0.3 1.09 ( 0.02 1.07 ( 0.03 1.13 ( 0.03 1.02 ( 0.01 15.4 ( 0.2 15.4 ( 0.1 15.3 ( 0.2 15.3 ( 0.1

a

The chemical shift and J coupling values were measured from the 1D 1H-31P HSQC spectra of the detection limit test samples.

of untreated decontamination solutions for degradation products of sarin, soman, and VX at low part-per-million levels. EXPERIMENTAL SECTION Sample Preparation. Degradation products IPMPA (purity >99%), PMPA (>95%), and EMPA (>95%) were synthesized by Spiez laboratory, Switzerland, and MPA (98%) was purchased from Fluka, Switzerland. Deuterated acetone (99.5 d%), deuterated methanol (99.8 d%), deuterium oxide (99.9 d%), and 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt, TSP-d4 (98 d-%), were purchased from Aldrich. Polyethylene glycol 300 (practical), 2-methoxyethanol (99.5%), and tetramethylsilane, TMS (>99.8%), were purchased from Fluka, Switzerland. Sodium hydroxide (>99%) and acetone (SupraSolv) were purchased from Merck, Germany. Sodium hypochlorite solution (10% w/v NaOCl) was purchased from Oy FF-Chemicals Ab, Finland. Potassium hydroxide (p.a.) was purchased from J. T. Baker, U.K. Diethylenetriamine (97%) was purchased from Acros, Belgium. Ethanol (technical grade) was purchased from Alko, Finland. Constituents of the decontamination solutions, NaOCl, PEG, KOH/EtOH, and DS2, are presented in Table 1. Commercially available hypochlorite solution (pH ca. 13.1) was used in this study as the NaOCl solution. The PEG solution (pH ca. 13.8) was prepared by mixing 11 parts (by volume) of 30% w/v sodium hydroxide solution, 7 parts of PEG, and 16 parts of water. The KOH/EtOH solution (pH ca. 14.8) was prepared by mixing 1 part (by volume) of 20% w/v potassium hydroxide solution with 1 part of ethanol. The DS2 solution (pH ca. 15.6) was prepared by dissolving 10 g of ground sodium hydroxide in 350 g of diethylene triamine. The solution was sonicated for several hours, and 140 g of 2-methoxyethanol was added when the sodium hydroxide was completely dissolved. NMR samples from the decontamination solutions were prepared by adding 0.8 mL of the studied solution and 0.2 mL of deuterated solvent, D2O, for the aqueous solutions and MeOH-d4 for the DS2 solution, in an NMR tube. 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. For the detection limit studies, NMR samples were prepared containing six different concentration (2, 5, 10, 20, 50, or 100 µg/ mL) of one degradation product in a decontamination solution. The total number of the detection limit samples was 96 after all combinations of four degradation products and four decontamination solutions. Stock solutions of IPMPA, PMPA, EMPA, or MPA 9100 Analytical Chemistry, Vol. 79, No. 23, December 1, 2007

Figure 1. Pulse sequence for 1D 1H-31P HSQC. Narrow and wide bars represent 90° and 180° pulses, respectively. Open rectangles represent gradient pulses. Phase cycles are φ1 ) {x,-x}, φ2 ) {x,x,x,-x}, φR ) {x,-x,-x,x}. Polarization transfer delay ∆ (=1/(2Jopt)) is optimized for Jopt ) 24 Hz. The delay δ equals the sum of the gradient pulse duration (1 ms) and the recovery delay (0.1 ms). Gradient strengths are 44.8 and 17.9 G/cm for g1 and g2, respectively.

