A Simple Method for Simultaneous Determination of Basic Dyes Encountered in Food Preparations by ReversedPhase HPLC

1874  Dixit et al.: Journal of AOAC International Vol. 94, No. 6, 2011
FOOD COMPOSITION AND ADDITIVES

A Simple Method for Simultaneous Determination of Basic Dyes
Encountered in Food Preparations by Reversed-Phase HPLC
Sumita Dixit, Subhash K. Khanna, and Mukul Das1
Council of Scientific and Industrial Research, Indian Institute of Toxicology Research, Food Toxicology Division,
Mahatma Gandhi Marg, PO Box 80, Lucknow – 226001, Uttar Pradesh, India
The present method utilizes a simple
pretreatment step, cleanup on polyamide SPE
cartridges, and HPLC resolution on reversedphase C18 for the detection of the three
basic nonpermitted dyes encountered in food
matrixes. Polyamide cartridges were chosen
because both acidic and basic dyes can be
cleaned up due to their amphoteric nature.
Analysis was performed on a reversed-phase
C18 µ-Bondapak column using the isocratic
mixture of acetonitrile–sodium acetate with a
flow rate of 1.5 mL/min and a programmable
λmax specific visible detection to monitor
colors, achieving higher sensitivity and
expanded scope to test multicolor blends. All
the colors showed linearity with the regression
coefficient, from 0.9983 to 0.9995. The LOD and
LOQ ranged between 0.107 and 0.754 mg/L and
0.371 and 2.27 mg/L or mg/kg, respectively.
The intraday and interday precision gave good
RSDs, and percentage recoveries in different
food matrixes ranged from 75 to 96.5%.
The study demonstrates that the use of a
combination of a simple SPE cleanup and HPLC

resolution with UV-Vis end point detection was
successful in screening the presence of these
three basic nonpermitted dyes individually
or in blend, in a variety of food matrixes.

C

olors are added to food to restore the original
shade lost during processing, as well as to make
them aesthetically and psychologically more
attractive to consumers. Synthetic colors are considered
superior to natural colors due to their tinctorial value,
uniformity, and availability in different shades. Food
colors are comprehensively screened for safety, and
their use is governed by individual countries (1–3). The
Prevention of Food Adulteration Act of India permits
eight food colors, namely, brilliant blue FCF, carmoisine,
erythrosine, fast green FCF, indigo carmine, ponceau 4R,
sunset yellow FCF, and tartrazine (3). These colors
Received November 16, 2010. Accepted by SG January 12, 2011.
1
Corresponding author’s e-mail:
DOI: 10.5740/jaoacint.10-450

are water-soluble acidic dyes and belong to the azo,
triarylmethane, xanthene, and indigoid groups. In addition
to the permitted food colors, indiscriminate use of various
nonpermitted colors has been reported from time to time,
and poses serious health concerns (4–14). Among the six
nonpermitted colors encountered, three are acidic dyes

(metanil yellow, orange II, and blue VRS) and the others
(auramine, malachite green, and rhodamine  B) are
basic dyes. Though these are meant for nonfood
applications (15–17), their use in foodstuffs is presumably
due to their lower cost and, at times, to a lack of awareness.
In an earlier report from the state of Uttar
Pradesh, India, Khanna et al. (18) found Rhodamine B,
auramine, and malachite green in 24% of food samples.
In subsequent surveys from this state, these basic dyes
were found in 5 to 10% food samples (7, 14, 19). Among
other reports, use of these dyes has been encountered in the
states of Andhra Pradesh (9, 13), Karnataka (11), and West
Bengal (5). The use of Rhodamine B in food preparation
is also reported by other developing countries, such as
China (20), Israel  (21), Malaysia (22), Pakistan  (23),
and Vietnam (24). Through increasing awareness and
regulatory measures, the use of nonpermitted basic dyes
is decreasing; nonetheless, these are still encountered.
The survey studies undertaken on the use pattern of
food colors in Indian markets utilized the conventional
paper chromatographic method, now considered to be
a dated methodology. TLC or column chromatography
methods coupled with spectrophotometric detection
have been among other methods used. HPLC is the
preferred technique, as it offers relatively high resolution
potential. The presently available HPLC methods are
for Rhodamine B in ballpoint pen inks  (16,  25) and
cosmetic products  (15), and for malachite green in
fish tissue  (17,  26, 27). Andersen et al.  (28) involve
an additional step converting leuco malachite green

metabolite to the parent compound. These methods are
for individual colors in a single matrix and may not be
applicable for a variety of food preparations. No method
is reported for the detection of auramine. In the absence
of a sensitive method to simultaneously detect these
three basic dyes in various food commodities, an HPLC
method incorporating a polyamide SPE cartridge as a
cleanup step has been developed.


Dixit et al.: Journal of AOAC International Vol. 94, No. 6, 2011  1875



Table  1.  Common names, Color Index (CI)
names, CI numbers, and λmax of three basic dyes
encountered in foods
Common name

CI name

CI No.

