JPC – Journal of Planar Chromatography – Modern TLC (2020) 33:413–418

[Link]

ORIGINAL RESEARCH PAPER

Detection of synthetic food color “Metanil Yellow” in sweets:

a systematic approach
Khushboo Kourani 1 & Neeti Kapoor 2 & Ashish Badiye 2 & Ritesh K. Shukla 1

Received: 8 May 2020 / Accepted: 27 July 2020 / Published online: 26 August 2020
# Akadémiai Kiadó, Budapest, Hungary 2020

Abstract
Food adulteration is a primary global concern for public health, especially in developing countries, due to the lack of monitoring
and appropriate policy developments and implementations. Synthetic colors and dyes are used to enhance the appearance of food
products. Metanil Yellow (MY), a non-permitted, toxic, chief additive adulterant is majorly used in sweets, pulses, and turmeric
in India owing to its color that ranges from yellow to orange. Owing to this, we have assessed the presence of MY in sweets like
“jalebi” and “laddu” from branded and local vendors. We present a method to isolate the additive from the sweets with the help of
acetic acid, followed by screening through colorimetric assay. The chemical characterization of MY was conducted using a multi-
plate ultraviolet–visible (UV–Vis) spectrophotometer and attenuated total reflectance–Fourier transform infrared spectroscopy
(ATR–FTIR). The appearance of magenta/pink color indicates the presence of MY in the chemical spot test. Also, thin-layer
chromatography (TLC) was applied to validate the presence of this adulterant by comparing the peaks of reference (MY) with the
extracted samples. TLC of food color extracted from the samples is an easy, efficient, and cost-effective method to detect toxic
adulterants like MY. Our study demonstrates a simple food color extraction procedure, with easy and sensitive chemical assays,
for the detection of MY in sweets. It also offers reasonable sensitivity and can be used to screen large numbers of samples in a
short time.

Keywords Adulterant . Metanil Yellow . Food color . Non-permitted . Toxic . Thin-layer chromatography

1 Introduction availability compared to their branded counterparts, con-

sumers are attracted to these unbranded food products more
Food adulteration is a significant challenge in food safety often. Generally, these unbranded food products do not meet
and regulation in many countries of the world, including the standards of FSSAI, as various non-permitted food colors
India. The Food Safety and Standards Authority of India and dyes are used to enhance the outer appearance of the
(FSSAI) is functioning to check the food quality in the product to attract consumers [1]. These types of food prod-
Indian market. However, it is mostly limited only to branded ucts are commonly known as adulterated food. Non-
food products [1]. India is a country of service marketing, permitted food colors are generally synthetic colors or dyes
and unbranded food products are widely circulated into the prepared and/or used in laboratories for testing purposes and
markets here. Moreover, due to their lower price and easy may pose adverse effects on human health [2]. Hence, the
consumption of these food products in the presence of non-
permitted color or dye may lead to ill health, disease, or even
* Ashish Badiye death. Most of the time, the food vendors indulge in such
[Link]@[Link] malpractices with callous disregard for the health interest of
* Ritesh K. Shukla the consumer.
[Link]@[Link] The Indian sub-continent is known worldwide for its wide
1 variety of festivals as well as food. Apart from the festivities,
Biological and Life Sciences, School of Arts and Sciences,
Ahmedabad University, Ahmedabad, Gujarat 380009, India the routine diet, as well as meet-and-greet, also include differ-
2 ent confections. Two of the most popular sweets are “jalebi”
Department of Forensic Science, Government Institute of Forensic
Science, R.T. Road, Civil Lines, Nagpur, Maharashtra 440001, India (also known as zalabia, zulbia, and jilapi) and “laddu”(also
known as laddoo or avinsh; the name originates from the
414 JPC-J Planar Chromat (2020) 33:413–418

