Analysis of Chemical Warfare Agents in Food Products by

Sep 27, 2007 - Flow injection high field asymmetric waveform ion mobil- ity spectrometry ... 1-33 ng/mL in canola oil, 1-34 ng/g in cornmeal, and. 13-...
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Anal. Chem. 2007, 79, 8257-8265

Analysis of Chemical Warfare Agents in Food Products by Atmospheric Pressure Ionization-High Field Asymmetric Waveform Ion Mobility Spectrometry-Mass Spectrometry Beata M. Kolakowski,† Paul A. D’Agostino,‡ Claude Chenier,‡ and Zolta´n Mester*,†

NRC Institute for National Measurement Standards, 1200 Montreal Road, Ottawa, ON, Canada, and DRDC Suffield, P.O. Box 4000 Station Main, Medicine Hat, AB, Canada

Flow injection high field asymmetric waveform ion mobility spectrometry (FAIMS)-mass spectrometry (MS) methodology was developed for the detection and identification of chemical warfare (CW) agents in spiked food products. The CW agents, soman (GD), sarin (GB), tabun (GA), cyclohexyl sarin (GF), and four hydrolysis products, ethylphosphonic acid (EPA), methylphosphonic acid (MPA), pinacolyl methylphosphonic acid (Pin MPA), and isopropyl methylphosphonic acid (IMPA) were separated and detected by positive ion and negative ion atmospheric pressure ionization-FAIMS-MS. Under optimized conditions, the compensation voltages were 7.2 V for GD, 8.0 V for GA, 7.2 V for GF, 7.6 V for GB, 18.2 V for EPA, 25.9 V for MPA, -1.9 V for PinMPA, and +6.8 V for IMPA. Sample preparation was kept to a minimum, resulting in analysis times of 3 min or less per sample. The developed methodology was evaluated by spiking bottled water, canola oil, cornmeal, and honey samples at low microgram per gram (or µg/mL) levels with the CW agents or CW agent hydrolysis products. The detection limits observed for the CW agents in the spiked food samples ranged from 3 to 15 ng/mL in bottled water, 1-33 ng/mL in canola oil, 1-34 ng/g in cornmeal, and 13-18 ng/g in honey. Detection limits were much higher for the CW agent hydrolysis products, with only MPA being detected in spiked honey samples. Rapid, reliable, analytical methods for the detection and identification of chemical warfare (CW) agents and their hydrolysis products are required to determine the presence of these chemicals following release, to assess and treat exposed first responders/victims more effectively, to support forensic investigations, and for site remediation purposes. Samples taken for analysis under these scenarios would typically be environmental, man-made materials, or biological fluids. Deliberate contamination of food or consumer products with CW agents (or other toxic chemicals) represents another analytical challenge, as methods for the determination of CW agents in typical food or consumer products have not been previously investigated. * To whom correspondence should be addressed. [email protected]. Fax: 613 9932451. Phone: 613 9935008. † NRC Institute for National Measurement Standards. ‡ DRDC Suffield. 10.1021/ac070816j CCC: $37.00 Published 2007 Am. Chem. Soc. Published on Web 09/27/2007

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Hand held and field-deployable detectors are useful in many operational settings for provisional CW agent identification but these portable instruments lack the sensitivity and specificity of laboratory-based analytical techniques.1 The most frequently used method for the analysis of CW agents such soman (GD), sarin (GB), tabun (GA), and cyclohexyl sarin (GF) is gas chromatography/mass spectrometry (GC/MS).2 However, GC/MS is not suitable for the direct analysis of low-volatility CW hydrolysis products. These compounds generally require sample derivatization prior to GC/MS analysis3 and frequently involve significant sample preparation. These limitations spurred the development of LC-MS methods for direct determination of CW hydrolysis products in aqueous samples and extracts. Read and Black4 compared electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) for the LC-MS determination of hydrolysis products and found that ESI provided enhanced sensitivity compared to APCI but found that APCI was more robust for screening purposes. LC-MS has the added benefit that it may also be utilized for the determination of organophosphorus CW agents and related compounds during the same analysis.5-7 Additional work has focused on the development of analytical methodologies for the detection of CW agents or their degradation products in environmental and biological matrixes including water,4-6,8-14 soil,10,11,15-17 urine,18,19 serum,20-22 and plasma.23 These and other analytical methods used for the analysis of CW agents (1) Hill, H. H., Jr.; Martin, S. J. Pure Appl. Chem. 2002, 74, 2281. (2) Mesilaakso, M., Ed. Chemical Weapons Convention Chemical Analysis. Sample Collection, Preparation and Analytical Methods,; Wiley: Chichester, 2005. (3) Black, R. M.; Muir. B. J. Chromatogr., A 2003, 1000, 253. (4) Read, R. W.; Black, R. B. J. Chromatogr., A 1999, 862, 169. (5) D’Agostino, P. A.; Provost, L. R.; Hancock, J. R. J. Chromatogr., A 1999, 840, 289. (6) Creasy, W. R. J. Am. Soc. Mass Spectrom. 1999, 10, 440. (7) Creasy, W. R.; Stuff, J. R.; Williams, B.; Morrissey, K.; Mays, J.; Duevel, R.; Durst, H. D. J. Chromatogr. 1997, 774, 253. (8) D’Agostino, P. A.; Hancock, J. R.; Provost, L. R. J. Chromatogr., A 1999, 837, 93. (9) Black, R. M.; Read, R. W. J. Chromatogr., A 1998, 794, 233. (10) Hooijschuur, E. W. J; Kientz, C. E.; Hulst, A. G. Anal. Chem. 2000, 72, 1199. (11) D’Agostino, P. A.; Hancock, J. R.; Provost, L. R. Adv. Mass Spectrom. 2001, 15, 297. (12) D’Agostino, P. A.; Chenier, C. L.; Hancock, J. R. J. Chromatogr., A 2002, 950, 149. (13) Liu, Q.; Hu, X. Y.; Xie, J. W. Anal. Chim. Acta 2004, 512, 93. (14) Mercier, J.-P.; Morin, Ph.; Dreux, M.; Tabute´, A. J. Chromatogr., A 1999, 849, 197.

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and their degradation products were recently reviewed by Mesilaakso.2 In general, the chromatography-based methods offer good selectivity and sensitivity for the analytes of interest. However, all of these methods are time- and labor-intensive and often require significant sample preparation. There have been no previous reports of CW agent analyses in food matrixes by atmospheric pressure ionization mass spectrometry (API-MS). Therefore, sample preparation technologies were selected from pesticide analysis protocols, except for bottled water, which was subjected to the protocols developed for flow injection high field asymmetric waveform ion mobility spectrometry (FAIMS)based analysis of drinking water. The water was diluted with organic solvent to reduce salt concentrations and to ensure a better ESI spray.24-31 Analysis in flour or cornmeal requires removal of the complex carbohydrates. Sample cleanup strategies include boiling32 or freezing,33 followed by filtration or liquidliquid or solid-phase extraction. Analysis in vegetable oils usually involves dilution with organic solvents34-36 or removal of the fats by successive liquid-liquid or solid-phase extractions.37 From honey samples, removal of the carbohydrates requires liquidliquid extraction38 or solid-phase extraction,39 and subsequent sample concentration requires filtration through a concentrator.40 Sonication has been used for sample dilution when spiking.41-43 (15) D’Agostino, P. A.; Hancock, J. R.; Provost, L. R. J. Chromatogr., A 2001, 912, 291. (16) D’Agostino, P. A.; Hancock, J. R.; Chenier, C. L. Eur. J. Mass Spectrom. 2003, 9, 609. (17) D’Agostino, P. A.; Hancock, J. R.; Chenier, C. L. J. Chromatogr., A 2004, 1058, 97. (18) Read, R. W.; Black, R. M. J. Anal. Toxicol. 2004, 28, 346. (19) Read, R. W.; Black, R. M. J. Anal. Toxicol. 2004, 28, 352. (20) Noort, D.; Hulst, A. G.; Platenburg, D. H. J. M.; Polhuijs, M.; Benschop, H. P. Arch. Toxicol. 1998, 72, 671. (21) Katagi, M.; Tatsuno, M.; Nishikawa, M.; Tsuchihashi, H. J. Chromatogr., A 1999, 833, 169. (22) Noort, D.; Fidder, A.; Hulst, A. G.; Woolfitt, A. R.; Ash, D.; Barr, J. R. J. Anal. Toxicol. 2004, 28, 333. (23) Smith, J. R. J. Anal. Toxicol. 2004, 28, 390. (24) Barnett, D. A.; Guevremont, R.; Purves, R. W. Appl. Spectrosc. 1999, 53, 1367. (25) Handy, Barnett, D. A.; Purves, R. W.; Horlick, G.; Guevremont, R. J. Anal. At. Spectrom. 2000, 15, 907. (26) Ells, B.; Barnett, D. A.; Purves, R. W.; Guevremont, R. J. Environ. Monit. 2000, 2, 393. (27) Ells, B.; Barnett, D. A.; Froese, K.; Purves, R. W.; Hrudey, S.; Guevremont, R. Anal. Chem. 1999, 71, 4747. (28) Ells, B.; Barnett, D. A.; Purves, R. W.; Guevremont, R. Anal. Chem. 2000, 72, 4555. (29) Gabryelski, W.; Wu, F.; Froese, K. L. Anal. Chem. 2003, 75, 2478. (30) Delinsky, A. D.; Bruckner, J. V.; Bartlett, M. G. Biomed. Chromatogr. 2005, 19, 617. (31) Sultan, J.; Gabryelski, W. Anal. Chem. 2006, 78, 2905. (32) Kosee, J. S.; Yeung, A. C.; Gil, A. I.; Miller, D. D. Food Chem. 2001, 75, 371. (33) Riediker, S.; Obrist, H.; Varga, N.; Stadler, R. H. J. Chromatogr., A 2002, 966, 15. (34) Gliszczyn´ska, A.; Sikorska, E. J. Chromatogr., A 2004, 1048, 195. (35) Sanchez, R.; Vasquez, A.; Andini, J. C.; Ville´n, J. J. Chromatogr., A 2004, 1029, 167. (36) Janssen, H.-G.; Boers, W.; Steenbergen, H.; Horsten, R.; Flo¨ter, E. J. Chromatogr., A 2003, 1000, 385. (37) Barranco, A.; Alonso-Salces, R. M.; Bakkali, A.; Berrueta, L. A.; Gallo, B.; Vicente, F.; Sarobe, M. J. Chromatogr., A 2003, 988, 33. (38) Rezic´, I.; Horvat, A. J. M.; Babic´, S.; Kasˇtelan-Macan, M. Ultrason. Sonochem. 2005, 12, 477. (39) Herrera, A.; Pe´rez-Arquillue´, C.; Conchello, P.; Bayarri, S.; La´zaro, R.; Yagu ¨ e, C.; Arin ˜o, A. Anal. Bioanal. Chem. 2005, 381, 695.

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FAIMS separates and focuses ions formed by API ion sources. A rigorous description is provided in the original papers by Buryakov et al.44 and by Guevremont et al.45-47 and is augmented by recent theoretical studies.48-51 Here, a brief summary is given. The mobility of an ion in an electric field can be described mathematically as52

Kh (E) ) K0[1 + f (E)]

(1)

where Kh is the ion mobility at high field, K0 is the ion mobility at zero field, and f(E) describes the ion mobility as a function of electric field strength. If no other voltage is applied, the ions will eventually collide with one of the plates and be lost. If a constant dc voltage, termed the compensation voltage, or CV, of the correct magnitude and polarity is applied, the ions will continue to travel between the two plates and will be transmitted to the detection device (a mass spectrometer in this study). This effectively traps the ions between the plates. In an ideal situation, each ion’s mobility and associated compensation voltage value will be unique. To determine the actual compensation voltage of an ion or an ion in a mixture, the CV is scanned over a range while monitoring the m/z of the target analytes. If the CV is indeed unique, the ion of interest will traverse the FAIMS device and be detected to the exclusion of other ions. This selective transmission could provide ion focusing with the accompanying benefits of better signal-tonoise ratios and better detection limits. The polarity and the magnitude of the compensation voltage is determined by the magnitude and polarity of the dispersion voltage,45,46 the gas composition,47,53,54 the gas flow rate and pressure,43 the purity of the gas,55,56 the temperature,46,47 and the compound identity. The FAIMS parameters are experimentally manipulated to optimize ion separation, to maximize analyte response, and to minimize the noise. Recently, the FAIMS behavior of CW hydrolysis products and simulants was discussed as a function of solvent load.57 (40) Albero, B.; Sa´nchez-Brunete, C.; Tadeo, J. L. J. Agric. Food Chem. 2004, 52, 5828. (41) Jime´nez, J. J.; Bernal, J. L.; del Nozal, M. J.; Martı´n, M. T.; Mayorga, A. L. J. Chromatogr., A 1998, 829, 269. (42) Yu, J.; Wu, C.; Xing, J. J. Chromatogr., A 2004, 1036, 101. (43) Blasco, C.; Ferna´ndez, M.; Pico´, Y.; Font, G. J. Chromatogr., A 2004, 1030, 77. (44) Buryakov, I. A.; Krylov, E. V.; Nazarov, E. G.; Rasulev, U. Kh. Int. J. Mass Spectrom. Ion Processes 1993, 128, 143. (45) Purves, R. W.; Guevremont, R.; Day, S.; Pipich, C. W.; Matyjaszczyk, M. S. Rev. Sci. Instrum. 1998, 69, 4094. (46) Guevremont, R.; Purves, R. W. Rev. Sci. Instruments 1999, 70, 1370. (47) Guevremont, R. J. Chromatogr., A 2004, 1058, 3. (48) Shvartsburg, A. A.; Tang, K.; Smith, R. D. J. Am. Soc. Mass Spectom. 2005, 16, 2. (49) Shvartsburg, A. A.; Tang, K.; Smith, R. D. J. Am. Soc. Mass Spectom. 2004, 15, 1487. (50) Tang, K.; Li, F.; Shvartsburg, A. A.; Strittmatter, E. F.; Smith, R. D. Anal. Chem. 2005, 77, 6381. (51) Shvartsburg, A. A.; Tang, K.; Smith, R. D. J. Am. Soc. Mass Spectom. 2005, 16, 1447. (52) Mason, E. A.; McDaniel, E. W. Transport Properties of Ions in Gases; Wiley: New York, 1988. (53) Barnett, D. A.; Ells, B.; Guevremont, R.; Purves, R. W.; Viehland, L. A. J. Am. Soc. Mass Spectrom. 2000, 11, 1125. (54) Shvartsburg, A. A.; Tang, K.; Smith, R. D. Anal. Chem. 2004, 76, 7366. (55) Krylova, N.; Krylov, E.; Eiceman, G. A.; Stone, J. A. J. Phys. Chem. A 2003, 107, 3648. (56) Eiceman, G. A.; Krylov, E. V.; Krylova, N. S.; Nazarov, E. G.; Miller, R. A. Anal. Chem. 2004, 76, 4937. (57) Kolakowski, B. M.; McCooeye, M. A.; Mester, Z. Rapid Commun. Mass Spectrom. 2006, 20, 3319.

The aim of this study was to develop simplified sample preparation methods coupled with flow injection API-FAIMS-MS to allow rapid, semiquantitative identification of CW agents and their hydrolysis products in food products, including bottled water, honey, cornmeal, and canola oil. EXPERIMENTAL SECTION Materials and Equipment. Soman (GD), sarin (GB), tabun (GA), cyclohexyl sarin (GF), isopropyl methylphosphonic acid, and pinacolyl methylphosphonic acid were provided by DRDC Suffield, (Medicine Hat, AB, Canada). Methylphosphonic and ethylphosphonic acid were purchased from Sigma-Aldrich (St. Louis, MO). Bottled water, canola oil, cornmeal, and honey were obtained from local grocery stores. OmniSolv grade acetonitrile and methanol were obtained from EMD Chemicals (Gibbstown, NJ). Distilled water was obtained from an in-house water purification system. ESI-MS and APCI-MS analyses were performed with an Applied Biosystems (Foster City, CA) API 3000 triple-quadrupole mass spectrometer operating in turbospray or heated nebulizer mode at NRC Institute for National Measurement Standards. The flow injection analysis made use of the injector of the Agilent 1100 series HPLC system (Palo Alto, CA). A FAIMS device consisting of temperature-controlled electrodes, a waveform generator, a temperature control module, and integrated Analyst-Selectra software were obtained from Ionalytics Corp. (now ThermoFisher, San Jose, CA). Sample Preparation. Calibration Curve. Standard solutions of each of the chemical warfare agents (GA, GB, GD, GF) or hydrolysis products were prepared by serial dilution of the stock solutions with 75:25 acetonitrile/water. The concentrations ranged between 0.0019 and 5 µg/mL for the CW agents and from 0.5 to 40 µg/mL for the CW agent hydrolysis products. Spikes into Bottled Water. Bottled water was spiked with individual CW agents at the 1.250 µg/mL level. One milliliter of the bottled water was pipetted out into a 4-mL amber vial. This was then spiked with 25 µL of 0.050 mg/mL CW agent in dichloromethane and vortexed. After 10 min, 2 mL of 75:25 acetonitrile/water was added. The vial contents were vortexed and transferred to an HPLC vial for immediate analysis. The same process was repeated with the blank bottled water. A similar procedure was undertaken with the hydrolysis products with a spiking level of 2.5 µg/mL. The hydrolysis products were prepared in water, and 25 µL of a 0.1 mg/mL hydrolysis product was directly spiked into the bottled water. Two milliliters of 75:25 acetonitrile/water was added. The vial contents were vortexed and transferred to an HPLC vial for analysis. The same process was repeated with the blank bottled water. Spikes into Canola Oil. Canola oil was spiked at the 3.125 µg/ mL level with CW agents or at the 6.250 µg/mL level with hydrolysis products. A 400-µL sample of oil was pipetted into a 4-mL amber vial. This was then spiked with either 25 µL of 0.050 mg/mL CW agent in dichloromethane or 25 µL of 0.1 mg/mL hydrolysis product in dichloromethane. The solvent was allowed to evaporate (10 min) and then 3 mL of 75:25 acetonitrile/water was added. The vial contents were mixed in a vortex. The upper layer was removed and filtered through a 0.45-µm syringe filter disk. The filtrate was transferred to an HPLC vial for immediate

Table 1. Transitions Used in Positive and Negative Ion Mode API-MS/MS

compound tabun sarin cyclohexyl sarin soman methylphosphonic acid ethylphosphonic acid isopropyl methylphosphonic acid pinacolyl methylphosphonic acid

transition monitored in positive mode

transition monitored in negative mode

m/z 163 f 135 m/z 141 f 99 m/z 181f 99 m/z 183 f 99 m/z 97 f 79 m/z 111 f 79 m/z 139 f 97

not applicable not applicable not applicable not applicable m/z 95 f 77 m/z 109 f 79 m/z 137 f 79

m/z 181 f 97

m/z 179 f 95

analysis. The same process was repeated with the blank solution except that no spike was added. Spikes into Cornmeal or Honey. Cornmeal and honey were also spiked at the 3.120 µg/mL level with CW agents or at the 6.250 µg/mL level with hydrolysis products. A 400-mg sample of cornmeal or honey was weighed into a 4-mL amber vial. This was then spiked with 25 µL of 0.050 mg/mL CW agent in dichloromethane or 25 µL of 0.1 mg/mL hydrolysis product in dichloromethane. The solvent was allowed to evaporate (10 min), and then 3 mL of 75:25 acetonitrile/water was added. The vial contents were mixed in a vortex. The supernatant was removed and filtered through a 0.45-µm syringe filter disk. The filtrate was transferred to an HPLC vial for immediate analysis. The same process was repeated with the blank samples. Analysis of CW Agents and Hydrolysis Products. Optimization of Response. The transition used for MS/MS analysis of each compound (Table 1) was determined prior to analysis of standards or spiked food product samples by analyzing standards in full scanning mode. Using these transitions, the MS and FAIMS parameters were optimized for separation and response. Analysis. All analyses were done by flow injection. A 10-µL injection of standard solution or sample extract was introduced into a flowing stream (400 µL/min of 75:25 acetonitrile/water + 0.1% formic acid). All connections between the HPLC system and the instrument were via 0.005-in.-i.d. PEEK tubing. Optimal MS and FAIMS methods were used for each of the analytes. Calibration curves were generated by triplicate analysis of blanks and prepared solutions in the 1.25-9.4 µg/mL (or g) range for CW agents and 2.5-18.8 µg/mL (or g) range for CW hydrolysis products. This was followed by replicate injections of blank water, spiked water, blank cornmeal, spiked cornmeal, blank oil, spiked oil, blank honey, and spiked honey samples. Analyses were performed with single ion monitoring (one ion/analyte), full scan detection, tandem mass spectrometry, and multiple reaction monitoring (one transition per analyte). The signal intensity from 0.05 to 0.45 min was averaged and was used to generate the calibration curve and to determine recoveries of the spikes. LC-ESI-MS Conditions for CW Analysis. LC-ESI-MS and MS/ MS data were acquired using a Waters (Milford, MA) Q-ToF Ultima tandem mass spectrometer equipped with a Z-spray electrospray interface at DRDC Suffield. The electrospray capillary was operated at 3 kV with a sampling cone voltage of 35 V. The collision energy was maintained at 5 V for LC-ESI-MS operation Analytical Chemistry, Vol. 79, No. 21, November 1, 2007

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and was varied from 3 to 10 V (depending on the precursor ion selected) for LC-ESI-MS/MS operation. Argon was continually flowing into the collision cell at 9 psi during both LC-ESI-MS and LC-ESI-MS/MS operation. Nitrogen desolvation gas was introduced into the interface (80 °C) at a flow rate of 300 L/h, and nitrogen cone gas (100 °C) was introduced at a flow rate of 50 L/h. ESI-MS data were acquired from m/z 70 to 500 (0.4 s with a 0.1-s interscan delay), and ESI-MS/MS (product mass spectra) data were acquired for the protonated molecular ions of the spiked compounds (0.9 s with a 0.1-s interscan delay) during the same LC-MS analysis. All data were acquired in the continuum mode with a resolution of 8000 (V-mode, 50% valley definition). Chromatographic separations were performed with an Aglient 1100 using a 5-50% B gradient over 15 min and a flow rate of 10 µL/min. The following solvent compositions were prepared for the mobile phase: solvent A (0.1% trifluoroacetic acid in water) and solvent B (acetonitrile). Separations were performed with an Agilent 50 mm × 0.3 mm i.d. glass-lined tube packed with Zorbax SB-C18 (1.8-µm particle size). The Agilent 1100 autosampler was used to introduce 1-µL samples of the spiked bottled water and blanks. RESULTS AND DISCUSSION API-FAIMS-MS Analysis of CW Agents. The CV scan of the four CW agents was obtained during FAIMS-ESI-MS and FAIMSAPCI-MS analysis of a 0.5 µg/mL standard mixture. Figure 1 illustrates the differences in relative responses between analytes during FAIMS-ESI-MS and FAIMS-APCI-MS using optimized conditions. Both API techniques exhibited the same range of responses for the analytes studied, either in the presence or in the absence of the FAIMS device. This indicates that this differential response is probably related to ionization/fragmentation processes and not to the FAIMS process. It is also apparent that there is a distinct shift in CVs with the use of the APCI source relative to the ESI source. These profiles were obtained on the same instrument platform so the difference is not related to the mass analyzer. The shift is likely due to differing desolvation efficiencies in the two sources. FAIMS was not able to separate the compounds, regardless of the API source used. This is both an advantage and a disadvantage. A nonunique CV simplifies detection as the parameters are static and all CW agents will be detected under a single set of conditions, making scanning of transmission parameters unnecessary. However, a compound-specific CV value would provide a separation orthogonal to the mass spectrometer that would serve to better identify the analyte of interest. The major benefit of the use of the FAIMS device is the potential reduction in chemical background. This usually leads to an improvement in signal-to-background (S/B) ratio. This improvement in S/B ratio is best seen from the full scan mass spectral data presented in Figure 2. The background signal of canola oil is significantly higher with the use of conventional electrospray mass spectrometry (A) than with ESI-FAIMS-MS (B). Comparing the spectra of tabun-spiked canola oil by ESI-MS (C) and by ESI-FAIMS-MS (D), it is clear from both spectra that tabun is detected. However, the ESI-MS spectra are far more complex than that obtained with ESI-FAIMS-MS. The ESI-FAIMS-MS spectra could potentially be used for the unequivocal identification 8260 Analytical Chemistry, Vol. 79, No. 21, November 1, 2007

Figure 1. Mixture of 10 µg/mL tabun, sarin, soman, and GF infused at 2 µL/min into a flow of 400 µL/min of mobile phase (75:25 acetonitrile/water + 0.1% formic acid). The compensation voltage was scanned with positive ion mode ESI-FAIMS-MS and positive ion mode APCI-FAIMS-MS detection. All FAIMS conditions were optimal.

of the CW agent, but not so the conventional ESI-MS spectra. Table 3 lists the signal to background ratios of the analytes. This ratio was determined by dividing the average intensity of the calibration solution (0.5 µg/mL) by the average intensity of the blank solution. The results presented in Table 3 indicate that the S/B levels were higher for APCI-MS/MS than for ESI-MS/MS. This difference was usually associated with a lower background signal for APCI-MS during blank analysis, as the signal intensities for the compounds themselves were comparable or lower in APCI mode using the same instrument platform. Incorporation of the FAIMS device reduced both the analyte and background signal intensity, likely due to reduced transmission efficiency through the FAIMS device However, addition of FAIMS device improved the S/B ratio significantly in APCI mode. It appears that the shift to more positive CVs with APCI ionization improved the S/B by up to 2 orders of magnitude over APCI MS, a difference that may be attributed to more efficient declustering in the APCI source. Subsequent CW agent analyses were conducted using the APCI source for these reasons. A method performance evaluation for APCI-FAIMS-MS and for APCI-MS was carried out by determining the figures of merit (linear dynamic range, limits of detection) for the analysis of the four CW agents in acetonitrile/water. (Table 4).

Table 2. Mass Spectrometric and FAIMS Parameters for the Analysis of CW Agents and Hydrolysis Productsa ionization mode ESI, positive

ESI, negative

APCI, positive

APCI, negative

MS parameters nebulizer gas ) 15 curtain gas ) 15 IS ) 4 kV source T ) 400 °C collision Gas ) 7 DP ) 10 V FP ) 128 V EP ) 9.8 V CE ) 5-15 V 4 transitions dwell time ) 248 ms interscan delay ) 2 ms nebulizer gas ) 15 curtain gas ) 15 IS ) -4 kV source T ) 400 °C collision gas ) 12 DP ) -27 V FP ) -162 V EP ) -12.1 V CE ) -17 to -22 V 4 transitions dwell time ) 248 ms interscan delay ) 2 ms Nebulizer gas ) 10 curtain gas ) 10 NC ) 4.5 µA source T ) 450 °C collision gas ) 7 DP ) 12.6 to 22.7 V FP ) 117 to 145 V EP ) 4.2 to 9.0 V CE ) 8 to13 V 4 transitions dwell time ) 248 ms interscan delay ) 2 ms nebulizer gas ) 10 curtain gas ) 10 NC ) -4.5 µΑ source T ) 450 °C collision gas ) 7 DP ) -21 to -31 V FP ) -110 to -187 V EP ) -11 to -14 V CE ) -20 to -33 V 4 transitions dwell time ) 248 ms interscan delay ) 2 ms

FAIMS parameters DP ) -3000 V 50% He/50% N2 gas flow ) 3.50 L/min outer bias voltage ) 11-14 V inner heater ) outer heater setting ) 70 °C

CV values GF, -4.4 V GD, -3.2 V GB, -4.2 V GA, -4.2 V

EPA, +4.0 V MPA, +3.4 V PinMPA, +3.0 V IMPA, +4.8 V DP ) -4000 V 25% He/75% N2 gas flow ) 6.50 L/min outer bias voltage ) -10 V inner heater ) outer heater setting ) 70 °C

GF, ND GD, ND GB, ND GA, ND

EPA, +18.2 V MPA, +25.9 V PinMPA, -1.9 V IMPA, +6.8 V DP ) -3500 V 100% N2 gas flow ) 5.00 L/min outer bias voltage ) 25 V inner heater ) outer heater setting ) 70 °C

GF, +7.2 V GD, +7.2 V GB, +7.6 V GA, +8.0 V

EPA, ND MPA, ND PinMPA, ND IMPA, ND DP ) -4000 V 25% CO2/75% N2 gas flow ) 5.00 L/min outer bias voltage ) 25 V inner heater ) outer heater setting ) 70 °C

GF, ND GD, ND GB, ND GA, ND

EPA, +12.6 V MPA, +15.6 V PinMPA, +1.6 V IMPA, +6.0 V

a IS, ion spray voltage; DP, declustering potential; FP, focusing potential; EP, entrance potential; CE, collision energy; NC, nebulizer current; ND, not detected.

The limit of detection was improved for all four CW agents with the addition of the FAIMS device with limits of detection in the 0.5-1.4 ng/mL range (5-140 pg injected). The dynamic range was linear over 2-3 orders of magnitude and exhibited no evidence of saturation even at the highest concentration studied. These detection limits compare well with the selected reaction monitoring (SRM) detection limits attainable by GC/MS/MS analysis (30-70 pg injected on-column)58,4 and in the hundreds of picogram range with GC-flame ionization detection.59 During LC-ESI-TOF-MS analysis, the CW agents were readily detected in solids spiked at the 10 µg/g level.15 (58) Kientz, Ch. E. J. Chromatogr., A 1998, 814, 1. (59) Witkiewicz, Z.; Mazurek, M.; Szulc, J. J. Chromatogr. 1990, 503, 293.

API-FAIMS-MS Analysis of the Hydrolysis Products. Figure 3 illustrates a CV scan following ESI analysis of the four hydrolysis products. The four hydrolysis products, introduced simultaneously, were fully resolved by the FAIMS device. The compensation voltage values of the hydrolysis products in negative ion mode seem to be correlated with the molecular weight, where pinacolyl methylphosphonic acid (largest molecule) appears at the most negative CV value and methylphosphonic acid (smallest) appears at the most positive CV values. In addition, there was signal intensity for m/z 97 below each of the larger peaks, arising from in-source fragmentation of the parent hydrolysis product. The difference in CV values allowed FAIMS separation of the signal from methylphoshphonic acid from the in-source fragment(s) with the same m/z value. Analytical Chemistry, Vol. 79, No. 21, November 1, 2007

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Figure 2. Full scan mass spectra of blank canola oil (ESI-MS, spectrum A and ESI-FAIMS-MS, spectrum B) and canola oil spiked with 3 µg/mL tabun (ESI-MS, spectrum C and ESI-FAIMS-MS, spectrum D). In all cases, 10 µL of analyte was injected (400 µL of canola oil diluted with 3 mL of 75:25 acetonitrile/water). Mass spectrometric conditions used for analysis: positive mode electrospray, ion spray voltage 4 kV, nebulizer gas setting ) curtain gas setting, 15 arbitrary units, collision gas setting 7 arbitrary units, source temperature 400 °C, declustering potential 10 V, focusing potential 128 V, entrance potential 9.8 V, and collision energy between 5 and 15 V. The FAIMS parameters used wereas follows: dispersion voltage -3000 V, 50% helium/50% nitrogen at a total gas flow rate of 3.50 L/min, outer bais voltage settings between 11 and 14 V, and inner heater setting ) outer heater setting, 70 °C. Table 3. Signal-to-Background Ratios Obtained during Flow Injection API-MS/MS Analysis of CW Agents in the Presence and Absence of FAIMSa species

ESI-MSMS

tabun GF sarin soman

1 ()11.1/8.26) 5()212/41.1) 9 ()23.8 /2.66) 86 ()84.6/0.986)

ESI-FAIMS-MSMS 0 ()1.00/2.56) 1 ()2.9/2.1) 5 ()1.8/0.36) 7 )(2.7/0.37)

APCI-MSMS

APCI-FAIMS-MSMS

69 ()10/0.15) 21 ()129/6.1) 21 ()53/2.5) 22 ()8.5/0.38)

326 ()2.8/0.00867) 395 ()30/0.077) 1251 ()9.3/0.024) 47 ()1.6/0.033)

a Average signal intensity for 5 ng (0.5 µg/mL; 10 µL Injected) of CW agent was divided by the average signal intensity for the blank solution under optimized FAIMS-MS conditions. Intensities are in parentheses and are in units of 1000 counts/s.

Several trends were observed during analysis of the CW agent hydrolysis products (Table 5). The addition of the FAIMS device during ESI-MS results in a slight improvement in the S/B ratio, with the greatest increase being observed for MPA in solvent. The difference between the two ionization modes is likely related to the relatively high polarity of the hydrolysis agents, which would favor the ESI source over the APCI source. These results are not believed to be related to the CV settings as the hydrolysis products were equally well-separated by both ESI and APCI and their CV settings are virtually identical. Chemical noise was observed to be lower in negative ion mode than in positive ion mode. As the principal advantage of the use of the FAIMS device was the reduction in chemical noise with no significant change in signal. The effect of FAIMS on S/B ratio was greater in positive mode than in negative mode (data not shown). The results in Table 6 indicate that the use of the FAIMS device somewhat increases the linear dynamic range (the effect is quite small).The detection limits for the CW agent hydrolysis products were best with the ESI sourcewith limits in the 2-7 ng 8262

Analytical Chemistry, Vol. 79, No. 21, November 1, 2007

Table 4. Figures of Merit for the Analysis of CW Agents as a Function of Analyte and Ionization Sourcea limit of detection analyte

mode

FAIMS (Y/N)

linear dynamic range (ng/mL)

concn (ng/mL)

amt injected (ng)

tabun

APCI APCI APCI APCI APCI APCI APCI APCI

N Y N Y N Y N Y

398-1350 3-1350 203 to 1350 5-1350 129-1350 2-1350 33-1350 8-1350

10 0.9 61 1.4 39 0.5 10 2

0.10 0.009 0.61 0.14 0.39 0.005 0.1 0.02

GF sarin soman

a CW agent concentrations ranged from 2 to 1350 ng/mL. The lower level of the linear dynamic range during flow injection MS/MS analysis was defined as the limit of quantitation, namely, 10 times the standard deviation of the blank. The upper limit of the range was the highest concentration analyzed.

range when the FAIMS device was used during ESI-MS/MS analysis. These detection limits were 1 order of magnitude higher

Table 5. Signal-to-Background Ratios for the Hydrolysis Products in the Presence and Absence of FAIMSa ESI-MS

EPA IMPA MPA PinMPA

ESI-FAIMS-MS

APCI-FAIMS-MS

S

W

S

W

S

W

7 ()1830/247) 2 ()663/413) 6 ()1800/287) 2 ()637/310)

4 ()200/57) 6 ()257/44) 5 ()170/32) 8 ()210/27)

13()143/11) 2 ()5/2.1) 97 ()213/2.0) 3 ()25/8)

5 ()11.7/2.3) 8 ()10/1.2) 9 ()15/1.8) 13 ()7.6/0.6)

2 ()15/10) 1 ()29/21) 1 ()6/4) 2 ()49/32)

1 ()3.2/3.3) 3 ()18/5.5) 1 ()0.8/0.7) 3 ()19/7.6)

a Average signal intensity for 20 ng (2 µg/mL; 10 µL Injected) of CW agent hydrolysis product divided by the average signal for the blank solution under optimized FAIMS-MS conditions. Intensities are in parentheses and in units of 1000 counts/s. S, 75:25 acetonitrile/water; W; bottled water. APCI-MS was not performed as there was no clear advantage to the use of this technique for the analysis of the hydrolysis products.

Table 7. Limits of Detection (ng/mL for Water and Oil or ng/g for Honey and Corn Meal) of CW Agent Hydrolysis Products in Solvent and Four Food Products

analyte EPA

Figure 3. Mixture of methylphosphonic acid, ethylphosphonic acid, ethylmethylphosphonic acid, isopropylmethylphosphonic acid, and pinacolylmethyl phosphonic acid infused into a 400 µL/min of mobile phase (75:25 acetonitrile/water + 0.1% formic acid) to a final concentration of 1.25 ng/mL analyte. The compensation voltage was scanned with negative ion mode ESI-FAIMS-MS detection. The following FAIMS conditions were used: DV of -4000 V, OBV set at -10 V, and heater temperature settings of 70 °C. Table 6. Linear Dynamic Range and Limits of Detection (ng/mL) as a Function of Analyte and Ionization Source for the Hydrolysis Productsa limit of detection species EPA

FAIMS linear dynamic concn amt injected mode (Y/N) range (ng/mL) (ng/mL) (ng)

ESI ESI APCI IMPA ESI ESI APCI MPA ESI ESI APCI PinMPA ESI ESI APCI

N Y Y N Y Y N Y Y N Y Y

2000-2500 600-2500 1600-5000 2000-2500 800-2500 3900-4000 600-2500 300-1000 10000-11000 2500-2800 2000-2500 2000-7000

600 200 500 750 200 1200 170 260 3400 840 700 2200

6.0 2.0 5.0 7.5 2.0 120 1.7 2.6 34 8.4 7.0 22

a The lower point of the linear dynamic range is equal to the limit of quantitation (i.e., 10 times the standard deviation of the noise). The upper point refers to the highest concentration that shows a linear dependence of the intensity with concentration.

than those reported by Read and Black,4 but are considered sufficient for food spiking experiments. API-FAIMS-MS Analysis of CW Agent Hydrolysis Products in Spiked Food Products. Preliminary method development work for the CW agent hydrolysis products enabled the selection of optimal detection conditions for the analysis of spiked food products. The limits of detection by SRM were determined for each of the CW agent hydrolysis products spiked into bottled

75:25 FAIMS MeCN/ mode (Y/N) H2O water corn

ESI ESI APCI IMPA ESI ESI APCI MPA ESI ESI APCI PinMPA ESI ESI APCI

N Y Y N Y Y N Y Y N Y Y

600 200 500 229 200 1200 170 60 3400 245 282 1460

263 318 ns 99 1081 512 144 94 ns 208 576 705

nsa 1222 ns ns 2206 ns ns 1090 ns ns 9615 ns

oil

honey

NS 1229 ns ns 3940 ns ns 180000 ns ns 454 2267

ns ns ns ns ns 1409 ns 7544 ns ns ns ns

a ns, could not be successfully determined at the 2.50 µg/mL (water), 6.25 µg/mL (oil), or 6.25 µg/g (corn, honey) level, likely due to matrix suppression.

water, corn meal, canola oil, and honey at the 2.6 and 6 µg/mL (or g) level. The signal intensities for a 10-µL injection of the spiked food products and blanks acquired by API-MS and APIFAIMS are presented in Table 7. Table 7 indicates that CW agent hydrolysis products could not be analyzed at the spiking levels studied. This could be due to the poor ionization efficiency of these hydrolysis products combined or matrix interference effects. The best results were obtained with ESI-FAIMS-MS, although all approaches were unsuccessful for honey or canola oil analyses. These represent the most complex matrixes and so are expected to cause the most ion suppression. API-FAIMS-MS Analysis of CW Agents in Spiked Food Products. Optimized APCI-FAIMS-MS/MS methodology was used for the determination of CW agents spiked into food products at the 3 µg/mL level in canola oil, the 3 µg/g level in cornmeal and honey, and the 1.3 µg/mL level in bottled water. Results were similar to those obtained for the same CW agents in standard solution (75:25 acetonitrile/water + 0.1% formic acid). Detection limits were in general poorer for the spiked food products, likely due to chemical interferences. The LOD results for the four CW agents in each of the matrixes (Table 8) were in the 1-35 ng/mL (or g) range for all the spiked media, and in all cases, it was possible to determine the presence of the CW agent in spiked solvents and food products. The LODs in solvent were in most cases within 1 order of magnitude of the results with bottled water. This difference is Analytical Chemistry, Vol. 79, No. 21, November 1, 2007

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Table 8. APCI-MS/MS Limits of Detection (ng/mL for Water and Oil or ng/g for Honey and Corn Meal) of CW Agents Spiked into Canola Oil at the 3 µg/mL Level, Cornmeal and Honey at the 3 µg/g Level, Bottled Water and Solvent (Define Composition) at the 1.3 µg/mL Level

analyte

mode

FAIMS (Y/N)

tabun

APCI APCI APCI APCI APCI APCI APCI APCI

N Y N Y N Y N Y

GF Sarin Soman

75:25 MeCN/ water 10 0.9 61 1.4 39 0.5 10 2

water

corn

oil

honey

42 3 173 1.5 592 4 103 15

59 12 512 1.3 957 6 377 33

28 15 552 0.9 126 9 52 34

123 17 1349 1.5 971 13 163 18

likely related to the salts present in bottled water, causing suppression of analyte signal and enhancement of background during flow injection analysis. In the case of sarin, which is the smallest of the CW agents, this signal suppression is almost complete, leading to very high LODs. Comparison to Conventional LC-ESI-MS. LC-ESI-MS was evaluated at DRDC Suffield for the bottled water sample spiked with GB, GD, GF, and TEP at 10 µg/mL (10 ng of each compound injected), the level typically used during proficiency testing by the Organization for the Prohibition of Chemical Weapons. Total ion current (m/z 70-500) data were collected for one-third of the scan time (0.4 s with a 0.1-s interscan delay) with the chromatograms illustrated in Figure 4 being typical of spiked bottled water and its corresponding blank. The remaining two-thirds of the scan time (0.9 s with a 0.1-s interscan delay) were spent acquiring product mass spectra for the spiked compounds. The product mass spectra illustrated in Figure 5 were acquired with collision energies that resulted in the acquisition of both the [M + H]+ ion and structurally significant product ions. Similar ESI-MS data were acquired with the Sciex instrument, enabling selection of ions for SRM experiments during FAIMS analyses. Quadrupole time-of-flight sensitivity was estimated based on the acquisition of an interpretable full scan MS or MS/MS data. A conservative sample detection limit for the acquisition of full scan ESI-MS was estimated to be 0.5 µg/mL (0.5 ng per compound injected) based on the present acquisition. TEP, a compound that resists hydrolysis, was detected with the best full scan sensitivity, with detection limits estimated to be