On-Flow Pulsed Field Gradient Heteronuclear Correlation

Hyphenation of liquid chromatography with nuclear magnetic resonance ... The Chemical Weapons Convention (CWC)(1) prohibits the development, productio...
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Anal. Chem. 2009, 81, 1262–1269

On-Flow Pulsed Field Gradient Heteronuclear Correlation Spectrometry in Off-Line LC-SPE-NMR Analysis of Chemicals Related to the Chemical Weapons Convention Harri Koskela,*,† Mia Ervasti,‡ Heikki Bjo¨rk,† and Paula Vanninen† VERIFIN, University of Helsinki, P.O. Box 55, FIN-00014 Helsinki, Finland, and Technology and Transport, Helsinki Metropolia University of Applied Sciences, P.O. Box 4000, FIN-00079 Helsinki, Finland Hyphenation of liquid chromatography with nuclear magnetic resonance spectroscopy (LC-NMR) is a useful technique in the analysis of complex samples. However, application of on-flow 1H NMR spectrometry during the LC-NMR analysis usually suffers from high intensity of eluent resonances. The poor dynamic range can be improved either with use of deuterated eluents or with various signal suppression schemes. Deuterated eluents are expensive, and peak-selective signal suppression schemes are often unsatisfactory when detection of chemicals at low concentration is needed. If the analytes have a common heteronucleus, on-flow pulsed field gradient heteronuclear correlation spectrometry can offer several benefits. The analytes can be monitored selectively, while the intense nondeuterated eluent and impurity background can be effectively eliminated. In our study, on-flow one-dimensional (1D) 1H-31P heteronuclear single quantum coherence (HSQC) spectrometry was utilized in the analysis of characteristic organophosphorus degradation products of nerve agents sarin and soman during chromatographic separation. These chemicals were not detectable by UV, so their retention times were monitored using on-flow 1D 1H-31P HSQC. This enabled application of LC-NMR combined with solidphase extraction (LC-SPE-NMR) in analysis of these organophosphorus chemicals in an alkaline decontamination solution. The analytes were extracted from the SPE cartridges with deuterated eluent, and the off-line NMR analysis was performed using a mass-sensitive microcoil probe head. The used on-flow 1D 1H-31P HSQC approach offered a high dynamic range and good detection limit (ca. 10 µg/55 nmol) with a high sampling frequency (1 point per 2 s) in the acquired pseudo-twodimensional spectrum. No significant impurity background was present in the off-line NMR samples, and identification of the extracted analytes was straightforward. The Chemical Weapons Convention (CWC)1 prohibits the development, production, stockpiling, and use of chemical weap* To whom correspondence should be addressed. E-mail: Harri.T.Koskela@ helsinki.fi. Fax: +358-9-191 50437. † University of Helsinki. ‡ Helsinki Metropolia University of Applied Sciences.

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ons. The Technical Secretariat of the Organisation for Prohibition of Chemical Weapons (OPCW)2 is the governing body that implements the CWC internationally. Designated laboratories, nominated by the Director General of the OPCW, must be capable of verifying the presence of scheduled chemicals in samples taken during OPCW inspections. Verification of a scheduled chemical in the sample requires confirmation with at least two analytical, preferably spectrometric, techniques. NMR spectroscopy as one of the most important structural elucidation methods has demonstrated its usefulness in analysis of organophosphorus compounds, a major chemical group listed in the CWC.3-5 However, unambiguous NMR identification of the relevant chemical directly from the complex samples can be demanding due to the overlapping background resonances. Therefore, separation techniques like gas and liquid chromatography combined with mass spectrometry (GC/MS, LC/MS) are more common in the verification analysis.6-8 HyphenationofliquidchromatographywithNMR(LC-NMR)9-11 has intrigued analytical chemists due to its potential in analysis of complex samples. In LC-NMR the sample is injected to a highperformance liquid chromatography (HPLC) system, and the (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. (3) Albaret, C.; Loeillet, D.; Auge´, P.; Fortier, P.-L. Anal. Chem. 1997, 69, 2694– 2700. (4) Meier, U. C. Anal. Chem. 2004, 76, 392–398. (5) Koskela, H.; Grigoriu, N.; Vanninen, P. Anal. Chem. 2006, 78, 3715–3722. (6) 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, Finland, 1994. (7) Kientz, Ch. E. J. Chromatogr., A 1998, 814, 1–23. (8) Mesilaakso, M., Ed. Chemical Weapons Convention Chemical Analysis. Sample Collection, Preparation and Analytical Methods; John Wiley & Sons Ltd.: Chichester, U.K., 2005. (9) Watanabe, N.; Niki, E. Proc. Jpn. Acad., Ser. B 1978, 54, 194–199. (10) Albert, K. On-Line LC-NMR and Related Techniques; Wiley: Chichester, U.K., 2002. (11) Keifer, P. A. Annu. Rep. NMR Spectrosc. 2007, 62, 1–47. 10.1021/ac802407t CCC: $40.75  2009 American Chemical Society Published on Web 01/07/2009

