Method for Analyzing Lewisite Degradation Products Using Cysteine

Abstract

The present invention relates to a method for analyzing Lewisite degradation products using cysteine. Specifically, the invention relates to a method for analyzing Lewisite degradation products comprising: (a) a derivatization step of reacting cysteine ​​with a Lewisite degradation product contained in a sample to form a cysteine ​​derivative of the degradation product; (b) a step of separating the derivative; and (c) a step of determining the presence or concentration of the Lewisite degradation product from the separated derivative; an application for analyzing Lewisite degradation products to evaluate the presence or concentration of Lewisite degradation products in an environmental sample using the above method; and a kit for analyzing Lewisite degradation products used for analyzing Lewisite degradation products based on mass spectrometry after converting the Lewisite degradation products in the sample into cysteine ​​derivatives. The method for analyzing Lewisite degradation products according to the present invention overcomes the problem of low ionization efficiency under ESI conditions in LC-MS analysis through derivatization using cysteine, thereby enabling the detection of CVAA, a major degradation product of Lewisite, with high sensitivity and reproducibility. Accordingly, by complementing the limitations of existing GC-MS and LC-MS-based analytical methods, this invention provides an analytical technique capable of rapidly and accurately evaluating the presence or concentration of Lewisite degradation products in various environmental samples.

Description

Technology Field

[0001] The present invention relates to a method for analyzing Lewisite degradation products using cysteine. Specifically, the invention relates to a method for analyzing Lewisite degradation products comprising: (a) a derivatization step of reacting cysteine ​​with a Lewisite degradation product contained in a sample to form a cysteine ​​derivative of the degradation product; (b) a step of separating the derivative; and (c) a step of determining the presence or concentration of the Lewisite degradation product from the separated derivative; an application for analyzing Lewisite degradation products to evaluate the presence or concentration of Lewisite degradation products in an environmental sample using the above method; and a kit for analyzing Lewisite degradation products used for analyzing Lewisite degradation products based on mass spectrometry after converting the Lewisite degradation products in the sample into cysteine ​​derivatives. Background Technology

[0003] Lewisite (L) is an organoarsenic chemical agent containing arsenic (As) and is one of the representative chemical weapon substances exhibiting potent blistering and systemic toxicity. Lewisite was produced and stockpiled in large quantities during World War I and World War II, and to this day, it is classified as a substance subject to strict international control and disposal under the Chemical Weapons Convention (CWC). However, a significant number of chemical weapon warheads produced in the past have not been fully recovered and remain buried in inland and coastal areas around the world; as time passes, concerns continue to be raised regarding the potential release of chemical agents into the environment due to the corrosion of the warheads or marine activities.

[0004] Leakage of chemical agents from such buried chemical weapon warheads can cause bioaccumulation or histological lesions, and is recognized as a serious risk factor in terms of environmental and human safety. Therefore, there is a need for the development of analytical technologies to assess whether there is leakage from buried chemical weapon warheads and to rapidly and accurately identify the types of chemical agents present.

[0005] Lewisite is relatively unstable in the environment and undergoes rapid hydrolysis upon contact with atmospheric moisture or water. During this hydrolysis process, Lewisite forms various arsenic-containing degradation products, which have also been reported to exhibit high toxicity. Consequently, in the analysis of actual environmental samples, samples suspected of exposure, or post-hoc analyses, it is considered a more reliable approach to analyze the degradation products rather than the raw Lewisite material itself.

[0006] Approximately 90% of inorganic Lewisites exist in the L1 form, and L1 and L2 rapidly hydrolyze in the presence of moisture to produce 2-chlorovinylarsonous acid (CVAA) and bis(2-chlorovinyl)arsonous acid (BCVAA) (Non-patent Literature 1). These degradation products can be converted into various oxidized forms through additional dehydration or oxidation reactions, but CVAA is recognized as a key indicator degradation product for determining the presence of Lewisite exposure. Accordingly, analytical techniques for accurately and sensitively detecting CVAA have been continuously studied.

[0007] Conventionally, various chromatography-based analytical techniques, such as gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), and LC-ICP-MS, have been utilized for the analysis of Lewisite degradation products, including CVAA. In the case of GC-MS analysis, CVAA requires a derivatization process due to its low volatility, rapid hydration reaction, high reactivity with alcohols and thiol compounds, and adsorption characteristics to GC liners and columns. However, this derivatization process has limited practical analytical applications due to reduced analytical reproducibility, column contamination, and difficulties in equipment maintenance.

[0008] Meanwhile, although LC-MS analysis has been recognized as a technique advantageous for the analysis of non-volatile and polar compounds, it has been reported that CVAA exhibits low ionization efficiency under electrospray ionization (ESI) conditions, which are the most commonly used in LC-MS. As a result, it is difficult to secure sufficient detection sensitivity for CVAA in LC-MS analysis; on the other hand, since the oxidized form, CVAOA, exhibits relatively high ionization efficiency, LC-MS-based analytical methods have been developed primarily focusing on CVAOA (Non-patent Literature 2).

