Title of Invention	" A METHOD FOR ESTIMATION OF LEAD IN FOOD SAMPLES"
Abstract	The invention relates to a method for quick estimation of lead in food sample, the said method comprising ashing of food sample; adding test solutions sequentially to the ashes, observing and comparing the color developed with the standard color chart to estimate the lead concentration in food sample. The invention also provides a method of preparing test solutions and a kit for estimation of lead in food sample. The method for estimation of lead in the food sample disclosed in the present invention does not require any sophisticated equipment, technical expertise or involve laborious procedure.

Title of Invention

" A METHOD FOR ESTIMATION OF LEAD IN FOOD SAMPLES"

Abstract

The invention relates to a method for quick estimation of lead in food sample, the said method comprising ashing of food sample; adding test solutions sequentially to the ashes, observing and comparing the color developed with the standard color chart to estimate the lead concentration in food sample. The invention also provides a method of preparing test solutions and a kit for estimation of lead in food sample. The method for estimation of lead in the food sample disclosed in the present invention does not require any sophisticated equipment, technical expertise or involve laborious procedure.

Full Text	A METHOD FOR ESTIMATION OF LEAD IN FOOD SAMPLES FIELD OF INVENTION The present invention relates to a method for estimation of lead in food samples. The method disclosed in the invention does not require any sophisticated equipment, technical expertise or involve laborious procedure. The invention also provides a kit for quick test to estimate lead in the food samples including processed food. BACKGROUND OF THE INVENTION Lead contamination leads to the toxic effects on the central nervous system and causes renal damage. Lead gets into foods through metallic pesticides/insecticides or when food is processed in lead lined tank used for storing water or cooking in soldered containers. Lead is harmful in food samples and maximum permissible limit of lead in food sample especially in processed food is 5 ppm except in dehydrated onions (not more than 10 ppm). For estimating lead present in food samples, generally colorimetric procedure is adopted and in the recent years atomic absorption spectrophotometric (AAS) method has been used. These methods are quite laborious, time consuming though very accurate. Colorimetric method takes about 7-8 hr and atomic absorption spectrophotometric method takes 6-7 h for estimation of lead in food sample. Codex Elementarius Commission ((CEC) Codex alimentarius 1997, 14(7), metal screening test, page 182-184) has specified metal screening test which is applicable for detection of lead. In this method for identifying the presence of lead in the food sample, three sample solutions were compared with a standard solution. The CEC method is qualitative, by which only presence or absence of lead in sample can be predicted but, quantity of lead cannot be estimated. There are few references in terms of patents for detection of lead and mostly in non-food field. These are only qualitative methods. Gould John patented (US5010020, US4873197 and WO9004783) a quick color test to detect lead released from glazed and enamel coatings. The method disclosed in these patents use citric acid solution on filter paper to extract lead from the ware. Presence of lead on the paper was detected by a lead sensitive chromogen. This is again a lead detection test used for enamel coatings and is not suitable for lead estimation in food materials. Cole Sandralipin patented (US6489170, US 2003203496 and US 2003049852) a chemical spot test for the presence of lead in paints. The test is based upon the reaction of lead with sodium rhodizonate under strong acidic conditions to develop an intense purple colored complex. This test is reported for non-food system. Decastro et al patented (US5437772) a portable, hand held detector system and method of operating the system for detection of trace amount of lead as well as other metals. The limitation of this method is requirement of sophisticated equipment and expertise. In CEC method, sulfuric acid is used which is very hazardous acid, whereas in the method disclosed in the present sulfuric acid is not required at all. In the present invention estimation of lead in food is carried out within 25-30 minutes. Color comparison with a standard concentration makes the test semi-quantitative. This is very simple, easy and quick method and does not require sophisticated equipment or expertise. OBJECTS OF THE INVENTION The main object of the present invention is to develop a method for estimation of lead in food sample which is simple, easy and does not require sophisticated equipment, and specially trained man power and can be achieved within 25-30 minutes by the addition of test solutions to ashes of food samples. Yet another object of the present invention is to develop a method for estimation of lead present in food sample which require different color development based on the concentration of lead in food sample. Still yet another object of the present invention is to provide the composition of the test solutions used for the estimation of the lead present in the food sample. Still another object of the present invention is to provide the kit comprising the test reagents necessary for the estimation of the lead present in the food sample. SUMMARY OF INVENTION The present invention describes a method of estimation of lead in food sample. The method comprises preparation of test solution, ashing of food sample on direct flame, development of color and comparison of color with standard color chart to determine the concentration of lead. DETAILED DESCRIPTION OF THE INVENTION A method has been, developed for estimation of lead in food materials. The method comprises preparing test solutions containing chloroform, ammonia solution, potassium cyanide, citric acid, nitric acid, and dithizone; ashing of food samples on direct flame; addition of test solution to the ashes of food sample. Comparing the color developed with standard color chart to estimate the concentration of lead in the tested food sample. After color development, the concentration of lead was measured