Sanskrit word lattika) [3–5]. The production of sweets in bulk,

especially during a festive season while maintaining purity
and sound quality is a difficult task for the makers. To pull
the crowd to their shops, most of the sweet shop vendors use
food colors, to make their products visually more attractive
and esthetically pleasing [2]. Even though natural and permit-
ted food colors (such as Ponceau 4R, Carmoisine,
Erythrosine, Tartrazine, Sunset Yellow, Indigo carmine,
Brilliant Blue, Fast Green) are available, they are costlier as
compared to the dyes and non-permitted food colors (such as a
Metanil Yellow [MY], Quinoline Yellow, Auramine,
Malachite green, Rhodamine B, and Orange II). Among these,
MY, a non-permitted food color has been the most commonly
used in Indian sweets such as laddus and jalebis, primarily due
to its easy availability at meager rates and, secondarily, for the
attractive yellowish golden color that it imparts [6].
Chemically, MY is an azo dye synthesized from the cou-
pling of metanilic acid and diphenylamine [7]. MY itself is not
toxic, but its metabolite, diphenylamine, is a suspected muta-
gen and carcinogen, which may lead to cancer. Because of its
genotoxic and carcinogenic nature, MY has been classified as
a non-permitted food color [1, 8, 9]. Its consumption for a long
time may cause degenerative changes in the stomach lining,
kidney, and liver. Weakness, giddiness, and food poisoning b
are common symptoms from the consumption of MY over Fig. 2 Characterization of Metanil Yellow dye, using a UV–Vis spec-
troscopy, b Fourier transform infrared spectroscopy (FTIR) (absorption
some time [10].
maxima at 1140 cm−1)
Some previous studies confirmed that MY is widely used
in food products in Indian cities as well as in rural areas [11,
2 Experiment
12]. Sweets are an essential part of Gujarati food, and their
daily intake is significantly higher than in other states of India.
2.1 Samples
Hence, the use of non-permitted synthetic food color in sweets
may result in severe health implications in the sweet-loving
A total of 100 sweet samples (i.e., 50 jalebi samples and 50
population. Till date, no scientific report was found to study
laddu samples weighing 50 g and 50–60 g respectively) were
the presence of MY in the sweets in this region. Hence, in this
collected from 10 different sweet shops over 5 days from
study, we attempted to detect the non-permitted dye MY in
Ahmedabad city, Gujarat state in India.
yellow-orange colored sweets (i.e., laddu and jalebi) pur-
chased from the sweet shops in Ahmedabad, city of Gujarat,
India. 2.2 Materials

MY dye (to be used as standard) was purchased from SD Fine

y = 0.0044x + 0.0521 Chemical Ltd. (Mumbai, India). Ethanol, hexane, propyl al-
0.15
0.14 R² = 0.9927 cohol, ethyl acetate, acetic acid, ammonia, and concentrated
0.13
0.12 hydrochloric acid were procured from HiMedia (Mumbai,
Absorbance at 420

0.11
0.1 India). Pre-coated thin-layer chromatography (TLC) alumi-
0.09 num silica gel sheets were purchased from Merck (Mumbai,
0.08
0.07 India). Guaranteed reagent (GR) grade chloroform and meth-
0.06
0.05 anol were purchased from HiMedia. Glassware and plastic-
0.04
0.03 ware were procured from the local vendors.
0.02
0.01
0
0 2 4 6 8 10 12 14 16 18 20 22 2.3 Standard preparation
Concentration of Metanil Yellow ( g/mL)

Fig. 1 Standard curve of Metanil Yellow dye at different concentrations Standards of MY (1–20 μg/mL) were prepared from the
(1–20 μg/mL) stock solution (500 μg/mL), and the absorptions maxima
JPC-J Planar Chromat (2020) 33:413–418 415

Fig. 3 Representative image

showing chemical (spot) test for
a) Extracted sweet samples (S1
the detection of Metanil Yellow
dye. a Extracted sweet sample
(S1–S5) before chemical test. b
Extracted sweet sample (S1–S5)
after chemical test showing posi-
tive colorimetric changes (red to b) Samples showing posive result
magenta color on the addition of aer spot test (S1
conc. HCl)