postcolumn flow is connected to an NMR flow probe head. The analytes are then analyzed with NMR using on-flow (or continuous-flow), stopped-flow, or loop storage/loop transfer modes.12 Ideally, if the relevant chemical can be isolated from the sample, a simple 1H NMR experiment can give a verification of the chemical structure. The quality of 1H NMR spectra in stoppedflow or loop storage/loop transfer modes can still be compromised due to intense background of eluent or coeluting impurities. One solution to extract the analyte to a matrix that is more suitable for NMR analysis is to combine HPLC separation with postcolumn solid-phase extraction (SPE) and elute the analytes from the SPE cartridge with deuterated eluents (LC-SPE-NMR).13,14 The method has shown its potential in studies of plant extracts,15,16 metabolism,17 in drug research,18 and in food analysis.19 In LC-SPE-NMR analysis the elution of the analytes from the HPLC column is detected with a suitable detector in order to determine the correct peak picking times for SPE enrichment. As most of the organic analytes contain hydrogens, the on-flow 1 H NMR is useful in spotting the presence of chemicals of interest, and it gives also an overview of the compounds present in the sample. If the amount of the target chemical is low and nondeuterated HPLC eluents are used, dynamic range in the on-flow 1H NMR spectrometry can be insufficient even with sophisticated signal suppression schemes. A solution to this problem is to use deuterated HPLC eluents, but the high cost of the deuterated solvents can make this approach sometimes impractical. A typical LC-NMR system is equipped with a ultraviolet or diode-array detector (UV/DAD) as a complementary detector, as they are useful in LC due to their robustness and good sensitivity.20 However, if the analytes lack a chromophore, UV detection is not useful; indirect detection21 may give some help in this respect. There is also a selection of other techniques like mass spectrometry (MS),22 infrared (IR),23 refractive index (RI),24 and evaporative light scattering (ELS),25 which are also usable as detectors, but they are not usually a part of typical LC-NMR systems. When the proton background is severe, it is often advantageous in the on-flow NMR spectrometry to exploit other nuclei than (12) Braumann, U.; Spraul, M. In On-Line LC-NMR and Related Techniques; Albert, K., Ed.; Wiley: Chichester, U.K., 2002; pp 23-43. (13) Griffiths, L.; Horton, R. Magn. Reson. Chem. 1998, 36, 104–109. (14) Nyberg, N. T.; Baumann, H.; Kenne, L. Magn. Reson. Chem. 2001, 39, 236–240. (15) Clarkson, C.; Stærk, D.; Hansen, S. H.; Jaroszewski, J. W. Anal. Chem. 2005, 77, 3547–3553. (16) Miliauskas, G.; van Beek, T. A.; de Waard, P.; Venskutonis, R. P.; Sudho¨lter, E. J. R. J. Nat. Prod. 2005, 68, 168–172. (17) Krauser, J. A.; Voehler, M.; Tseng, L.-H.; Schefer, A. B.; Godejohann, M.; Guengerich, F. P. Eur. J. Biochem. 2004, 271, 3962–3969. (18) Sandvoss, M.; Bardsley, B.; Beck, T. L.; Lee-Smith, E.; North, S. E.; Moore, P. J.; Edwards, A. J.; Smith, R. J. Magn. Reson. Chem. 2005, 43, 762–770. (19) Christophoridou, S.; Dais, P.; Tseng, L.-H.; Spraul, M. J. Agric. Food Chem. 2005, 53, 4667–4679. (20) Fielden, P. R. J. Chromatogr. Sci. 1992, 30, 45–52. (21) Crommen, J.; Herne´, P. J. Pharm. Biomed. Anal. 1984, 2, 241–253. (22) Pullen, F. S.; Swanson, A. G.; Newman, M. J.; Richards, D. S. Rapid Commun. Mass Spectrom. 1995, 9, 1003–1006. (23) Lenz, E.; Taylor, S.; Collins, C.; Wilson, I. D.; Louden, D.; Handley, A. J. Pharm. Biomed. Anal. 2002, 27, 191–200. (24) McCrossen, S. D.; Bryant, D. K.; Cook, B. R.; Richards, J. J. J. Pharm. Biomed. Anal. 1998, 17, 455–471. (25) Petritis, K.; Gillaizeau, I.; Elfakir, C.; Dreux, M.; Petit, A.; Bongibault, N.; Luijten, W. J. Sep. Sci. 2002, 25, 593–600.

Table 1. Characteristic Degradation Products of Sarin and Somana

a The CAS numbers are shown in square brackets. Schedule codes are from the CWC (ref 1).

proton that are common to the analytes of interest. There are some good demonstrations of how on-flow 19F NMR can be utilized in order to detect chromatographic separation of metabolites with fluorine.26,27 Use of the other, less sensitive NMR nuclei like carbon-1328,29 and phosphorus30 is, however, less common for direct on-flow detection, but there are examples how sensitivity can be improved with dynamic nuclear polarization.31 Also, heteronuclear decoupling can be used during on-flow 1H NMR in order to identify chemicals with phosphorus,32 but the problem with the intense proton background still prevails in these kinds of decoupling experiments. If the analytes, either naturally or due to isotopic labeling, contain a common NMR-active heteronucleus (e.g., 13C, 15N, or 31 P), on-flow analysis can be accomplished using one-dimensional (1D) inverse detected polarization transfer experiments. The signal suppression schemes like WET33 rely on selective suppression of a limited number of peaks present in the spectrum, whereas coherence selection with pulsed field gradients offers a nonselective method to suppress all background resonances, including solvents as well as nonrelevant chemicals. This means that the LC can be performed with nondeuterated eluents, which reduces cost of the analysis. Transfer of chromatographic methods with other hyphenated LC techniques is also easier. In order to study the feasibility of the on-flow detection approach, it was tested on LC-SPE-NMR analysis of degradation products of the nerve agents sarin and soman, namely, isopropyl methylphosphonic acid (IPMPA), pinacolyl methylphosphonic acid (PMPA), and methylphosphonic acid (MPA) (Table 1). As these (26) Spraul, M.; Hofmann, M.; Wilson, I. D.; Lenz, E.; Nicholson, J. K.; Lindon, J. C. J. Pharm. Biomed. Anal. 1993, 11, 1009–1015. (27) Shockcor, J. P.; Unger, S. E.; Savina, P.; Nicholson, J. K.; Lindon, J. C. J. Chromatogr., B 2000, 748, 269–279. (28) O’Leary, D. J.; Hawkes, S. P.; Wade, C. G. Magn. Reson. Med. 1987, 5, 572–577. (29) Bayer, E.; Albert, K. J. Chromatogr., A 1984, 312, 91–97. (30) Chapman, B. E.; Kuchel, P. W.; Lovric, V. A.; Raftos, J. E.; Stewart, I. M. Br. J. Haematol. 1985, 61, 385–392. (31) Stevenson, S.; Dorn, H. C. Anal. Chem. 1994, 66, 2993–2999. (32) Preiss, A.; Godejohann, M. In On-Line LC-NMR and Related Techniques; Albert, K., Ed.; Wiley: Chichester, U.K., 2002; pp 142-178. (33) Smallcombe, S. H.; Patt, S. L.; Keifer, P. A. J. Magn. Reson., Ser. A 1995, 117, 295–303.