by AAS. The present invention provides a method for quick estimation of lead in food sample, the said method comprising a) ashing of food sample on direct flame, b) adding test solutions sequentially to the ashes of the food sample as follows: i. Solution A containing 1 % nitric acid ii. Solution B containing 3.3% citric acid, 8.33% ammonia solution and 1.6% potassium cyanide iii. Solution C containing dithizone (0.02 %) in chloroform and, iv. Solution D containing chloroform and c) Observing and comparing the color developed with the standard color chart as shown in the table 1 to estimate the lead concentration in food sample. In another embodiment, the present invention wherein the ashing of the food sample is carried out for about 10 to 20 minutes, preferably for about 15 minutes. In another embodiment, the invention provides a kit for the estimation of lead in food, the said kit comprising nitric acid, ammonia, potassium cyanide, dithizone in chloroform, chloroform, crucible, spatula, graduated glass test tube, color chart and protocol. This method also describes standardization of lead concentration using pure lead nitrate solution. Results obtained by this method are compared with AAS results and found comparable. Estimation of the lead in the food sample was carried our by ashing the food sample on direct flame and adding the test solutions to the charred food sample. Color development was observed and compared with the standard color chart as given in table 1. The development of color occurs due to replacement of hydrogen ion of secondary amino group with the metal i.e. lead that forms a lead dithizone color complex. Table: 1 Color and Concentration profile of lead with dithizone reagent(Table Removed) EXAMPLE It should be understood that the following examples described herein are for illustrative purposes only and that various modifications or changes in light will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims. Example 1 1) Preparation of test solution A: One ml nitric acid mixed with 99 ml distilled water (1% nitric acid). Solution was mixed well and transferred to a glass bottle. 2) Preparation of test solution B: Test solution B was prepared by mixing 100 ml citric acid (10%), 100 ml. ammonia solution (25%) and 100 ml potassium cyanide (5%). 3) Preparation of test solution C: Dithizone reagent (0.02%) was prepared by dissolving 0.02 gm dithizone in 100 ml chloroform. Before preparing the dithizone reagent dithizone was extracted with ammonia solution 3 times to remove oxidized portion of dithizone. Dithizone was extracted with ammonia solution three times to remove the oxidized form of dithizone. 4) Test solution D: Chloroform 5) Standard lead solution: Standard lead solution was prepared by using pure lead nitrate solution in distilled water. Lead concentration varies from 0-20 ppm in the standard lead solution. Example 2 Quick test method for estimation of lead in food materials was developed based on color development with the help of test solutions and ashes of food sample. To standardize the method, lead concentration was measured by atomic absorption spectrophotometer (AAS) in sample solution and found out how quantity of lead varies with the color change. Standard lead solution (0 to 20 ppm) was also prepared and the lead concentration was measured by AAS and compared with sample solution to standardize the method. Various processed food samples were tested for lead content ad the results are presented in table 2. One gram of tuna fish sample was taken in silica crucible and heated over direct flame for 15 min. Ash of tuna fish sample was transferred to a graduated glass tube containing 5 ml of solution A. To this 6 ml solution B, 1 ml solution C and 20 ml solution D were added and the mixture was shaken vigorously. Color development was observed and compared with the standard color chart to estimate the concentration of lead in tuna fish sample. The blue color (Table 2) indicated that concentration of lead in tuna fish sample is 2 ppm. One gram of canned Sardine was taken in a silica crucible, heated over direct flame for 15 min and transferred to a graduated glass tube containing 5 ml of solution A. To this 6 ml solution C and 20 ml solution D were added and mixture was shaken vigorously. Color developed was observed and compared with standard color chart to determine lead concentration in canned Sardine sample. The color developed was red and hence the lead concentration was more the 10 ppm (Table 2). Statistical analysis of the Lead Content in various food Samples is provided in table 3. Example 3 Components of Kit for quick estimation of lead present in the food sample 1. 250 ml solution A containing 1 % nitric acid in glass bottle. 2. 300 ml solution B containing citric acid (3.3%), ammonia solution (8.33%) and potassium cyanide (1.6%) in glass bottle or plastic bottle. 3. Dithi/one (0.02 g) in plastic tube (100 ml solution D to be added prior to test to get dithizone reagent). 4. 1 liter solution D containing only chloroform, in glass bottle. 5. 1 crucible, one spatula, graduated glass test tube of 50 ml capacity, 5 nos. 6. One standard color chart. All the components of kit are packed in a corrugated box of size 30 cm x 30 cm x 21 cm weighing 3 kg. This kit is suitable for analysis of 50 samples. I/We Claim: 1) A method for quick estimation of lead in food sample, the said method comprising a) ashing of food sample on direct flame, b) adding test solutions sequentially to the ashes of the food sample as follows: i. Nitric acid (1 %), ii. Solution containing citric acid (3.3%), ammonia solution (8.33%) and potassium cyanide (1.6%), iii. Dithizone (0.02 %) in chloroform and, iv. Chloroform and c) Observing and comparing the color developed with the standard color chart as shown in the table given below to estimate the lead concentration in food sample.(Figure removed) 2) A method as claimed in claim 1, wherein the food sample is processed food. 3) A method as claimed in claim 1, wherein the ashing of the food sample is carried out for about 10 to 20 minutes, preferably for about 15 minutes. 4) A kit for the estimation of lead in food, the said kit comprising nitric acid, ammonia, potassium cyanide, dithizone in chloroform, chloroform, crucible, spatula, graduated glass test tube, color chart and protocol. 5)