(λmax) were recorded using a UV–Vis spectrophotometer 2.6 Screening test

(BioSpectrometer, Eppendorf, Hamburg, Germany; funda-
mental, single beam). A calibration curve (shown in 2.6.1 Chemical test
Fig. 1) was drawn to determine the unknown quantity of
MY in sweet samples. In this study, a chemical spot test was used as the screening
method for MY detection in the extracted samples of sweets
(jalebis and laddus). In this test, the extracted sample (0.5 mL)
2.4 Characterization was solubilized in 2.450 mL of propanol, and an aliquot of
50 μL of 1 N hydrochloric acid was added into it, followed by
The chemical characterization of MY was conducted the observation of the solution for color changes. If the solu-
using a multi-plate ultraviolet–visible (UV–Vis) spectro- tion turns to a magenta pink color, it indicates the presence of
photometer and attenuated total reflectance–Fourier trans- MY [1, 13].
form infrared spectroscopy (ATR–FTIR). The distinct
peak of MY found at the particular wavelength 2.7 Confirmatory test
(430 nm) in water as well as the bathochromic shift were
observed when MY reacts with HCl [13, 14]. Similarly, 2.7.1 UV–Vis spectrophotometry
FTIR also provides a distinct peak at 1140 cm−1 for con-
firmation of MY [15]. MY has characteristics to turn into pink/magenta color under
high acidic condition, and UV–Vis spectrophotometry is the
superlative instrument to analyze the colored compound/solu-
2.5 Extraction of food color from samples tion. These colorimetric changes give spectral peak on a spe-
cific wavelength. Therefore, to confirm the presence of MY in
Food color from jalebis and laddus were extracted under the samples, UV–Vis spectrophotometry was conducted [6].
acidic condition. The samples were dissolved in 10 mL In this test, MY was used as the positive control, while the
acetic acid solution, and the mixture was incubated over- extracted samples from sweets have been tested for the pres-
night. The samples were then centrifuged at 5000 rpm for ence of MY.
5 min; the supernatant was collected and was used for
further analysis. 2.7.2 Thin-layer chromatography

MY in sweets was qualitatively analyzed by TLC. Aliquots of

2 mL jalebis/laddus extracts (500 ng/spot), along with stan-
dard MY, were applied on a pre-coated TLC plate and re-
solved by using solvent systems (ethyl acetate–methanol–
ammonia (25%)–water [Link], v/v]). Finally, the spots
were visualized using chloroform-methanol (90:10, v/v) as
the developing solvent for MY [15, 16].

3 Results

Fig. 4 Detection of Metanil Yellow dye in the extracted samples of In this study, the characterization of MY was performed
sweets using UV–Vis spectroscopy using a multiplate UV–Vis spectrophotometer and ATR–
416 JPC-J Planar Chromat (2020) 33:413–418

Fig. 5 Detection of Metanil

Yellow dye in the extracted
sample of sweet using thin-layer
chromatography

Representative image (not to scale)

M = Metanil Yellow dye (standard); S2 = extracted sweet sample RF = retardation factor

FTIR spectroscopy. Our spectroscopic results depicted confirm the colorimetric results, UV–Vis spectrophotom-
that the standard (MY) gives a spectral peak at 430 nm etry was performed, and a spectral peak at 430 nm in the
in UV–Vis and a distinct peak at 1140 cm−1 in ATR– extracted sample resembled the standard peak of MY
FTIR (shown in Fig. 2a, b). These findings are in agree- (shown in Fig. 4). The pure and spiked sample of MY
ment with earlier reported peaks indicating the presence served as control. Absorptions maxima (λmax) for MY
of MY [19]. In the present study, the samples were incu- and samples containing MY (S1, S2, and S3) were ob-
bated overnight under acidic condition, and the food color served at 430 nm. This result confirms the presence of
was successfully extracted from the samples. This soluble MY in the extracted samples. For further validation,
content (extracted) was then tested by a chemical spot test TLC was carried out, and our results (shown in Fig. 5)
that resulted in a pink color indicating the possibility of visibly demonstrate that the band and retention factor val-
the presence of MY in the sample (shown in Fig. 3). To ue of the extracted sample are equivalent to the standard

Table 1 The total number of sweet samples collected (N = 100) and the final result for the presence or absence of Metanil Yellow

The presence/absence of Metanil Yellow

Jalebi samples (n = 50) Laddu samples (n = 50) Total (N = 100)

Day Day
1 2 3 4 5 1 2 3 4 5 n
Shop A – – – – – – – – – – 10
Shop B – – – – – – – – – – 10
Shop C – – – – – – – – – – 10
Shop D – – – – – – – – – – 10
Shop E – – – – – – – – – – 10
Shop F + + + + + + + + + + 10
Shop G – – – – – – – – – – 10
Shop H – – – – – – – – – – 10
Shop I – – – – – – – – – – 10
Shop J – – – – – – – – – – 10

‘–’ denotes negative results, i.e., the absence of Metanil Yellow in the sample
‘+’ denotes positive results, i.e., the presence of Metanil Yellow in the sample
n = the number of samples; N = the total number of all samples
JPC-J Planar Chromat (2020) 33:413–418 417

(MY). The RF was calculated to be 0.56 for the standard 5 Conclusion

MY sample (M). These results demonstrated that out of
100 extracted samples of sweets (jalebi and laddu), 10 In the present study, a systematic procedure of MY detection
samples (five each of jalebi and laddu) of the same sweet in food products was elucidated. Our preliminary (colorimet-
shop showed the presence of MY dye. The overall result ric assay) results depicted that all tested samples may contain
is shown in Table 1. MY, but further confirmatory test (TLC) exhibited that the
samples from only one shop contained the prohibited dye,
while the rest yielded a negative result. In this study, we de-
veloped a simple extraction procedure through which MY
4 Discussion could be successfully extracted from fat-containing products
such as sweets (jalebis and laddus). This MY extraction pro-
Although food colors do not possess any nutritional value, cedure from sweets is relatively simple, offers reasonable sen-
they can undoubtedly enhance the appearance and luster sitivity, and can be used to screen large numbers of samples in
of the food products that attract consumers. Hence, man- a short time. Further research work is needed for enhanced
ufacturers always bear in mind the impact that it has on testing of adulteration in the food products. Our future work
the sale of products like sweets [2]. Different kinds of would also address the biological consequence of adulterated
synthetic colors and dyes such as Tartrazine, Sudan, and materials.
Rhodamine B, and Allura Red are available in the market,
that may be used in food [2, 17, 18]. Typically, these Acknowledgments K. K. and R. K. S. are thankful to the Department of
Biological and Life Sciences, School of Arts and Sciences, Ahmedabad
synthetic colors are highly toxic, and food regulatory
University, Ahmedabad. N. K. and A. B. are thankful to the Director,
agencies restrict most of them for human consumption Government Institute of Forensic Science, Nagpur, for their consistent
[2]. However, various synthetic coloring chemicals are support and encouragement.
often being used in various food products, MY being
one of them.
Author’s contribution statement All authors contributed to the study
In this study, we focused on detecting MY adulteration conception and design. Material preparation, data collection, and analysis
in sweets (jalebi and laddu). We have chosen sweets as were performed by K. K. and R. K. S. The first draft of the manuscript
our sample because sweets are not monitored quite often was written by K. K.. N. K., A. B., and R. K. S. critically reviewed and
by food safety agencies. They use surveillance for such wrote the final draft. All authors read and approved the final manuscript.
food products only in the festive season when the demand
Availability of data and material Not applicable.
for supply and consumption for sweets is high; neverthe-
less, regular monitoring is usually scarce. Code availability Not applicable.
Different extraction procedures were carried out to ex-
tract the food color from the sweet samples in this study. Compliance with ethical standards
In a previous study, Nath et al. demonstrated that sweet
sample (laddu) could easily be dissolved in distilled water
[1], while in this study, all extraction procedures conduct-
ed using water and other polar solvents failed because of a Conflict of interest The authors declare that they have no conflicts of
considerable amount of fat content present in the sweets, interest.
especially in jalebis. These fat contents were insoluble in
water and other polar solvents. Therefore, to solubilize the
Ethical approval The study does not involve any human participation.
fat content, the samples were incubated overnight under
acidic condition. On centrifugation, the insoluble content
settles down, while soluble contents, including food color, Consent Not applicable.
come in the supernatant. Our results showed that sweet
samples from one of the ten shops tested from the city
contained MY. In the previous study, Nath et al. reported References
that food samples from the unorganized (street shop) sec-
tors contain a significant amount of MY while organized 1. Nath PP, Sarkar K, Mondal M, Paul G (2015) Practice of using
sectors (branded shop) did not [1]. This study revealed metanil yellow as food colour to process food in unorganized sector
of West Bengal – a case study. Int Food Res J 22(4):1424–1428
that a conclusive remark on any food product’s adultera-
2. Dilrukshi PGT, Munasinghe H, Silva ABG, De Silva PGSM (2019,
tion could not be made by only one or two tests. Instead, 2019) Identification of synthetic food colours in selected confec-
a full battery of tests should be performed before provid- tioneries and beverages in Jaffna District, Sri Lanka. J Food Qual:
ing the report. 1–8 Article ID 7453169
418 JPC-J Planar Chromat (2020) 33:413–418

