Direct Derivatization and Rapid GC-MS Screening ... - ACS Publications

Aug 11, 2010 - were between 5 and 10 ng/mL APA in aqueous sample, and for identification using full scan EI 100 ng/mL. The development of analytical ...
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Anal. Chem. 2010, 82, 7452–7459

Direct Derivatization and Rapid GC-MS Screening of Nerve Agent Markers in Aqueous Samples ¨ stin*,†,§ Raja Subramaniam,†,‡ Crister Åstot,† Lars Juhlin,† Calle Nilsson,† and Anders O The Swedish Defence Research Agency, FOI CBRN Defence and Security, SE-901 82 Umeå, Sweden, Department of Chemistry, Umeå University, SE-901 87 Umeå, Sweden, and Swedish Armed Forces, National CBRN-Defence Centre, SE-901 76 Umeå, Sweden A rapid screening and identification method based on derivatization and gas chromatography mass spectrometry (GC-MS) has been developed for the detection of alkylphosphonic acids (APAs), the degradation products of organophosphorus nerve agents. The novel method described involves rapid (5 min) and direct derivatization of 25 µL aqueous sample using highly fluorinated phenyldiazomethane reagents (e.g., 1-(diazomethyl)-3,5-bis(trifluoromethyl)benzene). The APA derivatives are then screened by GC-MS negative ion chemical ionization (NICI) and identified by electron ionization (EI) mode. The conditions for the derivatization were optimized using statistical experimental design and multivariate data analysis. Method robustness was evaluated using aqueous samples from an official OPCW Proficiency Test and all APAs present in the sample were conclusively identified. Limits of detection for rapid screening using SIM NICI were between 5 and 10 ng/mL APA in aqueous sample, and for identification using full scan EI 100 ng/mL. The development of analytical methods for the detection of chemical warfare agents (CWAs) and related chemicals has gained momentum since the establishment of the Chemical Weapons Convention (CWC) in 1997. Much of this work has focused on the identification of the major classes of CWAs, for example, nerve agents and their degradation markers. Nerve agents have a short life span and are hydrolyzed in the environment to form stable, specific degradation markers, including alkylphosphonic acids (APAs). Rapid screening methods for APAs are versatile tools used in situations where a large number of samples need to be analyzed. Examples of these include where CWAs have been illicitly used,1 suspected production or spread/leakage from old CWA storage areas, and during the decommissioning of CWA facilities according to the CWC. Such methods facilitate better management of incident sites and allow the intensity and contours of the contaminated area to be determined and possible sources to be located. * To whom correspondence should be addressed. Phone: +46(0)90-106684. Fax: +46(0)90-106800. E-mail: [email protected]. † The Swedish Defence Research Agency. ‡ Umeå University. § Swedish Armed Forces. (1) Barr, J. R.; Driskell, W. J.; Aston, L. S.; Martinez, R. A. J. Anal. Toxicol. 2004, 28, 372–378.

