Detection of Chemical Warfare Agent-Related Phenylarsenic

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Detection of chemical warfare agent-related phenylarsenic compounds in marine biota samples by LC-HESI/MS/MS Hanna Niemikoski, Martin Soderstrom, and Paula Vanninen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03429 • Publication Date (Web): 06 Sep 2017 Downloaded from http://pubs.acs.org on September 11, 2017

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Analytical Chemistry

Detection of chemical warfare agent-related phenylarsenic compounds in marine biota samples by LC-HESI/MS/MS Hanna Niemikoski*, Martin Söderström, Paula Vanninen VERIFIN, Finnish Institute for Verification of The Chemical Weapons Convention, Department of Chemistry, P.O. Box 55, FI-00014 University of Helsinki, Finland ABSTRACT: A new method has been developed to determine oxidation products of three chemical warfare agent (CWA) related phenylarsenic compounds from marine biota samples by a liquid chromatography-heated electrospray ionization/tandem mass spectrometry (LC-HESI/MS/MS). The target chemicals were oxidation products of Adamsite (DM[ox]), Clark I (DPA[ox]), and triphenylarsine (TPA[ox]). Method was validated within the concentration range of 1-5 ng/g, 0.2-5 ng/g and 0.2-5 ng/g for DM[ox], DPA[ox] and TPA[ox], respectively. The method was linear, precise and accurate. Limits of quantification (LOQ) were 2.0, 1.3 and 2.1 ng/g for DM[ox], DPA[ox] and TPA[ox], respectively. A total of ten fish samples and one lobster sample collected from near Swedish coast, Måseskär dumpsite were analysed. Trace concentrations below LOQ values were detected in three samples and the elemental composition of oxidized form of Clark I and /or II was confirmed by LC-HESI/HRMS. To our knowledge, this is the first study that provides the presence of CWA related chemicals in marine biota samples.

Clark I (diphenylchloroarsine, DA), Clark II (diphenylcyanoarsine, DC) and Adamsite (10-Chloro-5,10dihydrophenarsazinine, DM) were designed as riot control agents during World War I.1 Currently, this type of chemicals would be classified as chemical warfare agent (CWA) by Chemical Weapons Convention.2 Triphenylarsine (TPA) is one component of technical Clark I, so-called arsine oil.3 Structures of these arsenic containing compounds are shown in Figure 1.

Scheme 1. Natural hydrolysis and oxidation of Clark I. Figure 1. Chemical structures of Clark I (DA), Clark II (DC), Adamsite (DM) and triphenylarsine (TPA)

In aqueous environment, Clark I and II degrade into diphenylarsinous acid (DPA[OH]), which dimerizes into bis(diphenylarsinic)oxide (BDPAO) or oxidizes further to form diphenylarsinic acid (DPA[ox]).4; 5 Degradation pathway of Clark I is presented in Scheme 1. Clark I and II form same hydrolysis and oxidation product. Adamsite degrades in a similar way as Clark forming hydrolysis and oxidized products.4; 5 In this paper, oxidation products of Clark I and II, Adamsite and triphenylarsine are referred as DPA[ox], DM[ox] and TPA[ox], respectively. Chemical structures of oxidation products of these compounds are presented in Figure 2.

Figure 2. Chemical structures of oxidation products of Clark I and II (DPA[ox]), Adamsite (DM[ox]) and triphenylarsine (TPA[ox])

DA, DC, DM and TPA were produced in large scale during World War I and II. After World War II their disposal began by sea dumping.6; 7 The Baltic Sea and Skagerrak between Norway and Denmark were areas where the largest dumping

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operations of chemical weapons took place. According to Russian, UK and US reports the total amount of chemical ammunitions (consisting mainly sulphur mustard and arsenic containing agents) dumped within Baltic Sea area by Allies was 40,000 tonnes and in Skagerrak 168,000 tonnes.8 This includes ships sunk near Måseskär, in the eastern Skagerrak, off the Swedish coast.6; 9 Dumped chemical warfare munitions have received international attention during last decade. Two EU projects MERCW (2005-2009) (http://www.mercw.org), CHEMSEA (20112014) (http://www.chemsea.eu) and the NATO SPS Project MODUM (2013-2016) (http://www.iopan.gda.pl/MODUM), have provided information on location of munitions and sediment pollution in Baltic Sea and Skagerrak areas. During these projects chemical analysis were developed and performed from sediment samples collected from these dumping areas.3; 10

