ARGEMONE OIL DETECTION KIT FOR MUSTARD OIL
The present invention is directed to a kit and a method of detecting argemone oil adulteration/contamination in mustard oil.
Argemone oil is very common adulterant in mustard oil and it is generally used for its pungent smell quite similar to mustard oil. However, this results in health hazard to the consumers. Musrard oil is consumed by population groups in Asian, European and North American countries. The toxic species of argemone oil is identified as quaternary benzo[c]phenanthridine alkaloid, known as sanguinarine. It is responsible for dropsy syndrome in humans. Several chromatographic, colorimetric and spectroscopic methods have been developed for sanguinarine detection. Most of the methods require concentrated acids and costly chemicals. In our study, argemone oil was detected by simple column based method, where the user has to pour the oil {a few ml) on the top of a column packed with beads of cupric acetate-chitosan beads and the adulterated oil will change it's colour to green as it passes out of the column. Detection occurs at the contamination level >0.1% in mustard oil. A column can be used 20 times for testing by washing with diethyl ether and water.
According to the present invention, the inventors have applied a novel approach to detect argemone oil by cupric acetate-chitosan beads packed bed column. The most interesting observation in this research has been the high sensitivity of the beads towards argemone oil. The column based detection kit displayed a qualitative determination of argemone oil adulteration in edible oils mainly mustard oil of more than 0.1%. The study was based on a number of elution experiments with adulterated mustard oils collected from the.local market. In the present invention, the cupric acetate cross-linked chitosan gel bead has been used as a packed bed. Argemone oil detection by cupric acetate-chitosan packed column is also new. The present invention discloses an easy column preparation and detection procedure which has made it very handy since any layman can do the test. The qualitative detection is confirmed for >0.1% of adulterant, whereas the adulterant is used in much higher proportion. Reuse of one column is possible for 20 tests.

The main object of the present invention is to develop a detection kit for argemone oil adulteration/contamination in edible oils mainly mustard oil by using cupric acetate-chitosan gel beads packed column.
The other object is to develop a new and simple method for detecting argemone oil adulteration/contamination in mustard oil.
According to the invention there is provided a kit for detecting argemone oil adulteration/contamination in edible oils comprising:
i) One or more tightly capped cupric acetate-chitosan bead packed column/tube with
provision for inlet and outlet for the test oil,
ii) One or more oil collecting tubes and
iii) One or more air tight bottles containing diethyl ether.
The inventive kit additionally has one or more airtight bottles of distilled water required for storing the packed column/tube by filling with distilled water when not in use.
In one embodiment of the invention, the cupric-acetate chitosan bead packed column/tube(s) used is/are of 15 ml capacity falcon tube(s) and the oil collecting tube(s) is/are of about 5 ml capacity preferably made of plastic and the bottle(s) containing ether is preferably of 50 ml capacity.
The kit of the invention is required to be suitably protected from breakage or damage for storage, transport etc. For that it is necessary that the same is packaged with a protective cover/box. The cover/box should also contain necessary printed directions or instructions for use of the kit either on the box itself or inside so that a layman can use it.
A used cupric acetate-chitosan beads column/tube can be reused by first washing the column/tube with diethyl ether and then with distilled water. The process can

be repeated for reusing the packed column/tube up to twenty times for maintaining its efficiency.
The advantage of this kit is that it is very handy and economical also. The following table shows the estimated cost of different reagents used and the set up used in the kit which comes about Rs. 35/- i.e much less than a US dollar.


