Polymeric Sorbent with Controlled Surface Polarity: An Alternate for

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Polymeric Sorbent with Controlled Surface Polarity: An Alternate for SolidPhase Extraction of Nerve Agents and their Markers from Organic Matrix Kanchan Sinha Roy, Ajay Kumar Purohit, Buddhadeb Chandra, D. Raghavender Goud, Deepak Kumar Pardasani, and Devendra K. Dubey Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01428 • Publication Date (Web): 26 Apr 2018 Downloaded from http://pubs.acs.org on April 26, 2018

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Analytical Chemistry

Polymeric Sorbent with Controlled Surface Polarity: An Alternate for Solid-Phase Extraction of Nerve Agents and their Markers from Organic Matrix. Kanchan Sinha Roy, Ajay Kumar Purohit, Buddhadeb Chandra, Raghavender Goud D., Deepak Kumar Pardasani, Devendra Kumar Dubey*. VERTOX Laboratory, Defence Research and Development Establishment, Jhansi Road, Gwalior-474002, Madhya Pradesh, India. ABSTRACT: Extraction and identification of lethal nerve agents and their markers in complex organic background have prime importance from the forensic & verification view point of the Chemical Weapons Convention (CWC). Liquid-liquid extraction with acetonitrile and commercially available solid phase silica cartridges are extensively used for this purpose. Silica cartridges exhibit limited applicability for relatively polar analytes and acetonitrile extraction shows limited efficacy towards relatively nonpolar analytes. The present study describes synthesis of polymeric sorbents with tunable surface polarity and their application as a solid-phase extraction (SPE) materials against nerve agents and their polar as well as nonpolar markers from nonpolar organic matrices. In comparison with the acetonitrile extraction and commercial silica cartridges the new sorbent showed better extraction efficiency towards analytes of varying polarity. The extraction parameters were optimized for the proposed method which included the ethyl acetate as extraction solvent and n-hexane as washing solvent. Under optimized conditions method linearity ranged from 0.10 – 10 μg mL-1 (r2 = 0.9327 – 0.9988) for organophosphorus esters and 0.05 – 20 μg mL-1 (r2 = 0.9976 – 0.9991) for nerve agents. Limits of detection (S:N=3:1) in SIM mode were found in the range of 0.03 – 0.075 μg mL-1 for organophosphorus esters and 0.015 – 0.025 μg mL-1 for nerve agents. Limit of quantification (S:N=10:1) were found in the range of 0.100 – 0.25 μg mL-1 for organophosphorus esters and 0.05 – 0.100 μg mL-1 for nerve agents in SIM mode. The recoveries of nerve agents and their markers ranged from 90% to 98% and 75.0% to 95.0% respectively. The repeatability and reproducibility (with relative standard deviations (RSDs) %) for organophosphorus esters were found in the range of 1.35 – 8.61% and 2.30 – 9.25% respectively. For nerve agents, the repeatability range from 1.00 to 7.75% and reproducibility were found in the range of 2.17 – 6.90%.

1. Introduction: Nerve agents such as sarin, cyclosarin, soman, tabun and Vx are considered to be the most powerful and deadliest class of the chemical warfare agents (CWAs)1-6 . These CWAs are most powerful inhibitors of the enzyme acetylcholinesterase, resulting impaired nerve impulses due to accumulation of neurotransmitter acetylcholine7-9. These synthetic organophosphate neurotoxins are highly potent, volatile, and colourless liquids and have deliberately been used in many incidents related to the military and terrorist activities10-13. Syria and Malaysia are the examples of recent event 14,15. To prohibit the development, production, stockpiling, transfer and usage of chemical warfare agents, an international treaty known as Chemical Weapon Convention 16,17 (CWC) has become operational since 1997. The Organization for Prohibition of Chemical Weapons (OPCW) located at The Hague, The Netherlands administers the CWC through its strict verification mechanism18. The verification regime involves unambiguous identification of CWAs and their markers (including precursors, starting compounds, and degradation products) in environmental as well as synthetic sample18,19. These samples are collected from production, storage, and suspected sites of CW activity 20, and contain complex matrices such as soil, water and organic liquids. Identification of aforementioned analytes is highly desired in these matrices1820 .