[0009] To overcome these limitations, some prior studies have proposed derivatization-based LC-MS analysis strategies to improve the ionization efficiency of CVAA. However, existing reported analytical methods have had limitations in being utilized for the analysis of actual environmental samples or as standardized analytical methods because they have not been sufficiently systematically verified regarding derivatization reaction conditions, reactant stability, reproducibility, and matrix interference effects.

[0011] Therefore, in order to rapidly and accurately determine the presence of Lewisite exposure, there is a need to develop a new analytical approach that targets Lewisite degradation products such as CVAA, overcomes the limitations of existing analytical techniques, and ensures ionization efficiency, reproducibility, and reliability. In particular, there is a growing need for a systematic and validated analytical strategy that can overcome the ionization limitations under ESI conditions while maintaining the advantages of LC-MS-based analysis.

[0013] Against this backdrop, the inventors have made research efforts to overcome the technical limitations of Lewisite degradation products, particularly 2-chlorovinylarsonous acid (CVAA), exhibiting low ionization efficiency under electrospray ionization (ESI) conditions based on LC-MS, and to develop an analytical method applicable to actual environmental samples. As a result, considering the chemical characteristics of CVAA, derivatives using cysteine ​​are stably detected in LC-MS analysis and exhibit improved ionization efficiency under ESI conditions. Furthermore, by applying the analytical method to environmental samples, it was confirmed that CVAA can be reliably detected even within various matrices, thereby completing the present invention. Prior art literature

[0015] Journal of Chromatography A Volume 1000, Issues 1-2, 6 June 2003, Pages 253-281Journal of Analytical Toxicology, Volume 40, Issue 6, July / August 2016, Pages 419-426 The problem to be solved

[0016] The object of the present invention is a method for analyzing Lewisite degradation products,

[0017] (a) a derivatization step of reacting cysteine ​​with a Lewisite degradation product contained in a sample to form a cysteine ​​derivative of the degradation product;

[0018] (b) a step of separating the above derivative; and

[0019] (c) a step of determining the presence or concentration of the Lewisite degradation product from the separated derivative; comprising,

[0020] This provides a method for analyzing Lewisite degradation products.

[0021] Another objective of the present invention is to provide a use for analyzing Lewisite degradation products to evaluate the presence or concentration of Lewisite degradation products in an environmental sample using the above method.

[0022] Another objective of the present invention is to provide a kit for analyzing Lewisite degradation products, which is used to analyze Lewisite degradation products in a sample after converting them into cysteine ​​derivatives and then analyzing them based on mass spectrometry.

[0024] The problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art from the description below. means of solving the problem

[0026] One aspect of the present invention for achieving the above objective is a method for analyzing Lewisite decomposition products, wherein

[0027] (a) a derivatization step of reacting cysteine ​​with a Lewisite degradation product contained in a sample to form a cysteine ​​derivative of the degradation product;

[0028] (b) a step of separating the above derivative; and

[0029] (c) a step of determining the presence or concentration of the Lewisite degradation product from the separated derivative; comprising,

[0030] A method for analyzing Lewisite degradation products is provided.

[0031] The above Lewisite degradation product may be one or more selected from the group consisting of 2-chlorovinylarsonous acid (CVAA), bis(2-chlorovinyl)arsonous acid (BCVAA), 2-chlorovinylarsonic acid (CVAOA), bis(2-chlorovinyl)arsenic acid (BCVAOA), and oxides, hydrates, or solvates thereof.

[0032] The above derivatization step is characterized by being performed at 25°C under stirring conditions.

[0033] The above derivatization step may be performed under conditions in the presence of formic acid (FA).

[0034] The above sample may be a liquid sample.

[0035] The above sample may be a solid sample, and the solid sample is characterized as being sand or soil.

[0036] Step (b) above may be performed by liquid chromatography.

[0037] Step (c) above may be performed by mass spectrometry.

[0038] Here, the mass spectrometry is characterized by including liquid chromatography-mass spectrometry (LC-MS or LC-MS / MS).

[0039] In addition, another aspect of the present invention for achieving the above objective provides a use for analyzing Lewisite degradation products, for evaluating the presence or concentration of Lewisite degradation products in an environmental sample using the above method.

[0040] The above environmental sample is characterized by containing water, soil, or sand.

[0041] The above Lewisite degradation product is characterized by containing 2-chlorovinylarsonous acid (CVAA) and / or bis(2-chlorovinyl)arsonous acid (BCVAA).

[0042] The above evaluation may be intended to determine whether there is Lewisite exposure or environmental contamination.

[0043] In addition, another aspect of the present invention for achieving the above objective provides a kit for analyzing Lewisite degradation products, which is used to analyze Lewisite degradation products in a sample by converting them into cysteine ​​derivatives and then analyzing them based on mass spectrometry.

[0044] The above mass spectrometry may be liquid chromatography-mass spectrometry (LC-MS).

[0045] The above Lewisite degradation product may contain 2-chlorovinylarsonous acid (CVAA).

