WO2004081561A1

WO2004081561A1 - Method for detection mustard

Info

Publication number: WO2004081561A1
Application number: PCT/GB2004/001083
Authority: WO
Inventors: Alison Bussey; Adrian Clarke; John Lambert
Original assignee: The Secretary Of State For Defence
Priority date: 2003-03-12
Filing date: 2004-03-11
Publication date: 2004-09-23
Also published as: WO2004081561A8; GB0305589D0

Abstract

This invention relates to a method suitable for detecting nitrogen mustard, sulphur mustard or a mixture of nitrogen and sulphur mustard in a sample solution comprising mixing the sample solution with a reagent wherein the reagent comprises 4-(4'-nitrobenzyl) pyridine or analogues thereof, and an additive selected from the group consisting of mercuric cyanide, a group I or group H metal perchlorate and mixtures thereof; gently warming the mixture; and adding a base. This invention also relates to the use of a kit comprising a reagent comprising 4-(4'-nitrobenzyl) pyridine or analogues thereof, and an additive selected from the group consisting of mercuric cyanide, a group I or group II metal perchlorate and mixtures thereof in such a method.

Description

METHOD FOR DETECTION MUSTARD

This invention relates to a method of detection of chemical contaminants. This method is especially useful for the detection of mustard in water. This invention also relates to the associated apparatus and use of such apparatus to conduct the method.

Many materials and substances are deposited on land or water for a variety of different reasons and if they are unwanted and / or dangerous they become contaminants, pollutants and toxic to the land. To effectively remediate the area it is important to accurately detect and identify such contaminants. Recently a new concern has arisen that during a time of conflict, or as an act of terrorism, unknown pollutants, including chemical and / or biological warfare agents, may be deliberately used to contaminate and pollute land or water. It is particularly important that, prior to troops or civilians entering such an area, the contaminant is identified so that personnel are not subjected to unnecessary danger, appropriate protective clothing can be used, antidotes can be administered as necessary, and the land and water can be safely and successfully remediated.

To this end it is necessary to consistently improve the detection mechanisms that are in place to identify potential land and water contaminants. Many different types of detection methods exist, some utilising complex equipment. However, when designing tests for use in the field and by personnel with little or no scientific training such as troops or civilians, it is important that the tests are as simple to conduct as possible and that they employ a clear end point to indicate a positive result. One particularly useful group of tests in this respect are those that involve a clearly identifiable reagent colour change. This is because they can be readily used in the field without the need for additional equipment. It is also useful if such reagents have a strong chromophore so that any colour change can be readily observed even when only a small amount of contaminant or reagent is present. This can both enhance the sensitivity of the test and can also reduce the cost of each individual test since a lower level of chromophore is required.

One current area of focus is the improvement of methods for detecting the chemical warfare agent mustard gas. Mustard gas has two forms, nitrogen mustard (formula I) and sulphur mustard (formula II).

. CH₂CH₂C1 .CHTCE CI

ID /

N-CH₂CH₂C1

\ CH₂CH₂CI \

CH,CH,C1 GD

One example of a known reagent that is currently used to detect mustard gas is 4-(4' nitrobenzyl) pyridine (DB-3, formula III):

The mechanism of action of DB-3 reagent is known to be as follows. The mustard gases themselves exist in equilibrium with their cyclic ions. The pyridinium nitrogen of DB-3 reacts with the cyclic ion (shown as A) to form a colourless intermediate pyridinium salt (formula IV). Upon the addition of strong base the intermediate (formula IV) is deprotonated to produce a highly coloured red-blue or mauve anion

(formula V).

The change of colour from colourless DB-3 to a blue / red product after reaction with mustard gas provides a distinct colour change that is ideal for use in a detection test. In the absence of colour, the interpretation must be that mustard agent is absent. Another advantage of this DB-3 colour change is that, due to the high molar absorptivity of the final anion, it can be seen when only low concentrations of the anion are present. Furthermore, although DB-3 may interact with other chemical agents that may also be used to contaminate land, for example other alkylating agents, this interaction does not produce the distinctive clear to blue/red colour change. These properties make DB-3 an ideal candidate for use in a highly sensitive and selective positive for both nitrogen or sulphur mustard.