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Rapid screening with a minimum of sample preparation of aqueous samples with no requirement for derivatization of APAs is currently performed using liquid chromatography-mass spectrometry (LC-MS).2-4 An alternative technique for the screening of APAs might be to employ GC-MS, which is more commonly available and often the method of choice in mobile laboratories. The GC-MS technique requires derivatization of the APAs and the derivatives give characteristic mass spectra, and demonstrate better sensitivity. To date, no method has been published for the application of GC-MS to the rapid screening of APAs primarily because sample preparation of APAs for GC-MS analysis requires complex and slow derivatization of the involatile APAs generally by methylation,5 silylation,6 or pentafluorobenzylation.7,8 Among these derivatization techniques, pentafluorobenzlylation has been adopted as the preferred technique for trace analysis, since fluorinated derivatives have high electron affinity and this enhances sensitivity in NICI.7,9 Limits of detection in the range of femtomole to attomole level are often achieved.10 This is particularly important when the availability of samples is limited. The case for the use of pentafluorobenzyl derivatives of APAs is further strengthened by the inclusion of their EI spectra in NIST11 and OPCW Central Analytical Database (OCAD)12 MS libraries. Unfortunately, the derivatization of APAs with the use of pentafluorobenzylbromide (PFBBr) involves a laborious procedure and a long reaction time. The reaction of PFBBr in a homogeneous solvent system (Acetonitrile) requires a base to promote the reaction and the introduction of water in this alkaline solution will hydrolyzes the reagent. The alternative phase transfer reaction (2) Black, R. M.; Read, R. W. J. Chromatogr., A 1997, 759, 79–92. (3) D’Agostino, P. A.; Chenier, C. L.; Hancock, J. R. J. Chrom. A 2002, 950, 149–156. (4) Liu, Q.; Hu, X.; Xie, J. Anal. Chim. Acta 2004, 512, 93–101. (5) Driskell, W. J.; Shih, M.; Needham, L. L.; Barr, D. B. J. Anal. Toxicol. 2002, 26, 6–10. ¨ stin, A. J. Chromatogr., A 2009, (6) Subramaniam, R.; Åstot, C.; Nilsson, C.; O 1216, 8452–8459. (7) Palit, M.; Gupta, A. K.; Jain, R.; Raza, S. K. J. Chromatogr., A 2004, 1043, 275–284. (8) Black, R. M.; Muir, B. J. Chromatogr., A 2003, 1000, 253–281. (9) Fredriksson, S.Å.; Hammarstro¨m, L. G.; Henriksson, L.; Lakso, H.Å. J. Mass Spectrom 1995, 30, 1133–1143. (10) Hofmann, U.; Holzer, S.; Meese, C. O. J. Chromatogr., A 1990, 508, 349– 356. (11) National Institute of Standards and Technology. http://www.nist.gov/. (Accessed May 28, 2010). (12) Organisation for the Prohibition of Chemical Weapons. http://www. opcw.org/. (Accessed May 28, 2010). 10.1021/ac101604n  2010 American Chemical Society Published on Web 08/11/2010

Figure 1. The fluorinated phenyldiazomethane reagents evaluated in this study.

method13 is generally considered to be too laborious. Large amounts of unwanted side-products are also produced by PFBBr10 and excess reagent has to be removed to prevent deterioration of column material. Also, derivatization of APAs with this reagent, especially at low concentrations, is very difficult and selectivity is compromised.14 As a consequence, unlike LC-MS, GC-MS in combination with PFBBr is not popular among analysts for screening APAs. This has led to the discovery of a number of alternative fluorinated reagents.8 However these reagents do not offer any significant advantages over PFBBr and, therefore, are also not widely used. Therefore, we undertook the challenge to define a nonlaborious sample preparation procedure using a reagent that produces rapid and direct derivatization of aqueous samples, which are subsequently detected with high sensitivity in screening and identification procedures for APAs. For that reason, we investigated the potential of four highly fluorinated phenyldiazomethane reagents (Figure 1). Our procedure was optimized using experimental design and multivariate data analysis. As far as we are aware, this is the first published report on rapid screening of APAs by GC-MS. EXPERIMENTAL SECTION Synthesis of Reagents. The reagents were prepared according to the procedure described by X.J.Creary.15 The concentration of the reagents was approximately 0.4 M. The purity of the reagents was checked using 1H NMR spectroscopy and estimated to be between 90% and 96% without distillation. The reagents should be handled with care.10 Synthesis of Reference Derivatives of Reagent R3. Alkyl methylphosphonic acid (10.0 mg) was dissolved in 500 µL acetonitrile (ACN) in a screw-cap glass vial. Reagent R3 (150 µL, (13) Miki, A.; Katagi, M.; Tsuchihashi, H.; Yamashita, M. J. Anal. Toxi. 1999, 23, 86–93. (14) Saha, M.; Saha, J.; Giese, R. W. J. Chromatogr., A 1993, 641, 400–404. (15) Creary, X. J. J. Am. Chem. Soc. 1980, 102, 1611–1618.