[0046] The above kit is

[0047] (a) Cysteine ​​derivatization reagent,

[0048] (b) reaction buffer solution, and

[0049] (c) It may include at least one of the mass spectrometry solvents. Effects of the invention

[0051] The method for analyzing Lewisite degradation products according to the present invention overcomes the problem of low ionization efficiency under ESI conditions in LC-MS analysis through derivatization using cysteine, thereby enabling the detection of CVAA, a major degradation product of Lewisite, with high sensitivity and reproducibility. Accordingly, by complementing the limitations of existing GC-MS and LC-MS-based analytical methods, this invention provides an analytical technique capable of rapidly and accurately evaluating the presence or concentration of Lewisite degradation products in various environmental samples. Brief explanation of the drawing

[0053] Figure 1 is a schematic diagram showing the overall analysis flow of the present invention, which detects Lewisite degradation products in a sample by mass spectrometry after derivatizing them into cysteine. Figure 2 is a graph showing the results of comparing the cysteine ​​derivatization reaction efficiency of Lewisite decomposition products according to cysteine ​​concentration, formic acid concentration, and reaction time conditions. Figure 3 is a calibration curve showing the correlation between concentration and peak area for Lewisite degradation products derivatized using cysteine, showing the results confirming excellent linearity in the quantitative analysis of the present invention. Figure 4 is a chromatogram showing the result of separating the Lewisite degradation product derivative formed by reaction with cysteine ​​using UPLC, in which CVAA derivatives and BCVAA derivatives are clearly separated and detected at different retention times. Figure 5 is a mass spectrum showing the results of mass spectrometry performed on cysteine-derivated Lewisite degradation products, indicating that Cys-CVAA and Cys-BCVAA derivatives were detected at specific m / z values, respectively. Specific details for implementing the invention

[0054] Preferred embodiments according to the present invention will be described in detail below with reference to the attached drawings.

[0055] The advantages and features of the present invention and the method for achieving them will become clear by referring to the embodiments described in detail below together with the accompanying drawings.

[0056] However, the present invention is not limited by the embodiments disclosed below but may be implemented in various different forms, and these embodiments are provided merely to make the disclosure of the present invention complete and to fully inform those skilled in the art of the scope of the invention, and the present invention is defined only by the scope of the claims.

[0057] In addition, in describing the present invention, if it is determined that related known technologies, etc., may obscure the essence of the present invention, a detailed explanation thereof will be omitted.

[0058] The present invention will be described in detail below.

[0060] Analysis method of Lewisite decomposition products

[0061] One aspect of the present invention for achieving the above objective is a method for analyzing Lewisite decomposition products, wherein

[0062] (a) a derivatization step of reacting cysteine ​​with a Lewisite degradation product contained in a sample to form a cysteine ​​derivative of the degradation product;

[0063] (b) a step of separating the above derivative; and

[0064] (c) a step of determining the presence or concentration of the Lewisite degradation product from the separated derivative; comprising,

[0065] A method for analyzing Lewisite degradation products is provided.

[0066] The term “Lewisite (L)” in the present invention refers to an organic arsenic chemical agent containing arsenic (As) that may refer to a chemical inorganic substance exhibiting strong blistering, mucosal irritation, and systemic toxicity. Generally, it contains a chlorine-substituted vinyl group, and inorganic Lewisites can be classified into Lewisite 1 (L1), Lewisite 2 (L2), and Lewisite 3 (L3) based on structural differences, and among these, L1 is known to exist in the highest proportion.

[0067] In the present invention, the term "Lewisite" may be used as a concept including chemical agents that exist or may have existed in natural environments, industrial environments, or military and non-military situations, and may encompass not only pure Lewisite raw material but also all chemical forms that may exist during manufacturing, storage, distribution, exposure, decomposition, and disposal processes. Furthermore, unless otherwise specified in the context, the term Lewisite may be used as a concept including, but is not limited to, substances that are the source of decomposition products generated by decomposition in the environment by moisture, oxygen, oxidizing agents, or other chemical factors.

[0068] Additionally, Lewisite degradation products may refer to one or more compounds produced by Lewisite undergoing chemical hydrolysis, dehydration, oxidation, or structural transformation upon exposure to moisture, air, solvents, oxidizing agents, or other chemical or physical conditions.

[0069] In the present invention, the Lewisite degradation product may include a marker compound for determining whether Lewisite exposure has occurred, the state of environmental contamination, or the presence of residual chemical agents, and the As 3+ ) or Oga(As 5+ It can be used as a concept that includes all compounds containing arsenic, their hydrates, oxides, condensates, or reaction intermediates.

[0070] Specific examples include 2-chlorovinylarsonous acid (CVAA), bis(2-chlorovinylarsonous acid, BCVAA), and CVAO (2-chlorovinylarsonous oxide), which are trivalent arsenic (As 3+It is a form containing ). The decomposition products are obtained through additional dehydration or oxidation reactions to form pentaarsenic (As) such as CVAOA (2-chlorovinylarsonic acid) and BCVAOA (bis(2-chlorovinylarsonic acid)). 5+ It can be converted into oxidative decomposition products of the form (Table 1).

[0071] The term “cysteine ​​(cys)” of the present invention may refer to an amino acid containing a sulfur (SH) group or a derivative thereof, which can chemically react with a compound containing arsenic to form a covalent bond, a coordinate bond, or a stable complex.

