INTRODUCTION:

Food analysis is a discipline dealing with the development, application and study of analytical procedures for characterizing the properties of food products and its constituents. These analytical procedures are used to provide information about a wide variety of different characteristics of food including their composition, structure, physiochemical properties of food and sensory attributes. This information is critical to our rational understanding of the factors that determine the properties of food, as well as the ability of our country to economically produce foods that are consistently safe, nutritious and desirable and for consumers to make informed choices about their diet. [1]A food additive is a substance that is added to the food for a specific purpose.

Direct or indirect food additives are those that become part of the food (if by default) in trace amount due to agricultural, packaging, storage, handling or processing technique.[2]

Health hazards or deterioration of health of common people takes place due to the presence of these additives or their presence in above/below the permissible limit.[3] Thus, the food products need to be analyzed for the presence of additives in the right quantity in order that they are only advantageous and do not turn disadvantageous for consumption.[4] Analysis of the food products for various parameters can be carried out by various methods. These involve various qualitative, quantitative and instrumentation methods.

SUDAN DYE IN RED CHILLI POWDER:

Chilli (botanically known as Capsicum annum L.; Capsicum frutescens L.) Is one of the most important commercial crop of India. It is grown almost throughout the country.[5] Indian chilli is considered to be world famous for two important commercial qualities-its colour and pungency levels. Sudan dyes (1-[E]-Phenyldiazenyl]-2-naphthol) are a family of lipophilic azo dyes, extensively used in industries for various purposes, but banned for use as a food colorant due to its potential risk of developing carcinogenicity. It is suspected to be fraudulently added to enhance and maintain the chilli colour as over the time a plant product looses it’s natural colour and the price of the chilli powder is largely linked to the intensity of its colour.[6] There has been concern about contamination of chilli powder and other spices with 1-amino-2-naphtol-based azo dyes (Sudan I, II, III, and IV and Para Red). There is evidence that Sudan dyes have genotoxic effects and that ingestion of food products contaminated with Sudan I, II, III and IV and Para Red could lead to exposure in the human gastrointestinal tract.[7] The use of these dyes in food products is banned as per Indian standards.[8] The dye can be detected by different chromatographic, colorimetric and spectrophotometric methods.

Figure-1: Structural Formula of Sudan I dye

Preservatives in Tomato Ketchup:

Tomato ketchup, also known as 'catsup' occupies an important place among processed tomato products. Preservatives due to their general property of avoiding spoilage of food products by micro- organisms, increased enzyme activity, insects, etc are commonly added in specific amounts. Ketchup contains the natural, commonly available and generally un-harmful preservatives such as salt, sugar and vinegar that come under class 1 of preservatives. No other class 2 preservative except for benzoic acid and its salts are permitted by the Food Safety and Standard Authority of India(FSSAI) and it’s permissible limit is up to 750 ppm.[9,Bureau of Indian Standard] Both benzoic acid and methyl paraben are bacteriostatic and bactericidal[10] and hence extensively used in ketchups as preservatives. However, their presence should not be in a quantity that is deteriorating to human health.

Figure-2: Structural Formula of Benzoic Acid

MATERIAL AND METHOD:

Detection Of Sudan I Dye In Red Chilli Powder :- [11]:

A] Standard (chilli):- 2.5 grams of raw chilli powder samples (10 different varieties) was weighed and solvent extraction was carried out using 20 ml ethyl acetate. The volume was made in a 25ml volumetric flask after which these standards were stored in amber coloured bottles.

B] Standard (Sudan 1 dye):- Sudan stock solution of 100 ppm was prepared using ethyl acetate.

C] Chilli sample:- 38 different chilli powder samples (branded and non-branded) were collected, weighed (2.5 gms) and their extraction was carried out in the same manner as for standard chilli powders.