0.4 M) in hexane was added through a syringe. The vial was loosely closed so that nitrogen produced would be released; the solution was left overnight at room temperature. The resultant yellow solution was then quenched with formic acid until the color disappeared. The solvent was removed and the residue was dissolved in 2.00 mL CDCl3. Some solution (∼0.6 mL) was removed for NMR spectroscopy in order to determine that the reaction was complete and that no phosphorus containing byproduct were present. Finally, a known volume of the solution was withdrawn and diluted with ACN in order to acquire a solution containing 1.00 mg/mL of derivative. Materials and Chemical Standards. HPLC grade ACN was purchased from Merck (Darmstadt, Germany). All aqueous solutions were prepared using purified distilled water from a Millipore Milli-Q water generator (Billerica, MA). The commercial reagent, PFBBr was purchased from Sigma Aldrich (Steinheim, Germany). Standard solutions (1.00 mg/mL) of methylphosphonic acid 98% (MPA), ethylphosphonic acid 98% (EPA), propylphosphonic acid 95% (PPA), and ethyl methylphosphonic acid 98% (EMPA) were prepared by dissolving commercial standards from Aldrich (Milwaukee, WI). Isopropyl methylphosphonic acid (IMPA), butyl methylphosphonic acid (BMPA), pinacolyl methylphosphonic acid (pinMPA), cyclohexyl methylphosphonic acid (CMPA), and ethyl methylphosphonothioic acid (EMPTA) were prepared from in-house synthesized standards16 and their chemical purities estimated by 1H NMR to be above 95%. A standard solution (1.27 mg/mL in ACN) of the internal standard, deuterated ethyl methylphosphonic acid (CD3-EMPA, also synthesized inhouse16,17) was prepared in ACN. The APAs targeted were selected on the basis of their prevalence as degradation products of nerve agents and their relevance to the CWC. The systematic chemical name, CAS number, structure and acronym for each (16) Petrov, K. A.; Baksova, R. A.; Korkhoyanu, L. V.; Sinogeikina, L. P.; Skudina, T. V. J. Gen. Chem. USSR 1965, 35, 723. (17) Kinnear, A. M.; Perren, E. A. J. Chem. Soc. 1952, 3437–3445.

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Figure 2. Flowchart to show sample preparation procedure.

target compound are presented in Supporting Information (SI) Table S-1. Silylated vials were used throughout unless otherwise stated. The vials were soaked in 5 M nitric acid for two hours followed by oven-drying at 110 °C for 30 min. The dried vials were then soaked in dichloromethylsilane (DCMS) (5%, v/v, in toluene) for about one hour. Finally the vials were rinsed three times with methanol and dried at 110 °C for 30 min. Nitric acid (65% w/v) was purchased from Merck (Darmstadt, Germany), whereas DCMS was purchased from Fluka (Buchs, Switzerland). Sample Preparation. Optimization of the sample preparation procedure (described in the Method Optimization section) resulted in the conditions described below (see also Figure 2). Rapid Screening. The following were mixed in a 2 mL vial: An aqueous sample of APAs (25 µL), 475 µL ACN, 1.0 µL internal standard CD3-EMPA and 4 µL reagent R3 (0.4 M in hexane). The mixture was allowed to react at room temperature with ultrasonic assistance for 5 min. The solution was directly analyzed by GCMS NICI SIM (CH4) in order to screen for APAs rapidly. 7454

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Verification. The same solution was then taken from the GC autosampler and quenched with 1.5 µL acetic acid for 10 min at 40 °C. The solution was then evaporated to dryness under nitrogen. The residue was dissolved in 25 µL hexane and analyzed by GC-MS EI and PICI to identify structures present. GC-MS Analysis. The derivatized samples were analyzed using an Agilent 7890A gas chromatograph coupled to an Agilent 5975C inert XL mass selective detector (MSD) with a triple axis detector. Splitless injections (1 µL) were performed at an injection temperature of 200 °C. The gas chromatograph was fitted with a 2 mm i.d. silanized linear for splitless injections and a DB5-MS capillary column (J&W, 30 m × 0.25 mm i.d., 0.25 µm film thickness). The carrier gas was Helium at a flow rate of 1 mL/ min. The transfer line temperature was 280 °C. The mass spectrometer was operated in the NICI, EI or PICI mode. In NICI and PICI modes, CH4 and i-C4H10 were used as reagent gases. Source temperatures were NICI, 150 °C; EI, 230 °C; and PICI, 300 °C. The ions were acquired in scan mode (m/z 40-600) or selected ion monitoring (SIM) mode. The target ions