3. Davidson A (2014) The Oxford companion to food. Oxford 14. Dhakal S, Kuanglin C, Schmidt W, Qin J, Kim M, Chan D (2016)
University Press, Oxford, pp 424–425 Evaluation of turmeric powder adulterated with Metanil yellow
4. Padgaonkar D (2010) Journey of the Jalebi. [Link] using FT–Raman and FT–IR spectroscopy. Foods 5(36):1–15
[Link]/Journey-of-the-jalebi/articleshow/[Link] 15. Yadav DS, Jaiswal S, Mishra MK, Gupta AK (2016) Analysis of
5. Dhairya D (2020) Sach or Soch. [Link] non-permitted dyes in bakery and dairy products for forensic con-
indian-dishes/ sideration. Int J Dev Res 6(7):8775–8779
6. Bachalla N (2016) Identification of synthetic food colors adultera- 16. Jaiswal S, Yadav DS, Mishra MK, Gupta AK (2016) Detection of
tion by paper chromatography and spectrophotometric methods. Int adulterants in spices through chemical method and thin-layer
Arch Integr Med 3(6):182–191 chromatography for forensic consideration. Int J Dev Res
7. Ghosh D, Singha PS, Firdaus SB, Ghosh S (2017) Metanil yellow: 06(08):8824–8827
the toxic food colorant. Asian Pac J Health Sci 4(4):65–66 17. Singh S, Shah H, Shah R, Shah K (2017) Identification and estima-
8. Khan IS, Ali MN, Hamid R, Ganie SA (2020) Genotoxic effect of tion of non-permitted food Colours (Sudan and Rhodamine-B dye)
two commonly used food dyes metanil yellow and carmoisine in Chilli and curry powder by rapid colour test, thin layer chroma-
using Allium cepa L. as indicator. Toxicol Reports 7:370–375 tography and spectrophotometry. Int J Curr Microbiol App Sci 6(7):
9. Prevention of Food Adulteration (5th Amendment) Rules (2008) 1970–1981
[Link] 18. Purba MK, Agrawal N, Shukla SK (2015) Detection of non-
10. Chandra SS, Nagaraja T (1987) A food poisoning outbreak with permitted food colors in edibles. J Forensic Res S4:S4–003
chemical dye – an investigation report. Med J Armed Forces India
19. Iqbal Z, Eriksson M (2013) Classification and quantitative optical
43:291–293
analysis of liquid and solid samples using a mobile phone as
11. Khanna SK, Singh GB, Dixit AK (1985) Use of synthetic dye in
illumination source and detector. Sens Actuators B: Chem 185:
eatable in rural areas. J Food Sci Technol 22:269–273
354–362
12. Khanna SK, Singh GB, Singh SB (1987) Non-permitted colours in
food and their toxicity. J Food Sci Technol 10:33–36
13. Nath PP, Sarkar K, Tarafder P, Paul G (2013) Development of a Publisher’s note Springer Nature remains neutral with regard to jurisdic-
visible spectrophotometric method for the quantitative determina- tional claims in published maps and institutional affiliations.
tion of Metanil Yellow in different food samples. Int J Pharm Biosci
4(2):685–692