D] Protocol:- I) TLC method:-TLC plates were prepared using silica gel powder and chloroform. The plates were activated and were spotted with sample, dye, and standard extract (different extracts were used alternately). The plates were allowed to develop in the chamber containing n-hexane and acetonitrile (6:4).plates were allowed to dry and R_f values were determined. II) Colorimetric method:- stock solution of dye was diluted in the range 50, 20, 5 and 1 ppm with ethyl acetate and λ-max was determined. 1/50 dilution of the sample extract was carried out. O.D was obtained at λ-max (470nm).The 10 chilli standard extracts were diluted into 100,50,20,5 and 1 ppm and their O.D was taken before spiking and after spiking with dye graph of concentration v/s O.D. Was plotted to spot the dye concentration in sample.

Detection Of Benzoic Acid and Methyl Paraben From Tomato Ketchups[12]:

A] Extract preparation:10 gms. Of ketchup sample was extracted with methanol (20ml).The mixture was vortex and centrifuged at approximately 2630rpm for 5 mins. and volume was made up in a 100 ml volumetric flask with methanol. The steps were repeated twice with further portions of methanol. The extracts were combined and the volume was made in a 100ml volumetric flask.

B]High Performance Liquid chromatography analysis the homogeneous solution was filtered through a filter paper, 1st few ml was rejected. About 15ml of the filtrate was collected and filtered through 0.45µm filter. : RP-HPLC was performed using simultaneous detection method. Mobile phase was 20% acetonitrile and 80% of 10 mm sodium citrate buffer. Column was C₁₈Phenomenex column, Injection volume was 10µl, Detector was Photodiode Array, Wavelength for benzoic acid: 223nm;methyl paraben:258nm.

OBSERVATION AND RESULT:

1) FOR CHILLI POWDER:

Figure 3

Figure 4

Table 4 :Cont....

Name of chilli standard	Lavangi			Bor
Concentration (ppm)	Before spiking (O.D)	After spiking (O.D)	Dye (O.D)	Before spiking (O.D)	After spiking (O.D)	Dye (O.D)
100	0.19	0.70	0.51	0.06	0.66	0.60
50	0.11	0.62	0.51	0.03	0.29	0.26
20	0.02	0.41	0.39	0.00	0.87	0.87
5	0.00	0.87	0.87	0.00	0.87	0.87
1	0.00	0.87	0.87	0.00	0.87	0.87

Figure 7:Chilli Standard - Lavangi. Figure 8: Chilli Standard - Bor

RESULT BY GRAPH:

Table 5 :After spiking, dye and chilli amount (ppm) of the 4 samples with respect to 10 chilli standards.

Chilli standard	Reshampatti	Lavngi	Bor	Madras pari	Kashmiri
Sample 1 A.F.	5.00	17.00	39.0	3.30	3.40
Sample 2 A.F.(ppm)	-	20.00	46.0	4.20	4.10
Sample 3 A.F.(ppm)	4.00	14.00	34.0	2.80	2.80
Sample 4 A.F.(ppm)	3.10	11.50	30.0	2.20	2.30
Sample 1 DYE (ppm)	4.60	16.00	38.0	3.20	3.20
Sample 2 DYE (ppm)	-	19.00	44.5	4.00	4.00
Sample 3 DYE (ppm)	3.80	13.50	33.5	2.70	2.60
Sample 4 DYE (ppm)	3.00	11.00	29.5	2.10	2.20
Sample 1 Colour of Chilli (ppm)	0.40	1.00	1.00	0.10	0.20
Sample 2 Colour of Chilli (ppm)	-	1.00	1.50	0.20	0.10
Sample 3 Colour of Chilli (ppm)	0.20	0.50	0.50	0.10	0.20
Sample 4 Colour of Chilli (ppm)	0.10	0.50	0.50	0.10	0.10

*A.F:- After Spiking.

Table 5 : Cont...