in acetone were used for the spiking. As IPMPA, PMPA, and EMPA can further decompose to MPA in decontamination solutions, the experiments were conducted immediately after addition of the chemical in order to minimize the effect of decomposition to the quantification. For quantification tests, a mixture NMR sample containing IPMPA, PMPA, EMPA, and MPA, 100 µg/mL each, in acetoned6 was prepared. In addition, four decontamination solution NMR samples, containing the same mixture of the chemicals in different decontamination solutions, were prepared in the same manner. A set of four samples were prepared for external proton chemical shift calibration by adding ca. 1 mg of TSP-d4 in the aqueous decontamination solution NMR samples (NaOCl, PEG, KOH/EtOH) and ca. 1 µg of TMS in the DS2 solution NMR sample. NMR Experiments. The experiments were carried out using a Bruker DRX 500 NMR spectrometer equipped with a 5 mm inverse z-gradient broad-band probe head at 300 K. The pulse sequence for the 1D 1H-31P HSQC experiment is shown in Figure 1. The sequence itself is a 1D version of pulsed-field-gradient HSQC experiment,25 where acquisition is conducted without X nucleus decoupling. The preserved JHP coupling makes the identification of the resonance easier, e.g., against library spectra, and the sample heating during the decoupling, which can alter the chemical shifts, is avoided. The 90° phosphorus pulse prior to acquisition is used to transform any antiphase proton magnetization with respect to phosphorus to multiple quantum coherence, thus assuring a clean pure-phase line shape in the spectrum. The proton and X nucleus coils were carefully tuned for each sample. Tuning of the proton coil needed, as expected, some effort, but was still feasible. Proton pulse length was calibrated for each sample with 1H NMR using the 360° pulse method. The 90° pulse lengths were about 50-170% longer than normal (Table 4), and the increase of the pulse length reflected the ionic strength of the solution (see Table 1). In all cases, the pulse length was sufficient to excite the whole proton spectrum range with reasonably uniform efficiency. Tuning of the X coil was straightforward due to the lower Q factor. The 90° phosphorus pulse length was optimized by observing proton resonance intensities in the 1D (25) Palmer, A. G., III; Cavanagh, J.; Wright, P. E.; Rance, M. J. Magn. Reson. 1991, 93, 151-170.

Table 4. Measured 90° Proton and Phosphorus Pulse Lengths (µs) in the Studied Solutions acetone

NaOCl

PEG

KOH/EtOH

DS2

p90(1H) 6.40-6.57 16.55-17.75 13.45-15.00 12.70-14.18 9.20-10.05 p90(31P) 13.5 20.0 16.5 15.5 14.0

1H-31P

HSQC spectra. The pulse lengths were about 3-50% longer than normal (Table 4), but the pulse had no significant variation between samples containing the same decontamination solution. Thus, after definition of typical 90° pulse lengths in studied decontamination solutions the pulse length was selected based on the decontamination solution in the sample. According to the tests, a slightly better signal-to-noise ratio for the studied chemicals would have been obtained if the Jopt has been set for values of 18-19 Hz (data not shown), but for the sake of generality of the experiment, the polarization transfer delay was set for Jopt ) 24 Hz in order to accommodate compromise between polarization transfer efficiency for all possible 1-3JHP values encountered among the relevant degradation products with phosphorus and loss of signal intensity due to relaxation and possible JHH coupling evolution during the INEPT steps. 24 The repetition time (acquisition time + relaxation delay) was set to 10 s, which corresponds 5 times the longest T1 of Ha protons of the studied degradation products. The transmitter frequencies for proton and phosphorus were 4.7 and 26 ppm, respectively. The phosphorus transmitter frequency is a typical phosphorus chemical shift for alkyl methylphosphonic acids in aqueous alkaline solutions and for MPA in organic solutions. The largest deviations of phosphorus chemical shift from this value may be expected for MPA in alkaline conditions (ca. 20 ppm) and alkyl methylphosphonic acids in organic solutions (ca. 34 ppm) so that offset effects to the RF pulse performance can be neglected. The spectrum width was set for 12 ppm. 1D 1H-31P HSQC spectra were acquired with 128 scans (8 dummy scans), giving a total acquisition time of 23 min. Processing was conducted using spectrometer operating software (TopSpin 1.3 PL6, Bruker BioSpin, Germany). The line widths of the resonances in the 1D 1H-31P HSQC spectra varied in range of 1-2 Hz. A line broadening (LB) of 1 Hz was used in apodization to give a slight emphasis on the spectral resolution over the signalto-noise ratio. For signal-to-noise calculations, the noise region was set in the signal-free area, typically at 8-10 ppm (noise window 500 Hz), and the calculation was performed with the specific calculation tool in TopSpin. The chemical shift calibration samples and the mixture sample of the chemicals in acetone-d6 were analyzed with 1H NMR. Experiments were carried out using 90° excitation pulse. The number of scans was 32; other relevant acquisition parameters were as with 1D 1H-31P HSQC. Spectra were weighted with an exponential window function (LB ) 0.1 Hz). External chemical shift calibration for all decontamination solutions spectra was obtained by correcting the SF parameter () 0 ppm frequency of the spectrum in MHz) with values taken from the 1H NMR spectra of the external chemical shift calibration samples. RESULTS AND DISCUSSION The aim of the 1D 1H-31P HSQC experiment was to facilitate the analysis of decontamination solutions without the need for extensive sample preparation and within reasonable time (half an

