IDENTIFICATION AND ESTIMATION OF NON-PERMITTED FOOD

www.wjpps.com Vol 6, Issue 8, 2017.

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Shah et al. World Journal of Pharmacy and Pharmaceutical Sciences

IDENTIFICATION AND ESTIMATION OF NON-PERMITTED FOOD

COLOURS (METANIL YELLOW AND ANILINE DYES) IN

TURMERIC POWDER BY RAPID COLOR TEST AND THIN LAYER

CHROMATOGRAPHY

Shruti Singh1, Himani Shah

1, Krishna Shah

1 and *Ritika Shah

1

1Department of Biotechnology, Mithibai College, Vile Parle (W), Mumbai-400056,

Maharashtra, India.

ABSTRACT

A study was carried out determine the presence of metanil yellow and

aniline dyes, both qualitatively, in nine turmeric samples collected

from the western region of Mumbai. For the purpose of this study, both

branded as well as non-branded turmeric samples were chosen at

random to test for adulteration. The standard (metanil yellow) and

turmeric samples were prepared of desired concentrations using

isopropyl alcohol as a diluent for thin layer chromatography. Out of the

nine turmeric samples, all samples (100%) showed positive results for

metanil yellow and for aniline dyes and 89% tested positive by TLC.

The present study indicates the presence of metanil yellow and aniline

dyes in spices. According to various governmental regulations, the use

of metanil yellow and aniline dyes in spices in strictly prohibited because metanil yellow is

known for affecting the brain adversely while aniline dyes have been known to cause

hyperkinesis in children. In this study, we have presented a simple, convenient, and

expeditious method that includes rapid color test and thin layer chromatography to prove the

presence of metanil yellow and aniline dyes in spices. The results thus obtained were

compared to multiple external standard methods. The aim of this study is to bring about

awareness among the people on the subject of food adulteration and the various simple

methods to detect the same.

KEYWORDS: Non-permitted food colors, metanil yellow, turmeric powder, color test, thin

layer chromatography.

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

SJIF Impact Factor 6.647

Volume 6, Issue 8, 2034-2045 Research Article ISSN 2278 – 4357

Article Received on

13 June 2017,

Revised on 03 July 2017,

Accepted on 24 July 2017,

DOI: 10.20959/wjpps20178-9867

*Corresponding Author

Ritika Shah

Department of

Biotechnology, Mithibai

College, Vile parle (W),

Mumbai - 400056,

Maharashtra, India.


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INTRODUCTION

Synthetic food colors are used worldwide to avoid the loss of original color in processed

foods, as well as to make the products more attractive to consumers. Synthetic food colors are

considered superior to natural food colors in terms of their color value, uniformity, and

applicability in various processed foods. Synthetic food colors have been authorized and

regulated for use in food additives in many countries.[1-3]

Only eight coal-tar food colors are

permitted in certain food products under the provision of Food Adulteration Act (1954). They

include three red shades namely: Carmoisine, Ponceau 4R, and Erythrosine; two yellow

shades: Sunset yellow FCF and Tartrazine; two blue shades: Brilliant Blue FCF and Indigo

Carmine; one green shade: Fast green FCF. However, certain unpermitted colors such as

Metanil yellow, Rhodamine B, Orange G, Pararosaniline (PA), Auramine O (AO), Sudan

dyes, Blue VRS, and certain oil soluble colors are often added in food as adulterants.

Metanil yellow is a banned dye as per PFA Act (1954) by Government of India, because the

same dye has found to be carcinogenic in humans. A report on carcinogenesis[4]

stated that

the metabolism of azo dyes derived from benzidine converted to aromatic amines by

intestinal bacteria is potentially carcinogenic. According to a report recently published in

Current Sciences, some synthetic dyes like auramine, metanil yellow, lead chromate,

rohdamine, sudan-3 and 4, orange-2, and malachite green cause serious health hazards as they

are mutagenic and potentially carcinogenic.[5]

Metanil yellow, the most frequently used non-

permitted food colour is widely used in food items like “ladoos”; it causes insufficient

oxygen supply to skin and mucous membranes along with degenerative changes in the

stomach, liver, kidney, abdomen, and testes. It is found to cause cyanosis.[6]

It was also found

to cause toxic methaemoglobinaemia in adult human males 2-4 hours after the consumption

of rice coloured with it.[4]

Although whole, dried, or fresh turmeric is typically free of contamination, turmeric powder

can be adulterated with different chemical powders which are used as substitutes for

curcumin.[6]