acquired in SIM NICI were the phosphonate anion [M-R]where M and R represent the derivative molecular ion and reagent, respectively. The GC oven temperature program for rapid screening was: 60 °C for 1 min, increasing by 30 °C/min to 300 °C with a total run time of 9 min. The GC oven temperature program for verification was: 40 °C for 1 min, increased by 10 °C/min to 280 °C (held for 5 min) with a total run time of 30 min. Mass spectral data analysis was performed using Agilent MSD ChemStation, NIST MS Search and deconvolution software (AMDIS) with NIST, OPCW Central Analytical Database (OCAD) and our own MS libraries. Characterization and Selection of the Derivatization Reagent and CI Reagent Gas. A mixture of nine APAs (5.00 µg/ mL) in ACN were derivatized with reagents R1, R2, R3, and R4 at 40 °C for one hour and analyzed by NICI (CH4 and i-C4H10), GC-MS EI and PICI (CH4 and i-C4H10). The spectra of the derivatives were interpreted and tabulated for characterization. Our own MS library for all derivatives in EI and PICI was created in AMDIS. Also, the integrated TICs of the derivatives were compared to determine the relative sensitivity for selection of a CI reagent gas. Derivatization of aqueous samples was attempted in order to select a derivatization reagent. Reagent R3 was chosen (as stated in the Results and Discussion section) for subsequent experiments. The relative vertical electron affinities (EA) of the fluorobenzyl derivatives of a model compound (methyl methylphosphonic acid) were estimated using the molecular modeling program Spartan ′08 (Wave function Inc., Irvine, CA). The geometry of the neutral molecule was optimized at the B3LYP/6-31G(d) level followed by single point B3LYP/6-31+G(d,p) calculations on the optimized neutral molecule and its anionic form at the geometry of the neutral species. Vertical electron affinities were calculated as the energy difference between the single point energy of the neutral molecule and its anionic form, both corrected for zero-point energy. Method Optimization for Analysis of Aqueous Samples Using Statistical Experimental Design and Multivariate Data Analysis. Method optimization was carried out using design of experiments (DOE). The design of experiments18 software, MoDDE version 9.0, was sourced from Umetrics (Umea˚, Sweden). Screening Step. Parameters were investigated to identify those with a critical influence on the results of derivatization and to define appropriate ranges over which to assess the effects of these parameters. A two-level, full factorial screening design was employed to investigate four parameters critical to the method: 10-90% H2O in ACN, the reaction temperature used for derivatization (from 20 to 60 °C), the derivatization reaction time (from 5 to 115 min), and the concentration of reagent R3 which was in excess (from 10× to 190×) compared to APAs. The ranges over which the effects of these parameters were selected for study were based on the authors’ experiences of derivatization, and practical considerations. Their impact was assessed on the obtained results for a mixture of 5.00 µg/mL EMPA, MPA, and pinMPA, which were assumed to represent (18) Eriksson, L.; Johansson, E.; Kettaneh-Wold, N.; Wikstro¨m, C.; Wold, S. Design of Experiments, Principles and Applications, 3rd revision; Umetric AB: Umea˚/Malmo ¨, Sweden, 2008.

all of the studied APAs. Nineteen experiments (including three center points) were performed. Optimization StepsSurface Modeling. To optimize the derivatization reaction, a three-level full factorial design, representing a quadratic model was employed. Three parameters of the method were investigated. These were 0-10% H2O in ACN, derivatization reaction time (from 5 to 115 min), and concentration of reagent R3 in excess (from 50× to 200×) compared to APAs. The ranges of the parameters selected were based on the results of the screening steps and, again, on practical considerations. Thirty two experiments (including five center points) were performed. Selection of Sample Preparation for Aqueous Samples. Experiments were conducted to determine the most appropriate sample preparation method for aqueous samples. The yields when analyzing a 5.00 µg/mL mixture of EMPA, pinMPA and MPA in aqueous samples were compared following (i) direct derivatization in aqueous media (See Sample Preparation section) and (ii) evaporation followed by derivatization which involved evaporating 500 µL aqueous sample to dryness, adding 500 µL ACN and derivatization with reagent R3. The removal of excess reagent was tested using three common quenching acids, viz. neat forms of acetic, phosphoric, and formic acid, over a range of volumes between 1 and 10 µL. The reagent and its reaction product peaks in NICI and EI were interpreted and the intensities compared before and after quenching, in order to determine the effects of quenching on improving the chromatographic background in NICI and EI. Performance and Validation of the Method. Aqueous samples were prepared as described in the “Sample Preparation” section and were used to determine the following: (a) Screenings and identifications of a “real sample” and a control sample were made in order to verify the performance of the method. The real sample comprised an aqueous sample of OPCW Proficiency Test (PT) 19 (sample W1) fortified with MPA (5 µg/mL), pinMPA (6 µg/mL), 1-methylpentyl methylphosphonate (6 µg/mL) and 4-methylpentyl methylphosphonate (6 µg/ mL). Sample W1 contained the following potential chemical interferences: magnesium sulfate (100 µg/mL), calcium chloride (250 µg/mL), sodium bicarbonate (200 µg/mL), polyethylene glycol dimethyl ether 150 (150 µg/mL), polyethylene glycol dimethyl ether 250 (150 µg/mL), polyethylene glycol 200 (PEG 200) (150 µg/mL), PEG 300 (150 µg/mL), 1,3-propanediol (50 µg/ mL), N,N-diisopropylethylamine (50 µg/mL). The control sample was fortified with 1.00 µg/mL EMPA, pinMPA, and MPA respectively. Both samples were screened with GC-MS using NICI and verification was performed by EI. (b) Recoveries (n ) 3) of the MPA, EMPA, and pinMPA in aqueous samples were calculated by external standard calibration using the synthesized reference derivatives (see Synthesis of Reference Derivatives section), and analyzed by NICI GC-MS. (c) Linearity (n ) 2): aqueous solutions containing MPA, EMPA, and pinMPA at concentrations ranging from between 1 ng/mL and 1000 ng/mL were prepared and analyzed by NICI SIM (CH4). (d) LODs (n ) 3) were calculated based on signal amplitude being three times that of the background noise. (e) Reproducibility (n ) 3) was calculated from recovery data. Analytical Chemistry, Vol. 82, No. 17, September 1, 2010