[0072] In the present invention, the cysteine ​​may be used as a reaction reagent to react with Lewisite degradation products to improve the chemical stability, detectability, or signal characteristics of the substance to be analyzed, and may be used in a concept that includes L-cysteine, D-cysteine, salts thereof, derivatives, or functionally equivalent compounds. Furthermore, the cysteine ​​may be used in a free form, in a solution state, in an immobilized form, or in a form combined with other compounds, but is not limited thereto.

[0073] The term “derivative” in this invention may refer to a compound whose chemical structure, charge characteristics, molecular weight, or physical and chemical properties are altered as a substance to be analyzed reacts with a specific reagent.

[0074] In the present invention, the cysteine ​​derivative comprises a reaction product formed by the reaction of a Lewisite degradation product with cysteine, which may include a form that exhibits improved chemical stability compared to the original degradation product or improved ionization efficiency in liquid chromatography-mass spectrometry (LC-MS), particularly under electrospray ionization (ESI) conditions.

[0075] The above cysteine ​​derivatives may be used in the sense of including not only single reaction products but also various structural isomers, hydrates, salts, or complexes that may be produced depending on the reaction conditions, but are not limited thereto (Table 1).

[0077] division designation abbreviation structure arsenic oxidation water Lewisite species Lewisite 1 L1 As 3+ Lewisite 2 L2 As 3+ Lewisite 3 L3 As 3+ Lewisite decomposition products 2-chlorovinylarsonous acid CVAA As 3+ bis(2-chlorovinylarsonous acid) BCVAA As 3+ 2-chlorovinylarsonous oxide CVAO As 3+ 2-chlorovinylarsonic acid CVAOA As 5+ bis(2-chlorovinylarsonic acid) BCVAOA As 5+ Cysteine ​​derivatives (solution phase) 2Cys-CVAA 2Cys-CVAA As 3+ Cys-BCVAA Cys-BCVAA As 3+ Cysteine ​​derivative (gaseous phase) Cys-CVAA ion Cys-CVAA ion As 3+ Cys-BCVAA ion Cys-BCVAA ion As 3+

[0079] The above derivatization step is characterized by being performed at 25°C under stirring conditions, and specifically, may mean stirring at 1000 rpm for 30 minutes at 25°C, but is not limited thereto.

[0080] In addition, the above derivatization step is characterized by being performed under conditions in which formic acid (FA) is present, specifically, the range may be 0.10% FA to 2.00% FA, and more specifically, the concentration may be 1.5% FA, but is not limited thereto.

[0081] In one embodiment of the present invention, in order to derive the optimal reaction conditions for cysteine ​​derivatization, a derivatization reaction was performed while varying the cysteine ​​concentration, reaction time, and acidification conditions. As a result, a derivatization signal was stably obtained under conditions of a cysteine ​​concentration of 2.4 mg / mL to 4.8 mg / mL, a final formic acid concentration of 0.10% to 2.00% (v / v), and a reaction time of 30 minutes, and the above concentration range was set as the derivatization condition considering reaction reproducibility and consistency of the detection signal (Experimental Example 2 and Fig. 2).

[0082] In the present invention, the sample may be a liquid sample or a solid sample; specifically, the liquid sample may refer to tap water, but is not limited thereto. Additionally, the solid sample may specifically be sand or soil, but is not limited thereto.

[0083] In one embodiment of the present invention, for the qualitative analysis of a Lewisite degradation product using cysteine ​​on a liquid sample (tap water), 10 μL of 30.0% (v / v) formic acid (FA) was added to 170 μL of a filtered sample, and then 20 μL of a 2.4 mg / mL cysteine ​​solution was added to perform a derivatization reaction (Example 1-1).

[0084] In addition, in one embodiment of the present invention, for the qualitative analysis of Lewisite degradation products using cysteine ​​on solid samples (sand and soil), 10 μL of 4.5% (v / v) formic acid (FA) was added to 170 μL of a filtered sample, and then 20 μL of a 2.4 mg / mL cysteine ​​solution was added to perform a derivatization reaction (Examples 1-2).

[0085] In addition, in one embodiment of the present invention, for the quantitative analysis of Lewisite degradation products using cysteine ​​on a liquid sample (tap water), three 170 μL samples of filtered samples or standard solutions were prepared, 10 μL of LC-MS grade water, 4 μg / mL hydrolysis L solution, and 8 μg / mL hydrolysis L solution were added, 10 μL of 30.0% (v / v) formic acid (FA) was added thereto, and then 10 μL of 4.8 mg / mL cysteine ​​solution was added to perform a derivatization reaction (Example 2-1).

[0086] In addition, in one embodiment of the present invention, for the quantitative analysis of Lewisite degradation products using cysteine ​​on solid samples (sand and soil), three 170 μL samples or standard solutions of filter-prepared samples or standard solutions were prepared, 10 μL of LC-MS grade water, 4 μg / mL hydrolysis L solution, and 8 μg / mL hydrolysis L solution were added, 10 μL of 4.5% (v / v) formic acid (FA) was added thereto, and then 10 μL of 4.8 mg / mL cysteine ​​solution was added to perform a derivatization reaction (Example 2-2).