Full Text

A METHOD FOR ESTIMATION OF LEAD IN FOOD SAMPLES FIELD OF INVENTION
The present invention relates to a method for estimation of lead in food samples. The method disclosed in the invention does not require any sophisticated equipment, technical expertise or involve laborious procedure. The invention also provides a kit for quick test to estimate lead in the food samples including processed food.
BACKGROUND OF THE INVENTION
Lead contamination leads to the toxic effects on the central nervous system and causes renal damage. Lead gets into foods through metallic pesticides/insecticides or when food is processed in lead lined tank used for storing water or cooking in soldered containers. Lead is harmful in food samples and maximum permissible limit of lead in food sample especially in processed food is 5 ppm except in dehydrated onions (not more than 10 ppm). For estimating lead present in food samples, generally colorimetric procedure is adopted and in the recent years atomic absorption spectrophotometric (AAS) method has been used. These methods are quite laborious, time consuming though very accurate. Colorimetric method takes about 7-8 hr and atomic absorption spectrophotometric method takes 6-7 h for estimation of lead in food sample.
Codex Elementarius Commission ((CEC) Codex alimentarius 1997, 14(7), metal screening test, page 182-184) has specified metal screening test which is applicable for detection of lead. In this method for identifying the presence of lead in the food sample, three sample solutions were compared with a standard solution. The CEC method is qualitative, by which only presence or absence of lead in sample can be predicted but, quantity of lead cannot be estimated.
There are few references in terms of patents for detection of lead and mostly in non-food field. These are only qualitative methods. Gould John patented (US5010020, US4873197 and WO9004783) a quick color test to detect lead released from glazed and enamel coatings. The method disclosed in these patents use citric acid solution on filter paper to extract lead from the ware. Presence of lead on the paper was detected by a lead sensitive chromogen. This is again a lead detection test used for enamel coatings and is not suitable for lead estimation in food materials.
Cole Sandralipin patented (US6489170, US 2003203496 and US 2003049852) a chemical spot test for the presence of lead in paints. The test is based upon the reaction
of lead with sodium rhodizonate under strong acidic conditions to develop an intense purple colored complex. This test is reported for non-food system.
Decastro et al patented (US5437772) a portable, hand held detector system and method of operating the system for detection of trace amount of lead as well as other metals. The limitation of this method is requirement of sophisticated equipment and expertise.
In CEC method, sulfuric acid is used which is very hazardous acid, whereas in the method disclosed in the present sulfuric acid is not required at all. In the present invention estimation of lead in food is carried out within 25-30 minutes. Color comparison with a standard concentration makes the test semi-quantitative. This is very simple, easy and quick method and does not require sophisticated equipment or expertise.
OBJECTS OF THE INVENTION
The main object of the present invention is to develop a method for estimation of lead in food sample which is simple, easy and does not require sophisticated equipment, and specially trained man power and can be achieved within 25-30 minutes by the addition of test solutions to ashes of food samples.
Yet another object of the present invention is to develop a method for estimation of lead present in food sample which require different color development based on the concentration of lead in food sample.
Still yet another object of the present invention is to provide the composition of the test solutions used for the estimation of the lead present in the food sample.
Still another object of the present invention is to provide the kit comprising the test reagents necessary for the estimation of the lead present in the food sample.
SUMMARY OF INVENTION
The present invention describes a method of estimation of lead in food sample. The method comprises preparation of test solution, ashing of food sample on direct flame, development of color and comparison of color with standard color chart to determine the concentration of lead.
DETAILED DESCRIPTION OF THE INVENTION
A method has been, developed for estimation of lead in food materials. The method comprises preparing test solutions containing chloroform, ammonia solution, potassium cyanide, citric acid, nitric acid, and dithizone; ashing of food samples on
direct flame; addition of test solution to the ashes of food sample. Comparing the color developed with standard color chart to estimate the concentration of lead in the tested food sample. After color development, the concentration of lead was measured by AAS.
The present invention provides a method for quick estimation of lead in food sample, the said method comprising
a) ashing of food sample on direct flame,
