In-situ derivatization and headspace solid-phase microextraction for gas chromatography-mass spectrometry analysis of alkyl methylphosphonic acids following solid-phase extraction using thin film

Journal of Chromatography A, 1599 (2019) 17–24

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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma

In-situ derivatization and headspace solid-phase microextraction for gas chromatography-mass spectrometry analysis of alkyl methylphosphonic acids following solid-phase extraction using thin film Hyunsuk Kim a,b , Yungyeong Cho b , Bong Soo Lee a,∗ , Insung S. Choi a,∗ a b

Center for Cell-Encapsulation Research, Department of Chemistry, KAIST, Daejeon, 34141, South Korea Chemical and Biological Defense Department, Agency for Defense Development, Daejeon, 34186, South Korea

a r t i c l e

i n f o

Article history: Received 23 January 2019 Received in revised form 4 April 2019 Accepted 6 April 2019 Available online 8 April 2019 Keywords: In-situ derivative HS-SPME Nerve agents Solid-Phase extraction GC–MS SI-ARGET ATRP

a b s t r a c t A headspace solid-phase microextraction (HS-SPME) method, involving solid-phase extraction and in-situ derivatization using polymeric thin film, was developed for the gas chromatography-mass spectrometry (GC–MS) analysis of the degradation products of nerve agents. The solid-phase extraction (SPE) was performed using poly([2-(Methacryloyloxy)ethyl]trimethylammonium chloride) film on a gold plate prepared via surface-initiated polymerization. The extracted analytes were directly derivatized with N,Obis(trimethylsilyl)trifluoroacetamide (BSTFA) on the plate. Various parameters like fiber type, headspace time, temperature, and amount of BSTFA were optimized. Under the optimized conditions, the relative standard deviations (RSDs) were in the range 7.0–13.1% and the limits of detection (LODs) were measured to be between 10 and 20 pg mL−1 . The application of the developed method was tested using the 35th Organization for Prohibition of Chemical Weapons (OPCW) proficiency test sample. © 2019 Elsevier B.V. All rights reserved.

1. Introduction Alkyl methylphosphonic acids (AMPAs), the degradation products of nerve agents, are classified as Schedule 2 chemicals by the Chemical Weapon Convention and considered chemical markers of nerve agents for the verification of chemical weapons [1,2]. AMPAs can be detected in the wastewater of facilities that develop or produce nerve agents. They have been identified using gas chromatography (GC) [3], gas chromatography-mass spectrometry (GC–MS) [4], liquid chromatography [5], liquid chromatographymass spectrometry [6–8], capillary ion electrophoresis [9], and ion chromatography [10]. GC–MS is most widely used method for chemical weapon verification owing to its excellent separation ability and mass fragmentation reproducibility. AMPAs must be derivatized before GC–MS analysis to change their volatilities. Trimethylsilylation (TMS) [11], tertbutyldimethylsilylation (TBDMS) [12], methylation [13], and pentafluorobenzylation [14] are used for derivatization. Since most AMPA derivatizations are hampered by the presence of moisture, all water must be removed for application to aqueous samples.

∗ Corresponding authors. E-mail addresses: [email protected] (B.S. Lee), [email protected] (I.S. Choi). https://doi.org/10.1016/j.chroma.2019.04.010 0021-9673/© 2019 Elsevier B.V. All rights reserved.

The simplest method to remove water is by drying with nitrogen stream. However, this method is time-consuming and does not remove interfering substances like polyethylene glycol (PEG). Solid-phase extraction (SPE), an alternative method, involves several steps and the removal of the eluting solvent prior to derivatization of analytes. Recently, magnetic dispersive solid phase extraction (MDSPE) [15] and stir bar sorptive extraction [16] have been used for the SPE but still require the elution of analytes and removal of eluting solvent for derivatization. To overcome these drawbacks, elution-free methods, such as GC injection port derivatization combined with ultrasound-assisted dispersive liquid-liquid microextraction [17] and dispersive liquid-liquid microextraction [18], were investigated for improving the efficient derivatization of carboxylic acids. For AMPAs, in-situ derivatization via solidphase microextraction (SPME) [19] and solid-supported liquid extraction [20], have been proposed. However, these proposed methods are limited to tert-butyldimethylsilylation, which is least sensitive to moisture during derivatization. Trimethylsilylation, the most widely used derivatization method, is more useful than tert-butyldimethylsilylation in the identification of AMPAs using GC–MS because of its applicability of the spectral database provided by the Organization for Prohibition of Chemical Weapons (OPCW). The method using the headspace is useful for analyzing volatile analytes by taking only the upper layer in the sample vial. This