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Figure 3. Spectra of EMPA derivatives of R3. (A) NICI. (B) EI. (C) PICI. M and R represent the derivative molecular ion and reagent respectively.

(f) PFBBr performance for direct derivatization in aqueous media compared to that of reagent R3.

RESULTS AND DISCUSSION As APAs are polar and some (e.g., MPA) are barely soluble in any solvent except for water, their derivatization is demanding. The use of water tolerant reagents is therefore preferred in order to simplify the sample preparation procedure. The standard diazomethane reagent is water tolerant to some extent and is widely accepted as a reagent for methylation of APAs. However, this reagent offers no advantage in terms of increasing the sensitivity of detection, due to the electron capture mechanism of ionization in the NICI technique. However, fluorinated APA derivatives offer excellent sensitivity in GC-MS NICI. A series of fluorinated phenyldiazomethane reagents were therefore synthesized and used here for the direct derivatization of aqueous APA samples. Reagent Synthesis. Two different routes have been described for the synthesis of the reagents: first from the corresponding 7456

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aldehyde15 and second from the amine.19 Both of these routes were evaluated. As the intermediate in the amine route is a carcinogenic nitroso compound, and the method produced a lower yield of the diazomethane reagent, the aldehyde route was chosen. Characterization. The EI, PICI, and NICI fragmentation behaviors of the derivatives showed the same general fragmentation pattern as Pentafluorobenzyl ester derivatives described earlier20 and are extensively described in the Supporting Information. The derivatives exhibit characteristic fragmentation in EI, thus allowing identification by library matching using AMDIS or NIST MS Search software. Molecular ion information, critical for confirming the identity of derivatives, was generally absent during EI analyses, but was obtained by using i-C4H10, and CH4 PICI. Both reagent gases showed one predominant and well-defined quasi molecular ion [M+H]+. Very high sensitivity was obtained in NICI SIM, since the derivatives produced a single phosphonate anion. This phosphonate anion retained the alkoxy group (except for MPA) of the original nerve agent and is, (19) Overberger, C. G.; Anselme, J. P. J. Org. Chem. 1963, 28, 592–593. (20) Shih, M. L.; Smith, J. R.; McMonagle, J. D.; Dolzine, T. W.; Gresham, V. C. Biol. Mass Spectrom. 1991, 20, 717–723.