λmax, nm

Auramine

Basic Yellow 2

41000

430

Rhodamine B

Basic Violet 10

45170

525

Malachite green

Basic Green 4

42000

618

Materials and Methods
Chemicals
ExelaR grade liquor ammonia (specific gravity 0.91),
glacial acetic acid, and HPLC grade methanol were
procured from Merck Ltd (Mumbai, India). Acetonitrile
(HPLC grade) was purchased from Fisher Scientific (Fair
Lawn, NJ). The basic dyes auramine, malachite green,
and Rhodamine B were provided by Sigma Aldrich (St.
Louis, MO).
Polyamide cartridges (DPA-6S, 3 mL, 250 mg),
used as the SPE column, were purchased from Supelco

(Bellefonte, PA).
Preparation of Dye Standards
Standard stock of each dye was prepared by dissolving
10  mg dye in 10 mL methanol; subsequent working
standards (0.10–100 ppm) were obtained by appropriate
dilutions with methanol. The standards were stored at 4°C
in the dark and were stable for ≥2 months. The visible
spectrum of standard dyes was obtained in order to know
their respective λmax (Table 1).
Sample Procurement and Pretreatments
Earlier survey studies have shown that some of the loose
and nonbranded food commodities—branded samples
invariably do not use nonpermitted dyes—like candy floss,
sweetened puffed rice, cream biscuits, fruit cakes, colored

fried peas, sugar-coated fennels, cereal/pulse-based
sweets, sugar toys, and starch-based savory products
like sago papad, rice papad, and fryums show presence
of Rhodamine B, auramine, and malachite green. Five
loose, nonbranded samples each of pink-, yellow-, and
green-colored food samples of the above commodities
were collected from the local market. The solid samples
were crushed into fine powder and/or homogenized and
stored in refrigeration until further processing.
Fatty foods (cereal/pulse-based sweets, colored
fried peas, and bakery items) were found to require
a predefatting step to avoid interference of fat in color
extraction and subsequent resolution. An accurately
weighed sample (1 g) was taken in a conical flask; 10 mL
n-hexane was added and shaken for 1 h in a water bath

shaker maintained at room temperature (27–29°C) at a
moderate speed. The hexane layer was decanted and
the process was repeated four to five times, using fresh
solvent each time, until the sample was fully defatted
and the residue turned into a powdered and nonsticky
form after drying. It was then kept in a Petri dish at room
temperature for the removal of hexane.
As part of a treatment step for starchy foods (savory
items, such as sago and rice papad, sweetened puffed
rice, and fryums), hot water soaking was needed to enable
them to swell; otherwise, color extraction was hampered.
After the pretreatment of fatty and starchy foods, the
color was extracted with 10 mL 5%  ammonia in 75%
methanol, shaken well, centrifuged, and the supernatant
collected. The process was repeated with fresh solvent
until the color was completely extracted. Finally, the
supernatants were pooled and concentrated to dryness.
In the case of water-soluble, sugar-based food matrixes
(candy floss, sugar toys, and sugar-coated fennel seeds),
the samples were dissolved directly in 5%  ammonia in
75% methanol and concentrated to dryness. Finally, the
residue was dissolved in 5% ammonia.
Sample Cleanup
The polyamide cartridges were conditioned prior
to use by washing with methanol (2 mL) and Milli-Q
water (2 mL). The clear extract of colors in 5% ammonia
was applied to the polyamide SPE columns at the rate
of approximately 0.5 mL/min. The columns were
washed with Milli-Q water (2 mL), and the absorbed
dyes were eluted with 1 mL of eluting solution

(3%  acetic acid–methanol, 1 + 1, v/v). The eluted dyes
were concentrated to dryness, reconstituted in methanol,
and filtered prior to HPLC injection through a Millipore
(Bangalore, India) filter of 0.45 µm PVDF membrane.
Instruments

Figure  1.  Structures of the three basic nonpermitted
colors.

A double-beam spectrophotometer (PerkinElmer
Lambda Bio 20, PerkinElmer Instruments, Waltham,


1876  Dixit et al.: Journal of AOAC International Vol. 94, No. 6, 2011
Table  2.  Adsorption of colors on polyamide
cartridges at different pH concentrations
pH Concentration

Amount of color adsorbed, µg

a

8.0

100

8.5

350

9.0

500

9.5

700

10.0

1000

a

 Pure Millipore water has a pH of 7.7; therefore, 8.0 pH was
chosen as the starting concentration.

MA) was used for spectrophotometric measurements
using a quartz cell of 10 mm path length. A pH meter
(Cyberscan 510, Eutech Instruments Pte Ltd, Singapore)
having a combined glass–calomel electrode was used
for pH measurements. Milli-Q water was produced by
using a Milli-Q Simplicity Water Purification System,
with a water outlet operating at 18.2 Ω, from Millipore.
Chromatographic analysis was carried out with a
Waters LC module (Waters Associates, Vienna, Austria)
equipped with a dual pump (Model 510), Rheodyne
injector with 20 µL loop, and tunable absorbance detector
(Model 486). The chromatograms were recorded and
®