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method avoids the disturbance of high-boiling substances because only the volatile part is used for analysis [21]. Recently, headspace solid-phase microextraction (HS-SPME), which uses SPME fibers as the headspace method, has been used [22]. SPME fibers are coated with various materials to adsorb the analytes on the fiber. The application of SPME fibers in the headspace mode improved the selectivity and sensitivity of the analytes due to selective adsorption and the concentration of analytes on the fiber. The SPE of AMPAs using poly(META) thin film has been performed in a previous study [23]. The SPE procedure using thin film was simple and quickly removed water with nitrogen stream. In this study, in-situ derivatization and the HS-SPME method were used for the GC–MS analysis of AMPAs extracted using poly(META) thin film. The developed method was optimized, validated and applied to OPCW proficiency test samples. 2. Experimental section 2.1. Materials chloride [2-(Methacryloyloxy)ethyl]trimethylammonium (META, 80 wt% aqueous solution), copper(II) bromide (CuBr2 ), 2,2 -bipyridyl (bpy), l-ascorbic acid (AsAc), 11,11 -dithiobis[1-(2bromo-2-methylpropionyloxy)undecane (atom transfer radical polymerization (ATRP) initiator), ethyl methylphosphonate (EMPA), pinacolyl methylphosphonate (PMPA), poly(ethylene ˜ Da) pyridine (>99.0%) and Tween® glycol) (PEG, Mn: 200 20 were purchased from Aldrich (Saint Louis). Isopropyl methylphosphonate (IMPA, >99% purity determined using 1 H and 31 P NMR) and cyclohexyl methylphosphonate (CMPA, >99% purity determined using 1 H and 31 P NMR) were obtained from the Agency for Defense Development (South Korea). N,O(BSTFA, derivatization Bis(trimethylsilyl)trifluoroacetamide grade for GC) was purchased from Supelco Inc (Bellefonte, PA). Pure water (18.3 M cm) from Milli-Q direct 8 (Merck. Ltd.) was used. The SPME devices were obtained from Supelco Inc (Canada). The fibers used were coated with 100-␮m polyethylene glycol (PEG), 85-␮m polyacrylate (PA), 65-␮m polydimethylsiloxane/divinylbenzene (PDMS/DVB), and 50/30-␮m carboxen/divinylbenzene/polydimethylsiloxane (Car/DVB/PDMS). 2.2. Instruments All analyses were performed using an Agilent 6890 gas chromatograph equipped with a 5975C mass-selective detector (Agilent Technologies, WI, USA). An HP-5MS Ultra Inert capillary column (30 m × 0.25 mm i.d.; 0.25 ␮m) from Agilent (Folsom, CA, USA) was used for GC–MS. The GC oven temperature was set from 40 ◦ C (hold for 1 min) to 280 ◦ C (hold for 5 min) at a heating rate of 10 ◦ C min−1 . Helium was used as the carrier gas under constant flow mode at a flow rate of 1.1 mL min−1 . The transfer line temperature was maintained at 280 ◦ C. The samples were manually injected in the splitless injection mode into the split-splitless injector containing a deactivated a splitless single-taper liner at 250 ◦ C. The splitless time was 0.75 min. The electron ionization source was maintained at 230 ◦ C with 70-eV ionization energy and the quadrupole temperature was 150 ◦ C. In the selected ion monitoring (SIM) mode, the dwell time was 100 ms and in full-scan mode, the scan range was from m/z (mass-to-charge ratio) 30–550 (1.67 scans per second). The ion for monitoring the analytes was m/z 153. 2.3. Experimental procedure An anion-exchange SPE thin film was prepared using Surface Initiated Activators Generated by Electron Transfer Atom Transfer Radical Polymerization (SI-ARGET ATRP) with [2(methacryloyloxy)ethyl]trimethylammonium chloride according

to a reported procedure [23]. The self-assembled monolayers of ATRP initiator were prepared by immersing a 1 × 1−cm2 gold substrate in a 2-mM ethanolic solution. Polymerization was initiated by transferring the 1 mL of aqueous solution containing 50 ␮L of 2 mM CuBr2 / 4 mM bpy complex solution, 50 ␮L of 20 mM AsAc and 214 ␮L of META to a 24-well cell culture plate containing the substrate with the ATRP initiator, and the reaction was allowed to proceed for 6 h. 2.3.1. In-situ derivatization of AMPAs extracted using poly(META) thin film and HS-SPME The SPE of AMPAs was performed as follows under the optimized condition reported in a previous study [23]. 1 ␮g mL−1 of four AMPAs (EMPA, IMPA, CMPA, and PMPA) solution was prepared with pure water. The prepared poly(META) film coated plate was immersed in 1 mL of the prepared solution for 1 h at room temperature, washed with pure water, and then dried in nitrogen gas. 10 ␮L of BSTFA was dropped on AMPAs-extracted plate and HSSPME using 65-␮m PDMS/DVB fiber at 75 ◦ C and GC–MS analysis was performed. 2.3.2. Method optimization of HS-SPME For the optimization of proposed HS-SPME, AMPAs-extracted thin films were prepared by a subsequent SPE. Stock solutions (1 mg mL−1 ) of the analytes, i.e., EMPA, IMPA, CMPA, and PMPA, were individually prepared with pure water. These stocks were diluted with pure water to form 10 ␮g mL−1 spiked solutions. The prepared poly(META) film coated plate was immersed in 1 mL of the prepared solutions for 1 h at room temperature, washed with pure water, and then dried in nitrogen gas. 2.3.2.1. Fiber selection. Four different fibers were evaluated to determine their extraction efficiencies for the alkyl methylphosphonic acids (AMPAs) used. The fibers were 65-␮m PDMS/DVB, 85-␮m PA, 100-␮m PEG, and 50/30-␮m Car/DVB/PDMS. They were conditioned as recommended by the supplier before use. The plate coated with poly(META) thin film, on which AMPAs are extracted, were inserted into a 10-mL SPE vial and 20 ␮L of BSTFA was dropped on the thin film before the SPME fiber was exposed to the headspace of the sample. The extraction was performed for 30 min at 75 ◦ . This procedure was repeated for all the fibers and analytes using the fresh spiked solution. Triplicate analyses were performed for each analyte and fiber. The best fiber was used for all subsequent analyses. 2.3.2.2. Effect of temperature. Four sets of experiments were performed at 45, 60, 75, and 90 ◦ C using the 65-␮m PDMS/DVB fiber, with 20 ␮L of BSTFA each for HS-SPME. Equilibration time of HSSPME was 30 min. Triplicate analyses were performed at each temperature. The temperature that gave the best results was used for all subsequent analyses. 2.3.2.3. Effect of equilibration time of HS-SPME. Five sets of experiments were performed for 7.5, 15, 30, 45, and 60 min. The 65-␮m PDMS/DVB fiber and 20 ␮L of BSTFA for each experiment were used for HS-SPME. All experiments were performed at 75 ◦ C. Triplicate analyses were performed for each duration. The time that gave the best results was used for all subsequent analyses. 2.3.2.4. Effect of derivative agent amount. Five sets of experiments were performed with 1, 2.5, 5, 10, and 20 ␮L of BSTFA. All HSSPME experiments were performed using 65-␮m PDMS/DVB fiber with equilibration time of 30 min, and an HS-SPME temperature of 75 ◦ C. Triplicate analyses were performed for each BSTFA amount. The amount that gave the best results was used for all subsequent analyses.

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Fig. 1. (a) Degradation pathway of nerve agent and (b) structures of analytes selected for the study.

2.3.3. Validation of the developed method The solutions for linearity tests were prepared from the stock solutions of the AMPAs at six concentration levels ranging from 1.0–1,000 pg mL−1 . Linear regression equation and correlation coefficients were obtained by plotting the peak area vs. concentration for the experiment. The limits of detection (LODs) and limit of quantification (LOQs) of each analyte were estimated at a signalto-noise ratio of 3:1 and 10:1, respectively, by injecting a series of diluted solutions of known concentration. The experiments were performed three times each using the proposed procedure. Blank injections were performed after each injection to ensure that any carry over, if present, was negligible. To obtain the percentage relative standard deviation (%RSD) for the developed method, analysis of AMPAs was repeated seven times at two different concentrations under the optimized headspace parameters. The concentrations studied were 1.0 and 5.0 ␮g L−1 . The optimized parameters were the 65-␮m PDMS/DVB fiber, extraction at 75 ◦ C for 15 min, and derivatization with 1 ␮L of BSTFA. A recovery study was performed in triplicate with 1.0 ␮g L−1 of AMPAs spiked into the 1000 ␮g L-1 aqueous solutions of tween 20, pyridine and PEG. 2.3.4. Application of method The poly(META) film-coated plate was immersed in 1 mL of the 35th OPCW proficiency test sample at room temperature for 1 h. The subsequent processes were performed in the same manner as mentioned before. 3. Results and discussion 3.1. In-situ derivatization of AMPAs extracted using poly(META) thin film and HS-SPME Nerve agents are easily hydrolyzed to form AMPAs in water, as shown in Fig. 1(a). Therefore, AMPAs are used as an important marker to verify the development, storage, and use of nerve agents. In a previous study, we developed an SPE method using poly(META) thin film for organophosphonic acids [23]. Thin-film SPE has several advantages such as ease of use, short preparation steps, and rapid removal of water. The derivatization reaction is essential to analyze AMPAs using GC–MS. BSTFA is the most widely used derivatization reagent for the GC–MS analysis of AMPAs. However, since BSTFA is very moisture-sensitive, water should be completely removed from the sample for the derivatization reaction [24]. Hence, thin-film SPE was thought to be useful for the extraction of AMPAs and removal of water in a short time (1 min). However, it is problematic to perform derivatization and analyze the AMPAs extracted on the thin film using GC–MS. In general, analytes extracted via SPE are eluted for analysis. The solvent used for elution (e.g. methanol) usually reacts with BSTFA; therefore, the elution solution must be removed. The analytical efficiency can be improved if the derivatization reaction is performed without eluting the analytes. In this study, BSTFA

was dropped on AMPA-extracted thin film and the thin film was heated for the reaction to occur. In addition, the derivatized reactants were evaporated from the thin film and absorbed by the SPME fiber for GC–MS analysis. Fig. 2 shows a schematic of the developed method, where AMPAs extracted on the poly(META) film plate are directly derivatized, desorbed from the plate, adsorbed onto the SPME fiber, and analyzed using GC–MS. Four AMPAs were used, as shown in Fig. 1(b), and the SPE conditions for the poly(META) coated plates were the same as those used in our previous studies [21]. The developed method was applied to a 1 ␮g L−1 solution of AMPAs in pure water. As shown in Fig. 3, the peaks of EMPA-TMS, IMPA-TMS, PMPA-TMS, and CMPA-TMS were obtained at 8.43, 8.84, 11.83, and 14.08 min, respectively. In addition, electron ionization (EI) spectra corresponding to AMPA-TMS were obtained. All the spectra show a peak corresponding to the m/z 153 ion, which is the most abundant and representative peak for all the AMPAs, corresponding to the [(H3 C)P(O)(OH)(O = Si(CH3 )2 )]+ ion produced by the loss of the alkyl group from alkoxy and methyl groups of TMS. The presence of four trimethylsilylated AMPAs was also confirmed based on a search of mass spectra in the OPCW central analytical database (OCAD) library [25]. 3.2. Method optimization The method was optimized in terms of the type of fiber, temperature, headspace time, and amount of derivatization reagent used in SPME. 3.2.1. Fiber selection The amount of analyte adsorbed onto the SPME fiber, which affects the sensitivity of analytes in the analysis, depends on the interaction between the analyte and the fiber-coating material. Therefore, the selection of the SPME fiber is an important factor in the analysis. In the GC–MS analysis of the AMPAs (Fig. 4(a)), the PEG and PA fibers showed much lower total ion chromatography (TIC) intensities than the other two fibers used in this study. Generally, PEG and PA fibers have been used for the analysis of polar analytes. Although AMPAs are polar chemicals, their non-polar nature is increased upon trimethylsilyl derivatization. Therefore, PDMS/DVB or CAR/DVB/PDMS fibers that are excellent for the adsorption of nonpolar materials are suitable. In this experiment, the PDMS/DVB fiber showed higher GC–MS intensity than the CAR/DVB/PDMS fiber. 3.2.2. Effect of temperature Temperature plays two roles in this analytical method: one is to provide the reaction energy for the silylation of the AMPAs and the other is to volatilize the silylated AMPAs. To study the effect of temperature, BSTFA, a derivatizing reagent, was dropped on the AMPA-extracted thin film, which was heated at 45, 60, 75, and 90 ◦ C.

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Fig. 2. Schematic procedure of developed method including steps such as SPE, HS-SPME, and GC–MS analysis.

Fig. 3. Analysis results of AMPAs using developed method (a) TIC and EI mass spectra of EMPA-TMS (c) IMPA-TMS (d) PMPA-TMS, and (e) CMPA-TMS.

As shown in Fig. 5, the best adsorptions for all the analytes were observed at 75 ◦ C. The adsorption seems to be less at 45 and 60 ◦ C due to insufficient derivatization and evaporation of the AMPAs. On the other hand, at 90 ◦ C, the adsorbed analytes on the fiber were desorbed again owing to the high temperature, resulting in reduced TIC intensity compared to that at 75 ◦ C. HS-SPME was performed at 75 ◦ C and the temperature of the SPME vial was raised to

90 ◦ C before GC–MS analysis. The same result as that for HS-SPME at 90 ◦ C was obtained and it can be confirmed that desorption of analytes reduces the TIC intensity at high temperatures. As shown in Fig. 4(b), the decrease in the TIC intensity of CMPA was relatively less than that of the other AMPAs when the HS-SPME temperature was increased from 75 to 90 ◦ C. Based on the retention time in GC–MS, the boiling point of trimethylsilylated CMPA is the highest

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Fig. 4. Optimization of (a) SPME fiber, (b) temperature, (c) HS-SPME time, and (d) the amount of derivatizing reagents, respectively.

Fig. 5. Reaction mechanism of BSTFA and AMPAs extracted by thin film.

among all the analytes. These results show that desorption from the SPME fiber by heating depends on the boiling point of the analyte. These results could be explained by the partition coefficients (K) value. The K value at a fixed temperature for an analytes between the SPME fiber and air can be calculated using Eq. (1) [26]. K=C fiber /C air

(1)

Cfiber and Cair are the concentration of the analyte on fiber and in the air. From Eq. (1), it can be concluded that larger the K value, higher is the amount of analyte adsorbed on the fiber. logK = a

1 T

+b

(2)

The effect of temperature on the K value is described by Eq. (2), which indicates that the K value decreases as the temperature increases. Therefore, the reduction in the intensity of AMPAs-TMS at higher temperature could be explained by their K values. 3.2.3. Effect of equilibration time of HS-SPME In order to optimize the headspace time, HS-SPME was performed for 7.5, 15, 30, 45, and 60 min. As shown in Fig. 4(c), the best adsorptions for all the analytes were achieved after 15 min. As per the procedure recommended in the blue book of chemical weapon verification, the trimethylsilylation of AMPA using BSTFA was typ-

ically carried out for 1 h at 70 ◦ C [27]. In the developed method, the headspace time includes the time required for the silylation of AMPAs, evaporation of trimethylsilylated AMPAs from the plate, and their adsorption onto the SPME fiber. One of the reasons why the above-mentioned processes can occur in a short time is that the derivatization reaction of AMPAs extracted onto the plate is very fast due to the binding of AMPAs with ammonium groups on the poly(META) film. As shown in Fig. 5, the trimethylsilylation reaction occurs when the oxygen atom of the AMPA nucleophile attacks the silicon atom of BSTFA and the monotrimethylsilyl trifluoroacetamide group leaves the BSTFA. In particular, AMPA is extracted via deprotonation by the ammonium group in the thin film, which can increase the reactivity, shortening the reaction time. In addition, the reaction time may be further shortened because neat BSTFA is used without a solvent in the derivatization reaction.

3.2.4. Effect of derivative agent amount The optimal amounts of derivatizing reagents used in this analytical method were examined. In this study, BSTFA was placed on the thin film by using an autopipette (Photographs of the procedure are provided in Fig. S1). Various volumes of BSTFA (1, 2.5, 5, 10 and 20 ␮L) were used in this study, in which the entire surface of the thin film was covered with only 1 ␮L of BSTFA, as shown in Fig. S2. As shown in Fig. 4d, the TIC intensity of AMPA-TMS increased

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Table 1 Linear regression equation, correlation coefficients (R2 ), linear range, LOD, LOQ and repeatability of each AMPAs. Analytes

EMPA IMPA CMPA PMPA

Linear regression equation y = 1.13 × 104 y = 1.61 × 104 y = 3.81 × 104 y = 2.86 × 104

x – 2.30 × 105 x – 1.91 × 105 x – 1.06 × 106 x – 3.73 × 105

R2 0.9978 0.9976 0.9955 0.9987

Linear range (ng mL−1 )

LOD Full scan (ng mL−1 )

SIM (pg mL−1 )

1.0 – 1000 1.0 – 1000 1.0 – 1000 1.0 – 1000

30 30 20 60

10 10 20 10

with decreasing amount of derivatization reagent. Generally, with increasing amount of reagent used in a reaction, the amount of product increases. Therefore, the TIC intensity of AMPA-TMS should be improved if the derivatization reagent is increased. In this study, the TIC intensity increased with decreasing derivatization reagents because the derivatization reagent can also be adsorbed on the SPME fiber and the amount of AMPA-TMS adsorbed on the fiber is reduced. The results shown in Fig. S3 indicate the relation between the amount of the BSTFA dropped on the film and corresponding amounts of BSTFA on the SPME fiber. The peak at 5 min was corresponds to BSTFA and the peaks between 1 and 4 min are attributable to the degradation products of BSTFA in GC injector. The usage of 1.0 ␮L of BSTFA causes a lower abundance of peaks as compared to that of 2.5 ␮L of BSTFA, indicating that as the amount of BSTFA increase, the concentration of AMPA-TMS adsorbed on the SPME fiber was decreases. Therefore, based on by the GC–MS analysis, the absorbed amount of AMPA-TMS was higher because that of BSTFA was smaller. 3.3. Validation of developed method The developed method was validated by using the optimized condition, with 1 mL of the standard aqueous solution. The linear calibration of each AMPA was obtained by plotting the mean value of the peak area by varying the concentrations of the AMPA using GC–MS SIM- mode analysis. The molecular ion with m/z 153 ion was selected for the SIM-mode analysis for trimethylsilylated AMPAs. The correlation coefficient was >0.995 in the range of 1 – 1000 pg mL−1 . The RSDs obtained from seven GC–MS analyses of the trimethylsilylated AMPAs using the proposed method were found to vary from 9.8–13.1% for 1 ␮g mL−1 spiked solutions and from 7.0–8.0% for 5 ␮g mL−1 spiked solutions (Table 1). This indicates that this method has good reproducibility. LODs for the analytes using the proposed method were defined as the point when a GC–MS chromatographic peak with a signalto-noise ratio greater than 3 is obtained in the scan and SIM modes. The LODs of the silylated AMPAs using the proposed method were found to vary from 20 to 60 ng mL−1 in the scan mode and from 10 to 20 pg mL−1 in the SIM mode. The LODs of the proposed method satisfies the concentration criterion of the target, i.e., 1 ␮g mL−1 or more, in environmental samples like water, soil, and waste solvent for chemical weapon verification. These low LODs are due to the enrichment of the analytes on the SPME fiber. There have been studies on improving the analytical sensitivity of analytes by immersing the SPME fiber in a water sample to increase their concentration [19]. Similarly, in the headspace, the concentration of the derivatized AMPAs can be increased in the SPME fiber, leading to improved analytical sensitivity and LODs. The LOQs of the developed method were also defined as the concentration at which a signal-to-noise ratio higher than 10 was obtained for GC–MS chromatographic peaks in the SIM modes. Recovery study was also conducted using the 1.0 ␮g L−1 of AMPAs spiked into the solutions containing 1000 ␮g L−1 of interference such as tween 20, pyridine and PEG aqueous solutions. Pyridine is widely used as an aprotic polar and basic solution and Tween 20 is a commonly used as a detergent or an emulsifier.

LOQ (pg mL−1 )

Repeatability (%RSD, n = 7) 5 ␮g mL−1

1 ␮g mL−1

80 80 70 100

7.2 7.4 7.0 8.0

9.9 10.0 9.8 13.1

Fig. 6. Recovery study of various interfering substances.

Furthermore, PEG is practically used as an interference chemical in aqueous samples for OPCW proficiency tests. The recovery of each AMPAs with difference interfering substances using developed method is shown the Fig. 6. While the recovery of AMPAs in PEG and Tween 20 was 70–80%, the recovery of AMPAs in pyridine was 50–60%. This result is similar to the previous recovery study of poly(META) thin film SPE using IR [21]. 3.4. Application of method The developed method was applied to the 35th OPCW proficiency test sample to evaluate its robustness and applicability. Samples related to chemical weapons are difficult to obtain realistically. Therefore, the developed method was evaluated by using the OPCW proficiency test sample prepared under the assumption that it was taken from a chemical weapons inspection area. OPCW proficiency test is performed to qualify as an OPCW-designated laboratory. When the developed test method was applied to test sample 351, PMPA, a degradation product of the nerve agent Soman, was found, along with high concentrations of interfering substances like PEG. The result of the developed method was compared with those of a blank and reference chemical, PMPA. The result of the sample GC–MS scan mode analysis in accordance with the OPCW proficiency test report format is shown in Fig. 7. The absence of the PMPA in thin film, SPME fiber, and the instrument was seen in the blank sample (a thin film that was not used for SPE) analysis (Fig. 7a). As shown by the GC–MS analysis results in Fig. 7b, for the solid-extracted sample, the peak was clearly observed in the scan mode analysis, unlike the blank sample. This peak was confirmed by analysis of 10 ␮g mL−1 of PMPA as the reference chemical (Fig. 7c). In sample 351, 5 ␮g mL−1 of PMPA was spiked. A simple comparison of the analysis results of the sample and reference, indicated the presence of approximately 4.35 ␮g mL−1 of PMPA in the sample. The recovery was 87%, as 5 ␮g mL−1 of PMPA was actually spiked during sample preparation. 3.5. Comparison with SPE using SAX cartridge A commercially available cartridge filled with a strong anion exchange (SAX) or mixed anion exchange (MAX) resin has been

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methanol, drying of the solvent, and derivatization. The method followed in this study involved the immersion of a poly(META) film-coated plate into the sample, washing with pure water, drying with nitrogen stream, and HS-SPME with in-situ derivatization (Fig. 8(b)). This procedure is simple compared with that involving the SAX or MAX cartridge. In addition, this method does not require the use of a solvent, and is therefore compatible with green analytical chemistry, which has been of great interest in recent years [28]. The sample preparation time of the developed method for one analysis was approximately 1 h 16 min. In the case of the cartridge, the sample preparation time was not provided in the literature, but only derivatization time of 1 h was used for sample except the entire SPE procedure [27]. These results clearly show that the developed method is more efficient than using the cartridge. Furthermore, the simplicity of the developed method is advantageous for the treatment of large number of samples. In addition, LODs of SAX and MAX for IMPA were 100 and 50 ng mL−1 , respectively, for the scan mode analysis. These results demonstrate the improved sensitivity of the developed method (LOD of IMPA: 30 ng mL−1 in scan mode) in comparison with SAX or MAX. 4. Conclusion Fig. 7. GC–MS analysis results of 35th OPCW PT of (a) blank, (b) sample, and (c) reference (10 ␮g L−1 of PMPA-TMS).

An HS-SPME method coupled with SPE was developed using a poly(META) film-coated plate for GC–MS analysis of AMPAs. Insitu derivatization was used to desorb the AMPAs from the plate by dropping BSTFA onto the plate and heating it. Various HSSPME parameters were optimized. An analysis of the HS-SPME time showed that AMPA was derivatized on the plate in a short time and adsorbed onto the fiber. The LODs of the developed method were very low in both the scan and SIM modes of GC–MS analysis. It is believed that there is a high concentration of analytes on the SPME fiber. The developed method was successfully validated using samples provided by the OPCW via the 35th OPCW proficiency test. It is suitable for green analytical chemistry, which is currently of interest because it does not use solvents during sample preparation. Although this study focused on the analysis of AMPAs for chemical weapon verification, this method can also be used for the analysis of organic acids. The range of the analysis target can be expanded further by changing the material of the thin film. Acknowledgements This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (NRF-2017R1D1A1B03027858) and Agency for Defense Development. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.chroma.2019. 04.010. References

Fig. 8. The sample preparation procedure for GC–MS analysis of (a) SPE using commercially available SAX cartridge and (b) HS-SPME using poly(META) film coated plate.

usually used for sample preparation of AMPAs [27]. Sample preparation using SAX or MAX for GC–MS analysis involves complex steps (Fig. 8(a)), such as cartridge cleaning, conditioning, sample loading, washing with pure water, cartridge drying, elution with acidic

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