During CHEMSEA project, phenylarsenic CWAs were analysed from sediment samples collected from Bornholm deep.11 Phenylarsenic compounds were analysed as propanethiol derivative (TPA was analysed as intact chemical) by gas chromatography tandem mass spectrometry (GC-MS/MS) after dichloromethane extraction. Also oxidation products of phenylarsenic compounds were analysed by liquid chromatography tandem mass spectrometry (LC-MS/MS) after acetonitrile extraction and oxidation step with hydrogen peroxide. 13 samples of 88 contained phenylarsenic compounds. The highest concentration were 210, 1300, 61 and 41 µg/kg dried sediment for Adamsite, Clark-, TPA-and phenyldichloroarsine (PDCA)-related compounds, respectively. PDCA, also known as PFIFFIKUS, is one of the components of arsine oil. For the first time, CHEMSEA project focused on chemical analysis of marine biota. Exposure studies were performed with mussels (Mytilus edulis). Mussels were exposed to a mixture containing DA, DM and α-chloroacetophenone (tear gas) in three different concentration levels. Mussel tissues were analysed by LC-MS/MS. High concentrations of oxidized form of Clark I and Adamsite were detected from exposed mussel tissues. During CHEMSEA project also chemical analysis of fish samples collected from Bornholm deep were performed. The samples were analysed with respect to DM[ox], DPA[ox] and TPA[ox] by LC-MS/MS. One cod sample contained TPA[ox] at concentration level below the limit of quantification (LOQ).12 Investigation carried out by FFI (The Norwegian Defence Research Establishment) in 2002 showed that ammunition dumped in Skagerrak was pierced through by corrosion resulting leakage of CWAs into marine environment.13 Sediment samples collected close to the scuttled wrecks were extracted according to “Recommended Procedures for Sampling and Analysis in Verification of Chemical Disarmament” (ROP).14 This was followed GC-EI/MS analysis. Many of the samples contained DA and TPA. Also BDPAO was identified from some of the sediment samples. The highest concentration of Clark I, TPA and BDPAO were 178, 63 and 137 mg/kg dried sediment, respectively.13 The same samples were analysed by FFI couple years later.9 At this time phenylarsenic compounds were analysed as their propanethiol derivatives by GC-EI/MS. Most of the sediment samples contained these agents, the highest concentrations were 1100 mg/kg for propanethiol derivatives of Clark I and PDCA.

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Hydrolysis products of Clark I and II are reported to have an equal toxicity as the intact chemicals, predicting a long term threat to the marine environment.15 Several investigations have shown that sediment samples are contaminated with CWAs as a result of leaking containers posing a probable threat to marine environment. Semi-quantitative chemical analysis of target chemicals are needed to prove presence of degradation products of dumped chemical warfare agents in the aquatic biota and to support the risk assessment for possible accumulation in the food chain. One part of the ongoing EU-project DAIMON (2016-2019) (www.daimonproject.com) is assessment of marine munitions’ impact on biota. One aim is develop specific and sensitive methods to assess the fate and impact of toxic CWAs on marine biota, in order to improve risk assessment.16 In this paper we describe method development and validation for cod samples in order to investigate possible contamination with phenylarsenic CWAs in fish tissue samples. We applied this validated method for authentic fish and lobster samples collected from Måseskär dumping site which is located east of Skagerrak area. Traces of Clark compounds were found in three samples analysed by LC-MS/MS. Detected concentrations were below the limit of quantification (LOQ) values. Positive samples were analysed by liquid chromatography high resolution mass spectrometry (LC-HRMS) to confirm elemental composition of Clark I and/or II. To our knowledge there are no previous published studies concerning analysis of CWA-related phenylarsenic compounds in marine biota samples. In addition, to our knowledge this is the first time that CWA-related compound has been found in marine biota samples collected near CWA dump site. EXPERIMENTAL SECTION Chemicals and reagents Adamsite and Clark I were synthesized at VERIFIN and triphenyl arsine was obtained from Acros Organics (Belgium). Concentrations of reference chemical stock solutions was determined by NMR. Methanol, acetonitrile (ACN) and n-hexane (all HPLC grade) were obtained from Fisher Scientific (United Kingdom), Fluka (Germany) and VWR International (Belgium), respectively. Formic acid (98 %) and hydrogen peroxide (33 %) was purchased from Sigma Aldrich and VWR International, respectively. Water was purified using a Direct-Q3 UV system (Millipore, Germany). Specimen Biological samples were received from Marine Monitoring AB (Lysekil, Sweden) and contained one lobster (Nephrops norvegicus) and ten fish samples. Three of ten fish samples were collected from reference site (located approximately 20 km from dumping area). Fish samples consisted of four different spiecies: Atlantic cod (Gadus morhua), witch flounder (Glyptocephalus cynoglossus), Norway pout (Trisopterus esmarkii) and saithe (Pollachius virens). Samples were collected from Måseskär deep. All samples were delivered as cut fillet samples with the exception of Norway pout and lobster samples, which were delivered as uncut. The samples were shipped frozen on dry ice and stored at -80 °C prior to analysis. Sample preparation Fish and lobster samples (5 ± 0.5 g) were homogenized using Precellys 24-Dual Tissue homogenizer (Bertin Technologies, Montigny-de-Bretonneux, France). The homogenates were transferred into a 15 ml Fal-


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con tubes. Then 20 ml of ACN and 1.5 ml of hydrogen peroxide was added. Samples were shaken vigorously for 15 minutes followed by centrifugation using Heraeus Multiguge X1R centrifuge (Thermo Scientific, Osterode am Harz, Germany) for 4 minutes at 5000 G. The acetonitrile phase was separated and washed twice with 20 ml of n-hexane. The acetonitrile phase was evaporated to dryness at 45 °C under a gentle stream of nitrogen and reconstituted in 400 µl methanol-water 1:1 (v/v %). Samples were filtrated with 0.45 µm Millex Samplicity filter (Millipore, USA) followed by LCMS/MS analysis. LC-MS/MS analysis The LC-MS/MS analysis was performed on Shimadzu Nexera liquid chromatograph (Kyoto, Japan) coupled with Thermo Scientific TSQ Quantum Ultra triple quadrupole mass spectrometer (San Jose, USA). LC separation was achieved using a Waters XBridge BEH C18 column (2.1 x 100 mm, 2.5 µm) with Waters BEH C18 Vanguard PreColumn (2.1 x 50 mm, 2.5 µm) at 30 °C using a linear gradient of two mobile phases: 0.1 % formic acid in water (A) and 0.1 % formic acid in methanol (B). The gradient was run from 5 % B at 0 min to 100 % B at 3 min. After this the B eluent was kept 100% for 1 min and at 5% for 1 min. The flow rate was 0.4 ml/min and the injection volume was 5 µl. The mass spectrometer was operated in the positive ion mode using heated electrospray ionisation (HESI) technique followed by selected reaction monitoring (SRM). The instrumental parameters were set as follows: capillary voltage 3000 V, capillary temperature 300 °C, vaporizer temperature 275 °C, sheath gas (N2) pressure 50 (arbitrary units), auxiliary gas (N2) pressure 7.0 (arbitrary units) and collision gas pressure 1.0 (arbitrary units). LC-MS/MS method applied in this study was optimized using reference standards of studied chemicals. Transitions and collision energies for studied arsenic compounds are presented in Table 1. Protonated molecules were used as the precursor ions. Table 1. Chromatographic and spectrometric parameters (Q quantifier ion, q qualifier ion) Compound rt [min] [M+H]+ Product ion, m/z

Collision energy [ev]

DM[ox]

2.30

276

230 (Q), 154 (q)

20

DPA[ox]

2.78

263

141 (Q), 152 (q)

25

TPA[ox]

3.07

323

227 (Q), 154 (q)

38

LC-HRMS Analysis The LC-HRMS analysis was performed on Thermo Scientific Orbitrap Fusion mass spectrometer (San Jose, USA) connected to Thermo Scientific Dionex Ultimate 3000 ultra-high performance liquid chromatograph (Germering, Germany). LC separation was done using Waters XBridge BEH C18 column (2.1 x 50 mm, 1.7 µm) at 40 °C using a linear gradient of two mobile phases: 0.1 % formic acid in water (A) and 0.1 % formic acid in acetonitrile (B). The gradient was run from 5 % B at 0 min to 100 % B at 5 min. After this the B eluent was kept 100% for 1 min and at 5% for 2 min. The flow rate was 0.5 ml/min and the injection volume was 5 µl.

The ionisation was done using HESI in the positive ion mode. The instrumental parameters were set as follows: spray voltage 3000 V, source temperature 300 °C, ion transfer tube temperature 350 °C, sheath gas 30, auxiliary gas 10 and sweep gas 0. Mass measurement was done with mass range m/z 50–500 using RF lens at 60% and quadrupole isolation (m/z 50–500) at resolution of 120,000. Mass accuracy of the instrument using external calibration is specified to be ≤ 3ppm. LC-HRMS method was optimized using DPA[ox] reference standard. RESULT AND DISCUSSION

Method Development Different extraction solvents or solvent mixtures were used to optimize recoveries for the studied compounds. Used solvents and corresponding recoveries are summarized in Supporting information (Table S-1). Compared to other tested extraction solvents ACN turned out to be the best choice providing the best recoveries for all studied compounds. Due to tendency of arsenic compounds to react with cellular sulfhydryl groups e.g. free cysteine residues of proteins forming arsenic-protein complexes17-20, hydrogen peroxide was added to the tissue homogenates before extraction step in order to break these complexes and to convert the analytes to the oxidized forms. This improved the recovery compared to a method where hydrogen peroxide was added to fish extract at the end of the sample preparation procedure just before LC-MS/MS analysis (data not shown). Method Validation The method developed in this study was validated. For each studied phenylarsenic-containing compound, two different product ions were measured, the more intense one chosen as the so-called quantifier ion (Q) and the less intense one as the qualifier ion (q) (See Table 1). Both ions are considered as characteristic product ions for the protonated molecular ions of studied compounds. LC– HESI/MS/MS SRM total ion chromatograms for 5 ng/g calibration standard of DM[ox], DPA[ox] and TPA[ox] are shown in Figure 3. All calibration standards for validation studies were prepared in blank cod matrix which was pre-treated as described above. Target chemicals were spiked into cod extracts after sample preparation procedure. A six-point calibration curve for DPA[ox] and TPA[ox] and five-point calibration curve for DM[ox] were applied. Validation runs were performed on three consecutive days. Three calibration curves were obtained each day (total = 9). True values for the concentration of the calibration standards were back-calculated and, thereafter, the mean of measured concentrations in respective calibration samples their standard deviation (SD) and relative standard deviation (RSD) were calculated. Systematic error between measured and theoretical concentration was also determined as well as combined uncertainty. The validation results for DPA[ox] and TPA[ox] were calculated over the linear concentration range from 0.2 ng/g to 5 ng/g (average coefficient of determination R2 = 0,9993 and 0,9978 for DPA[ox] and TPA[ox], respectively). The validation results for DM[ox] were calculated over the linear concentration range 1 ng/g to 5 ng/g (average coefficient of determination R2 = 0.9967). Based on validation studies, the method is considered precise and accurate. The validation results are presented in Table 2. Average calibration curves are



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shown in Supporting information (Figure S1 to S3). Quality sample preparation and carry over from instrumental analysis. control samples (2 ng/g) were analysed between every calibraNo contamination was observed. tion standard batch to establish the performance of the LC-MS instrument. Both solvent and cod extract blanks were analysed to examine the possibility of cross-contamination arising from Table 2. Calculated validation results for DM[ox], DPA[ox] and TPA[ox] spiked in blank cod matrix ranges 1.0-5 ng/g, 0.2-5 ng/g and 0.2-5 ng/g, respectively. Chemical

DM[ox]

DPA[ox]

TPA[ox]

Standard concentration [ng/g]

Average concentration [ng/g]

SD

RSD (%)

Systematic error (%)

Combined uncertainty (%)

n

1.0

1.07

0.09

8.2

7.3

3.9

9

2.0

1.96

0.08

4.3

2.2

2.6

9

3.0

2.87

0.23

7.9

4.2

3.5

9

4.0

3.98

0.20

5.1

0.4

2.3

8

5.0

5.18

0.34

6.5

3.7

3.2

8

QC (2.0 ng/g)

1.92

0.21

11.1

4.2

3.9

9

0.2

0.24

0.09

36.4

21.4

7.6

9

1.0

1.01

0.05

5.4

0.5

2.4

9

2.0

1.95

0.13

6.9

2.4

3.0

9

3.0

2.97

0.12

4.0

0.9

2.2

8

4.0

3.96

0.23

5.8

0.9

2.6

9

5.0

5.06

0.17

3.3

1.3

2.1

8

QC (2.0 ng/g)

1.99

0.19

9.6

0.7

3.2

9

0.2

0.24

0.16

67.2

21.3

9.4

9

1.0

1.06

0.25

23.9

5.7

5.4

9

2.0

2.14

0.58

27.3

7.2

5.9

9

3.0

3.34

0.64

19.3

11.2

5.5

9

4.0

3.97

0.95

23.8

0.6

4.9

9

5.0

5.34

1.47

27.5

6.9

5.9

9

QC (2.0 ng/g)

2.61

0.61

23.6

30.5

7.4

9

LOQ was 2.0 ng/g, 1.3 ng/g, and 2.1 ng/g for DM[ox], DA [ox] and TPA[ox], respectively. The equation used for calculation of LOQ values is presented in Supporting information. Recovery studies were carried out at 3 ng/g concentration level. The recovery samples (n = 5) were analysed on the third validation day. The recoveries were calculated from average calibration curve obtained in the third validation day. Recoveries for studied compounds were 81 %, 53 % and 92 % for DM[ox], DPA[ox] and TPA[ox], respectively. The availability of internal standards (ISTD) (e.g. stable isotope labelled analogues) specific to the target chemicals would further enhance reliability and repeatability of analysis. Analysis of Fish and lobster samples The method developed in this study was optimised for cod tissue matrix. Due to similar type of matrix e.g. low fat content of flatfish (witch flounder) and lobster tissues, it was assumed that validated method can be applied also for these matrices in order to investigate possible uptake of studied CWA related compounds in aquatic biota. A total of ten fish samples and one lobster sample were analyzed by the extraction procedure followed by LC-MS/MS

analysis. Triplicate analysis were performed. Blank samples (cod extract and solvent) were analysed before and after authentic samples analysis. No cross-contamination arose from sample pretreatment and no carry over occurred during instrumental analysis. LC–HESI/MS/MS SRM total ion chromatograms for calibration standard (0.2 ng/g), blank cod extract sample, positive lobster sample and flatfish sample are shown in Figure 4. Despite concentration levels of DPA[ox] in positive samples were below the LOQ value of the validated method, the elemental composition of DPA[ox] was confirmed with high resolution mass spectrometry technique.


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Figure 3. LC–HESI/MS/MS SRM total ion chromatograms for 5 ng/g calibration standard of (A), DM[ox], (B) DPA[ox] and (C) TPA[ox]

Figure 4. LC–HESI/MS/MS SRM total ion chromatograms for DPA[ox] calibration standard (0.2 ng/g) (A), blank cod sample (B), lobster sample (C) and flatfish sample (D)

The qualitative identification of the DPA[ox] was based on EU guidelines.21 In LC analysis, retention time of the identified compound shall not differ more than ±0.2 min from calibration standard sample (see Figure 4). For MS/MS techniques, the maximum permitted tolerance for the ratio of the areas of the q and Q ion is ± 20 % when relative peak intensity is more than 50 % of Q peak. The relative intensity of base peak of 0.2 ng/g calibration standard is 87.4 % and based on EU guidelines the maximum permitted tolerance is 87.4 ± 17.5 %. Relative ion intensities of the lowest calibration standard (0.2 ng/g), lobster and flatfish samples and the identification criteria determined by HESI-LC/MS/MS SRM techniques are presented in Table 3.



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Table 3. Relative ion intensities calculated from ratios of peak areas forDPA[ox]calibration standard (1 ppb) in fish matrix and analysed lobster and flatfish samples. The maximum permitted tolerance of peak intensities is 87.4 ± 17.5 %. Transition m/z 263 →141 (Q)

m/z 263 Ion ratio →152 (q) (%)

Criteria

DPA[ox] calibration STD (1 ng/ml)

639586

558752

87.4

-

Lobster

44304

39070

88.2

OK

Flatfish #1

51807

38802

74.9

OK

Flatfish #2

46672

41059

88.0

OK

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Both chromatographic and spectrometric identification criteria were fulfilled for three authentic samples. The elemental composition (C12H12O2As) of the target chemical in flatfish sample was determined by LC-HRMS analysis and it was found to match the elemental composition of DPA[ox] in reference sample. Due to small amount of lobster sample, only flatfish samples were analysed by LC-HRMS method. LCHESI/HRMS chromatogram for protonated molecule at m/z 263.0048 of DPA[ox] in refenrence sample and in flatfish sample are presented in Figure 5.

Figure 5. LC–HESI/HRMS chromatograms for DPA[ox] in reference sample (A) and flat fish sample (B). The ion chromatogram are showing the target mass with 5 ppm mass accuracy.

To our knowledge, this is the first study that provides a description of the occurrence of CWA related compound found in marine biota samples. In this study, tissue samples were oxidized with hydrogen peroxide during extraction step to confirm pentavalent oxidation state of arsenic containing compounds and only these oxidized arsenic-compounds were analysed. In the case of arsenic related compounds, it’s possible that agents interact with biomolecules e.g. proteins forming stable adducts. Based on this, more studies is needed to evaluate total concentrations of arsenic-containing CWAs in aquatic biota.

ASSOCIATED CONTENT

* E-mail: [email protected]

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We thank Marine Monitoring AB for providing samples for this study and Swedish Agency for Marine and Water Management for funding this project. This publication has been produced with the assistance of the EU, BSR Programme, DAIMON project. The content of this publication is the sole responsibility of its Authors and can in no way be taken to reflect the views of the European Union.

REFERENCES

Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Methods and discussion: Table S-1: Details of solvent/solvent mixtures used in method development and corresponding recoveries. Figure S-1: Average calibration curve for DM[ox]. Figure S-2: Average calibration curve for DPA[ox]. Figure S-3: Average calibration curve for TPA[ox]. Equation 1: LOQ calculation (pdf)

AUTHOR INFORMATION Corresponding Author

(1) Szinicz, L. Toxicology 2005, 214, 167-181. (2) Response to the Director-General’s Request to the Scientific Advisory Board to Provide Consideration on Which Riot Control Agents are Subject to Declaration Under the Chemical Weapons Convention. Organisation for the Prohibition of Chemical Weapons, 2017, SAB-25/WP.1, OPCW web site. https://www.opcw.org/documents-reports/subsidiarybodies/scientific-advisory-board/reports/ (accessed May 2017) (3) Missiaen, T.; Söderström, M.; Popescu, I.; Vanninen, P. Sci. Total Environ. 2010, 408, 3536-3553. (4) Hanaoka, S.; Nagasawa, E.; Nomura, K.; Yamazawa, M.; Ishizaki, M. Appl. Organomet. Chem. 2005, 19, 265-275. (5) Haas, R.; Schmidt, T.C.; Steinbach, K; von Löw, E. Fresenius. J. Anal. Chem. 1998, 361, 313-318.


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Detection of Chemical Warfare Agent-Related Phenylarsenic

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