METAL IONS

TECHNICAL FIELD The present invention relates to a storage stable dithizone preparation for detecting transition metal ions in a sample, and to methods for detecting transition metal ions using storage stable dithizone preparations.

BACKGROUND ART

The presence of some transition metal ions in an animal's body can dramatically increase the production of free radicals in the body. Excess free radicals in a body may contribute to a number of chronic diseases and aging.

The detection of metals in a sample is typically determined using expensive laboratory-based instruments such as atomic absorption spectrophotometers. When using such instruments, the sample has to be ^Λ ashed' , whereby any organic matter in the sample is decomposed. As such, these techniques do not differentiate between chelated metals or metal ions in the original sample.

The presence of some transition metal ions in a sample may be detected using the laboratory dependent "dithizone assay", in which a solution of dithizone (diphenylthiocarbazone) dissolved in certain organic solvents (for example chloroform) changes colour when exposed to the transition metal ions. This assay does not detect chelated metal atoms. The dithizone assay has not been used outside of laboratories for a number of reasons, mainly because of the instability of dithizone solutions and the dangerous properties of the organic solvents required.

It would be advantageous to be able to provide an untrained person with a reliable, fast and convenient test

for detecting the presence of transition metal ions in a liquid sample (e.g. drinking water, saliva, urine, blood, and so on) or on materials which can be suspended in a liquid phase (e.g. dust, garden soil, paint and so on).

DISCLOSURE OF INVENTION

In a first aspect, the present invention provides a storage stable dithizone preparation, whereby the dithizone preparation is in a form that is ready for use to detect the presence of transition metal ions in a sample.

The present inventor has adapted the dithizone assay such that it can be used outside of a laboratory by any untrained person. Advantageously, the present invention can provide a dithizone preparation for reliably, quickly and conveniently detecting the presence of transition metal ions in a sample.

Furthermore, the dithizone preparation of the present invention is storage stable. As discussed above, solutions containing dithizone are unstable and decompose during storage, for example because the dithizone is oxidised in sunlight. However, the present invention advantageously provides a dithizone preparation which may be kept under typical household storage conditions (e.g. at room temperature) for some time before use.

In some embodiments of the first aspect of the present invention, the storage stable dithizone preparation is a solution comprising the dithizone dissolved in a solvent. The solvent may, for example, be low odour mineral turpentine or a component thereof.

Low odour mineral turpentine is mineral turpentine

(mineral turpentine is a complex mixture of highly refined hydrocarbon distillates, mainly in the C9-C16 range) in

which the more toxic aromatic hydrocarbons have been reduced or removed. The present inventor has surprisingly and unexpectedly found that dithizone does not decompose when dissolved in low odour mineral turpentine. As such, the present inventor has been able to produce a storage stable solution of dithizone.

Low odour mineral turpentine is additionally desirable because it has a lower toxicity, lower volatility and lower flammability than standard mineral turpentine.

As low odour mineral turpentine consists of a complex mixture of highly refined hydrocarbon distillates, it is to be understood that a component or mixture of components of the low odour mineral turpentine can also provide a storage stable dithizone preparation when the dithizone is dissolved in those component (s) .

If too much dithizone is present in such preparations, then a large number of metal ions will need to be present in the sample in order to enable any colour change to be detected. However, if too little dithizone is present, the colour of the solution may not be strong enough. Such dithizone preparations may contain between about 0.005 and about 1.0% w/v dithizone dissolved in low odour mineral turpentine or a component thereof. The preparation may, for example, contain between about 0.1 and about 0.5% w/v dithizone .

In other embodiments of the first aspect of the present invention, the storage stable dithizone preparation is in the form of a coating of dithizone on the inside of a test container. For example, the storage stable dithizone preparation may be in the form of a coating of dithizone located on the inside surface of a cap of the test container, for example, in a test tube lid or stopper.

In such embodiments, a person can be provided with a container containing an appropriate amount of dithizone which is ready for use when the person has a test sample which they wish to test for transition metal ions. As will be described in detail below, all the person needs to do is to place the required amount of a solvent into the test container, shake the container until the dithizone dissolves (resulting in a green solution) , and then add the test sample into the container.

In one embodiment, the test container may contain between about O.OOOlg and about 0.00Ig of dithizone, for example, between about 0.0005g and about O.OOlg of dithizone. This small quantity of dithizone may be deposited into the test container by dissolving about 0.75g of dithizone into about 250 mL of a volatile solution (such as chloroform) . One drop (ca 1/30 ^th of a mL) of the resultant solution may then be deposited into the test container (or the container lid) and allowed to evaporate, leaving a coating of dithizone on the inside of the test container.

In a second aspect, the present invention provides for the use of the preparation of the first aspect of the present invention to detect the presence of transition metal ions in a water sample, or the blood, saliva or urine of an animal such as a human being.

Typically, the transition metal ions are one or more of the group comprising zinc, lead, copper, cadmium and mercury ions. The presence of other transition metal ions, such as iron and nickel, may also be detected.

In a third aspect, the present invention provides for the use of the preparation of the first aspect of the present invention to detect the presence of one or more ions from the group comprising zinc, lead, copper, cadmium, iron, nickel and mercury ions in the sample.

In a fourth aspect, the present invention provides a storage stable dithizone solution. In one embodiment, the storage stable dithizone solution comprises dithizone dissolved in a solvent. In one form, the solvent is low odour mineral turpentine or a component thereof.

In a fifth aspect, the present invention provides a method for detecting the presence of transition metal ions in a sample comprising the step of exposing a storage stable solution of dithizone to the sample, whereby a change in the colour of the dithizone solution indicates the presence of transition metal ions in the sample.

In some cases, the colour of the solution resulting from the exposure may be indicative of the transition metal (s) ions in the sample .

In one embodiment, the storage stable solution may be a solution of dithizone- in low odour mineral turpentine or a component thereof .

In a sixth aspect, the present invention provides a container having at least a portion of an inside surface thereof coated with dithizone whereby, in use, when a user adds a solvent to the container, the dithizone dissolves in the solvent to form a solution for use to detect the presence of transition metal ions in a sample.

The inside surface may, for example, be defined on a lid for the container such that, in use, the user adds the solvent to and shakes the container so that the solvent contacts the lid inside surface to dissolve the dithizone.

In other embodiments, a portion or patch located on an interior side wall of the container may be coated with dithizone.

- S -

The solvent added to the container to dissolve the dithizone may, for example, be low odour mineral turpentine or a component thereof, mineral turpentine or a component thereof, terpinyl acetate, canola oil, or a mixture of any of these solvents. As those skilled in the art will appreciate, these solvents are not as dangerous as the solvents (such as chloroform) previously used when performing the "dithizone assay" in a laboratory.

The solvent added to the container to dissolve the dithizone may, for example, consist of 50% canola oil, 40% mineral turpentine and 10% terpinyl acetate.

The container may, for example, be a test-tube.

In a seventh aspect, the present invention provides a kit comprising a first container that is the container of the sixth aspect of the present invention and a second container that contains the solvent into which the dithizone is dissolvable to form a solution for use to detect the presence of transition metal ions in a sample.

The kit may further comprise a colour chart that helps to identify the transition metal ions present in the sample.

In an eighth aspect, the present invention provides a method for detecting the presence of transition metal ions in a sample comprising the step of exposing the dithizone solution formed in the container of the sixth aspect of the present invention to the sample, whereby a change in the colour of the dithizone solution indicates the • presence of transition metal ions in the sample.

In one embodiment, the colour of the solution resulting from the exposure is indicative of the transition metal (s) ions in the sample.

BRIEF DESCRIPTION OP THE DRAWING

Preferred embodiments of the container and kit of the present invention will now be described by way of example only, with reference to the accompanying figure which shows a kit in accordance with a preferred embodiment of the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

The prior art "dithizone assay" referred to above provides an effective method for detecting the presence of some transition metal ions in a sample. In summary, the compound diphenylthiocarbazone (dithizone) is dissolved in a solvent such as chloroform or carbon tetrachloride to produce a characteristic green coloured solution. This solution is unstable when exposed to sunlight, which causes the dithizone to quickly oxidise and the solution to turn yellow.

When the green coloured dithizone solution is exposed to a sample containing transition metal ions (e.g. zinc, lead, copper, mercury, cadmium etc) , the solution changes colour. The colour change is distinctive of the particular metal ion, for example, if zinc ions are present in the sample, the solution will turn pink, whereas if mercury ions are present, the solution will turn yellow.

This assay, however, is not generally suitable to use outside of a laboratory, given the instability of the solution and the nature of the solvents. Furthermore, the assay requires at least some experience with scientific methods and may not give reliable results if incorrectly applied. For example, the assay would be expected to be dependent on the amount of dithizone and solvent used, the time allowed for reaction, how vigorously the solution was shaken, and so on.

In light of such issues, the present inventor has discovered a way of providing a dithizone preparation to a person in a form whereby the person can reliably, quickly and conveniently test for the presence of transition metal ions.

The inventor has developed a storage stable dithizone preparation, whereby the dithizone preparation is in a form that is ready for use to detect the presence of transition metal ions in a sample.

The dithizone preparation may, for example, be used to detect transition metal ions in the fields of health, the environment and industry. The dithizone preparation can, for example, enable a person to determine whether they can chelate transition metal ions inside their bodies, which can provide a general indication of their health. Unchelated transition metal ions are difficult to eliminate from a human body and may build up to toxic levels. Furthermore, transition metal ions can increase the production of free- radicals via the metal mediated Fenton reaction.

The present inventor has surprisingly and unexpectedly found that dithizone is stable when dissolved in some solvents, resulting in a dithizone solution that is storage stable. Such solutions have been found to be stable for in excess of 2 years under typical storage conditions in glass or polyethylene terephthalate (PET) containers.

The container used to store the solution may be made from any material which does not cause the dithizone in the solution to decompose. Suitable materials include glass and PET, however, it has been observed that the presence of other materials such as high density polyethylene (HDPE) or low density polyethylene (LDPE) (e.g. in the

_ g _ seal of the container) can cause the dithizone to decompose to some extent .

Because the storage stable dithizone solution is provided in the form of a solution containing an appropriate amount of dithizone for detecting the presence of transition metal ions, all a person needs to do in order to test for the presence of transition metal ions is to add a defined quantity of the stable (green) dithizone solution (e.g. 10 drops) to the liquid sample, shake the resultant mixture and visually observe any colour change. This dithizone solution can therefore provide reliable results without the person needing specialised equipment or training.

As noted above, the colour change can be indicative of the types of transition metal ions present in the sample, however, in practice, if a number of transition metal ions are present, the colour of the resultant solution will be a mixture of colours and identification of the individual metal ions may not be possible or may be difficult.

The solvent used in the stable dithizone solution is preferably low odour mineral turpentine or a component thereof.

The amount of dithizone dissolved in a litre of solvent (e.g. low odour mineral turpentine) may be between about 0.05 g (.005% w/v) and about 10 g (1.0% w/v) . For example, the amount of dithizone dissolved in a litre of solvent may be between about 0.1 g and about 0.3 g.

In other embodiments, the storage stable dithizone preparation is in the form of a coating of dithizone on the inside of a test container, preferably, inside a cap of the test container. This preparation provides to a person a container having an appropriate quantity of dithizone, which is ready for use to test a sample for

transition metal ions. The dithizone inside the container is storage stable because it is not dissolved in a solvent .

The container may be a test tube with the dithizone distributed on the inside of the cap of the test tube. Alternatively, the dithizone may be distributed as a coating on the inside of the walls of the container.

Other containers which may also be used include sample jars, small glass bottles, etc.

In order to coat the dithizone on the inside of the test container, one method involves placing a pre-determined amount of dithizone in a suitable solvent, such as chloroform, and dissolving it therein. The container is then charged with the resultant solution and the solvent is allowed to evaporate, leaving a residue of dithizone around a specified area of the inside of the container.

In order for a person to test for the presence of transition metal ions in a sample, all the person needs to do is add a suitable solvent to the test container (the solvent would usually be provided in a kit with the test container, as will be described below) and shake the test container until the dithizone coating the inside of the test container dissolves and forms a green solution. Whilst this solution is not necessarily a storage stable solution and the dithizone in this solution may degrade over time, it is expected that the person will only add the solvent to the test container shortly before the test for transition metal ions is to be performed. Hence, the dithizone preparation provided to the person is storage stable, but is immediately ready to use for detecting the presence of transition metal ions.

It is anticipated that any solvent which will dissolve

dithizone may be used. Suitable solvents which may be added to the container include vegetable oils such as canola oil and olive oil, grape seed oil, mineral turpentine, odourless mineral turpentine, Shellite, terpinyl acetate, and mixtures thereof. A particularly preferred solvent contains a mixture of 50% canola oil, 40% mineral turpentine and 10% terpinyl acetate.

The quantity of dithizone provided inside the container will govern the sensitivity of the test. In one example, about 0.75 g of dithizone will be dissolved in about 250 mL of chloroform. One drop of this solution would then be added to the cap of the test container, depositing about O.OOOlg of dithizone in the container.

The storage stable dithizone preparation is to be used to detect the presence of transition metal ions in a sample. In one example, in order to test a sample for the presence of transition metals, approximately 15 drops (ca 0.5 mL) of solvent is added to a test tube containing dithizone inside the cap of the test tube. The cap is then pushed in to seal the tube, and the tube shaken such that the solvent is brought in contact with the dithizone in the cap, resulting in a green solution. The liquid sample to be tested is then added to the test tube using a pipette, and the test tube is again shaken vigorously. If transition metal ions are present in the sample, the green solution will change colour.

The preparation can be used to detect the presence of transition metal ions in any liquid sample, for example in drinking water, saliva, urine, blood, and so on, or on materials which can be suspended in a liquid phase (e.g. dust, garden soil, paint and so on) .

As discussed above, a colour change from green indicates the presence of certain transition metals ions in the

sample, and the resultant colour is indicative of the metal ions present .

A colour chart may also be provided in order to help to identify the metal ions present in the test sample .

A kit in accordance with a preferred embodiment of the present invention is shown in Figure 1 in the form of testing kit 10. Testing kit 10 has a number of containers in the form of test tubes 12, which may be mounted on a rack 14 for convenience. Testing kit 10 also has a second container in the form of bottle 16, which contains the solvent (for example a mixture of 50% canola oil, 40% mineral turpentine and 10% terpinyl acetate) into which the dithizone is dissolvable (as described above) to form a solution for use to detect the presence of transition metal ions in a solution.

Testing kit 10 also has a colour chart 18 that helps identify the transition metal ions present in the sample, based on the colour of the resultant solution.

The test tubes 12 each have a cap 20 which contains a coating of dithizone inside it (not shown) . When the solvent from bottle 16 is added to test tube 12 and test tube 12 is shaken, the dithizone in the cap dissolves to give a green solution. As described above, this solution can be used to test for the presence of transition metal ions in a sample .

EXAMPLES

Example 1 - Stable dithizone solution

A storage stable solution of dithizone may be prepared by dissolving between 0.1 to 0.3 g of dithizone into 1 L of low odour mineral turpentine and storing the solution in a glass or PET container. Such solutions have been found to

be stable (i.e. the dithizone does not decompose and the solution remains a green colour) under normal storage conditions for at least 2 years.

Example 2 - Dithizone impregnated cap

Dithizone (0.75 g) is dissolved in chloroform (250 mL) and one drop of the dissolved reagent is placed into a test tube cap. The chloroform is then allowed to evaporate, leaving a coating of dithizone on the inside of the cap. This cap may be stored for an indefinite amount of time.

Shortly before use, about 0.5 ml (ca 15 drops) of a solvent consisting of 50% canola oil, 40% mineral turpentine and 10% terpinyl acetate is added to the test tube, the cap is fitted on the tube, and the is solvent brought in contact with the dithizone in the cap by lightly shaking the test tube until a green solution is produced.

Example 3 - Testing methods

0.5 mL (ca 15 drops) of a dithizone solution (either a storage stable dithizone solution as described in Example 1, or a dithizone solution which has been freshly prepared as described in Example 2) is prepared in a test tube. The substance to be tested (prepared in accordance with the sample preparations described below) is then added to the tube until the tube is approximately ³ Λ full, and the mixture is vigorously shaken. The colour change (if any) may then be observed, and the resultant colour compared with the colour chart in order to identify any transition metal ions which are in the sample.

Often a mixture of colours will be observed due to the fact that more than one metal ion is present in the

sample. In general, the more intense the colour, the more metal ions there are in the sample.

A colour chart can help to identify ionic metals present in a test sample. Such a colour chart may be provided as part of the kit of the seventh aspect of the present invention.

Sample preparation Some test substances such as tap water, tank water, urine and other aqueous solutions may be added to the dithizone solution without further preparation, however, other substances to be tested must be first prepared using the methods described below.

House dust or garden soil

Take a small amount of solid house dust from a vacuum cleaner, window still, air conditioning etc. and place it in a clean glass jar with a small amount of distilled or demineralised water and shake well.

Paint

Remove some specks of paint and place them in a glass jar together with distilled or demineralised water and shake well.

Ceramic dinner ware

Pour hot water into the cup, on the plate etc. and let stand until water is cool.

Amalgam filings

Chew some (sugar free) chewing gum and collect a small amount of saliva in a sample jar. Mix the saliva with

approximately twice the volume of demineralised water. Transfer approximately 2 ml of the mixture to the test tube.

Babies nappies

Take a wet nappy and add about 10 to 15 drops of the dithizone solution to the wet nappy and observe any colour change .

Although the present invention has been described with reference to particular embodiments, it will be appreciated by those skilled in the art that the invention may be embodied in many other forms .

In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.