Chilli standard	Guntur	Pandi	Sankeshwari	Thai bird	Byadagi
Sample 1 A.F.	37.00	14.00	3.00	14.00	4.00
Sample 2 A.F.(ppm)	44.00	17.00	4.00	17.0	4.90
Sample 3 A.F.(ppm)	33.00	11.50	2.20	11.50	3.30
Sample 4 A.F.(ppm)	29.50	9.50	1.70	10.00	2.30
Sample 1 DYE (ppm)	36.00	13.00	2.80	13.50	3.80
Sample 2 DYE (ppm)	43.00	16.00	3.80	16.00	4.80
Sample 3 DYE (ppm)	32.50	11.00	2.10	11.00	3.00
Sample 4 DYE (ppm)	29.00	9.00	1.60	9.50	2.20
Sample 1 Colour of Chilli (ppm)	1.00	1.00	0.20	0.50	0.20
Sample 2 Colour of Chilli (ppm)	1.00	1.00	0.20	1.00	0.10
Sample 3 Colour of Chilli (ppm)	0.50	0.50	0.10	0.50	0.30
Sample 4 Colour of Chilli (ppm)	0.50	0.50	0.10	0.50	0.10

*A.F:- After Spiking.

OBSERVATION AND RESULT -

2) FOR TOMATO KETCHUP:


Figure 9 : HPLC chromatogram - Standard at 223nm.	Figure 10 : HPLC chromatogram - Sample 1.

Figure 11 : HPLC chromatogram - Sample 2.	Figure 12 : HPLC chromatogram - Sample 3.

Figure 13 : HPLC chromatogram - Sample 4.	Figure 14 : HPLC chromatogram - Standard at 258nm.

Table 6 : Concentration of Benzoic acid in the 4 samples.

Name of analyte	Peak area	Peak area of std.	Conc. Of std.(ppm)	Conc. Of benzoic acid (ppm)
Sample 1	1494607	3411824	100	43.80 = ~44
Sample 2	1346193	3411824	100	39.45 = ~39
Sample 3	623288	3411824	100	18.26 = ~18
Sample 4	713125	3411824	100	20.90 = ~30

CONCLUSION:

1) FOR SUDAN 1 DYE IN CHILLI POWDER:

The absorbance values of the chilli samples considered is taken into account for being present probably because of the original chilli colour (from which the chilli powder has been made) and the colour of the dye (if present).However, there is a possibility that the high absorbance values in the 4 samples detected for the presence of the dye may be due to the original colour of the chilli (contributed by the high concentration of natural colouring pigments in it i.e capsanthin) from which it is made that gave the same/near R_fvalue as that of the dye and which accounted to give high concentration value when plotted on graph with their respective standards. Also, the 4 samples could be produced from the mixture of different chillies of the 10 used here or other varieties which lead to give a high concentration value. The samples can thus be detected further to spot their original source (raw chilli or chillies) from which they have been developed and the dye (if present) can be quantified using HPLC (high performance liquid chromatography) method or other methods so as to confirm the results.

2) FOR PRESERVATIVES IN TOMATO KETCHUP:

On the basis of the above results obtained by HPLC analysis for the particular plant matter i.e. Tomato ketchup, benzoic acid being detected was quantified whereas methyl paraben ( methyl 4-hydroxybenzoate ) was not detected at both the maximum wavelengths i.e. 223nm and 258nm in the samples. Thus, the 4 samples of sauces selected for analysis of preservatives (benzoic acid and methyl paraben) contain benzoic acid and not methyl paraben as a class 2 preservative. According to Food Safety and Standard Authority of India (FSSAI) and the Bureau of Indian Standards, permissible limit of benzoic acid concentration in tomato sauces is 750 ppm max. As the benzoic acid concentration in all the 4 samples were within the permissible limit they can be considered to be safe for consumption on the basis of the preservative concentration. Tomato ketchups selected can further be analyzed for presence of other commonly added preservatives such as sorbic acid and other derivatives of benzoic acid such as ethyl 4-hydroxy benzoate and propyl 4-hydroxy benzoate, which are also permissible up to 750ppm max. Other tomato sauces of different brands can be tested for presence and quantification of preservatives

ACKNOWLEDGEMENT:

We are thankful to SHP (Science Honors Program), K.C. College, for providing research opportunity. We would like to acknowledge Head of the Department of Chemistry and Life-Sciences, Mr. R.C. Moorjani and Mrs. Yamini Tayal for providing suitable work place for this project. We would also like to acknowledge Dr. P. S. Ramanathan Advanced Instrumentation Centre-Ramnarain Ruia College and University Of Mumbai-Kalina for their help in accomplishing our results. Also we would like to thank Consumer Guidance Society Of India(CGSI) and Bureau Of Indian Standards(BIS) for guiding us in our project.