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organophosphorus chemicals are only weak chromophores, their HPLC separation cannot be monitored with a UV detector. The common structural feature of the studied degradation products (Table 1) is that they contain a methyl group bonded to phosphorus. This methyl gives a doublet at characteristic proton chemical shift with a distinct JHP splitting. As demonstrated earlier, one-dimensional proton-phosphorus heteronuclear single quantum coherence (1D 1H-31P HSQC)34 is an excellent tool to screen untreated decontamination solutions for the presence of organophosphorus compounds related to the CWC. Therefore, the detection of nerve agent degradation products during chromatographic separation was tested using on-flow 1D 1H-31P HSQC. The approach should offer a selective and sensitive way to detect organophosphorus compounds and, therefore, enables determination of their retention times. After the retention times were determined, the fractions containing target chemicals from the next injection(s) were collected in separate loops for temporary storage. The fractions were then transferred from the loops to SPE cartridges, where the target chemicals were trapped. Fractions containing the same analyte taken from several sample injections were transferred to the same SPE cartridge, and in this way the chemical was enriched. The final step was to elute the chemical from the SPE cartridge with a small amount of deuterated solvent to a capillary NMR tube. After that the off-line structural verification of the target chemical was accomplished with the NMR spectrometer equipped with a mass-sensitive microcoil probe head.35 The proposed LC-SPE-NMR approach was finally tested on analysis of the aforementioned nerve agent degradation products in a decontamination solution. EXPERIMENTAL SECTION Materials. Degradation products IPMPA (purity >99%) and PMPA (>95%) were synthesized by Spiez laboratory, Switzerland, and MPA (98%) was purchased from Fluka. Deuterated methanol (99.8 D%) and deuterium oxide (99.9 D%) were purchased from Aldrich. Acetonitrile (HPLC grade S) was purchased from Rathburn. Formic acid (Suprapur), ammonium acetate (>98%), and methyl violet (for microscopy) were purchased from Merck. Potassium hydroxide (p.a.) and ammonium hydroxide (25% solution) were purchased from J. T. Baker. Ethanol (technical grade) was purchased from Alko, Finland. Water was purified with Elgastat UHQ system (Elga, U.K.). Sample Preparation. On-flow detection limit was tested with samples containing a mixture of MPA, IPMPA, and PMPA in HPLC eluent. The detection limit was found with a sample containing each of the chemicals at level 10 µg in 20 µL injections () 0.5 µg/µL); the corresponding amounts of substance in one injection were 81, 72, and 55 nmol, respectively. For decontamination solution tests, a solution containing 10% potassium hydroxide in 50%/50% (v/v) water/ethanol mixture was prepared as described previously.34 The solution was spiked with MPA, IPMPA, and PMPA (150 µg/mL each). The pH of the solution was above 14, which was not suitable for the used HPLC column (recommended pH range of 2-8). In order to adjust the (34) Koskela, H.; Rapinoja, M.-L.; Kuitunen, M.-L.; Vanninen, P. Anal. Chem. 2007, 79, 9098–9106. (35) Schlotterbeck, G.; Ross, A.; Hochstrasser, R.; Senn, H.; Ku ¨ hn, T.; Marek, D.; Schett, O. Anal. Chem. 2002, 74, 4464–4471.

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sample pH, one part (1 mL) of the decontamination sample was diluted with nine parts (9 mL) of water. Two strong cationexchange (SCX) cartridges (Varian BondElut SCX, 500 mg) were each conditioned with 5 mL of methanol and 5 mL of water. The diluted decontamination solution was then cation-exchanged using these two SCX cartridges in series. The resulting solution had pH 2.2. To enrich the sample to the level needed for LC-SPE-NMR, the solution was evaporated down to 100 µL with TurboVap LV evaporator (Caliper LifeSciences). The sample matrix was made compatible with the HPLC eluent by adding corresponding amounts of formic acid and acetonitrile (see the HPLC Conditions section). In order to quantify the amounts of the analytes in the resulting off-line NMR samples, external references in quantification were prepared from solutions containing 0.5 µg/µL MPA, IPMPA, or PMPA in HPLC eluent. Amounts of 20 µL of each solution and 10 µL of D2O were transferred to 1.7 mm NMR tubes (Bruker BioSpin). Analyte recoveries in the concentrated decontamination solution sample were determined by taking 20 µL of the sample and adding 10 µL of D2O. The solution was then transferred to a 1.7 mm NMR tube. The analytes were quantified against an external reference with the quantitative 1D 1H-31P HSQC method described elsewhere.34 The level of the analytes in the concentrated decontamination solution sample was ca. 1 µg/ µL. LC-NMR Instrumentation. The NMR experiments were carried out at 11.75 T using a Bruker DRX 500 NMR spectrometer (Bruker BioSpin) equipped with a Bruker LC equipment, which consists of an LC-22 HPLC pump with an LC225 gradient former, a BSFU-O/BPSU-36 stop-flow and peak sampling unit, and a Bischoff Lambda 1010 UV detector. The injector loop volume was 20 µL. A Waters Atlantis T3 polar-embedded reversed-phase HPLC column (4.6 mm × 150 mm, sorbent particle size 5 µm) was chosen for this study. The NMR spectrometer was equipped with a 4 mm inverse z-gradient triple-resonance (1H, 13C, 31P) flow probe head (flow cell volume 250 µL, active volume 120 µL) for on-flow NMR analysis. Typical 90° high-power pulse lengths for 1H, 13 C, and 31P were 6.8, 13.0, and 20.0 µs, respectively. Experiments were carried out at 290 K. The NMR spectrometer was controlled with TopSpin 1.3, and the LC equipment was controlled with HyStar 1.2. On-Flow LC-NMR. The pulse sequence for the on-flow 1D 1 H-31P HSQC experiment was the same as used previously.34 The transmitter frequencies for proton and phosphorus were 4.7 and 30 ppm, respectively. The spectrum width was set for 12 ppm. The experiment was optimized according to the average 2JHaP coupling of MPA, IPMPA, and PMPA (see Table 1) by setting the polarization transfer delay ∆ to 1/[(2)(16.8 Hz)]. Acquisition time was 0.34 s, and repetition time of the experiment was set so that one complete scan (including the relaxation delay, pulse and evolution times, and acquisition time) was performed in 1 s. For the on-flow analysis the 1D spectra were recorded in a two-dimensional (2D) matrix so that the 1D spectra can be plotted with respect to time in the pseudo-2D spectrum. The number of scans was two per spectrum so that the sampling frequency of the pseudo-2D spectrum in the retention time dimension was one point per 2 s. The acquisition and processing

parameters were set so that the time presented on the y-axis of the 2D on-flow spectrum was synchronous with the actual retention time. After the system was stabilized, the flow probe head was tuned, and gradient shimming was performed using proton detection. The on-flow 1D 1H-31P HSQC experiment was carried out unlocked. The acquisition was started simultaneously with the injection. The duration of the LC run was 10 min, so the acquired 2D data matrix size was 4K × 300 points. The 2D on-flow spectrum was processed in phase-sensitive mode in the observed dimension; line broadening (LB) of 0.1 Hz was used prior to Fourier transform with 4K real points. The on-flow 1H NMR experiment was acquired using the standard Bruker pulse sequence (lc2wetdc). A two-band WET suppression33 was set for the experiment. The base pulse shape for WET suppression was sinc-pulse with single cycle; the excitation band was 200 Hz. Proper pulse length and power was calculated with the TopSpin stdisp tool. The frequencies of the two most intensive eluent peaks (water and acetonitrile) were saved in the frequency list, and the two-band excitation shape pulse for WET was calculated from the sinc-pulse with the TopSpin automation program lcwetcalc. Carbon decoupling with GARP36 was applied during WET suppression and acquisition. The transmitter frequencies for proton and carbon were 4.7 and 50 ppm, respectively. The spectrum width was set for 12 ppm. Acquisition time was 0.34 s, and repetition time of the experiment was modified so that one complete scan (including the relaxation delay, pulse and evolution times, and acquisition time) was performed in 1 s. Otherwise the acquisition and processing parameters were as with on-flow 1D 1H-31P HSQC. For the loop storage of the analytes from the next injection (see Figure 1) the peak sampling unit (BPSU-36) that contained 36 loops with volume of 250 µL was programmed within HyStar 1.2 according to the observed NMR retention times. The Bruker LC unit was equipped with a UV detector located after the column, and the peak sampling unit used UV retention times for peak picking. The UV retention times were calculated by subtracting the time difference the eluent took to flow from the UV detector to the NMR flow probe head in 1 mL/min flow rate (26 s). The storage loops were washed prior to the injection with the HPLC eluent. SPE Interface. A hardware interface was constructed for the SPE enrichment of the analytes (see Figure 1). The PEEK tubing that was used to connect the Bruker LC unit to the flow probe head was connected to a Rheodyne valve (LabSource H.S.Valve 7000E). With the use of a T-junction, an auxiliary HPLC pump (Merck Hitachi L-6200A Intelligent Pump) was also connected to the same valve inlet. The usefulness of aminopropyl (NH2) sorbent in enrichment of phosphonic acids has been demonstrated earlier;37 therefore, a Varian BondElut NH2 Prospekt (2 mm × 10 mm) SPE cartridge was chosen for this study. The SPE cartridge was secured to a cartridge clamp (Spark Holland, part no. 0795.306), and the clamp was connected to another valve inlet using standard 1/16 in. HPLC PEEK tubing (i.d. 0.25 mm) and fittings. Proper tubing lines were also made to a waste container. The auxiliary HPLC pump was used to deliver suitable conditioning solvents to the SPE cartridge. The fraction (36) Shaka, A. J.; Barker, P. B.; Freeman, R. J. Magn. Reson. 1985, 64, 547– 552. (37) Tørnes, J. Å.; Johnsen, B. A. J. Chromatogr., A 1989, 467, 129–138.

Figure1.SchematicsofthehardwaresetupfortheusedLC-SPE-NMR approach. Step one, retention time determination: The retention times of relevant chemicals were determined from the first sample injection with on-flow 1D 1H-31P HSQC spectrometry. Step two, peak picking: The peak sampling unit was programmed to store the fractions to loops at correct retention times obtained from the previous run. Fractions from the next injection(s) were stored in separate loops. Step three, analyte enrichment: The fractions were transferred from storage loops to an SPE cartridge. The SPE cartridge is secured to a cartridge clamp, and the direction of the eluent flow is controlled with a Rheodyne valve. The auxiliary HPLC pump is used to deliver the SPE conditioning eluents prior to the fraction transfer. The valve is set to waste in the beginning of the fraction transfer from the peak sampling unit so that the tubing can be washed prior to the fraction transfer. The flow is manually diverted to the SPE when the fraction frontier reaches the valve. After the analyte is enriched in the SPE cartridge, the analyte is eluted with a suitable deuterated eluent, and the recovered solution is then used for the NMR sample. The NMR sample is analyzed off-line using a microcoil probe head.

transfer from the loop was performed with the HPLC pump of the Bruker LC unit and controlled within HyStar 1.2. The fraction transfer time from the loop to the SPE cartridge was optimized with 8 µg/µL methyl violet solution. With 1 mL/ min flow rate it took 11 s before the fraction frontier reached the SPE cartridge; the end of the fraction reached the SPE cartridge in 28 s. Later on, the flow was manually controlled during the fraction transfer with the Rheodyne valve so that in the beginning of the transfer the valve diverted the flow to the waste and only during the last 17 s the flow was diverted to the SPE cartridge. This configuration facilitated enrichment of multiple fractions containing the same analyte into the same SPE cartridge. The flow path from the peak sampling unit to the Rheodyne valve was washed with water for 60 s with flow Analytical Chemistry, Vol. 81, No. 3, February 1, 2009

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rate 1 mL/min between fraction transfers in order to prevent cross contamination. After the analyte was enriched in the SPE cartridge, the clamp with the cartridge was detached from the SPE interface. A N2 gas tubing was secured to the clamp, and N2 pressure (ca. 1.5 bar) was turned on for 2 min in order to purge the excess solvent. The analyte was back-eluted from the SPE cartridge with 50 µL of deuterated eluent, and the cartridge was purged with N2 gas. The collected eluent was transferred to a 1.7 mm NMR tube for the off-line NMR analysis. Off-Line NMR. The off-line NMR analyses were carried using a 1.7 mm inverse z-gradient triple-resonance (1H, 31P, 13C) microcoil probe head (Bruker BioSpin) at 290 K. The 1H NMR spectra were measured using 90° excitation pulse, and the repetition time (acquisition time plus relaxation delay) was set for 9.6 s in order to satisfy quantitative conditions for the studied organophosphorus compounds. The transmitter frequency for proton was 4.7 ppm, and the spectrum width was set for 12 ppm. If not stated otherwise, presaturation (γ i B2 ) 50 Hz) was applied on water resonance during the relaxation delay (6 s). Eight dummy scans were used prior to acquisition. The number of scans was 32, resulting in total measurement time of 6 min. The 1D 1H-31P HSQC experiment was used for quantitative estimation of amounts of the analytes in the NMR samples against external reference samples. The details of the experimental setup and quantification are described elsewhere.34 31 1 P{ H} NMR spectrum from the concentrated decontamination solution sample was acquired using a 45° excitation pulse angle. The repetition time was set for 1.2 s according to the Ernst angle equation38 based on the average of T1(31P) of the studied organophosphorus compounds. The transmitter frequency for phosphorus was 30 ppm, and the spectrum width was set for 60 ppm. Eight dummy scans and 256 scans were acquired, resulting in total measurement time of 5 min. The 2D 1H-31P fast-HMQC (heteronuclear multiple quantum correlation) spectrum5 from the concentrated decontamination solution sample was measured with eight scans per increment; the number of dummy scans was 64. Spectral widths in the proton and phosphorus dimensions were 10 and 20 ppm, respectively. The transmitter frequency for proton was 4.7 ppm and for phosphorus 30 ppm. The 2D spectrum was acquired with 8K × 200 points. Total measurement time was 29 min. Further experimental details are described elsewhere.5 HPLC Conditions. HPLC runs were performed under isocratic condition at 30 °C using 1 mL/min flow rate. Eluent ratio (v/v) was 30% of acetonitrile and 70% of water with 0.1% formic acid (pH 2.5). SPE Conditions. The SPE sorbent was conditioned using an auxiliary HPLC pump with 1 mL of methanol (flow rate 1 mL/ min) and then with 2 mL of 50 mM ammonium acetate buffer (pH 7.0). The fraction transfer from the loops to the SPE cartridge was performed using water as the transfer solvent. The analytes were back-eluted from the SPE cartridges manually with 50 µL of 1% ammonium hydroxide in 50%/50% (v/v) D2O/MeOH-d4 mixture (pH 11.6). (38) Ernst, R. R. Adv. Magn. Reson. 1966, 2, 1–135.

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RESULTS AND DISCUSSION Detection Limit. The detection limit of the proposed on-flow 1D 1H-31P HSQC approach was at first tested using mixture samples containing MPA, IPMPA, and PMPA in the chosen HPLC eluent. One important factor that affects the attainable sensitivity is the repetition time of the experiment. During onflow conditions,39 the observed longitudinal relaxation is 1 1 1 ) + T1flow T1 τ

(1)

where T1flow is the observed longitudinal relaxation time in onflow conditions, T1 is the relaxation time in static conditions, and τ is the residence time, that is, how long the sample stays in the flow cell during the on-flow mode. The repetition time needs to be considered from the point of Ha protons of the studied chemicals (see Table 1), as they will be detected by the on-flow 1D 1H-31P HSQC experiment. The relaxation times can vary in some degree depending on the sample matrix, but, for example, Ha protons of MPA can have a T1 time of 1.44 s.43 The detection volume of the used flow probe head is 120 µL, so the residence time τ is 7.2 s, when flow rate of 1 mL/min is used.39 Therefore, the optimal repetition time would be 1.52 s [) (1.269)(T1flow)].40 One point in the proposed approach was to satisfy a decent sampling frequency in the chromatographic dimension. For that reason a part of the optimal sensitivity was sacrificed, and the repetition time was set for 0.81 s. In this way, the recycle time of one scan was exactly 1 s, which was also convenient considering the presentation of the on-flow 1D 1 H-31P HSQC data with respect to time in the pseudo-2D spectrum. Two-scan phase cycle was used to suppress spectral artifacts in sufficient degree. The resulting sampling frequency was one point per 2 s. With the use of this acquisition setup the analytes were detected clearly at level of 10 µg in a 20 µL injection (Figure 2B). It is common that a large number of scans (e.g., 8-24) are used per transient in on-flow 1H NMR analysis, resulting in rather poor sampling frequency.12 The peak sampling unit contained 250 µL storage loops, which means that the time to fill one loop is 15 s with 1 mL/min flow rate. If the chromatographic sampling frequency is close to or worse than one point per 15 s, it means that one may face difficulties to determine the midpoint of the eluted peak. The high sampling frequency is important in the proposed on-flow 1D 1 H-31P HSQC approach so that the peak picking times can be determined accurately, and the most concentrated part of the fraction is stored in the storage loop. If a lower flow rate than 1 mL/min is used, a larger number of scans can be used without sacrificing the chromatographic resolution, as discussed by Godejohann et al.41 This would also result in a lower detection limit, but the total time of LC would also increase accordingly. One critical point in the used on-flow experiment was how well the pulsed field gradient coherence selection performs with (39) Albert, K. In On-Line LC-NMR and Related Techniques; Albert, K., Ed.; Wiley: Chichester, U.K., 2002; pp 1-22. (40) Ernst, R. R.; Bodenhausen, G.; Wokaun, A. Principles of Nuclear Magnetic Resonance in One and Two Dimensions; Clarendon Press: Oxford, U.K., 1990; Chapter 4.3. (41) Godejohann, M.; Preiss, A.; Mu ¨ gge, C.; Wu ¨ nsch, G. Anal. Chem. 1997, 69, 3832–3837.

Figure 2. Pseudo-2D spectra from on-flow 1H NMR acquisition (A) and on-flow 1D 1H-31P HSQC acquisition (B) of 20 µL injections of the detection limit test sample that contained 10 µg of MPA, IPMPA, and PMPA in the HPLC eluent. Both spectra are plotted close to the noise level. On top of pseudo-2D spectra are the observed 1D spectra at the MPA retention time. The on-flow 1H NMR experiment was carried out using two-band WET suppression on water and acetonitrile peaks. The retention time is shown on the y-axis in seconds. The residual eluent peaks (water peak at 4.7 ppm and acetonitrile peak at 2.2 ppm) dominate the on-flow 1H NMR spectrum; the Ha resonances of MPA, IPMPA, and PMPA at 135, 157, and 289 s, respectively, are barely noticeable among the intense background peaks. The same resonances are clearly shown in the on-flow 1D 1 H-31P HSQC spectrum, and the eluent peaks are almost completely suppressed. The receiver gain settings in the experiments were 256 and 8192 for on-flow 1H NMR and on-flow 1D 1H-31P HSQC, respectively.

flowing sample. Diffusion spectroscopy42 applies pulsed field gradients in various pulse experiments to monitor diffusion transportation of molecules. The observed signal intensity decay in these experiments is proportional to the rate of diffusion. In static samples the collective diffusion is typically slow, but in NMR flow cells the molecule transportation is caused by flowing eluent, making it much faster. It can be reasoned that there are two important effects that this unidimensional fast transportation causes during the pulsed field gradient coherence selection: (i) loss of polarization of the detected analytes and (ii) leakage of unwanted coherences (background signals). If we look at the on(42) Johnson, C. S., Jr. Prog. Nucl. Magn. Reson. Spectrosc. 1999, 34, 203–256. (43) Koskela, H.; Vanninen, P. Anal. Chem. 2008, 80, 5556–5564.

Figure 3. Direct NMR analysis of the concentrated decontamination solution sample containing a mixture of chemicals MPA, IPMPA, and PMPA at a level of ca. 1 µg/µL. The 2D 1H-31P fast-HMQC spectrum and expansions are shown; the 1H NMR and 31P{1H} NMR spectra are plotted as projections. The 2D 1H-31P fast-HMQC spectrum shows cross peaks for the relevant organophosphorus compounds; the cross peak for Hc(IPMPA), Hc(PMPA), and Hd(PMPA) are missing due to absence of JHP couplings. The 1H NMR spectrum demonstrates the intense level of overlapping impurity resonances in the sample, and the peaks of the relevant chemicals are overlapping in both 1H NMR and 31P{1H} NMR spectra, which makes resonance assignment difficult.

flow 1D 1H-31P HSQC spectrum in Figure 2B, it is clear that the leakage of unwanted coherences is negligible. The loss of polarization of the detected analytes was not found to impair the sensitivity of the experiment to a significant degree. For comparison, detection of the analytes was studied using the on-flow 1H NMR experiment. In order to suppress the intense eluent peaks, the experiment was carried out using twoband WET suppression33 on the water and acetonitrile peaks. Other acquisition and processing parameters were as with the on-flow 1D 1H-31P HSQC experiment. As can be seen from Figure 2A, the residual water and acetonitrile peaks dominate the pseudo-2D spectrum. Due to the intense solvent peaks, the receiver gain was not optimal for detection of the analytes (32fold lower receiver gain), and the analyte peaks were barely observable among the intense background peaks. LC-SPE-NMR Tests. The on-flow 1D 1H-31P HSQC detection was tested in LC-SPE-NMR analysis of a test sample that contained each of the analytes (MPA, IPMPA, and PMPA) at the detection limit (0.5 µg/µL). The important factor in this test was to monitor how selectively the analytes can be trapped and what is the recovery. It was found that the low-pressure gradient former of the used HPLC pump was not able to produce stabile eluent ratio, which caused fluctuation in the retention times. Therefore, the HPLC eluent was mixed in one bottle in order to guarantee a high reproducibility of the retention times in consecutive injections. Small differences in the retention times were observed in experiments performed Analytical Chemistry, Vol. 81, No. 3, February 1, 2009

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Figure 4. Spectra from the LC-SPE-NMR analysis of the concentrated decontamination solution sample. The sample contained ca. 1 µg/µL MPA, IPMPA, and PMPA. The pseudo-2D spectrum from the on-flow 1D 1H-31P HSQC analysis, which was used for the determination of the retention times of MPA, IPMPA, and PMPA, is shown at the top. The 1H NMR spectra from the off-line NMR analyses are shown at the bottom. The off-line NMR sample concentrations are shown in Table 3. The residual HPLC eluent/SPE conditioning background is shown in the 1H NMR spectra; the water peak at 4.7 ppm is suppressed with presaturation. The MPA fraction contained most of the impurities that were in the concentrated decontamination sample, and some of the impurities were carried along in the NMR sample (resonances marked with asterisks). The fractions that contained IPMPA and PMPA were quite pure; no significant coeluting impurities from the decontamination solution sample were seen in the 1H NMR spectra.

on different days due to evaporation of the HPLC eluent mixture (cf., Figures 2 and 4). The retention times of the analytes determined from the first injection were used in programming the peak sampling unit. The analytes were collected from the next injections into the storage loops and, after that, enriched to SPE cartridges. The analytes were then manually back-eluted from the SPE cartridges using a deuterated eluent, and the extracted solution was transferred to a 1.7 mm NMR tube. The contents of the NMR samples were analyzed off-line using the microcoil probe head, and the analyte amounts were quantified against external reference samples containing 10 µg of each analyte with the quantitative 1D 1H-31P HSQC method.34 The results indicated that all analytes were extracted selectively; no cross contamination was detected in 1268

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the NMR samples. On the basis of the analysis of the pseudo2D spectrum of the on-flow 1D 1H-31P HSQC acquisition (Figure 2B), the base of the peaks in the chromatographic dimension was ca. 20 s wide. The storage loop fill takes 15 s, which means that a part of the analytes will be lost. Still, recoveries in single trapping were acceptable for IPMPA and PMPA (Table 2), but the recovery of MPA was lower than expected. Additional tests (data not shown) confirmed that the used small eluent volume (50 µL) was not enough to elute MPA completely. On the other hand, the analyte concentration decreased accordingly with increased eluent volume, so this did not give any benefit for the NMR sample. Still, the amount of MPA in the NMR sample was more than sufficient for the mass-sensitive microcoil probe head.35,43 The tests with double and triple trapping demonstrated that the chosen SPE sorbent retained well IPMPA and PMPA; no significant breakthrough was occurring during the additional introduction of the analyte fractions (Table 2). The low recovery of MPA in the double and triple trapping tests was again due to poor elution. Decontamination Solution Analysis with LC-SPE-NMR. As demonstrated previously, the presence of trace amounts of phosphorus-containing CWA degradation products in decontamination solutions can be rapidly determined even at low-parts-permillion levels with 1D proton-phosphorus correlation experiments.34 A good approach for NMR identification is to concentrate the analytes into a small volume and use the high mass sensitivity of microcoil probe heads.43 However, sometimes the high level of impurities in the concentrated sample can prevent assignment of the proton resonances of the relevant organophosphorus compounds. Overlapping of the peaks of the relevant chemicals can occur even in 2D proton-phosphorus correlation experiments,3,5 which makes identification difficult. This situation was demonstrated in analysis of a decontamination solution containing 150 µg/mL MPA, IPMPA, and PMPA. A concentrated decontamination solution sample was prepared where the amount of analytes was slightly above the detection limit of the on-flow 1D 1H-31P HSQC (ca. 20 µg in 20 µL injections). At first, a part of the concentrated decontamination solution sample was analyzed directly using a microcoil probe head.43 The level of analytes was sufficient, but the sample contained a high level of impurities so that the 1H NMR spectrum was not useful in identification (Figure 3). Furthermore, the peaks of the relevant chemicals were overlapping both in 1H NMR and 31P{1H} NMR spectra so that even a 2D proton-phosphorus correlation experiment failed to give unambiguous assignments (Figure 3). Therefore, the LC-SPE-NMR with application of the on-flow 1D 1H-31P HSQC approach in identification of the analytes was tested. The LC-SPE-NMR procedure was as described above, and single trapping was used for the off-line NMR samples. According to the 1H NMR spectra, the off-line NMR samples were not absolutely pure (Figure 4). The most intense peak in the spectrum was water, but it was effectively suppressed with a simple presaturation. The main organic component peaks present in the 1H NMR spectra came from the SPE cartridge conditioning (methanol) and from the HPLC eluent (acetonitrile, formic acid). The level of the organic components could have been reduced using a suitable SPE cartridge washing procedure