b) adding test solutions sequentially to the ashes of the food sample as follows:
i. Solution A containing 1 % nitric acid
ii. Solution B containing 3.3% citric acid, 8.33% ammonia solution and
1.6% potassium cyanide
iii. Solution C containing dithizone (0.02 %) in chloroform and, iv. Solution D containing chloroform and
c) Observing and comparing the color developed with the standard color chart
as shown in the table 1 to estimate the lead concentration in food sample.
In another embodiment, the present invention wherein the ashing of the food sample is carried out for about 10 to 20 minutes, preferably for about 15 minutes.
In another embodiment, the invention provides a kit for the estimation of lead in food, the said kit comprising nitric acid, ammonia, potassium cyanide, dithizone in chloroform, chloroform, crucible, spatula, graduated glass test tube, color chart and protocol.
This method also describes standardization of lead concentration using pure lead nitrate solution. Results obtained by this method are compared with AAS results and found comparable.
Estimation of the lead in the food sample was carried our by ashing the food sample on direct flame and adding the test solutions to the charred food sample. Color development was observed and compared with the standard color chart as given in table 1. The development of color occurs due to replacement of hydrogen ion of secondary amino group with the metal i.e. lead that forms a lead dithizone color complex. Table: 1 Color and Concentration profile of lead with dithizone reagent(Table Removed)
EXAMPLE
It should be understood that the following examples described herein are for illustrative purposes only and that various modifications or changes in light will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims. Example 1
1) Preparation of test solution A: One ml nitric acid mixed with 99 ml distilled water
(1% nitric acid). Solution was mixed well and transferred to a glass bottle.
2) Preparation of test solution B: Test solution B was prepared by mixing 100 ml citric
acid (10%), 100 ml. ammonia solution (25%) and 100 ml potassium cyanide (5%).
3) Preparation of test solution C: Dithizone reagent (0.02%) was prepared by
dissolving 0.02 gm dithizone in 100 ml chloroform. Before preparing the dithizone
reagent dithizone was extracted with ammonia solution 3 times to remove oxidized
portion of dithizone.
Dithizone was extracted with ammonia solution three times to remove the oxidized form of dithizone.
4) Test solution D: Chloroform
5) Standard lead solution: Standard lead solution was prepared by using pure lead
nitrate solution in distilled water. Lead concentration varies from 0-20 ppm in the
standard lead solution.
Example 2
Quick test method for estimation of lead in food materials was developed based on color development with the help of test solutions and ashes of food sample. To standardize the method, lead concentration was measured by atomic absorption spectrophotometer (AAS) in sample solution and found out how quantity of lead varies with the color change. Standard lead solution (0 to 20 ppm) was also prepared and the lead concentration was measured by AAS and compared with sample solution to standardize the method. Various processed food samples were tested for lead content ad the results are presented in table 2.
One gram of tuna fish sample was taken in silica crucible and heated over direct flame for 15 min. Ash of tuna fish sample was transferred to a graduated glass tube containing 5 ml of solution A. To this 6 ml solution B, 1 ml solution C and 20 ml solution D were added and the mixture was shaken vigorously. Color development was observed and compared with the standard color chart to estimate the concentration of
lead in tuna fish sample. The blue color (Table 2) indicated that concentration of lead in tuna fish sample is 2 ppm.
One gram of canned Sardine was taken in a silica crucible, heated over direct flame for 15 min and transferred to a graduated glass tube containing 5 ml of solution A. To this 6 ml solution C and 20 ml solution D were added and mixture was shaken vigorously. Color developed was observed and compared with standard color chart to determine lead concentration in canned Sardine sample. The color developed was red and hence the lead concentration was more the 10 ppm (Table 2).
Statistical analysis of the Lead Content in various food Samples is provided in table 3. Example 3 Components of Kit for quick estimation of lead present in the food sample
1. 250 ml solution A containing 1 % nitric acid in glass bottle.
2. 300 ml solution B containing citric acid (3.3%), ammonia solution (8.33%)
and potassium cyanide (1.6%) in glass bottle or plastic bottle.
3. Dithi/one (0.02 g) in plastic tube (100 ml solution D to be added prior to test
to get dithizone reagent).
4. 1 liter solution D containing only chloroform, in glass bottle.
5. 1 crucible, one spatula, graduated glass test tube of 50 ml capacity, 5 nos.
6. One standard color chart.
All the components of kit are packed in a corrugated box of size 30 cm x 30 cm x 21 cm weighing 3 kg. This kit is suitable for analysis of 50 samples.