REFERENCES:

[1] people.umass.edu/~mcclemen/581introduction.html - cited on 25/07/2014.

[2] http://www.fda.gov/food/ingredientspackaginglabeling/ foodadditivesingredients/ucm094211.htm- cited on 25/07/2014.

[3] http://www.fda.gov/food/ingredientspackaginglabeling/ foodadditivesingredients/ucm094211.htm- cited on 25/07/2014.

[4] http://www.fssai.gov.in/portals/0/pdf/foodact.pdf- cited on 25/07/2014

[5] http://agmarknet.nic.in/preface-chhilli.pdf - cited on 04/09/2014.

[6] http://ec.europa.eu/food/food/chemicalsafety/fraudulent/ qanda_en.pdf - cited on 03/09/2014

[7] Haiyan Xu, Thomas M. Heinze, Siwei Chen, Carl E.Cerniglia And Huizhong Chen - Anaerobic Metabolism Of 1-Amino-2-Naphthol-Based Azo Dyes (Sudan Dyes) By Human Intestinal Microflora, Oct 12, 2007.

[8] Stiborova M, Martinek V, Rydlova H , Koblas T, Hodek P.- Expression Of Cytochrome P450 1a1 And Its Contribution To Oxidation Of A Potential Human Carcinogen 1-Phenylazo-2-Naphthol (Sudan I) In Human Livers, 2005 Apr 8.

[9] http://fssai.gov.in/portals/0/pdf/final_lab_parameters%2821-08-2012%29.pdf-

[10 ]Howard. Philip H.-Handbook Of Environmental Fate And Exposure Data For Organic Chemicals,1989.

[11] Sana Mustafa, Nughza Nasir, Talat Mahmood, Iffat Mahmood, Rashid Ali Khan, Saima Khaliq-Spectrophotometric Determination of Sudan I-IV Dyes in Selected Chilli Samples: A Survey of Karachi City, Pakistan, Volume I, Issue 6, September 2013.

[12] Roger Wood, Lucy Foster, Andrew Damant And Pauline Key- Analytical Methods For Food Additives,2004.

Received on 19.08.2016 Modified on 25.08.2016

Asian J. Research Chem. 2016; 9(8): 359-365.

DOI: 10.5958/0974-4150.2016.00054.7

Table 1
Sample	Solvent Front (cm)	Sample Flow (cm)	Dye Flow (cm)	Rf (cm)
Sample 19	5.50	5.00	5.20	0.90
Guntur (O)	5.50	3.00	5.20	0.54
Table 2
Sample	Solvent Front (cm)	Sample Flow (cm)	Dye Flow (cm)	Rf (cm)
Sample 23	5.30	4.70	5.00	0.88
Guntur (O)	5.30	2.90	5.00	0.54

Sample no.	Solvent front (cm)	Sample flow (cm)	Dye flow (cm)	Rf (cm)	O.D
11	4.00	3.60	3.80	0.90	0.67
17	5.50	4.80	5.20	0.87	0.59
19	5.50	5.00	5.20	0.90	0.36
23	5.30	4.70	5.00	0.88	0.50

Name of chilli standard	Pandi			Guntur
Concentration (ppm)	Before spiking (O.D)	After spiking (O.D)	Dye (O.D)	Before spiking (O.D)	After spiking (O.D)	Dye (O.D)
100	0.15	0.48	0.33	0.05	0.38	0.33
50	0.06	0.39	0.33	0.01	0.22	0.21
20	0.01	0.23	0.22	0.00	0.87	0.87
5	0.00	0.87	0.87	0.00	0.87	0.87
1	0.00	0.87	0.87	0.00	0.87	0.87