[0087] In addition, the above-mentioned derivatized derivative can be separated by liquid chromatography, specifically, UPLC can be used, but is not limited thereto.

[0088] In addition, the Lewisite degradation products of the above-mentioned isolated derivatives were analyzed by determining the presence or concentration of Lewisite degradation products through mass spectrometry.

[0090] Analytical Uses of Lewisite Decomposition Products

[0091] Another aspect of the present invention for achieving the above objective provides a use for analyzing Lewisite degradation products to evaluate the presence or concentration of Lewisite degradation products in an environmental sample using the above method.

[0092] The above Lewisite and (Lewisite) decomposition products are as described above.

[0093] Unless otherwise specified in the context, the term “environmental sample” in this invention refers to any sample collected or taken from a natural or artificial environment in which Lewisite or its decomposition products may be present. Such environmental samples may include, for example but not limited to, water matrix samples such as surface water, groundwater, tap water, seawater, rainwater, etc., solid or semi-solid samples such as soil, sand, sediment, dust, etc., and liquid extracts obtained by extraction, elution, or pretreatment from these samples.

[0094] In addition, the environmental samples of the present invention may include not only samples in their natural state, but also samples that are artificially prepared or to which standard substances have been added for environmental monitoring, pollution assessment, response to chemical accidents, military or terrorism response, or experimental verification.

[0095] The term “evaluation” in the present invention refers to any analytical judgment act for confirming or determining the presence, concentration, content, relative level, or change of Lewisite or its degradation products in a sample, and may include qualitative analysis, semi-quantitative analysis, or quantitative analysis.

[0096] In the present invention, the evaluation may be performed based on the presence or absence, intensity, peak area, peak height of a signal obtained through chromatography and / or mass spectrometry after a derivatization reaction using cysteine, comparison with a calibration curve, calculation results according to the standard addition method, etc., but is not limited thereto.

[0097] In one embodiment of the present invention, it was confirmed that the presence or concentration of Lewisite degradation products can be evaluated quickly and accurately using tap water, sand, and soil samples (Tables 4 and 5).

[0098] In addition, in one embodiment of the present invention, UPLC analysis results of CVAA derivatives (Cys-CVAA) and BCVAA derivatives (Cys-BCVAA) formed by reacting cysteine ​​with standard solution, tap water, sand, and soil samples confirmed that they were detected as distinct single peaks separated at retention times of approximately 1 minute and approximately 4 to 5 minutes, respectively (Example 3-3 and Fig. 4).

[0099] In addition, in one embodiment of the present invention, mass spectrometry results for each derivative separated by UPLC showed that the Cys-CVAA derivative [Cys+CVAA+H-2H2O] at m / z approximately 255.9 + It was detected as an ion corresponding to (Fig. 5a), and the Cys-BCVAA derivative [Cys+BCVAA+H-H2O] at m / z approximately 317.9. + It was detected as an ion corresponding to (Fig. 5b).

[0101] These results demonstrate that cysteine ​​reacted with Lewisite degradation products to form stable derivatives, suggesting that the generated cysteine ​​derivatives can be specifically detected by mass spectrometry.

[0103] Analysis kit for Lewisite decomposition products

[0104] Another aspect of the present invention for achieving the above objective provides a kit for analyzing Lewisite degradation products, which is used to analyze Lewisite degradation products in a sample after converting them into cysteine ​​derivatives and then analyzing them based on mass spectrometry.

[0105] The above Lewisite, (Lewisite) decomposition products, cysteine, and derivatives are as described above.

[0106] The term “kit” in the present invention refers to a combination of one or more components used to analyze, detect, quantify, or evaluate Lewisite or its degradation products, said components may be provided in the same packaging unit or in separate packaging forms, and may be used together or sequentially.

[0108] The above kit may include at least one of a derivatization reagent containing cysteine ​​or a derivative thereof, an acidic solution (e.g., formic acid), a buffer solution, a diluent solution, a standard substance, a standard addition reagent, an internal standard, a reaction vessel, a filtration means, a separation column, a solvent suitable for analysis, and an instruction manual, but is not limited thereto.

[0109] In addition, the kit may be configured to convert Lewisite degradation products in a sample into cysteine ​​derivatives and then analyze or evaluate them through chromatography and / or mass spectrometry.

[0111] Experimental Example 1. Preparation of Environmental Samples

[0112] 1-1. Preparation of Tap Water Samples

[0113] 3 mL of tap water sample was taken and diluted to 300 ng / mL, 1000 ng / mL, and 3000 ng / mL using CVAA or Lewisite (L) stock solution, and then stirred in a horizontal shaker at 350 rpm for 10 minutes. The stirred solution was filtered through a PTFE syringe filter and then transferred to a 2 mL microcentrifuge tube to be used as a sample.