hour). The only sample pretreatment was an addition of a small amount of deuterated solvent to obtain a field lock. The detection limit of the experiment was tested for each degradation product, IPMPA, PMPA, EMPA, and MPA, in each decontamination solution. Figure 2 shows the high background level that is seen in the 1H NMR spectrum of a decontamination solution. The background resonances are eliminated almost completely in the 1D 1H-31P HSQC spectrum, and the Ha resonance of the degradation product (MPA) is clearly visible. On the basis of the analysis of the detection limit test samples (Table 5), detection of these chemicals was feasible at low part-per-million levels in all studied solutions. Signal-to-noise ratio exceeding 3:1 (the limit of detection)26 was attained for all chemicals at a concentration level of 10 µg/mL in all solutions, whereas in some cases the limit of detection was fulfilled even at a level of 2 µg/mL. One point that was observed during the tests was that solubility of MPA was limited to the DS2 solution. MPA is a highly polar compound, which explains the poor solubility to nonaqueous solutions. Therefore, further studies with DS2 were limited to chemicals IPMPA, PMPA, and EMPA. Although the main factors affecting the attainable detection limit are sample matrix related, such as the high ion strength and electrical conductivity of the sample,27 and even the presence of paramagnetic ions, other factors must also be considered. Certain instrumental factors that affect the performance of the spectrometer, like magnetic field strength, are normally not alterable by users. The factors that can and should be optimized by the user are the pulse calibration and shimming. Nonoptimal pulses can cause a significant loss of signal-to-noise ratio; therefore, proper pulse calibration is essential for valid results. Poor shims lead to broad resonances, causing also the loss of signal-to-noise ratio. Good magnetic field homogeneity can be obtained with manual adjustment of shim coil currents, but it can take a considerable amount of time, particularly when studying complicated samples. Gradient shimming offers a quick way to get almost an optimal shim for a sample. Only a small gain, if any, in signal-to-noise ratio was achievable with manual optimization of shims. Therefore, the best option for the shimming of these samples was to use automated gradient shimming for the Zn shims. NMR spectroscopy can deliver accurate quantitative information about the content of the sample.28 While the most accurate quantitative information is obtained using traditional NMR experiments, like 1H NMR, it has been recently demonstrated that multipulse experiments, when certain requirements are fulfilled, can be used for quantification.29-31 Therefore, the reliability of quantitative information derived from 1D 1H-31P HSQC spectra was investigated. For that, the repetition time in the 1D 1H-31P HSQC experiment was beforehand adjusted to g5 T1(Ha) in order to avoid saturation of proton magnetization. The effect of trans(26) Ingle, J. D., Jr.; Cruch, S. R. Spectrochemical Analysis; Prentice Hall Inc.: Englewood Cliffs, NJ, 1988. (27) Kelly, A. E.; Ou, H. D.; Withers, R.; Do¨tsch, V. J. Am. Chem. Soc. 2002, 124, 12013-12019. (28) Maniara, G.; Rajamoorthi, K.; Rajan, S.; Stockton, G. W. Anal. Chem. 1998, 70, 4921-4928. (29) Koskela, H.; Va¨a¨na¨nen, T. Magn. Reson. Chem. 2002, 40, 705-715. (30) Heikkinen, S.; Toikka, M. M.; Karhunen, P. T.; Kilpela¨inen, I. J. Am. Chem. Soc. 2003, 125, 4362-4367. (31) Koskela, H.; Kilpela¨inen, I.; Heikkinen, S. J. Magn. Reson. 2005, 174, 237244.