This invention also includes a method of detection of argenome oil adulteration/contamination in mustard oil which comprises passing about 2 ml test oil through a column/tube packed with cupric acetate-chitosan beads by gravity and looking for the change of colour of the eluted oil as green showing positive contamination of argemone oil in the test oil.
The detection method is very well accomplished by using the kit as per the
instruction/directions given therewith. The packed colum/tube is filled with distilled .
water when not in use and the water is drained out before use.
The edible oils which are tested for contamination/adulteration includes mustard
oil.
Methodology:
Cupric acetate-chitosan bead preparation:
Reagent A: Chitosan solution is prepared by dissolving 2.5% chitosan in 2% acetic acid solution. The mixture is allowed to homogenize overnight at 80°C temperature with vigorous stirring at 800 rpm using a magnetic stirrer bar. Triton X-100 (neutral surfactant) is first added to chitosan solution (10% by volume) to reduce surface tension and enhance porosity.
Reagent B: Cupric acetate solution is prepared simply dissolving 6% solid cupric acetate in ultrapure water at room temperature with 150rpm stirring condition.

Beads: Chitosan gel beads are prepared by adding chitosan solution drop by drop from 5cm height at the rate of 20 drops per minute. The system is controlled by a peristaltic pump with 5rpm rotor speed. 50 ml of this mixture is used for bead preparation by dispensing drop wise into 0.5M NaOH solution. The beads are separated by a strainer. The same is washed with reverse osmosis water and subsequently dropped into 500 ml of Reagent B. Beads are maintained in dispersed within Reagent B for 24 hours for maximum cross-linking. Beads are then washed thoroughly with distilled water to remove all the uncross-linked reagents.
Cupric acetate-chitosan bead packed-column preparation:
The usual 15 ml capacity falcon tubes with an outlet can be used to pack the column. A total 9gm (wet bead weight) is required to tightly pack the column. Both the inlet and outlet channels are covered with screens. The column is stored at room temperature filled with distilled water when not in use.
Measurement procedure:
Total 2ml of oil sample is poured from top of the column and allowed to elute normally at atmospheric pressure and temperature. If the oil is contaminated with argemone oil, it will turn green while coming out of the packed column. It is a qualitative measurement procedure. The presence of argemone oil is confirmed immediately by passing the test oil through the packed column and checking the colour of the eluted oil as green. It will take 45 min to elute totally from the column and reproducibility of column is found to be twenty tests per column. After every testing, column should be washed with diethyl ether and then with water immediately to remove residual oil of previous test.

We claim:
1. A kit for detecting argemone oil adulteration/contamination in edible oil
comprising:
i. One or more tightly capped cupric acetate-chitosan bead packed
column/tube with provision for inlet and outlet for the test oil,
ii. One or more oil collecting tubes and
iii. One or more air tight bottles containing diethyl ether.
2. A kit as claimed in claim 1 wherein the kit additionally has one or more air tight bottles of distilled water required for storing the packed column/tube by filling with distilled water when not in use.
3. A kit as claimed in claims 1 and 2 wherein the cupric acetate- chitosan bead packed column/tube (s) is/are of 15 ml capacity, falcon tube{s) and the oil collecting tubes is/are of about 5 ml capacity preferably made of plastic and the bottle(s) containing ether is preferably of 50 ml capacity.
4. A kit as claimed in any of the claims 1 to 3 which is suitably packaged with a protective cover/box printed thereon/inside inter alia necessary directions/instructions for use of the kit.
5. A kit as claimed in any of the claims 1 to 4 wherein the column/tube is adaptable to be reused up to 20 times by washing the beads each time after use with first diethyl ether and then with distilled water.

6. A kit as claimed in any of the claims 1 to 5 costing up to about Rs. 35/- only (i.e much less than an USD) inclusive of all chemicals and set up.
7. A method of detection of argemone oil adulteration/contamination in mustard oil comprising passing about 2 ml test oil through a column/tube packed with cupric acetate-chitosan beads by gravity and looking for the change of colour of the eluted oil as green showing positive contamination of argemone oil in the test oil.
8. A method as claimed in claim 7 wherein the method is carried out by using the kit as claimed in any of the claims 1-6.
9. A method as claimed in claims 7&8 wherein the packed column/tube is filled with distilled water when not in use and the water is drained out before use.
10. A method as claimed in claims 7 to 9 wherein the edible oils include mustard oil, sunflower oil and sesame oil.