[0115] 1-2. Preparation of Sand Samples

[0116] 3 g of sand was placed in a 15 mL centrifuge tube, and 4.5 μL, 15 μL, or 45 μL of L or hydrolyzed L stock solution were dropped onto the surface of the sand. After dropping, the lid of the tube was opened to allow the solvent to evaporate inside a fume hood, and the mixture was stirred for 10 minutes at 25°C and 350 rpm. Subsequently, 3 mL of 1.5% (v / v) aqueous formic acid solution (approx. pH 2.0) was added, and the mixture was stirred for an additional 10 minutes under the same conditions (25°C, 350 rpm). After stirring, the solution was filtered through a PTFE syringe filter and transferred to a microcentrifuge tube.

[0118] 1-3. Preparation of Soil Samples

[0119] The soil was dried at 65°C for 48 hours, cooled to room temperature, and sieved through a mesh size of 30-60 for use. 3 g of the prepared soil was placed in a 15 mL tube, and 4.5 μL, 15 μL, or 45 μL of L or hydrolyzed L stock solution was added dropwise and spiked. The solvent was evaporated in a fume hood, followed by mixing at 25°C and 350 rpm for 10 minutes. Subsequently, 3 mL of 1.5% (v / v) aqueous formic acid solution was added and extracted at 25°C and 350 rpm for 10 minutes. The mixture was then centrifuged at 4500×g for 5 minutes, and the supernatant was filtered through a PTFE filter and transferred to a microcentrifuge tube.

[0121] Experimental Example 2. Optimization of Cysteine ​​Derivatization Reaction Conditions

[0122] To determine the optimal reaction conditions for improving the derivatization efficiency of Lewisite degradation products using cysteine, the derivatization reaction was performed while varying the cysteine ​​concentration, formic acid concentration, and acidification conditions.

[0123] Specifically, for samples containing Lewisite degradation products, a derivatization reaction was performed by adjusting the cysteine ​​concentration differently in the range of 0.8 mg / mL to 5.6 mg / mL.

[0124] Next, the reaction was carried out by varying the reaction time in 15-minute increments up to 120 minutes under stirring conditions of 25℃ and 1000 rpm.

[0125] In addition, the effect of acidification conditions on the derivatization reaction was evaluated by adjusting the final concentration of formic acid (FA) to 0.10% (v / v), 0.50% (v / v), 1.50% (v / v), and 2.00% (v / v).

[0126] The cysteine ​​derivatives produced after the derivatization reaction were detected through UPLC-MS analysis, and the reaction efficiency was compared based on the peak area and signal intensity of the produced derivatives.

[0127] As a result, as shown in Figure 2, there was a significant difference in the amount of derivative produced depending on the cysteine ​​concentration. In particular, a signal of derivative production was stably obtained under conditions of a cysteine ​​concentration of 2.4 mg / mL to 4.8 mg / mL, a final formic acid concentration of 0.10% to 2.00% (v / v), and a reaction time of 30 minutes. Considering the reaction reproducibility and the consistency of the detection signal, the above concentration range was set as the derivatization condition.

[0128] Accordingly, in subsequent experiments, a cysteine ​​concentration of 2.4 mg / mL or 4.8 mg / mL, a reaction time of 30 minutes, and a final formic acid concentration of 1.5% (v / v) were selected as representative conditions and experiments were conducted.

[0130] Example 1. Derivatization for qualitative analysis of Lewisite degradation products using cysteine

[0131] To qualitatively analyze the Lewisite degradation products contained in liquid and solid samples using cysteine, the following procedures were performed depending on the type of sample.

[0133] 1-1. Liquid Sample Derivatization

[0134] The liquid sample used was tap water pretreated in Experimental Example 1-1 above.

[0135] First, 170 μL of the prepared tap water was dispensed and transferred into a microcentrifugal tube.

[0136] Next, to adjust the reaction conditions of the sample, 10 μL of 30.0% (v / v) formic acid (FA) was added to the filtered sample, followed by the addition of 20 μL of a cysteine ​​(Cys) solution at a concentration of 2.4 mg / mL to proceed with the derivatization reaction. After adding both the FA and Cys solutions to the sample, the reaction was carried out at 25°C with stirring at 1000 rpm for 30 minutes to ensure sufficient derivatization proceeded.

[0138] 1-2. Derivatization of Solid Samples

[0139] The solid samples used were sand and soil pretreated in Experimental Examples 1-2 and 1-3, respectively.

[0140] The filtered sand and soil filtrate was transferred by dispensing 170 μL into a microcentrifugal tube in the same manner as the qualitative analysis method of the liquid sample above.

[0141] Next, 10 μL of 4.5% (v / v) formic acid (FA) was added to each filtered sample, followed by the addition of 20 μL of a 2.4 mg / mL cysteine ​​(Cys) solution to proceed with the derivatization reaction. After adding both the FA and Cys solutions to each sample, the reaction was carried out at 25°C for 30 minutes while stirring at 1000 rpm.

[0143] Example 2. Derivatization for quantitative analysis of Lewisite degradation products using cysteine

[0144] To quantitatively analyze Lewisite degradation products contained in liquid and solid samples using cysteine, the following procedures were performed depending on the type of sample.

[0146] 2-1. Liquid Sample Derivatization

[0147] The liquid sample used was the tap water sample pretreated in Experimental Example 1-1 above.

[0148] Since verified internal standards such as stable isotope-labeled CVAA are not commercially available, the standard addition method was applied to the quantitative analysis of CVAA.

[0149] Specifically, 170 μL of the filtered sample or standard solution was dispensed into three 1.5 mL microcentrifuge tubes, and 10 μL each of (i) LC-MS grade water, (ii) 4 μg / mL hydrolyzed L solution, and (iii) 8 μg / mL hydrolyzed L solution were added to each tube. Subsequently, 10 μL of 30% (v / v) FA solution was added to each tube, followed by the addition of 10 μL of cysteine ​​solution at a concentration of 4.8 mg / mL. After adding all reagents, the mixture was stirred using a ThermoMixer C at 1000 rpm at 25°C for 30 minutes to ensure that the derivatization reaction proceeded sufficiently.

[0151] 2-2. Derivatization of Solid Samples

[0152] The solid samples used were the sand and soil samples pretreated in Experimental Examples 1-2 and 1-3 above.

[0153] Liquid samples extracted and filtered from each solid sample were dispensed in 170 μL portions into three microcentrifuge tubes. Subsequently, 10 μL portions of (i) LC-MS grade water, (ii) 4 μg / mL hydrolyzed L solution, and (iii) 8 μg / mL hydrolyzed L solution were added to each tube to establish standard addition conditions.

[0154] Next, to proceed with the derivatization reaction, 10 μL of 4.5% (v / v) formic acid (FA) was added to each tube, followed by the addition of 10 μL of a cysteine ​​(Cys) solution at a concentration of 4.8 mg / mL.

[0155] After adding all reagents, the derivatization reaction was carried out by stirring at 25°C and 1000 rpm for 30 minutes.

[0157] Example 3. Derivative Separation and Detection

[0158] Separation and detection of the generated derivatives were performed using the derivatized samples from Examples 1 and 2 above.

[0160] 3-1. UPLC Analysis

[0161] To separate and analyze the cysteine ​​derivatives produced after the derivatization reaction, chromatographic analysis was performed using a UPLC system. A CORTECS UPLC C18+ column (particle size 1.6 μm, inner diameter 2.1 mm × length 100 mm) was used as the analytical column, and the column oven temperature was maintained at 30℃. The mobile phase consisted of A: 0.1% aqueous formic acid (FA) and B: 0.1% acetonitrile formic acid (FA), and the flow rate was set to 0.3 mL / min.

[0162] The above mobile phase composition was analyzed under gradient conditions that varied the ratio of mobile phase B as shown in Table 2 below.

[0164] 0-0.5 minutes 0.5-3.0 minutes 3.0-8.0 minutes 8.0-9.0 minutes 9.0-9.5 minutes 9.5-14.5 minutes 13% B 13% → 50% B 50% → 90% B 90% B 90% → 13% B 13% B

[0166] 3-2. MS Analysis

[0167] The derivatives separated by UPLC were detected by mass spectrometry. The analysis was performed with the ionization conditions set as shown in Table 3 below.

[0169] Spray voltage Sheath gas flow Auxiliary gas flow Ion transfer tube temp. Vaporizer temp. 3400 V 35 arb 7 arb 300℃ 275℃

[0171] In addition, for MS / MS analysis, a target m / z value was set for the selective detection of derivatives; specifically, m / z 255.9175 is [Cys+CVAA+H-2H2O] + , m / z 317.9098 is [Cys+BCVAA+H-H2O] + It was set as the ion corresponding to.

[0172] Next, a calibration curve was constructed using standard solutions within a certain concentration range for the quantitative analysis of Lewisite degradation products.

[0174] As a result, as shown in Figure 3, excellent linearity between concentration and peak area was confirmed for the Lewisite degradation product derivatized using cysteine.

[0176] In addition, to evaluate the accuracy and precision of the quantitative analysis method of the present invention for liquid and solid samples, the recovery rate and repeated analysis results were reviewed, and the results are shown in Tables 4 and 5 below.

[0178] Specimen CVAA(ng / mL) Intra-day RSD (%) Inter-day RSD (%) Avg. Min. Max. Avg. Min. Max. Standard Solution 292.8 1.7 0.1 3.4 2.7 2.2 3.6 976.0 2.5 0.2 5.3 3.7 2.7 4.7 2928.0 3.4 1.6 5.8 3.1 1.9 4.3 Tap Water 292.8 4.2 1.7 5.1 3.7 2.5 4.5 976.0 0.3 0.2 0.4 1.2 0.3 2.5 2928.0 2.3 0.8 4.3 2.4 1.7 2.9 Sand 292.8 2.0 1.2 2.6 3.6 1.8 6.4 976.0 4.5 3.4 6.0 5.8 5.2 6.4 2928.0 4.2 1.8 6.6 5.5 4.0 6.7 Soil 292.8 1.0 0.2 2.2 4.3 1.1 7.9 976.0 1.6 0.4 2.6 2.4 1.7 3.8 2928.0 2.2 0.1 3.4 2.3 0.8 4.4

[0180] Day [CVAA] (ng / mL or ng / g) 292.8 976.0 2928.0 Tap Water 1 118.6 96.7 104.1 2 101.6 104.1 104.4 3 82.5 97.1 98.4 Sand 1 108.7 102.6 104.6 2 95.7 104.9 106.9 3 96.6 97.6 100.0 Soil 1 96.6 102.0 105.2 2 99.7 106.7 96.2 3 95.9 88.8 100.0

[0182] 3-3. Derivative Separation and Detection Results

[0183] For the Lewisite degradation products derivatized using cysteine ​​in Examples 1 and 2 above, the separation and detection results were confirmed using UPLC and mass spectrometry.

[0184] As a result, as shown in Figure 4, the CVAA derivative (Cys-CVAA) formed by reaction with cysteine ​​was detected as a single peak with a retention time of approximately 1 minute in UPLC analysis, and the BCVAA derivative (Cys-BCVAA) was detected as a distinct peak with a retention time of approximately 4 to 5 minutes. These retention times were observed identically in tap water, sand, and soil samples as well as in standard solutions.

[0185] Furthermore, the Cys-CVAA and Cys-BCVAA derivatives detected in each sample exhibited identical retention times and similar peak shapes regardless of the type of sample matrix, and almost no non-specific interference peaks were observed. This demonstrates that the derivatization and separation method of the present invention can be stably applied to various environmental samples.

[0187] Meanwhile, as a result of performing mass spectrometry on each derivative separated by UPLC, as shown in Fig. 5, the Cys-CVAA derivative [Cys+CVAA+H-2H2O] at m / z approximately 255.9. + It was detected as an ion corresponding to (Fig. 5a), and the Cys-BCVAA derivative [Cys+BCVAA+H-H2O] at m / z approximately 317.9. + It was detected as an ion corresponding to (Fig. 5b). In each ion signal, the theoretically expected isotope distribution pattern was observed together.

[0189] These mass spectrum results demonstrate that cysteine ​​reacted with Lewisite degradation products to form stable derivatives, and show that the generated derivatives can be specifically detected by mass spectrometry. Therefore, it has been confirmed that the method of the present invention can effectively separate Lewisite degradation products through cysteine ​​derivatization and reliably detect them using mass spectrometry.

[0191] From the above results, it was confirmed that the cysteine ​​derivatization method of the present invention can stably separate, detect, and quantify Lewisite degradation products in various sample matrices.

[0192] Although specific embodiments regarding the analysis of Lewisite degradation products using cysteine ​​according to one embodiment of the present invention have been described so far, it is obvious that various modifications are possible within the scope of the present invention.

[0193] Therefore, the scope of the present invention should not be limited to the described embodiments, but should be defined by the claims set forth below as well as equivalents thereof.

[0194] That is, the aforementioned embodiments should be understood as exemplary in all respects and not limiting, and the scope of the invention is defined by the claims set forth below rather than by the detailed description, and all modifications or variations derived from the meaning and scope of the claims and equivalent concepts thereof should be interpreted as being included within the scope of the invention.

Claims

Claim 1 A method for analyzing Lewisite degradation products, comprising: (a) a derivatization step of reacting cysteine ​​with a Lewisite degradation product contained in a sample to form a cysteine ​​derivative of the degradation product; (b) a step of separating the derivative; and (c) a step of determining the presence or concentration of the Lewisite degradation product from the separated derivative. Claim 2 The method according to claim 1, wherein the Lewisite decomposition product is one or more selected from the group consisting of 2-chlorovinylarsonous acid (CVAA), bis(2-chlorovinyl)arsonous acid (BCVAA), 2-chlorovinylarsonic acid (CVAOA), bis(2-chlorovinyl)arsenic acid (BCVAOA), and oxides, hydrates, or solvates thereof. Claim 3 A method according to claim 1, characterized in that the derivatization step is performed under stirring conditions at 25°C. Claim 4 A method according to claim 1, characterized in that the derivatization step is performed under conditions in which formic acid (FA) is present. Claim 5 A method according to claim 1, characterized in that the sample is a liquid sample. Claim 6 A method according to claim 1, characterized in that the sample is a solid sample. Claim 7 A method according to claim 6, characterized in that the solid sample is sand or soil. Claim 8 A method according to claim 1, wherein step (b) is performed by liquid chromatography. Claim 9 A method according to claim 1, wherein step (c) is performed by mass spectrometry. Claim 10 A method according to claim 9, characterized in that the mass spectrometry comprises liquid chromatography-mass spectrometry (LC-MS or LC-MS / MS). Claim 11 A method for evaluating the presence or concentration of Lewisite decomposition products in an environmental sample using the method according to paragraph 1. Claim 12 A method according to claim 11, characterized in that the above environmental sample comprises water, soil, or sand. Claim 13 A method according to claim 11, characterized in that the Lewisite degradation product comprises 2-chlorovinylarsonous acid (CVAA) and / or bis(2-chlorovinyl)arsonous acid (BCVAA). Claim 14 In paragraph 11, the above evaluation is a method for determining whether there is lewysite exposure or environmental contamination. Claim 15 delete Claim 16 delete Claim 17 delete Claim 18 delete

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