Studies have reported the mixing of Curcuma zedoaria, a wild relative of

turmeric, into turmeric powder due to its close resemblance with turmeric.[6,7]

Similarly,

metanil yellow (C18H14N3NaO3S) is a toxic azo dye that has been added to turmeric

powder to mimic the appearance of curcumin[8]

when the actual curcumin content is low.[9]

Toxicologically, metanil yellow is classified as a CII category substance by the Joint

FAO/WHO Expert committee on Food Additives, and it implies that it is a compound for


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which virtually no information on long-term toxicity is available.[10]

Toxicity is categorized

into four classes according to the toxicity of chemicals, where Category I chemicals are the

most toxic and poisonous, and Category IV chemicals are least toxic and poisonous. Studies

on rats show that long term consumption of metanil yellow causes neurotoxicity[11]

,

hepatocellular carcinoma[12]

, tumor development[8]

, deleterious effect on gastric mucin[13]

,

and lymphocytic leukemia.[14]

A variety of conventional methods have been effectively used

for detection of metanil yellow in food items. Ion-pair liquid chromatography detected with

99% linearity the presence of azo dyes, such as metanil yellow, in the range of 0.05 ppm to

10 ppm in food.[9]

Similarly other methods such as high performance liquid chromatography-

electrospray ionization tandem mass spectrometry[10]

, high performance capillary

electrophoresis[13]

, and micellar chromatographic method[12]

have been used for detection of

metanil yellow and other dyes in food and beverages. Despite their high accuracies and

satisfactory detection limits, these conventional methods are limited in practicality due to

their operational complexity and sample-destructive nature. In contrast, the relative simplicity

of optical methods has driven their increasing use for safety and quality detection of foods

and food products.[8-10]

Although FT-IR and FT-Raman spectroscopy have not been

previously reported for detection of metanil yellow adulteration in food, these spectroscopy

methods have been widely used for detection of other food adulterants. Thin Layer

Chromatography (TLC) was used to identify the presence of metanil yellow in turmeric

powder. TLC is one of the easiest and most versatile methods for identifying and separating

compounds due to its low cost, simplicity, short development time, high sensitivity and good

reproducibility.[10]

TLC simultaneously separates substances in space. The RF (retardation

factor, ratio of fronts, or retention index) value is the standard measure of retention. Given the

general difficulty of controlling absolute RF values, it is common to separate standards and

samples in the same system for identification purposes.

This study presents a comprehensive study to evaluate the presence of metanil yellow and

aniline dyes in the turmeric samples collected at random from the city of Mumbai

qualitatively by rapid color test, followed by thin layer chromatography.

The primary objectives of this study are to qualitatively determine the presence of metanil

yellow and aniline dyes in various turmeric samples by:

a. Rapid color test.

b. Thin layer chromatography.


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MATERIALS AND METHODS

Turmeric samples collected for testing the presence of non-permitted colors (metanil

yellow and aniline dyes)

For the purpose of the study, 9 turmeric samples (both branded and non-branded) were

purchased from different shops from the western part of Mumbai, India during the period of

2016-2017; these are mentioned in Table-2. From each shop, 50 grams of the sample was

purchased for the purpose of testing. Fresh turmeric and standards (metanil yellow) were also

tested for comparison.

Figure-1: Turmeric samples

Rapid Color Test

Rapid Color Tests are based on specific color changes in the presence of reagents such

concentrated hydrochloric acid and concentrated sulphuric acid. These tests get their name

from the fact that the color change is observed within 2-3 minutes on adding the specific

reagent. The following table (Table-1) shows the different color tests carried out to test the

presence of metanil yellow and aniline dyes in turmeric samples and the associated color

changes.[15]


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TABLE-1: METHOD FOR DETECTION OF METANIL YELLOW AND ANILINE

DYES IN TURMERIC POWDER

Spices Adulterants Test Inference

Turmeric

Samples

Metanil

Yellow

1. Take 2 grams of a sample in a test

tube. Add 1-2mL of distilled water

to it. To this, add 8-10 drops of

conc. Hydrochloric acid.

Immediate appearance of

pink/purple color which persists

on addition of distilled water

indicates the presence of metanil

yellow.



to it. To this, add 8-10 drops of 13N

Sulphuric acid.

Immediate appearance of red

color which persists on addition

of distilled water indicates the

presence of metanil yellow.




rectified spirit.

Immediate appearance of reddish

pink color which persists on

addition of distilled water

indicates the presence of metanil

yellow.

Aniline Dyes

Take 2 grams of sample in a test



rectified spirit.

Immediate separation of yellow

color in the spirit layer indicates

the presence of aniline dyes.

Thin layer chromatography (TLC)

Thin Layer Chromatography is a chromatographic technique that is used to separate mixtures

containing volatile elements. In the present study, we have used thin layer chromatography to

determine the presence of adulterant (metanil yellow) in the turmeric samples by comparing

their Rf (ratio of the distance travelled by the sample to the distance travelled by the solvent)

with the Rf of the standard.

Preparation of turmeric samples

5 grams of each turmeric sample were taken in a test tube and dissolved in 20mL of

chloroform. These were kept overnight in the rotating shaker. After 18 hours, these test tubes

were removed from the shaker and filtered. The filtrate obtained was used for thin layer

chromatography.

Preparation of standard solution

Stock solutions of the standard (metanil yellow) were prepared by dissolving 2mg of the

standard in 50mL of isopropyl alcohol (solvent). This was stored at 10°C. The working

standard solution was obtained from the stock solution by diluting it with the solvent (1:5) at

the time of analysis.


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Preparation of TLC plates

Silica gel slurry was prepared in distilled water and then applying it on the TLC plates (15cm

x 7cm) as a thin layer having a thickness of 0.25mm. These plates were then allowed to dry.

These were activated by keeping them at 1100C in the oven for 1 hour.

Procedure

The samples and standards were spotted onto the TLC plates with the help of capillaries. The

solvent used for the development of the TLC plates was a solution of ethyl acetate, methanol,

ammonia, and distilled water which were mixed in the ratio of 35: 11: 5: 5 respectively.

These plates were then allowed to develop in a TLC jar for 1 hour using the aforementioned

solvent system. The chromatograms obtained were then evaluated under normal light and the

distances travelled by the solvent were measured using a template scale. Their Rf values were

then calculated and compared to that of the standards.[16]

RESULTS AND DISCUSSION

The study was conducted to determine the presence or absence of non-permitted colours

metanil yellow and aniline dyes in spices. Metanil yellow is azo dye synthesized from the

coupling of metanilic acid and diphenylamine, and is carcinogenic. Metanil yellow is added

in spices only to enhance the colour of spices, their redness, and they are continuously

harming the human health.

In the present study nine turmeric samples were collected at random from different locations

from the western suburbs of Mumbai to determine the percentage of metanil yellow and

aniline dyes present. Based on the observations we obtained on performing the rapid color

test, all the samples tested positive for metanil yellow and aniline dyes; i.e. 100% of the

samples tested positive for the presence of metanil yellow and aniline dyes. (Fig.2-4, Table-

2).

FIGURE-2: TURMERIC SAMPLES SHOWING POSITIVE RESULTS FOR THE

PRESENCE OF METANIL YELLOW USING RAPID COLOR TESTS.


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FIGURE-3: TURMERIC SAMPLES SHOWING POSITIVE RESULTS FOR THE

PRESENCE OF ANILINE DYES USING RAPID COLOR TESTS.

FIGURE-4: TURMERIC SAMPLES SHOWING POSITIVE RESULTS FOR

PRESENCE OF METANIL YELLOW ALONG WITH POSITIVE AND NEGATIVE

CONTROLS.

The results obtained for the given turmeric samples after thin layer chromatography were

expressed in terms of their Rf (retardation factors) values. The Rf values of the standard

(metanil yellow) are also calculated in order to compare and determine the presence of the

adulterant; i.e. metanil yellow in the turmeric samples. The Rf values for the samples and the

standards are shown in Table-2. The samples were checked by internal standard procedure

and compared with the standard. It was found that metanil yellow was present in 8 samples

out of total 9 samples that were analyzed; i.e. 89 % of the samples showed similarities with

the standard on comparing the chromatograms (Fig -5, Table-2). Other samples which

showed colored spots but did not have an Rf value matching that of the standard may contain

other food colorants.[17]


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FIGURE-5: TLC CHROMATOGRAM SHOWING TURMERIC SAMPLES B-T1, T2,

T3, T4, T5 AND NB- T2, T3, T4.

TABLE-2: TURMERIC SAMPLES SHOWING RESULTS FOR COLOR TEST AND

TLC.