To date several kits have been developed which comprise DB-3 for the detection of mustard in gases, including in the atmosphere. One such kit comprises a paper ticket impregnated with macroporous silica gel to which a solution comprising DB-3 is added. The air to be analysed is drawn through the ticket using a simple pump. The ticket is then gently warmed for a short period, a solution of sodium hydroxide is added and, if either sulphur or nitrogen mustard gas are present in the air that has been sampled, a mauve colour will develop. In another kit, a tube has been developed in which the same reaction can occur. The tube comprises a finely divided substrate that has been impregnated with a solution of DB-3, and also a thin walled glass ampoule comprising sodium hydroxide solution. As before, a simple hand pump is used to pump the air to be sampled through the tube, the tube is then warmed and the ampoule crushed to allow the sodium hydroxide solution contents to come in contact with the DB-3 impregnated substrate. Again, if mustard agent is present in the vapour, a mauve / purple colour will develop. Although these test kits provide a useful means for the positive detection of both nitrogen and mustard gas in air or in a gaseous sample, there remains a need to develop a test method whereby it is possible to positively identify mustard contamination in a solution. This would allow, among other things, personnel to quickly and accurately identify if a water source has been contaminated with mustard agent. There have been several attempts to develop a water test kit for mustard gas, again based on the known reagent DB-3. The simple addition of DB-3 reagent to the solution in question was unfortunately unable to produce a positive result. A kit was therefore developed where any mustard gas in solution was first extracted into the gaseous phase, and then tested for the presence of mustard gas using DB-3. The extraction is conducted by dissolving a mixture of organic acid and sodium bicarbonate in the test sample. This liberates carbon dioxide that effervesces through the sample partitioning any mustard gas contaminant from solution into the gas. The gas then rises out of the solution and is directed into an atmospheric sampling tube as described above. The DB- 3 test is then conducted, again as described. Such a procedure has been used in the past to detect mustard gas in solution but there are several associated problems. These include that because of the low volatility of nitrogen mustard the test procedure can only be used to detect sulphur mustard with sufficient sensitivity; the test can be easily distorted by the presence of chlorine in the sample; and the gas evolution procedure is complicated for users to conduct in the field.

Due to the complexities and problems of the above water test method an alternative means of detecting sulphur mustard in solution has been developed for use in the field utilising a mixture of platinic chloride and potassium iodide. In order to ensure that nitrates in the water do not interfere with the test it is first necessary to add sulphamic acid to the water sample. The test reagent is then added followed by a starch indicator. The platinic chloride complexes with the sulphur atom of the sulphur mustard liberating a chloride ion. This in turn liberates an iodide ion from the potassium iodide that causes the indicator to turn from colourless to deep blue. Although this test no longer requires the collection of gas it still has several problems. These include that the use of several different chemicals, which need to be added in series, is complicated to administer; the test can take up to 10 minutes to complete; the test is not as sensitive as the DB-3 test; sample contaminants such as nitrates and chlorine can cause the test to fail; the presence of other sulphur agents in the sample can lead to a false positive result; and, as before, the test is unable to detect nitrogen mustard.

Other problems that exist with such test kits to date is that the rate of colour development can be very slow such that it can take a while for the test result to be known, the efficiency of colour development can be very poor such that it can be difficult to readily identify if a positive result has been achieved and the stability of the test kits over time is poor with reagents degrading during storage. There remains a need to improve the test kits to overcome these problems.

There is also a need for a test method that is able to detect the presence of nitrogen mustard, sulphur mustard or a mixture of nitrogen and sulphur mustard in a sample solution, preferably in water. The method should use a distinct coloured end point that can be readily visualised. The method should be designed such that it is sufficiently sensitive to detect the presence of a mustard agent even at very low concentrations. Furthermore it should be designed such that it does not fail or lead to a false positive result in the presence of other contaminants in the sample solution. Furthermore it should be simple to use, give a quick result, and be portable such that it can be used in the field by personnel with little or no scientific training. Finally it should be economical to produce and have a good shelf life. Ideally it should be based on the known reagent DB-3.

A test method has now been developed which addresses this need. The method comprises mixing the sample solution with a reagent comprising DB-3 and a sensitising additive chosen from the group consisting of mercuric cyanide, a group I or group 13 metal perchlorate, for example sodium perchlorate, and mixtures thereof. Experiments have shown that this method provides for the first time a detection test that can be used to positively detect the presence of nitrogen mustard, sulphur mustard or a mixture of nitrogen and sulphur mustard in a sample solution. This method has several further advantages. It is of sufficient sensitivity to positively detect the analyte when it is present at a level of less than 20mg/L. The test can be executed using only a very small volume of sample solution. This method requires only gentle warming of the sample and hence can be easily warmed in the field using the hand without the need for additional heating equipment. Furthermore a test kit can be readily developed comprising sufficient quantity of the required reagents which is very easy to use by personnel with little or no scientific training allowing for reduced user error, reduced waste, and also extends the shelf life of the kits themselves.

It is an object of this invention to develop a method that can be used to detect the presence of nitrogen mustard, sulphur mustard or a mixture of nitrogen and sulphur mustard in a sample solution with equal effect. It is another object of this invention that the method be sufficiently sensitive to be able to detect the contaminants when present at low levels and that the method should be sufficiently reliable such that the presence of other materials in the sample can not lead to a false positive result. Furthermore the test should be easy to execute in the field by personnel with little or no scientific training. These and other objects of this invention will become apparent in light of the following disclosure.

Summary of the Invention

According to a first aspect this invention relates to a method suitable for detecting nitrogen mustard, sulphur mustard or a mixture of nitrogen and sulphur mustard in a sample solution comprising mixing the sample solution with a reagent wherein the reagent comprises 4-(4'-nitrobenzyl) pyridine or analogues thereof, and an additive selected from the group consisting of mercuric cyanide, a group I or group II metal perchlorate and mixtures thereof; gently warming the mixture; and adding a base.

According to a second aspect this invention relates to the use of a kit comprising a reagent comprising 4-(4'-nitrobenzyl) pyridine or analogues thereof, and an additive selected from the group consisting of mercuric cyanide, a group I or group II metal perchlorate and mixtures thereof in a method according to the present invention.

Detailed Description of the Invention

All publications cited herein are hereby incorporated by reference in their entirety, unless otherwise indicated.

The elements of the method are described in more detail below. This invention relates to a method suitable for detecting nitrogen mustard, sulphur mustard or a mixture of nitrogen and sulphur mustard in a sample solution comprising mixing the sample solution with a reagent wherein the reagent comprises 4-(4'- nitrobenzyl) pyridine or analogues thereof, and an additive selected from the group consisting of mercuric cyanide, a group I or group II metal perchlorate and mixtures thereof; gently warming the mixture; and adding a base.

The sample solution to be used in the present method can be any solution in which the user wishes to detect the presence of nitrogen mustard, sulphur mustard or mixtures of nitrogen and sulphur mustard. Preferably the sample is an aqueous solution and may be a solution collected directly from the environment such as a stream, ditch or water supply. Alternatively the solution could be prepared by dissolving a solid sample which it is believed may be contaminated in a suitable solution of solvent, such as water, and then conducting the present method. The method can be used with only a very small volume of sample solution, preferably less than about 20ml, more preferably less than about lOmls, even more preferably less than about 5mls. This has the result that the test is very easy to use.

The method comprises mixing the sample solution with a reagent containing 4-(4'- nitrobenzyl) pyridine or analogues thereof. Such analogues should be designed to produce a strong colour change in the presence of nitrogen mustard, sulphur mustard or a mixture of nitrogen mustard or sulphur mustard. Suitable analogues of 4-(4'- nitrobenzyl) pyridine include, but are not limited to, those where the benzene ring is substituted with one or more substituents selected from the group consisting of methyl, ethyl, propyl, hydroxy, halo, nitro, amine, and the like. Similarly, suitable analogues of 4-(4'-nitrobenzyl) pyridine include those where the pyridine ring is replaced by an alternative heterocyclic ring such as those selected from the group consisting of, but not limited to, pyrrole, pyrazole, imidazole, pyrimidine, pyrazine, pyridazine, 1, 2, 4- triazine, 1, 2, 4, 5-tetrazine, furan, pyrylium, thiophene thiepine and the like. Further suitable analogues of 4-(4'-nitrobenzyl) pyridine also include those where the pyridine ring is substituted with one or more substituents selected from the group consisting of methyl, ethyl, propyl, hydroxy, halo, nitro, amine, and the like. Even further analogues of 4-(4'-nitrobenzyl) pyridine include those analogues which comprises more than one of the above mentioned modifications. It is preferred that the reagent is 4-(4'- nitrobenzyl) pyridine, known commercially as DB-3.

In order to ensure that the reagent has sufficient sensitivity and reactivity to detect both nitrogen mustard and sulphur mustard in solution, it is necessary that the reagent also comprises one or more additives. When the reagent is 4-(4'-nitrobenzyl) pyridine or analogues thereof the reagent should comprise one or more of the reagents selected from the group consisting of mercuric cyanide, a group I or group II metal perchlorate and mixtures thereof. It is preferred that the group I or group II metal perchlorate is selected from the group consisting of sodium perchlorate, magnesium perchlorate and mixtures thereof. The most preferred perchlorate is sodium perchlorate. Other optional reagent additives include sodium diethyldithiocarbamate. It is preferred that the weight ratio of 4-(4'-nitrobenzyl) pyridine or analogues thereof to mercuric cyanide is in the range of from about 1:1 to about 1:20, more preferably from about 1:5 to about 1:15, even more preferably from about 1: 10 to about 1 :15 and most preferably about 1:12.5. It is also preferred that the weight ratio of 4-(4'-nitrobenzyl) pyridine or analogues thereof to a group I or group II metal perchlorate is in the range of from about 1:1 to about 1:20, more preferably from about 1:5 to about 1:15, even more preferably from about 1: 10 to about 1:15 and most preferably about 1:12.5.

The method comprises mixing the sample solution with a sufficient quantity of reagent comprising 4-(4'-nitrobenzyl) pyridine or analogues thereof, such that the mustard gas can be positively identified at the required level of sensitivity. Such levels can be readily determined by one skilled in the art using routine trial and error. They will be determined by the desired sensitivity of the test, the reactivity of the reagent with the mustard gas and the strength of the chromophore so produced.

The reagent and additives can be used in the method as a dry mix, alternatively they can be dissolved in a suitable solvent prior to addition to the sample solution, or alternatively they can be impregnated into or coated onto a suitable substrate prior to use in the present method. It is preferred that prior to use the reagent is either dissolved in a solvent or impregnated onto a substrate. Suitable substrates can be easily determined by one skilled in the art, but include ceramic materials, glass materials or silica materials. It is preferred that one or more of the reagents is impregnated onto a substrate and that the substrate is a silica material, preferably a silica gel. The reagent can either be added to the substrate by a wide variety of means well known to those skilled in the art. These include preparing a solution of the reagent in a suitable solvent and then saturating the substrate in the solution, and then drying the substrate to drive off the solvent. This enables the reagent to be stored over an extended period of time. Alternatively the reagent mix can be provided as a solution which is ideally stored in a pre-dispensed ampoule until it is ready to be used. Any solvent capable of dissolving the reagent is suitable provided that the solvent does not impact the reaction of the mustard gas with the reagent itself. Where the reagent is 4-(4'-nitrobenzyl) pyridine or analogues thereof it is preferred that the reagent is dissolved in an organic solvent such as example toluene, methanol, preferably methanol. The concentration of reagent in methanol is preferably of the order of 5mg/l. Such ampoules are made of a material which can be readily opened in the field prior to use, for example a glass bottle with a snap top or a thin walled capsule which can be crushed. In order to prevent degradation of the reagent solution it is preferred that the ampoules are dried thoroughly prior to packing and that the reagents are packed and stored under an atmosphere of carbon dioxide.

The test method comprises gently warming the mixture of the sample solution with reagent. Incubation can be carried out by any suitable warming means. Because only low temperatures are required, for example in the region of about 25° to about 45 °C, preferably from about 25°C to about 35°C, the sample solution can be warmed by hand thus removing the need or complexity for use of any additional warming. The sample needs only be warmed for a short period such as less than 20 minutes, preferably less than 10 minutes and more preferably less than 5 minutes. Alternatively the warming can be provided by a reuseable pack heating which contains a gel which as it crystallises releases heat. Such packs are well known. After wanning, the method comprises the addition of a base to the sample. A wide variety of different bases could be used, again as readily determined by one skilled in the art. It is preferred to use a solution of an inorganic base, preferably an alkali metal hydroxide for example sodium hydroxide or potassium hydroxide. The base can either be added as a solid that is able to dissolve in the sample or as a solution. It is preferred that the base is added as a solution, preferably at least about 1M solution, more preferably at least about 5M, even more preferably at least about 10M. Again the base can be stored either as a bulk to be dispensed during the test, or preferably predispensed into an individual ampoule for use in each test. Again, to improve the shelf life of the test it is preferred that any ampoules are packed as described previously.

The method can be usefully used to detect the presence of nitrogen mustard in a sample solution at a concentration of less than 20mg/l, preferably less than lOmg/l, more preferably less than 5mg/l. The method can also be successfully used to detect the presence of sulphur mustard in a sample solution at a concentration of less than 20mg/l, preferably less than lOmg/l, more preferably less than 5mg/l. Equally the method can be used to detect the presence of a mixture of nitrogen mustard and sulphur mustard in a sample solution.

This invention also relates to the use of a kit comprising a reagent comprising 4-(4'- nitrobenzyl) pyridine or analogues thereof, and an additive selected from the group consisting of mercuric cyanide, a group I or group II metal perchlorate and mixtures thereof in a method according to the present invention. Such a kit could take many forms. In a simplistic form the kit could comprise each of the different regents supplied with use instructions as to how to use the reagents to test for mustard gas in solution. However, in order to make the test method as simple as possible to conduct in the field, it is preferred that the kit comprise the predispensed reagents for use in an individual test. It is also preferred that the kit is designed so that the test can be executed with as little interaction with the sample as possible to minimise the danger to personnel and also the risk of cross contamination.

In a preferred embodiment the kit comprises a tube that comprises the reagent, optionally impregnated onto a solid substrate or as a solution stored into a crushable ampoule, inside the tube. In the latter case the tube also optionally comprises some packing material onto which the reagent solution can be dispensed after the ampoule has been crushed. This provides a surface on which the reaction can take place. It is preferred that the tube comprise a solid substrate impregnated with the reagent. In order to minimise costs it is preferred that the tube is manufactured from plastic such as polyethylene. During storage the tube is sealed. Prior to use of the kit it is necessary to remove an end from the tube. It is preferred that the tube is pre-scored to allow the end to be readily removed. The open end of the tube is then inserted into the sample solution to be tested. The sample rises in the tube, either by means of capillary action or otherwise, for example by use of a pump, until it comes into contact with the reagent. The tube is then removed from the sample, dried and gently warmed for a short period. Base is then added to the reagent. The base is preferably stored within the tube either in a crushable ampoule or in a separate area of the tube with which the reagent can be mixed after warming. It is preferred that the kit is designed to have a shelf life of at least 1 year preferably 2 years when stored at less than 25°C. Figure 1 shows a preferred embodiment of a kit suitable of use in the present method. The kit comprises an outer deformable tube (1) which contains a thin walled glass ampoule comprismg a solution of strong base (2); a wire gauze plug (3); a silica gel substrate onto which is impregnated a reagent comprising DB-3, mercuric cyanide and sodium perchlorate (4); a further porous plug (5); and a score line which allows breaking of the tube (6). During use the end of the tube is removed along the score line (6). The tube is then inserted into a sample (not shown) such that the reagent impregnated substrate becomes immersed in sample solution. After a short time the tube (1) is removed from the sample solution (not shown) and warmed by hand. The glass walled ampoule (2) is then broken and the tube is orientated such that the strong base solution interacts with the reagent impregnated substrate (4). A change in colour to blue / red indicates a positive result.

Test Results

Silica gel was placed into a tube and the gel was then soaked with 0.5ml of a solution comprising lOg of DB-3, 125g of mercuric cyanide, 125g of sodium perchlorate, 0.25g of sodium diethyldithiocarbamate, dissolved in 2L of methanol. The reagents used were standard analytical grade and each measurement was made to within 5% error margin. 5ml of sample solution containing the mustard agent to be detected was then sucked up into the tube using a teat pipette until the sample solution came into contact with the silica gel. The tube was then removed from the solution, the outside dried and then warmed in the hand for 1 minute. The tube was then placed into a 1.2M solution of sodium hydroxide and this was drawn up into the tube again using a teat pipette. The colour change was observed. Each test was repeated up to four times.

These results demonstrate that this new method is a useful method of detecting both sulphur mustard and nitrogen mustard in water. The method has excellent sensitivity especially in the case of nitrogen mustard, being able to detect nitrogen mustard when present at less than 2mg/l.

Claims

1. A method suitable for detecting nitrogen mustard, sulphur mustard or a mixture of nitrogen and sulphur mustard in a sample solution comprising mixing the sample solution with a reagent wherein the reagent comprises 4-(4^s-nitrobenzyl) pyridine or analogues thereof, and an additive selected from the group consisting of mercuric cyanide, a group I or group II metal perchlorate and mixtures thereof; gently warming the mixture; and adding a base.

2. A method according to Claim 1 wherein the reagent is 4-(4'-nitrobenzyl) pyridine.

3. A method according to any of Claims 1 to 2 wherein the weiglit ratio of 4-(4'- nitrobenzyl) pyridine or analogues thereof to mercuric cyanide is in the range of from about 1:1 to about 1:20, more preferably from about 1:5 to about 1:15, even more preferably from about 1: 10 to about 1:15 and most preferably about 1:12.5.

4. A method according to any of Claims 1 to 3 wherein the weight ratio of 4-(4'- nitrobenzyl) pyridine or analogues thereof to a group I or group II metal perchlorate is in the range of from about 1:1 to about 1:20, more preferably from about 1:5 to about 1:15, even more preferably from about 1: 10 to about 1:15 and most preferably about 1 : 12.5.

5. A method according to any of Claims 1 to 4 wherein the reagent also comprises sodium diethyldithiocarbamate.

6. A method according to any of Claims 1 to 5 wherein the mixture is warmed to a temperature of about 25°C to about 45°C, preferably from about 25°C to about 35°C.

7. A method according to any of Claims 1 to 6 wherein the base is preferably a solution of an inorganic base, preferably an alkali metal hydroxide, more preferably sodium hydroxide or potassium hydroxide.

8. A method according to Claim 7 wherein the concentration of the solution of base is fat least about 1M solution, preferably at least about 5M, and more preferably at least about 10M.

9. A method according to any of Claims 1 to 8 wherein the sample solution is an aqueous solution.

10. Use of a kit comprising a reagent comprising 4-(4'-nitrobenzyl) pyridine or analogues thereof, and an additive selected from the group consisting of mercuric cyanide, a group I or group II metal perchlorate and mixtures thereof in a method according to Claim 1.

11. Use of a kit according to Claim 10 wherein the kit comprises a substrate that has been impregnated or coated with the reagent.

12. Use of a kit according to any of Claims 10 or 11 wherein the kit comprises a crushable ampoule that in turn comprises a basic solution.