Table 2. Recoveries of the Chemicals in the Off-Line NMR Samples from the LC-SPE-NMR Analysis of the Detection Limit Test Sample That Contained MPA, IPMPA, and PMPA at a Level of 0.5 µg/µL in the HPLC Eluenta recovery single trapping double trapping triple trapping

MPA

IPMPA

PMPA

1.6 µg (17 nmol)/17% 1.7 µg (18 nmol)/9% 3.3 µg (34 nmol)/11%

5.5 µg (40 nmol)/55% 12.6 µg (91 nmol)/63% 20.8 µg (151 nmol)/69%

5.4 µg (30 nmol)/54% 12.3 µg (68 nmol)/61% 16.1 µg (89 nmol)/54%

a The recoveries are shown as the absolute amounts in the sample and as percentages from the injected amount. Quantification was performed against external reference samples using the quantitative 1D 1H-31P HSQC experiment (ref 34).

Table 3. Recoveries of the Chemicals in the Off-Line NMR Samples from the LC-SPE-NMR Analysis of the Concentrated Decontamination Solution Samplea recovery MPA

IPMPA

PMPA

single trapping 4.9 µg 8.8 µg 7.4 µg (51 nmol)/25% (64 nmol)/44% (41 nmol)/37% a The recoveries are shown as the absolute amounts in the sample and as percentages from the injected amount. Quantification was performed against external reference samples using the quantitative 1D 1H-31P HSQC experiment (ref 34).

prior to elution, but it would have also affected the recovery of the analytes. None of these organic components overlapped the peaks of the analytes, and identification was straightforward. The MPA fraction contained most of the impurities present in the concentrated decontamination solution sample, and some of these are also present in the off-line NMR sample (Figure 4). They were still minor components and presented no complications in identification. The recoveries (Table 3) were under 50% for all analytes, but the amounts were still more than sufficient for NMR analysis with the microcoil probe head; further NMR experiments would have been feasible within reasonable total measurement time.43 The usefulness of the on-flow heteronuclear correlation spectrometry in detection of chromatographic separation was demonstrated with organophosphorus compounds, but it is not limited to these chemicals. Carbon-13 labeled compounds are commonly utilized in biomedical research in studies of metabolic pathways.44 The concept of indirect detection of carbon-13 labeled analytes has been demonstrated earlier by Albert et al.45 Their experiment based on 13C-edited spin-echo sequence, which uses phase cycling to suppress the carbon-12 bound proton magnetization. A more efficient method to suppress the carbon-12 bound proton magnetization should be achievable, e.g., with the onflow pulsed field gradient 1D 1H-13C HSQC. This technique (44) Mutlib, A. E. Chem. Res. Toxicol. 2008, 21, 1672–1689. (45) Albert, K.; Sudmeier, J. L.; Anwer, M. S.; Bachovchin, W. W. Magn. Reson. Med. 1989, 11, 309–315.

could be very useful in detection of carbon-13 labeled metabolites in biomedical samples during chromatographic separation. The pseudo-2D spectrum information could be directly used to supplement the LC/MS information about what is the carbon-13 distribution in the metabolites. The other possibility is to use the on-flow 1D 1H-13C HSQC for retention time determination like in the presented LC-SPE-NMR analysis. The organic components of the HPLC eluent can give a more intense background, but as they contain carbon-13 in natural abundance (1.1%), they should not cause too much problem in the dynamic range if the concentration of the carbon-13 labeled analytes in the sample is sufficient. CONCLUSIONS The on-flow 1D 1H-31P HSQC approach offered a good sensitivity and selectivity in detection of important nerve agent degradation products during chromatographic separation. The method facilitated determination of the retention times of these non-UV-detectable chemicals so that the LC-SPE-NMR technique was possible to use in selective enrichment of the chemicals for off-line NMR analysis. The presented on-flow heteronuclear correlation spectrometry can also have potential to monitor chromatographic separation of other small molecules, like isotope-labeled drug chemicals and their metabolites. ACKNOWLEDGMENT The supply of the reference chemicals from the Spiez Laboratory of the Federal Department of Defence, Civil Protection and Sports, Switzerland, is gratefully acknowledged. Our colleagues Marja-Leena Rapinoja and Marja-Leena Kuitunen are acknowledged for their advice in the solid-phase extraction development. Matti Keina¨nen from the Laboratory of Organic Chemistry, University of Helsinki, is acknowledged for his technical assistance with the instrumentation.

Received for review November 13, 2008. Accepted December 15, 2008. AC802407T

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