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Figure 2. 1H NMR spectrum (A) and 1D 1H-31P HSQC spectrum (B) from a sample containing 5 µg/mL MPA in the KOH/EtOH solution. The Ha resonance of MPA is shown in the 1D 1H-31P HSQC spectrum at 1.13 ppm with a signal-to-noise ratio of 13.9:1. Residual water resonance can be seen at 5 ppm.

verse relaxation during the pulse sequence will diminish the intensity of observed proton resonance, which was partially addressed by optimizing the INEPT delays. If the variation of T2’s between the observed protons is small, as expected for the studied compounds, this affect has been regarded as negligible to quantitativity.30 On the other hand, the efficiency of polarization transfer must be taken into account by correcting the integral values with an appropriate coefficient calculated with the product operator analysis.32 In the 1D 1H-31P HSQC experiment the polarization of the protons is transferred to the phosphorus and back via J coupling during the two INEPT steps, and a mismatch between the actual coupling constant and optimization of the INEPT delays reduces the transferred polarization. Corrected resonance integrals (Icorr) that take this loss into account, can be calculated with eq 1:

( ( ))

JHP Icorr ) Iobs sin π 2Jopt

-2

(1)

The term Iobs is the integral of the Ha resonance observed in the spectrum, JHP is the coupling constant between the Ha proton (32) Sørensen, O. W.; Eich, G. W.; Levitt, M. H.; Bodenhausen, G.; Ernst, R. R. Prog. Nucl. Magn. Reson. Spectrosc. 1983, 16, 163-192.

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and the phosphorus, and Jopt is the optimum coupling constant value (24 Hz) used for the INEPT delay calculation. The accuracy of the 1D 1H-31P HSQC quantification was compared with the 1H NMR quantification in determination of the concentration of compounds in a mixture sample. The 1H NMR and 1D 1H-31P HSQC spectra were measured from a sample containing a mixture of IPMPA, PMPA, EMPA, and MPA at a level of 100 µg/mL in acetone-d6. The 1D 1H-31P HSQC spectrum was acquired with the same experimental settings as above, and the 1H NMR spectrum was acquired using a sufficient repetition time for quantitative analysis. Assignments of the Ha resonances were confirmed with the 2D 1H-31P fast-HMQC experiment24 (data not shown). The Ha resonances of the chemicals were partially overlapping, and therefore the spectra were deconvoluted with the PERCH spectral simulation program (version 1/2005, PERCH Solutions Ltd., Kuopio, Finland) to get accurate integrals. The same integral calibration, which gave the value of 100.0 for the total integral for the first 1D 1H-31P HSQC spectrum from the acetone-d6 sample, was used for all the spectra. The integration values from the 1D 1H-31P HSQC spectrum were corrected using eq 1. The 1H NMR integration results were normalized by setting the sum of the integrals to 100% so that an individual integral

Table 5. Signal-to-Noise Ratios of Ha Resonance of the Chemicals in 1D 1H-31P HSQC Experiments signal-to-noise ratio (X:1) chemical IPMPA

PMPA

EMPA

MPA

concn µg/mL (µM)

NaOCl

2 (15) 5 (36) 10 (72) 20 (145) 50 (362) 100 (724)

PEG

KOH/EtOH

DS2

3.8 8.3 16.3 34.3 87.7 169.3

5.9 10.9 23.0 60.4

3.9 5.6 7.3 17.8 40.8 83.2

3.6 9.3 16.8 33.9 79.5 154.4

2 (11) 5 (28) 10 (56) 20 (111) 50 (278) 100 (555)

5.5 8.7 15.8 50.8 85.0

3.5 7.7 20.4 43.0

3.8 5.5 10.6 23.9 58.5

4.4 10.3 22.0 76.0 161.2

2 (16) 5 (40) 10 (81) 20 (161) 50 (403) 100 (806)

6.9 14.3 29.4 64.3 110.7

4.6 8.0 14.9 35.0 82.8

3.0 6.2 11.8 31.7 52.9

3.7 9.3 14.2 30.7 80.4 148.1

2 (21) 5 (52) 10 (104) 20 (208) 50 (521) 100 (1041)

3.9 8.8 20.8 32.2 84.8 127.4

4.3 7.3 15.2 33.6 88.6 171.8

13.9 14.5 25.6 82.6 189.5

4.0 8.4 7.4 9.4

represents the percentage of the molar proportion of the chemical in the sample. The same normalization was done to the corrected 1D 1H-31P HSQC integration results. The equivalence of these quantification results acquired with two different experiments in the same sample is evident, as shown in Table 6, on acetone-d6 results; the difference between the quantification results stays under 1%, and standard deviation for three separate measurements is below 5%. The effect of sample matrix on the quantification and resolution of the Ha resonances in the decontamination solutions was investigated with samples containing the same mixture of the chemicals at a level of 100 µg/mL. The 1D 1H-31P HSQC spectra from these mixture samples were acquired, processed, integrated, and the assignments confirmed with the same manner as above. The Ha resonances were partially overlapping in the aqueous solutions (Figure 3), but the resolution was sufficient for deconvolution of the resonances. However, in DS2 it was not possible to resolve the Ha resonances of IPMPA, PMPA, and EMPA from each other, and the solubility of MPA was limited, so the DS2 sample was excluded from this analysis. The 1H NMR quantification result of the acetone-d6 sample was used as the reference of molar proportions of the chemicals because quantification of the decontamination solutions with 1H NMR was not feasible due to the high level of background. The equivalence between molar proportions of the chemicals from the 1D 1H-31P HSQC quantification in the decontamination solutions compared to the 1H NMR quantification results is shown in Table 6. The deviation was higher, and systematic error can be suspected. One possibility for higher deviation in quantification of the chemicals in the decontamination solutions could be due to the lower signal-tonoise level in these spectra (4 times lower in average). Overlapping of the resonances may also give some uncertainty to the integra-

tion values, but deconvolution is generally regarded as a precise method to evaluate overlapping signals, so the error from this source should be negligible. A small error in the sample preparation could also explain the results, but this was not possible to confirm as quantification of the decontamination solutions with 1H NMR was not feasible. It is also possible that transverse relaxation times of the chemicals have more dispersion in these solutions due to differences in viscosity or contamination by paramagnetic ions, but the observed line widths did not support this assumption. Therefore, the most logical reason that would explain the deviation is partial degradation of the alkyl methylphosphonic acids to MPA. The level of precision and accuracy in the quantification of different compounds in a mixture sample was still acceptable when considering the challenging sample matrices. It is the usual practice in the CWC-related verification analysis to avoid any unnecessary addition of scheduled chemicals in the authentic sample; thus, the use of internal standards conflicts with this practice. In order to circumvent this restraint, the use of external standards in quantification was tested. The principle of reciprocity, which states that the NMR signal strength is inversely proportional to the 90° pulse length,33 has been recently applied in 1H NMR quantification of algal toxins34 and protein concentrations35 with external standards. Confidence of 1D 1H-31P HSQC quantification when applying the same principle was tested with the detection limit test samples. It is considered to be more precise to apply the approach where the chemical to be quantified is the same as in the external standard so that variation of integrals due to spin system dependent parameters, such as J couplings and relaxation, is minimal. The detection limit samples contained only one chemical, and the Ha resonance was integrated with the spectrum integration tool of TopSpin. The value of the integral obtained from the NaOCl sample with 100 µg/mL of MPA was calibrated for a constant value. The rest of the spectra from the detection limit test samples containing the same decontamination solution and degradation product were then integrated using the same calibration setting so that integral values in these spectra were comparable. The integrals were then corrected using eq 1. The unknown chemical concentration CU, which means the chemical concentration in the samples containing 2, 5, 10, 20, and 50 µg/mL, were calculated using eq 2:

CU ) CE

MUIUcorrpU90 MEIEcorrpE90

(2)

CU, MU, and IUcorr are the concentration level (µg/mL), molar mass, and the corrected Ha integral of the chemical in the unknown sample U, respectively. The pulse length (pU90) is included in eq 2 as the measure of the probe Q factor dampening,34,35 because the length of the proton pulse is reciprocally proportional to the Q factor, which again is proportional to the observed proton signal intensity. The terms CE, ME, pE90, IEcorr are the corresponding values from the external standard sample E containing the known concentration of the chemical, i.e., the sample with 100 µg/mL MPA. The same procedure was conducted (33) Hoult, D. I.; Richards, R. E. J. Magn. Reson. 1976, 24, 71-85. (34) Burton, I. W.; Quilliam, M. A.; Walter, J. A. Anal. Chem. 2005, 77, 31233131. (35) Wider, G.; Dreier, L. J. Am. Chem. Soc. 2006, 128, 2571-2576.

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Table 6. Confidence of 1D 1H-31P HSQC Quantification Results in Analysis of the Degradation Product Mixture Samples (IPMPA, PMPA, EMPA, and MPA, All at the Level of 100 µg/mL) Compared to 1H NMR Quantification of the Chemicals in the Acetone-d6 Samplea acetone

NaOCl

chemical

av integral (SD)

normalized integral

difference (to 1H NMR)

av integral (SD)

normalized integral

difference (to 1H NMR)

IPMPA PMPA EMPA MPA

27.9 (0.4%) 31.7 (2.6%) 27.8 (0.4%) 33.3 (3.6%)

23.1% 26.3% 23.0% 27.6%

-0.1% -0.2% -0.2% 0.5%

8.5 (5.0%) 9.7 (3.7%) 9.9 (6.2%) 14.8 (1.9%)

19.8% 22.6% 23.1% 34.5%

-3.4% -3.9% -0.1% 7.5%

chemical

av integral (SD)

normalized integral

difference (to 1H NMR)

av integral (ST)

normalized integral

difference (to 1H NMR)

IPMPA PMPA EMPA MPA

10.4 (1.9%) 11.4 (2.4%) 12.8 (3.8%) 18.4 (2.6%)

19.6% 21.5% 24.2% 34.6%

-3.6% -4.9% 1.0% 7.6%

12.0 (3.6%) 14.0 (1.3%) 14.6 (2.6%) 19.7 (0.6%)

20.0% 23.2% 24.2% 32.7%

-3.3% -3.3% 0.9% 5.6%

PEG

KOH/EtOH

a Average of corrected integrals with standard deviation (n ) 3), normalized integrals (sum of integrals ) 100%), and the difference of the normalized integrals with respect to the normalized integrals from the 1H NMR quantifications are shown.

Figure 3. 1D 1H-31P HSQC spectra of solutions NaOCl, PEG, KOH/EtOH, and DS2 containing a mixture of chemicals IPMPA, PMPA, EMPA, and MPA at the level of 100 µg/mL. In aqueous solutions (NaOCl, PEG, and KOH/EtOH) the chemicals gave Ha resonance at distinct chemical shifts, and the chemicals could be resolved and identified. In DS2 the Ha resonances of IPMPA, PMPA, and EMPA could not be resolved, and the identification must be concluded with other experiments (ref 24). The solubility of MPA was limited in DS2, which explains the low intensity of the resonance.

on the NaOCl samples containing IPMPA, PMPA, or EMPA. In each case, the sample containing 100 µg/mL the chemical was the reference for integrations and used as the external standard. This quantification procedure was also repeated for the remaining decontamination solutions. 9104 Analytical Chemistry, Vol. 79, No. 23, December 1, 2007

Figure 4 demonstrates that calculated concentrations of the chemical agreed reasonably well with actual concentrations. The linearity of the results was good (Table 7), and no systematic error was observed as in almost all cases the deviation of the calculated concentration from the true concentration is within the

Figure 4. Example of correlation of calculated concentrations with true concentrations of the degradation products using external standard quantification in NaOCl solution. The MPA concentrations were calculated from the integration results using the sample with 100 µg/mL of the degradation product as the external standard and plotted with error bars (SD, see Table 8). Table 7. Linearity of 1D 1H-31P HSQC Quantification of the Chemicals IPMPA, PMPA, EMPA, and MPA at Levels of 2, 5, 10, 20, or 50 µg/mL in the Solutionsa NaOCl chemical

R2

slope

IPMPA PMPA EMPA MPA

0.9984 0.9937 0.9952 0.9997

0.94 0.98 0.93 1.02

chemical

R2

IPMPA PMPA EMPA MPA

0.9950 0.9984 0.9996 0.9911

PEG intercept

R2

slope

intercept

0.21 0.07 0.93 0.12

0.9998 0.9991 0.9937 0.9994

0.82 1.01 0.94 0.97

0.72 -1.37 0.61 0.53

slope

intercept

R2

slope

intercept

0.88 0.95 1.24 0.94

0.63 1.76 -2.75 -0.37

0.9998 0.9894 0.9993

1.02 1.07 1.02

-0.15 -3.43 -0.10

KOH/EtOH

DS2

Table 8. Average of Calculated Concentrations and Standard Deviations in 1D 1H-31P HSQC Quantification of the Chemicals IPMPA, PMPA, EMPA, and MPA at Levels of 2, 5, 10, 20, or 50 µg/mL in the Solutionsa average of calculated concentration and standard deviation chemical level (µg/mL)

NaOCl

2 5 10 20 50

2.3 ( 0.8 5.3 ( 1.3 9.6 ( 1.2 20.0 ( 1.3 48.7 ( 1.9

PEG 1.7b

5.8 ( 0.1 9.3 ( 1.0 19.3 ( 1.9 46.3 ( 3.1

KOH/EtOH 3.0b

5.6 ( 1.8 9.1 ( 1.2 19.3 ( 2.4 50.2 ( 6.4

DS2 2.2 ( 0.4 4.9 ( 0.8 8.9 ( 1.3 18.2 ( 2.9 51.1 ( 0.2

a Standard deviations were calculated using the calculated concentrations of the chemicals with the same true concentration in the same decontamination solution, when more than one result was possible to determine (see Table 5). The samples with 100 µg/mL of the degradation product were used as the external standards. Because of poor solubility, MPA was omitted from the DS2 results. b n ) 1.

a The samples with 100 µg/mL of the degradation product were used as the external standards. Because of poor solubility, MPA was omitted from the DS2 results.

standard deviation (Table 8). One must bear in mind that the signal-to-noise ratio (see Table 5) is in the lower end of the concentration range is in some cases below the limit of quantification (10:1).26 Their accuracy is obviously compromised, and these quantification results should be used with caution. Quantification with external standards was demonstrated on decontamination solutions where it was possible to prepare an external standard sample with the identical constituent. It is possible to apply quantification of degradation products in decontamination solutions where a detailed constituent of the solution is not known, as differences in sample conductivity, the reason for Q factor dampening,34,35 in the studied sample and the prepared external standard can be taken into account in eq 2. However, more accurate results are achievable using the standard addition approach. The presence of relevant degradation products in an unknown decontamination solution is at first confirmed with the 1D 1H-31P HSQC experiment. If the relevant chemical is identified in the solution, the concentration of the chemical can be estimated with the standard addition of the same chemical. It

should be then straightforward to calculate the original chemical concentration by using the sample after standard addition as the external standard. It must be of course realized that the concentration of the chemical in this external standard is the sum of the original concentration and the standard addition. Standard addition can be also used for confirmation of the identification as there should be no additional resonance in the 1D 1H-31P HSQC spectrum if the added chemical was the same as the solution already contained (see Figure 3). While the 1D 1H-31P HSQC approach offers a way to analyze untreated decontamination solutions and at this point supersedes LC/MS and GC/MS techniques, it can be also used as a tool in development of sample pretreatment for these techniques. One application of the method is in recovery estimation of intermediate phases in the sample preparation development for LC/MS and GC/MS analysis. Although these intermediate phase solutions are in most cases not analyzable by LC/MS or GC/MS, information of recoveries in these steps would be crucial for the sample preparation development. These solutions are directly analyzable with the 1D 1H-31P HSQC quantification for valuable recovery information. The experiment can be optimized to better suit the Analytical Chemistry, Vol. 79, No. 23, December 1, 2007

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studied chemical, e.g., by setting the Jopt optimal for the JHP of the chemical. Use of phosphorus decoupling during acquisition would further improve signal-to-noise ratio. Accuracy of these recovery results can be improved with a calibration curve which has been defined by several external standards that cover the expected concentration range. These external standards are blank samples that have gone through the same sample preparation procedure and in which a known amount of the chemical has been added prior to analysis. In this way the intermediate steps of the sample preparation can be optimized for a good final recovery so that LC/MS and GC/MS analysis can reach even lower limits of detection than NMR analysis. The 1D 1H-31P HSQC quantification should be also applicable in kinetic studies. Degradation of nerve agents with the CH3-P bond can be monitored in decontamination solutions, which can be helpful in development of novel solutions. Total acquisition time must be set to meet expected degradation rates. When studying relatively fast degradation reactions, sufficient signal-to-noise ratio is achievable within a short experiment time (with a few scans) if the concentration of nerve agents in the sample is relatively high (e.g., 1 mg/mL). If degradation reactions are slow, a longer experiment time is applicable, and degradation products should be able to be monitored at trace amount levels. It should be noted that although the presented 1D 1H-31P HSQC experiment was optimized for the quantitative analysis, the method can also be used exclusively for screening when quantitativity of the experiment and the reliability of identification is sacrificed for the sake of sensitivity. A 2-fold increase in signalto-noise ratio is achievable when using phosphorus decoupling during acquisition. Further increase is attained when the repetition rate is shortened to obtain the best signal-to-noise ratio within a

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given total acquisition time. Degradation products should be detectable even at high part-per-billion levels, if the total acquisition time is extended for several hours. The magnetic field strength and probe head of the spectrometer are of course the main factors that dictate achievable limit of detection. CONCLUSIONS NMR spectroscopy is a suitable technique in the analysis of untreated decontamination solutions for trace amounts of phosphorus-containing CWA degradation products. Although the high ionic strength of decontamination solutions affects the pulse lengths and sensitivity, it is possible with the 1D 1H-31P HSQC experiment to detect CWA degradation products at low part-permillion levels. As the complete analysis of one decontamination solution sample can be conducted within half an hour, the proposed approach offers an advantage over other commonly used analysis techniques like LC/MS and GC/MS which are dependent on time-consuming sample pretreatments. The method is also suitable for the quantification of the degradation products, thus offering numerous applications for decontamination solution studies. ACKNOWLEDGMENT Funding and supply of chemicals from the Swiss NBC Defense Establishment, Spiez Laboratory, 3700 Spiez, Switzerland is gratefully acknowledged. Mari Granstro¨m is acknowledged for her assistance with the manuscript. Received for review June 21, 2007. Accepted September 19, 2007. AC0713196