Table 1. Relative TIC NICI (CH4) SIM Abundance in % for OPCW PT Aqueous Samples W1 (Normalized to Highest Abundance, R3) relative response, % R1 R2 R3 R4

MPA

pinMPA

1-methylpentyl MPA

4-methylpentyl MPA

7% 5% 100% 67%

7% 2% 100% 94%

9% 1% 100% 100%

0% 69% 100% 88%

therefore, useful for screening. Examples of NICI (CH4), EI, and PICI (CH4) spectra of EMPA derivatives with reagent R3 are illustrated in Figure 3. Selection of Derivatization Reagent and CI Reagent Gas. The sensitivity in negative ion CI mass spectrometry is known to be related to the electron affinity of a molecule. Thus the relative vertical electron affinities (EA) of the fluorobenzyl derivatives of a model compound (methyl methylphosphonic acid) were estimated: R1 ) -0.043, R2 ) 0.035, R3 ) 0.371, and R4 ) 0.161. The results shows an increasing electron affinity in the order R1 < R2 < R4 < R3. This coincides with the experimental data where reagent R3 performed best in direct derivatizations (Table 1) and thus, it was chosen for subsequent experiments. Also, the derivatization yields for R3, as discussed below, were generally good (Table 2). Mass spectrometry using NICI gave better sensitivity than PICI and EI, when fluorinated derivatives were analyzed.20 This was confirmed by our data. The comparison between i-C4H10 and CH4 as reagent gas for APAs show CH4 NICI was almost three times more sensitive than i-C4H10 NICI and was selected as the CI reagent gas (data not shown). Optimization of Methods Using Design of Experiments. Experimental design shows how to conduct and plan experiments in order to extract, in the fewest experimental runs, the maximum amount of information from the collected data. The basic idea was to vary all the relevant factors simultaneously over a set of planned experiments, and then to connect the results using an appropriate mathematical model. The model could then be used for further interpretation, optimization, and predictions.18 Based on the screening and optimization study (see SI), the predicted optimized conditions for derivatization in order to provide maximum yields are as follows: 4 µL (0.4 M) reagent, 5 min derivatization time, and 0% H2O in 500 µL total solution. The prediction data (see SI Figure S-1(C)) shows that derivatization of APAs in ACN is possible with reagent R3 at a 5% H2O content with an insignificant drop in yield for EMPA and pinMPA. However the problematic MPA observed a significant 20% drop in yield which is acceptable since the goal of the method is to speed up the analysis and the identification of the MPA is still possible.

Selection of Sample Preparation Method for Aqueous Samples. Evaporation followed by derivatization is the most common sample preparation approach used when determining APAs in aqueous samples. However, this approach is timeconsuming21 and reported losses in yield for some APAs due to irreversible adsorption on the surface of the glassware. Therefore, two approaches to sample preparation of OPCW PT 19 aqueous samples W1 were investigated and the yields compared: (i) the direct derivatization of aqueous samples; and (ii) evaporation of aqueous samples to dryness, solvent exchange, followed by derivatization. Experimental results showed that the standard derivatization technique (evaporation followed by derivatization) resulted in considerably lower yields as compared to the direct derivatization technique (data not shown). Glass vials used in the experiments were silylated to avoid irreversible adsorption of APAs. Comparing treated and untreated vials for the OPCW PT 19 aqueous samples W1 showed that silylation improved the yield of MPA by 20% and that the yield for the other APAs in the sample were unaffected. At ng/mL concentrations of all APAs, it is, therefore, essential to use silylated vials. Investigation of Background Interference Caused by the Reagent. Excess reagent creates large background peaks in the NICI scanning mode and to a lesser extent in the EI scanning mode. Furthermore it may also react with any free APA contamination in the GC-injector, and so contribute to false positive identifications. Thus, for verification analysis, the effect of post derivatization reagent quenching was investigated. Optimum reduction of background interferences were achieved using 1.5 µL acetic acid at 40 °C for 10 min. Performance and Validation of the Method. Rapid Screening. Under optimum conditions, the derivatization reaction proceeded rapidly and was complete within 5 min. The screening of the phosphonate anions of the APAs in NICI SIM was performed within 9 min analysis time. Figure 4 shows the NICI SIM chromatogram of the OPCW PT 19 aqueous sample W1. Verification. The sample obtained from positive screening was evaporated to dryness with a mild N2 flow and redissolved in 25 µL hexane for verification in EI. Figure 5 (A) shows the GC-MS EI chromatogram of the OPCW PT 19 aqueous sample W1. Samples from Proficiency Test 19 were used to evaluate the robustness of the method for APAs. All APAs were conclusively identified with a good match factor (AMDIS net match >94). Further identification was possible using molecular ion information obtained from PICI (CH4) as shown in Figure 5 (B).

Table 2. Validation Data for R3 Derivatives

analyte

EMPA pinMPA MPA

NICI SIM CH4 (m/z)

123 179 321

LODs (s/n ) 3), ng/mL NICI rapid screening (CH4) (n ) 3)

EI (full scan/ AMDIS) (n ) 3)

PICI scan (CH4) (n ) 3)

5 5 10

100 100 100

500 500 500

correlation coefficient (r2) (1-1000 ng/mL)

recovery at 5.00 µg/mL concentration (n ) 3)

RSD% of recovery (n ) 3)

0,9475 0,9943 0,9025

66% 98% 87%

12 3 11

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Figure 4. Rapid screening chromatogram of OPCW PT19 sample W1. NICI SIM of m/z 179 (pinMPA, 1-methylpentyl MPA, 4-methylpentyl MPA), and m/z 321 (MPA) ions representing the anions of the APAs.

Figure 5. Extracted ion chromatograms (EIC) from verification analysis of aqueous samples W1 OPCW PT in (A) EI and (B) PICI (CH4). EIC EI of m/z 80 (pinMPA, 1-methylpentyl MPA, 4-methylpentyl MPA), and m/z 321 (MPA) ions. EIC PICI of m/z 323 (pinMPA, 1-Methylpentyl MPA, 4-Methylpentyl MPA) and m/z 529 (MPA) ions.

A matrix that negatively influences the efficiency of derivatization of APAs would be noticed by the poor yield of the derivatization control (CD3-EMPA). In such cases, rapid screening in NICI may not be adequate to provide the sensitivity required. A repeat screening step of the concentrated aliquot in NICI would then be preferred. The optimized method achieved quantitative recoveries in the range from 66% to 98% (n ) 3, RSD from 3% to 12%). The method was sensitive, with LODs in SIM NICI mode ranging from between 5 ng/mL and 10 ng/mL, and demonstrated excellent linearity with an average r2 g0.90 over the concentration range from 1 ng/mL and 1000 ng/mL in SIM NICI mode. Summary data for linearity, recovery, reproducibility and LODs are presented in Table 2. 7458

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Comparison with PFBBr. To confirm further the intolerance of PFBBr to direct derivatization in aqueous solution and to demonstrate the advantage of using reagent R3, we used reagent PFBBr and reagent R3 for derivatization of aqueous APAs using the experimental conditions described in the sample preparation section and then compared the relative responses. As expected, results showed that direct derivatization in aqueous solution using PFBBr yielded little or no derivatives in comparison to reagent R3, emphasizing the fact that PFBBr requires the more laborious sample preparation method described by M.Rautio et al.22 (21) Dubey, D. K.; Pardasani, D.; Palit, M.; Gupta, A. K.; Jain, R. J. Chromatogr., A 2005, 1076, 27–33.

CONCLUSIONS In this study, we have demonstrated a novel and powerful derivatization reagent, 1-(diazomethyl)-3,5-bis(trifluoromethyl)benzene (R3), for the determination of APAs in aqueous samples by direct derivatization, rapid screening and their identification using CI and EI mass spectrometry. Only 25 µL aqueous sample is required with a 5 min sample preparation time to give ng/mL (ppb) level sensitivity in NICI MS. The advantage of this will even become more important when the technique is applied to the analysis of biomedical samples. For the unequivocal identification of APAs in aqueous samples, compound-specific ions in NICI and PICI spectra, together with library matching in EI were used. Verification of the identity of, for example, APAs in NICI according to OPCW criteria12 may also be supported by a retention index database which includes the new derivatives. The method described can be used to screen multiple samples collected from active and decommissioned chemical

weapons testing sites, as well as from sites where incidents have taken place. It allows for prompt and accurate decision making regarding the severity and the contour of contamination. It also makes it possible to select appropriate decontamination procedures and rescue operations without compromising on costs or resources.

(22) B.2 Identification of Degradation Products of Potential Organophosphorous Warfare Agents. An Approach for the Standardisation of Techniques and Reference Data; Enqvist, J., Rautio, M., Eds.; The Ministry of Foreign Affairs of Finland: Helsinki, 1980.

Received for review June 17, 2010. Accepted July 27, 2010.

ACKNOWLEDGMENT We are grateful to Lars-Gunnar Hammarstro¨m for calculations of the electron affinity of the reagents used in this study. This work was in part presented orally at the 10th CBW Protection Symposium, Kista, Stockholm, June 8-11, 2010. This study was financially supported by the Public Service Department of Malaysia (R.S) and the Swedish Armed Forces. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

AC101604N

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