I/We Claim:
1) A method for quick estimation of lead in food sample, the said method comprising
a) ashing of food sample on direct flame,
b) adding test solutions sequentially to the ashes of the food sample as
follows:
i. Nitric acid (1 %),
ii. Solution containing citric acid (3.3%), ammonia solution (8.33%)
and potassium cyanide (1.6%), iii. Dithizone (0.02 %) in chloroform and, iv. Chloroform and
c) Observing and comparing the color developed with the standard color
chart as shown in the table given below to estimate the lead concentration
in food sample.(Figure removed)
2) A method as claimed in claim 1, wherein the food sample is processed food.
3) A method as claimed in claim 1, wherein the ashing of the food sample is carried
out for about 10 to 20 minutes, preferably for about 15 minutes.
4) A kit for the estimation of lead in food, the said kit comprising nitric acid,
ammonia, potassium cyanide, dithizone in chloroform, chloroform, crucible,
spatula, graduated glass test tube, color chart and protocol.
5)

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Patent Number

271224

Indian Patent Application Number

339/DEL/2006

PG Journal Number

07/2016

Publication Date

12-Feb-2016

Grant Date

09-Feb-2016

Date of Filing

06-Feb-2006

Name of Patentee

DIRECTOR GENERAL, DEFENSE RESEARCH & DEVELOPMENT ORGANIZATION

Applicant Address

Ministry of Defence, Goverment of India, Room No 348, B-Wing, DRDO Bhawan, Rajaji Marg, New Delhi 110011 India

Inventors:

#	Inventor's Name	Inventor's Address
1	TAPAS KUMAR MAJUMDER	DEFENSE FOOD RESEARCH LABORATORY, SIDDARTHNAGAR, MYSORE-570011, INDIA.
2	AMARINDER SINGH BAWA	DEFENSE FOOD RESEARCH LABORATORY, SIDDARTHNAGAR, MYSORE-570011, INDIA.
3	KUNIGAL SRINIVASAIAH PREMAVALLI	DEFENSE FOOD RESEARCH LABORATORY, SIDDARTHNAGAR, MYSORE-570011, INDIA.

PCT International Classification Number

G01N 21/78

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA