DESC:Technical field of the Invention
The present invention provides a kit for determining creatine content in a creatine supplement sample by the end user consumer in a home setting. The kit is based on a method that works on the principle of volumetric analysis by titration wherein the colour change is used to determine the endpoint of the titration. The amount of the titrant used to achieve the end point provides the creatine content in the sample.

Background of the Invention
Creatine is an amino acid located mostly in body's muscles as well as in the brain. Most people get creatine through seafood and red meat — though at levels far below those found in synthetically made creatine supplements. The body's liver, pancreas and kidneys also can make about 1 gram of creatine per day.
Creatine dietary supplement is marketed as creatine monohydrate or as a combination with phosphorous. The majority of creatine i.e. about 95% is stored in skeletal muscle (fast twitch, type II) and is further classified as: two-thirds in a phosphorylated form (phosphocreatine) and one-third as free creatine. Creatine serves as an energy substrate for the contraction of skeletal muscle. The intention of creatine supplementation is to increase resting phosphocreatine levels in muscles, as well as free creatine, with the goal of postponing fatigue, even briefly, for sports-enhancing results. Cells with high energy requirements use creatine in the form of phosphocreatine. Phosphocreatine serves as a source of phosphate to produce adenosine triphosphate (ATP) from adenosine diphosphate (ADP). Skeletal muscle cells store enough ATP and phosphocreatine for approximately 10 seconds of high-intensity activity; short-term creatine supplementation leads to a total creatine increase of 10% to 30%, with phosphocreatine stores increasing by 10% to 40%.
Creatine is available in numerous grades and variants form different manufacturers for consumption by athletes and bodybuilders. These are usually not monitored for their quality by the food regulatory authorities and may lead to adulteration or products with less creatine content than the claimed labeled amounts. This means that the consumers will not be sure of the quality and contents. Also, they will not know if the product they are using are as per the quality standards mentioned on the label by the sellers.
It is often that the bodybuilders consult their gym instructors for guidance on bodybuilding supplements who themselves have limited knowledge. They would suggest supplements which are available at cheaper rates. These are usually unregulated products from the grey market and may be spurious. Usually the people from the bodybuilding fraternity buy supplements from local supplement stores or through online channels so the grey marketers directly distribute their products bypassing any quality check by regulatory authority.
The customers can check the authenticity of the products via unique authentication code inside or above the pack which they can use to verify their purchase, but still checking the quality of product is a big question for them. For them it is not possible to check their products as many of them are not aware about the labs, moreover it is not economically feasible to check the creatine content every time they buy the supplement as it would be a costly activity for the consumer.
There are different methods used in laboratories for the estimation of protein such as potentiometric titration by USP method, high performance liquid chromatography (HPLC), and liquid chromatography and mass spectroscopy (LCMS) methods. These methods are complex and can only be performed in a well-equipped laboratory and that too by a skilled analyst.
There are no readily available and consumer friendly methods to check the creatine content at their places. The present invention provides a kit and the method to make customers more aware and confident for the products they are using on routine basis. Furthermore, with the help of the kit, this method can be performed in a home setting even when the consumer has limited technical skills.

Summary of the Invention
The present invention provides a kit for determining creatine content in a creatine supplement sample for the end user consumer in a home setting. The kit is based on a method that works on the principle of volumetric analysis by titration wherein the colour change is used to determine the endpoint of the titration. The amount of the titrant used to achieve the end point provides the creatine content in the sample. Titration is one of the common and easiest method which determines the unknown concentration of the known analyte.
It is an object of the present invention to provide a kit for the determination of creatine content in a protein supplement by the consumer in a home setting.
It is another object of the invention to provide a method for determination of creatine content in a protein supplement wherein the method reduces requirement of complex laboratory apparatus and chemical reagents and the consumer can carry out the method in a simple setup such as a home.
It is another object of the invention to provide a kit and a method for testing if the creatine supplement is a genuine creatine supplement or a spurious creatine supplement. The method can be carried out by the consumer in a home setting.
In one general aspect, the present invention provides a kit for quantitative analysis of creatine content in a protein supplement wherein the kit comprises two glass containers each containing a different solution and a centrifugal tube, wherein:
(a) the first glass container contains the purple colored solution 1 which comprises 2 mL of formic acid, 1 drop crystal violet and 5 ml phenolphthalein indicator.
(b) the second glass container contains the solution 2 which is a titration solution of 0.1 N Perchloric acid.
(c) a centrifugal tube; and
(d) measurement chart.
In an embodiment of the above aspect, the method of using the kit comprises:
1. Taking one scoop (about 300mg) of creatine sample to be tested and adding it to the purple solution 1 and shaking the solution for 10-20 seconds.
2. To the above mixed solution is then added solution 2 in small increments with shaking till the purple color of the solution 1 turns to blue.
3. Measuring the amount of the solution 2 required to change the color of the solution 2 and compare it with the provided measurement chart to determine the amount of creatine in the creatine sample.

Detailed Description of the Invention
This method is fast, reliable, and can be done at the consumer end. The object of the invention was to transfer the complex laboratory method to the consumer’s home where anyone can confirm the quality and purity of the creatine supplements and in this way consumers can become more aware and confident of the quality of the creatine supplements.
The chemical reactions involved in the method are as follows:

Dissociation of perchloric acid in water:
HClO4 ? H+ + ClO4-
Reaction between perchloric acid and creatinine:
Creatine + H+ ? Creatinium ion (Cation) + H2O
Reaction between the indicator (crystal violet) and the perchloric acid:
Crystal Violet + H+ ? Crystal Violet-H+ (Purple to blue)

The method of the invention involves the use of the kit to perform the titration process with the gradual addition of perchloric acid to the creatine solution until all the creatine has reacted, resulting in the color change from purple to blue due to the phenolphthalein indicator. The volume of perchloric acid solution required to reach the endpoint is used to calculate the concentration of creatinine in the sample.
The kit has been used to determine the quantitative amount of creatine monohydrate in the sample and several marketed products were tested by this novel method (see Table 1).

Table 1. Testing of some commercially available creatine supplements by HPLC and the kit of the invention
S. No. Sample Name Claimed Creatine (grams) per 100 grams Creatine (grams) per 100 grams determined by HPLC Titration Solution 2 consumed till end point (mL) Creatine (grams) per 100 grams determined by Kit
1. As It Is Creatine Monohydrate s 100% 92% > 20 mL > 80%
2. MB Creatine Monohydrate 100% 99% > 20 mL > 80%
3. Fast N Up Creatine Monohydrate 100% 95% > 20 mL > 80%
4. Nutrabay Creatine Monohydrate 100% 66% 16 mL 60% - 80%
5. Muscle Asylum Creatine Monohydrate 100% 0.0 % 2 mL 0% -20%

The results were confirmed in the laboratory through quantitative analysis of the same samples using HPLC method for creatine monohydrate. The results were comparable, thus proving that the kit and the method are reliable for a home setting.

Measuring Chart: The amount of Titration Solution 2 corresponds to the amount of creatine present in the test sample.
S. No. Titration Solution 2 consumed (mL) Creatine Content
(weight %)
1. 0-5 0-20
2. 5-10 20-40
3. 10-15 40-60
4. 15 - 20 60-80
5. More than 20 mL > 80 ,CLAIMS:1. A kit for quantitative analysis of creatine content in a protein supplement wherein the kit comprises:
(a) the first glass container containing a purple colored solution 1 which comprises 2 mL of formic acid, 1 drop crystal violet and 5 ml phenolphthalein indicator.
(b) the second glass container containing the solution 2 which is a titration solution of 0.1 N Perchloric acid.
(c) a centrifugal tube; and
(d) measurement chart.

2. The kit as claimed in claim 1, wherein the method of using the kit comprises:
1) Taking one scoop (about 300mg) of creatine sample to be tested and adding it to the purple solution 1 and shaking the solution for 10-20 seconds.
2) To the above mixed solution is then added solution 2 in small increments with shaking till the purple color of the solution 1 turns to blue.
3) Measuring the amount of the solution 2 required to change the color of the solution 2 and compare it with the provided measurement chart to determine the amount of creatine in the creatine sample.

3. The method of using the kit as claimed in claim 1, wherein the method reduces requirement of complex laboratory apparatus and chemical reagents and the consumer can carry out the method in a simple setup such as a home.

4. The method of using the kit as claimed in claim 1, wherein the method is used for testing if the creatine supplement is a genuine creatine supplement or a spurious creatine supplement.