Turmeric Samples

Rapid color test Thin Layer

Chromatography

M Y

(Test-1) M Y (Test-2)

Aniline

Dyes

Rf of the samples with

respect to MY

NB-T1 + + + 0.79

NB-T2 + + + 0.66

NB-T3 ++ ++ + 0.66

NB-T4 + + ++ 0.66

B-T1 + + + 0.66

B-T2 + ++ ++ 0.66

B-T3 + + ++ 0.66

B-T4 + + + 0.66

B-T5 + + + 0.66

Fresh turmeric (rhizome) - - - 0.82

Metanil Yellow/ Aniline Dye

(Positive Control) + + + 0.66 (MY)

Water (Negative Control) - - - -

Key: NB- Non-branded samples.

B- Branded samples.

MY- Metanil Yellow.

From the results obtained above, it is seen that food adulterants like metanil yellow and

aniline dyes are being indiscriminately used by unorganized food organizing sectors in food

items like turmeric powder. With the help of rapid color test and thin layer chromatography,

metanil yellow and aniline dyes were found to be present in the turmeric samples which were

purchased at random from the western suburbs of Mumbai.


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Metanil yellow is an established food toxicant. From the literature, we found that a variety of

food items were usually adulterated with non-permitted colours such as auramine, rhodamine

B, congored, orangeII, melachite green and metanil yellow. Some information about the

metanil yellow induced health hazards have been reported discriminately in animal

models.[18-20]

We have examining the probable toxic effects of metanil yellow on female

reproductive system in rat model. We have seen that metanil yellow altered the reproductive

cycle in female rat and also altered the hormonal levels significantly (unpublished data). So,

in order to avoid the deleterious toxic effects in human physiological functions, the use of

metanil yellow should be stopped immediately. Otherwise, the people belonging to lower

economic stratum will suffer the most as a result of the consumption of metanil yellow-laden

food items. From a wide variety of literature, it has been proven by a number of studies that

metanil yellow is carcinogenic in nature. Metanil yellow, one of the most frequently used

adulterants, was found to cause insufficient oxygen supply to skin and mucous membranes,

coupled with degenerative effects to stomach, kidney, liver, and abdomen.[6]

It was also

found to cause methaemogobinaemia in adult human males 2-4 hours after consumption of

rice adulterated with it.[9]

Aniline dyes have the potential to damage haemoglobin, the oxygen carrier present in blood,

leading to a condition called methaemoglobinaemia. Acute high exposure to aniline leads to

cyanosis. Dizziness, headache, convulsions, coma, and death may also occur. The EPA has

determined that aniline is a possible human carcinogen.

In order to avoid the deleterious effects of metanil yellow and aniline dyes on human

physiological functions, the use of metanil yellow and aniline dyes in food items must be

stopped completely. We suggest that the state and district administrations should intervene

and supervise the processing of food with metanil yellow as per Prevention of Food

Adulteration Act of India (PFA, 2008).

CONCLUSION

From the present study, we can conclude that rapid color test and thin layer chromatography

are preliminary tests for the detection of food additives in various food items. Further

quantitative analysis can be carried out with the help UV Visible Spectrophotometer.

Nowadays, synthetic food colors are frequently used in a variety of food items to make them

look more appealing to the consumer. In such a scenario, the results of this study can be used

to prevent the malpractice of synthetic food color adulteration. Food adulterants pose a


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serious threat to human well-being, resulting in life-threatening diseases such as cancer. Even

at low concentrations, food additives have the capacity to disrupt normal body functions. The

harmful effects of food additives are found to be more pronounced in young children as their

immune system is still developing. This study is useful for Forensic Chemistry and

Toxicology. From a medico-legal standpoint, this study must be assessed carefully. This

study is focused on the detection of non-permitted food colors (metanil yellow and aniline

dyes) from selected food samples. The methods of detection employed in the present study

can be used in food quality control to detect the presence of food additives in a variety of

food items. The level of adulteration in edibles is still high and further research can be

conducted in order to detect various non-permitted food colours in other food products so that

effective measures can be undertaken for improving the quality of the food products.

ACKNOWLEDGEMENT

We would like to express our sincere thanks to our principal, Dr. Rajpal Shripat Hande,

Mithibai College, for his constant support and encouragement. We also wish to thank

S.V.K.M for the wonderful facilities provided to us in order to carry out this undergraduate

project.

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IDENTIFICATION AND ESTIMATION OF NON-PERMITTED FOOD