Organic matrix has high importance from the CWC verification view point because it can be used as vehicle for spread of CWAs, or be used as solvent during synthesis. Analysis of organic sample is also required from the suspected area or production plant. Analysis of organic samples from different sources like engine oil, machine fuel, silicon oil, vacuum pump oil or incineration waste also give information about the use or production of convention related compounds (CRCs). But detection and identification of CRCs from such organic samples is very challenging task because of the similar chemical nature of analytes and matrix 19,20. In addition, most of the organic samples generally contain high hydrocarbon background. Gas chromatography coupled to mass spectrometry (GC-MS) is the most sought after analytical technique for retrospective detection and identification of CWAs21-24. Straight analysis of such organic samples leads to false negative results because analytes peak get masked by the co-eluting background peaks 25. Therefore, extraction and sample clean-up are of utmost importance for analysis of a complex sample. Sample clean-up through extraction process is most reliable tool for chromatographic separations and analysis. However, only a few sample preparation methods are reported in the form of recommended operating procedures 26 (ROPs) for the analysis of CRCs in organic samples in the presence of high matrix

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background 20,25-29. These include concentration, dilution, solvent exchange, liquid–liquid extraction (LLE), and solid phase extraction (SPE). Each type of sample clean up procedure has its own limitations. The applicability of these extraction modes depend on the nature of matrix and background as well as the type of analytes. Liquid–liquid extraction (LLE) with acetonitrile (ACN) and solid phase extraction (SPE) using silica base sorbent are most common methods for extraction of CWAs from hydrocarbon containing organic matrix 25,28. The ACN extraction shows limited efficacy for relatively non polar analytes and further back extraction or solvent exchange is also required 26 . SPE delivers a straightforward and robust means for sample preparation that should chromatographically more superior than LLE. SPE plays an important role in eliminating background by trapping the analyte on sorbent used in the SPE column. Elimination of background chemicals is achieved by partitioning the analytes from background matrix to the sorbent surface. Silica based SPE sorbent (silanol) is generally used for the analysis of CWAs in presence of hydrocarbon and nonpolar background 25,26 . From silica cartridges, extraction of nitrogen-containing basic analytes is difficult due to their strong adsorption on acidic silica surface. Even in ROP, it is clearly mentioned that polar analytes are not properly extracted from silica- cartridges26. Presence of polar moieties such as P=O, P ̶ CN, P ̶ S and P ̶ F make nerve agents reactive towards hydroxyl functionality present on the sorbent and extractant, thereby causing reduction in recoveries. Methanol being a protic solvent is expected to react with labile bonds such as P ̶ F, P ̶ CN and P ̶ S. Therefore, use of methanol in silica cartridge as eluting or extracting solvent may result in hydrolysis of the real agents. Thus there is a desire need for development of new SPE materials that can be used for analytes of varying polarities. Many sorbents are commercially available and also prepared in-house for SPE. They are usually categorized into silica-based, carbon-based and polymeric sorbents30-34. Among these, polymeric sorbents have received great attention towards the SPE due to their stability and tunability for broad range analytes 30,32. For SPE, polymers with various functional characteristics had been developed35-37. The partition coefficients of these sorbents either do not favor the transfer of the target analytes to the sorbent bed or there is irreversible sorption 30. This suggest the use of compound-specific and class-specific sorbents need for complex sample containing huge interfering species. The tailored materials were successfully applied for the extraction of various compounds30-36. In this study, we have sequentially synthesized four different kind of polymers and evaluated them as extractant against polar as well as nonpolar CRCs/CWAs present in organic sample. To circumvent the problems associated with the conventional silica SPE, we selected the polymeric sorbent with higher surface polarity. The extraction efficiency of selected polymeric sorbent was compared with the existing silica based SPE. The polymeric sorbent was finally applied for the solid phase extraction of nerve agents and their markers from organic matrix containing high non-polar background.

2. Experimental Section: 2.1. Chemicals:

2,2-Azobis(2-methyl-propionitrile) (AIBN), 2-hydroxyethyl methacrylate (HEMA), methacrylic acid (MAA), divinylbenzene (DVB), ethylene glycol dimethacrylate (EGDMA), alumina column, tri-n-butyl phosphate (TBP) (chromatographic standard) were purchased from Sigma-Aldrich (New Delhi, India). AIBN was recrystallized twice from ethanol and dried in a vacuum oven for 24 hrs. HEMA, MAA, DVB and EGDMA were passed through alumina column and then vacuum distilled before use. HPLC grade solvents n-hexane, dichloromethane, chloroform, ethyl acetate, acetone, acetonitrile, methanol and silicon oil were purchased from E. Merck (Mumbai, India) and used as received. The normal phase SPE silica cartridges (200mg, 3mL) were obtained from Agilent Technologies (USA). The empty polypropylene SPE tube (3mL) with frits also procured from Sigma-Aldrich (New Delhi, India). The diesel was purchased from a local petrol filling station (Bharat Petroleum, Gwalior, India). CWAs simulants such as O,O-dimethyl methylphosphonate (DMMP), O,O-diethyl methylphosphonate (DEMP), O,O-diisopropyl methylphosphonate (DIMP), ), O,O-diethyl ethylphosphonate (DEEP), O,Odiethyl propylphosphonate (DEPP), O,O-dipropyl methylphosphonate (DPMP), O,O-dibutyl methylphosphonate (DBMP), O,O-diethyl N, N-diethylphosphoramidate (DEDEPA), O,O-dimethyl N,N-dipropylphosphoramidate (DEDEPA), O,O-dioctyl methylphosphonate (DOMP), O,O-dicyclohexyl methylphosphonate (DHMP), were synthesized in our laboratory in microgram quantities and purified as per the reported procedures 38,39. The real agents O-isopropyl methylphosphonofluoridate (sarin), O-pinacolyl methylphosphonofluoridate (soman), diisopropyl fluorophosphates (DFP), O-cyclohexyl methylphosphonofluoridate (cyclosarin), O-ethyl-N,N-dimethylphosphoramidocyanidate (tabun) were used in declared schedule-1 facility of our institute. Caution: Chemical warfare agents are very toxic in nature. These chemicals should be handled in presence of trained professionals in an efficient fume hood equipped with alkali scrubber and must wear appropriate protective gears. Decontamination solution (approximately 15% w/v solution of bleach and alkali) must be kept at the workplace and the solvent waste must be decontaminated proper way.

2.2. Instruments: All the GC–MS analyses were performed in the electron ionization (EI) mode with an Agilent 6890N gas chromatograph equipped with 5973N mass-selective detector (Agilent Technologies, USA). A DB-5MS (Agilent technologies) capillary column (30 m × 0.25 mm I.D., 0.25 µm film thickness) was used as stationary phase. The temperature for GC oven was programmed from 50°C (2 min) – 300°C @ 20°C min−1 (2 min). Helium was used as carrier gas at a constant flow rate of 1.0 mL min−1. The samples were analysed in the splitless mode at an injection temperature of 250°C. A 10 µL micro syringe was used to inject the samples and the injection volume was kept at 1 µL. The EI source was kept at 230°C; ionization energy was 70 eV and quadruple temperature was 150°C. All the qualitative studies were performed in fullscan mode scan range was from m/z 35 to 450 (3.47 scans per second) and selected ion monitoring (SIM) mode the dwell time was 100ms were used for quantitation studies. Infrared spectroscopy data were collected on Thermo FT-IR Nicolet 6700 (Madison, WI USA) spectroscope equipped with deuterated tri glycine sulfate (DTGS) detector between 4000


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Analytical Chemistry and 450 cm-1 as KBr pellets. The FT-IR spectrum (Figure-ESI1 in supporting information) showed presence of characteristic functional groups in polymer. Thermal stability of polymer was studied by thermo gravimetric analysis (TGA) using Pyris-1 TGA, (make Perkin Elmer, USA). P1 and P2 sorbents were separately treated with the heating rate of 20°C min-1 upto 800°C under N2 atmosphere. Thermograms (Figure-ESI-2 in supporting information) showed decomposition of P1 and P2 at 230 °C and 260°C respectively. Surface characteristics of polymer particles were characterized through BET-surface area, pore volume, and pore sizes. These are summarized in Table-2. The shape and size of polymer resins were visualized by the Environmental Scanning Electron Microscopy (ESEM) (FEIQuanta 400 ESEM FEG, FEI, Hillsboro, OR) operated at 10 kV. Images were recorded at 40000x magnification (Figure-ESI-3 in supporting information).

with suitable solvents. Elutate was concentrated up to 500 µL under gentle nitrogen stream. The tri-n-butyl phosphate was used as a chromatographic standard and added (25 µL of stock 100 μg mL-1 solution) to the eluate and 1 µL of that sample was analyzed with GC–MS in EI mode. Recoveries of all analytes were calculated by comparing the peak areas of analytes in control and treated samples. Control samples were prepared by adding analytes at concentration of 6 µg mL-1 (maximum possible extraction of analyte) to the blank extracting solvent just before the GC-MS analysis. Thus matrix matched control samples were prepared similar to treated samples (i.e. preconditioned, loaded, washed and eluted with extracting solvent) with blank n-hexane did not contain the analytes. All the experiments were performed in triplicate runs and the values are shown in figures are average of the triplicate runs

2.3. Synthesis of Materials:

3. Results and Discussion:

In a typical reaction, MAA (20.0 mmol) and EGDMA (40.0 mmol) was dissolved in 25 mL chloroform for ‘P1’ and 25 mL acetonitrile for ‘P2’. The mixture was stirred at room temperature and degassed with nitrogen for 15 mins to remove dissolve O2. Polymerization was induced by addition of AIBN (0.40 mmol) and heating the mixture at 65°C in a thermostat for 18 hrs after sealing the reaction vessel. The obtained bulk polymers was grounded manually with mortar. Unreacted monomers, homomers and oligomers were removed by Soxhlet extraction with methanol over period of 48 hrs. Finally, polymer powder was dried at 60°C in vacuum for overnight.

3.1. Synthesis and Selection of Sorbents:

2.4. Standard and Spiking Solution: Stock solutions of individual analytes (10000 µg mL-1) were prepared in n-hexane. A combined stock solution was prepared in n-hexane containing DMMP, DEMP, DIMP, DEEP, DEPP, DPMP, DEDEPA, DMDPPA, DBMP, DHMP and DOMP each at 500 µg mL-1 and used for the method optimization. Also a combined stock solution for real agents containing sarin, soman, DFP, tabun, and cyclosarin was prepared at 500 µg mL1 in n-hexane. A series of working standards were prepared by proper diluting the combined stock solution with n-hexane in a 10 mL volumetric flask. All the standard solutions were stored at -4°C in the dark. A stock of diesel (10000 µg mL-1) was also prepared in n-hexane for hydrocarbon background. Method optimization was performed in n-hexane matrix and the matrix was spiked with a concentration of 3 µg mL-1 for each analytes (nerve agents as well as their markers) using the intermediate stock (500 µg mL-1) of analytes. To mimic the real scenario, the matrix was spiked with 5000 µg mL-1 of hydrocarbon background using intermediate diesel stock (10000 µg mL-1).

2.5. Solid Phase Extraction Procedure: Two different types of SPE cartridges were home-made by separately packing polypropylene SPE tube with 50 mg of polymeric sorbent P1 and P2 particles. The pre-conditioning of P1, P2 and normal phase silica SPE cartridges were done with 1 mL acetone followed by equilibration with 2 mL n-hexane. Then 1mL of organic matrix sample spiked with selected analytes was loaded on all the pre-conditioned cartridges separately and passed through under the mild nitrogen flow. All the cartridges were then washed with n-hexane. Finally the elution was done

The study was aimed to develop polymeric sorbent that can overcome limitations of currently used sorbents. Generally, the hydrophobicity of polymeric backbone favours extraction of non-polar analytes. It becomes more challenging when background matrix is also hydrophobic. To tame the hydrophobicity of polymeric sorbent, its surface should be endowed with adequate number of polar groups to make it interactable with both polar as well as nonpolar analytes. The appropriate selection of the polymeric sorbent with balanced surface polarity have been achieved by the proper selection of monomer, cross-linker and solvent in the synthesis step. For this, we have first synthesized polymeric sorbent “A”, combination of methacrylic acid (MAA) and divinylbenzene (DVB) at molar ratio 1:2 in CHCl3 (Table-1). The performance of polymer “A” was tested against organophosphorus esters shown in Figure.1 (spiked concentration of each analytes was 3 μg mL-1). Results summarized in Figure.2 indicate less recovery of polar analytes than those of nonpolar analytes, which could be due to nonpolar surface of the sorbent “A”. Therefore, to impart surface polarity, different polymeric sorbents “B”, “C” and “D” were synthesized in CHCl3 by varying the functional monomers and cross-linkers (Table-1). The best extraction efficiency for polar analytes was found with the sorbent “D” (Figure.2). This is because of the higher surface polarity of sorbent “D” compared to other sorbents. This surface polarity was imparted by the presence of carboxyl and carbonyl functional groups of MAA and EGDMA respectively. Therefore, sorbent “D” was chosen for the further study. In addition to the functional groups of monomer and cross-linker, polarity of the solvent used for the synthesis of polymer, also plays an important role in deciding the surface polarity 40,41. Despite pore generating ability of the solvent, it also governs the prearrangement of monomers and cross-linkers according to its polarity 42,43. Nonpolar solvents make the polymer surface hydrophobic 42,43. This is because during synthesis, polar parts of the monomer and cross-linker are less solvated by the nonpolar solvent. As a result, relatively polar parts of monomer remains situated inside the bulk of polymer matrix and make the surface of polymer hydrophobic. Whereas, the polar solvent strongly solvate the polar functional groups of the monomer and cross-linker. It results in the exposure of polar portions towards outer periphery of polymer 42,43. Therefore for fine tuning of the surface polarity of the polymeric sorbent “D”, its synthesis was performed in more polar solvent (acetonitrile)



than CHCl3. The extraction efficiency of both the polymers which were synthesized in CHCl3 (P1) and in ACN (P2) was checked. The extraction ability of polymers P1 & P2 was compared with those of commercial solid phase silica cartridges (200 mg) (Figure.3).

than those obtained from sorbent P1. It could be attributed to high surface polarity of P2 over P1 despite having its lower surface area than P1 (Table-2). Therefore, sorbent P2 was selected for further method optimization.

(a) Table-1: Synthesis of different polymers with different monomers composition Monomers

MAA

HEMA

EGDMA

DVB

A

1

-

-

2

B

1

-

1

1

C

0.5

0.5

2

-

D

1

-

2

-

aPolymer

(b)

aAll the polymer was synthesized in chloroform using bulk polymerization techniques

(c)

Figure.3: Recovery % of selected organophosphorus ester in different extracting solvents using: (a) commercial silica cartridges, (b) polymeric sorbent P1 and (c) polymeric sorbent P2

3.2. Characterization of Poly(MAA-co-EGDMA) Sorbent: Figure.1: Structures of Nerve agents and their environmental markers as well as CWC related organophosphorus ester.

It is evident from the Figure.3 that P1 and P2 sorbents were more efficient than the silica sorbent obtained commercially. Only methanol and to some extent acetone were able to extract the analytes from silica. Whereas both P1 and P2 performed better with all type of extracting solvents. Close look at Figure.3b and Figure.3c, reveals that sorbent P2 gave better recoveries of the polar analytes with relatively more polar extractants

Figure.2: Extraction performances (%) of different polymeric sorbents in Chloroform for selected organophosphorus esters.

Synthesized P1 and P2 sorbents were characterized by the FT-IR analysis. FT-IR spectra are given in Figure. ESI-1 (see supporting information). Similarity of IR spectra indicates similar chemical contribution of polymer matrix of P1 and P2. The stretching vibration of (C=O) the carboxylic acid, aliphatic CH groups and (-C-O-C-) moiety shows the strong peaks at 1725 cm-1 2926 cm-1 and 1160 cm-1 respectively. Broad peaks at 3445 cm-1 is due to the stretching vibration of free -OH group of the carboxylic acid. It confirmed the formation of (MAA-coEGDMA) backbone for both P1 and P2. Thermal stability of polymeric sorbents were checked by thermo gravimetric analysis (TGA). Thermograms (Figure-ESI-2 in supporting information) showed decomposition temperature of 230 °C and 260°C respectively for P1 and P2. SEM images shown in Figure. ESI-3 (see supporting information) provided the information regarding the particle size and shape of P1 and P2. From SEM images, it is evident that the sorbent P1 and P2 have homogeneous regular morphology with average particle diameter of 150-250 nm. The results of BET surface area analysis are shown in Table-2, indicates higher surface area of P1 than that of P2. Sorbent P2 exhibit slightly higher porosity than P1 with


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Analytical Chemistry respective pore sizes of 9.667 nm and 12.451 nm. The total pore volume of P1 and P2 were found to be 0.26 cm3g-1 and 0.17 cm3g-1 respectively.

Table.2: The surface areas, total pore volumes, and average pore sizes Characteristics

P1

P2

BET Surface Area (m2g-1)

106.55

53.49

0.26

0.17

9.677

12.451

Pore Volume

(cm3g-1)

Pore Size (nm)

Surface properties of polymeric sorbent P1 and P2s was obtained from the BET studies

3.3. Optimizations of Extraction Conditions: To obtain the best enrichment factors from sorbent P2, the governing parameters including elution solvent, elution solvent volume, washing solvent and its amount were optimized. Considering the extreme toxicity of CWAs, the initial optimizations were done with the non-toxic organophosphorus esters.

Only acetone and ethyl acetate could recover them .These observations are ascribed to hydrogen bonding capability of fluorine and nitrogen containing agents with –OH group of silica. The DCM and CHCl3 were unable to break the H-bond whereas ethyl acetate (EtOAc) and acetone, owing to their hydrogen bond acceptor property, could recover nerve agents by disrupting the H-bonds. Results enumerated in Figure.4a further illustrate the fact, because the recoveries of nerve agents from silica were relatively inferior with acetone than those obtained with EtOAc (acetone has less H-bond accepting properties than EtOAc). It is important to note that all the extracting solvents were able to recover the nerve agents efficiently from sorbent P2. Here too, the EtOAc showed best performance amongst the all, hence it was used for subsequent extractions (Figure.4b). Further experiments were done to optimize the volume of eluting solvent. Different amounts of ethyl acetate (1 mL, 0.5 mL + 0.5 mL, 1 mL + 0.5 mL and 2 mL) were used to study the effect of eluting solvent volume. Results depicted in Figure.5 show that highest recoveries (75-98%) were achieved with 1 mL EtOAc when used in two portion of 0.5 mL each. No further increase was observed with higher amount of EtOAc. Therefore, 1 mL (0.5 mL + 0.5 mL) EtOAc was fixed as eluting solvent.

3.3.1 Optimization of Elution Solvent and its Volume: The elution of retentates is crucial to determine the overall recoveries of the analytes. To optimize this parameter, various eluting solvents (dichloromethane, acetone, ethyl acetate, chloroform acetonitrile and methanol) were tested to find the best solvent. Results depicted in Figure-3c indicate that ethyl acetate, acetone, acetonitrile and methanol showed good recoveries of the all analytes. However, owing to better chromatographic response in GC-MS analysis, ethyl acetate was selected as eluting solvent. Methanol being a protic solvent is likely to react with the labile bonds (P-F, P-CN and P-S) of real agents, hence it was not preferred as eluting solvent. When real agents were attempted for elution from commercial silica cartridges, their recoveries were almost nil with DCM and CHCl3 (Figure. 4a).

Figure.5: Recoveries (%) organophosphorus ester with different amount volume of EtOAc.

3.3.2. Background Elimination: Presence of complex background (such as diesel fuel) can mask the analytes in total ion current (TIC) chromatogram of GC-MS analysis, rendering difficulty in their identification (Figure.6a). Therefore elimination of background chemicals is essential and it is affected by the washing. Nature of the washing solvent and its volume must be such that it causes minimum or no loss of the desired analytes. A highly complex hydrocarbon background such as diesel fuel also remains adsorbed on the surface of polymer sorbent due to hydrophobic interactions. To eliminate the hydrocarbon background, n-hexane was considered as the washing solvent due to its affinity with hydrocarbon. Results of washings with 1 mL of n-hexane are depicted in Figure.6. After washings, sufficient removal of hydrocarbon background was observed for organophosphorus esters (Figure.6b) and nerve agents (Figure.6c). Further increase of washing solvent to 2 and 3 mL, did not result in further appreciable removal of background hence, 1 mL n-hexane was fixed as washing solvent.

(a)

(b)

Figure.4: Extraction performance of (a) commercial silica cartridges (b) polymeric sorbent P2 for nerve agents.



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3.4. Linearity, Sensitivity and Reproducibility (LOD, LOQ and RSD):

(a)

Linearity range, limits of detection (LODs) and limits of quantification (LOQs) were determined under the optimized conditions. Calibration curves were established with different concentration levels of analytes. Linearity range of the method were found in concentration range of 0.25-10.0 µg mL-1 for organophosphorus esters and 0.05-20.0 µg mL-1 for nerve agents with good correlation coefficients (r2) between 0.9327–0.9991 for all analytes. The recoveries of nerve agents and their markers ranged from 90% to 98% and 75.0% to 95.0% respectively. The LODs and LOQs for selected analytes were determined based on signal to noise (S:N) ratio of 3:1 and 10:1. Table.3 and Table.4 show the LODs and LOQs of all the analytes. Observed LODs of each analyte were re-evaluated by analysis of the identical spiked concentration in n-hexane. It was also observed that S:N ration in LOD of each analytes was more than 3. The intraday/inter-day precision and accuracy were determined by relative standard deviation for selected analytes at three quantification levels in triplicate for three consecutive days for three weeks. The intraday precision for organophosphorus esters and for nerve agents were found in the range of 1.31–8.61% and 1.10–7.75 % respectively. The corresponding intraday mean precision for organophosphorus esters and nerve agents were lies in the range of 2.03–9.47% and 2.04–6.90% respectively that indicates the decent applicability of methods towards the analysis of environmental samples.

(b)

(c)

3.5. Application of Developed Method: Figure.6: Total ion chromatogram obtained from GC-EI-MS analyses of (a) selected analytes spiked in organic matrix with diesel background (b) after washing the diesel background for organophosphorus ester (c) after washing the diesel background for nerve agents.

The developed SPE method was used for the extraction of nerve agents and their non-toxic organophosphorus esters from different organic liquids like silicon oil and vacuum pump oil. The samples were spiked at three different concentrations of the selected analytes. Polymeric sorbent P2 was used to extract the analytes under the optimized conditions. The organophosphorus esters were extracted with good recoveries from silicon oil

Table. 3: Analytical figures of merit for organophosphorus esters under the optimized SPE conditions Analytes

Linearity Range (µg mL-1)

Coefficient of determination (r2)

Intraday repeatability (RSD %)

Inter-day reproducibility (RSD %)

LOD in SIM (µg mL-1)

LOQ in SIM (µg mL-1)

DMMP

0.25-10

0.9959

2.05

2.98

0.07

0.250

DEMP

0.25-10

0.9988

1.82

3.83

0.07

0.250

DIMP

0.20-5

0.9927

2.45

4.60

0.06

0.200

DEEP

0.20-5

0.9327

2.02

3.22

0.06

0.200

DEPP

0.18-5

0.9833

2.00

3.14

0.05

0.180

DPMP

0.18-5

0.9924

2.43

2.94

0.05

0.180

DEDEPA

0.15-5

0.9632

2.16

2.28

0.04

0.150

DMDPPA

0.15-5

0.9741

2.30

2.03

0.04

0.150

DBMP

0.10-5

0.9894

2.57

3.14

0.03

0.100

DHMP

0.10-5

0.9889

2.98

4.68

0.03

0.100

DOMP

0.10-5

0.9349

3.89

5.63

0.03

0.100


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Analytical Chemistry (68.62–93.45%) with RSD ≤ 8.69% as well as from vacuum pump oil (65.26-90.61%) with RSD ≤ 8.62. The nerve agents were also extracted with excellent recoveries from silicon oil (90.82–98.96%) with RSD ≤ 7.20% as well as from vacuum pump oil (87.85-97.63%) with RSD ≤ 8.66% (Table-ESI-1 and Table-ESI-2 see in supporting information). The performance of the developed method also tested with the sample provided by the OPCW in 26th official proficiency test. This sample was successfully extracted using polymer sorbent P2 under the optimized extraction conditions. Results of the analysis are presented in Figure.7. The spiked compound VX was clearly detected in this sample. A comparison of the developed method with reported methods is also presented in Table-5. These data demonstrate that the present method has comparable recoveries and RSDs with other reported methods with better LODs in full scan mode.

Figure.7: Total ion chromatogram obtained from GC-EI-MS of 26th OPCW OPT sample 841 after solid phase extraction using P2 polymeric sorbent under optimized condition.

4. Conclusion: This study has resulted in development of a polymeric sorbent with optimized extraction parameters to extract and enrich the nerve agents from organic sample. By varying the proportions of monomer and cross-linker from 0.5 : 2 to 1 : 2 , the surface polarity was tuned. Role of porogen was also investigated as function of the surface polarity and pore size. Optimum composition of the sorbent was found to be methacrylic acid (MAA) as monomer and ethylene glycol dimethacrylate (EGDMA) as cross-linker in the ratio of 1 : 2 respectively using acetonitrile as porogen. The sorbent was evaluated for the extraction of nerve agents and their polar as well as nonpolar markers from organic samples in presence of hydrocarbon background. A comparison with commercially available silica was also made. The results indicated better performance of polymeric sorbent than that of silica in terms of overall recoveries. The recoveries of nerve agent’s markers ranged from 75.0% to 95.0% by the developed sorbent and 9.0 % to 18.0 % by silica. Various extraction parameters like ethyl acetate as eluting solvent, n-hexane washing solvent and their amounts were optimized for the best enrichment of the analytes. The LODs were obtained in the range of 0.03 – 0.075 μg mL-1 for organophosphorus esters and 0.015 – 0.025 μg mL-1 for nerve agents. These values meet the requirement of analysis of CRCs for verification of CWC. The clean-up and enrichment ability of developed polymeric sorbent towards the analytes of varying polarity, indicates applicability of the method in the real world samples.

ASSOCIATED CONTENT Supporting Information Experimental section and additional contents as noted in text had been also included in Supporting Information.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]

Fax: (+) 0751-2341148

Table. 4: Analytical figures of merits for nerve agents under the optimized SPE conditions Analytes

Linearity Range (µg mL-1)

Coefficient of determination (r2)

Intraday repeatability (RSD %)

Inter-day reproducibility (RSD %)

LOD in SIM (µg mL-1)

LOQ in SIM (µg mL-1)

Sarin

0.05-20

0.9976

2.97

2.72

0.015

0.050

DFP

0.100-20

0.9991

1.00

4.91

0.025

0.100

Soman

0.100-20

0.9996

1.50

2.75

0.030

0.100

Tabun

0.125-20

0.9936

3.13

6.28

0.035

0.150

Cy. Sarin

0.100-20

0.9981

2.46

3.87

0.025

0.100

Table. 5: Comparison of the current method with other reported methods Method

Sample

Analytes

Recovery (%)

LODs# (µg mL-1)

RSDs (%)

Ref.

*SE-GC-MS

n-Hexane

CWAs

80 - 99

-

2.30 - 3.31

28

*LLE-GC-MS

n-Hexane

CWAs

69 - 85

-

3.23 - 4.01

28

*SPE-GC-MS

n-Hexane

Phosphoramidates

70 - 85

1.0

8.2 - 10.3

25

Present study

n-Hexane

Nerve agents and Organophosphorus esters

75 - 98

0.200

1.00 - 9.28

—

*LLE: Liquid-liquid extraction, SE: solvent exchange, MS: Mass GC: Gas Chromatography, SPE: Solid Phase ExtracACS Paragon Plusspectrometry, Environment tion, # Full scan


Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT Kanchan Sinha Roy is thankful to DRDE, Gwalior and DRDO, New Delhi for funding and fellowship. We are thankful to Dr. Pratibha Pandey, Dr. G.K Prasad and Dr. P.K Gutch for their support for characterization of materials. This manuscript is assigned DRDE accession no. DRDE/VTX/005/2018.

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Polymeric Sorbent with Controlled Surface Polarity: An Alternate for

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