processed by Waters Millennium software.
Chromatographic Conditions
Analysis was performed on a 300 × 3.9 mm id
reversed-phase C18 µ-Bondapak column with a 90 mm
precolumn. The components of the mobile phase were
filtered under vacuum through a membrane filter with a
pore diameter of 0.45 µm. The injection volume was set
at 20 µL. The optimal mobile phase conditions consisted
of the isocratic flow of acetonitrile–sodium acetate
(20  mM, pH  4.0; 80 + 20, v/v) for a period of 10  min,
with a flow rate of 1.5 mL/min. The UV-Vis detector
was programmed to monitor the individual colors at
their respective maximum absorbance wavelength. For
single dyes, the elution was monitored at wavelengths
of 420 nm for auramine, 525 nm for Rhodamine B, and
600 nm for malachite green. For green blends, the elution
was monitored at 420 nm for auramine (0–6 min) and at
600 nm for malachite green (6–10 min).
Results and Discussion
Optimization of the Cleanup Process on SPE
Polyamide Cartridges
Many sorbents such as octadecyl silica (ODS; 29–31),
quaternary amine (32), aminopropyl (NH2; 33), and
polyamide (34, 35) SPE columns have been used for
the cleanup of acidic synthetic dyes in foods. Sorbents

Table  3.  Effect of different concentrations of acetic
acid on the elution pattern of three basic dyes from
SPE cartridges
Percentage of colors eluted

Acetic acid concn,
methanol (%, 1:1)

Auramine

Rhodamine B

Malachite
Green

0.0

25.3

40.4

18.9

0.5

48.8

72.3

62.2

1.0

67.3

73.2

62.3

2.0

74.4

84.9

68.5

3.0

88.6

93.0

77.8

4.0

88.0

93.2

77.6

5.0

88.4

93.0

77.7

like ODS (15), styrene divinylbenzene polymeric
surface (Strata-X and Strata-SCX; 17, 27), and
primary/secondary amines (36) have been used for the
cleanup of basic dyes in cosmetic products and fish tissue.
We have opted for polyamide, as it adsorbs synthetic
colors more specifically than other sorbents and because
its amphoteric nature allows both acidic and basic dyes
to be cleaned up by variations in the pH. Below the
isoelectric point (pH 4.2), polyamide has a positive
charge and reacts with anionic dyes (acid, direct, etc.):
+
–
–
+
H3N – Polyamide – COOH + Dye → Dye /H3N –
Polyamide – COOH (Ionic bond)

Above the isoelectric point, it has a negative charge
and reacts with cationic dyes; thus, a single column can
be used for both acidic and basic dyes:
–
+
H2N – Polyamide – COO + Dye → H2N –
–

+
Polyamide – COO Dye (Ionic bond)

The three basic colors involved in the present study
+
have N groups in their molecular structures (Figure 1).
The interaction between these colors (carrying positive
charges) and the polar polyamide is predominated by
hydrogen bonding when color samples are loaded on
polyamide columns at pH 7.0. Because the medium tends
to become basic, the polyamide carries more negative
–
charge on the COO group, thereby enhancing the
cation–anion attraction force.
In order to test the influence of pH on the adsorption
capacity of polyamide, the three colors were added
to Milli-Q water of different pH values. The results
indicated that the adsorption capacity increased with an
increase in pH value, the highest being at pH 10.0, where
approximately 1000 µg/mL of colors could be loaded, and
the capacity reduced to <100 µg/mL at pH 8.0 (Table 2).
In order to obtain higher recovery, the colored solutions
were adjusted to pH 10.0 before loading to polyamide
SPE cartridges.




Dixit et al.: Journal of AOAC International Vol. 94, No. 6, 2011  1877

Figure  2.  HPLC resolution of three basic nonpermitted colors encountered in standard (a) and food samples
(b, c, and d).

Effect of Acetic Acid on the Elution of Colors from
SPE Polyamide Cartridges
Pure methanol could elute 20–40% of the dyes adsorbed
onto the polyamide column, so methanol containing
0.5–5.0% acetic acid was tried (Table 3). The results
showed that with increasing acetic acid concentrations
of 0.5–3%, there was a nonproportional increase in the
elution pattern of dyes, and these remained virtually
unaltered upon any further increase of acetic acid (4 and

5%). Hence, 3% acetic acid in methanol was found to be
the optimal concentration for elution and was checked for
any background peaks in the chromatograms.
Optimization of the Separation on HPLC
Lyter (37) used acetonitrile–water (70 + 30, v/v) as
the mobile phase for the resolution of some basic dyes.
However, basic dyes used in the present study could not
be separated satisfactorily in this combination or with

Figure  3.  Calibration curves showing linearity of three basic nonpermitted colors. Equation: Y = b + mx.
Linearity range: auramine (0.2–25.0 mg/L), Rhodamine B (0.2–50.0 mg/L), and Malachite green (0.1–50.0 mg/L).


1878  Dixit et al.: Journal of AOAC International Vol. 94, No. 6, 2011
Table  4.  LOD and LOQ of three basic dyes in different food commodity groups
LOD, mg/L

LOQ, mg/L

Sugar-based
matrixes

Starch-based
matrixes

Fatty food
matrixes

Sugar-based
matrixes

Starch-based
matrixes

Fatty food
matrixes

Auramine

0.371

0.754

0.713

1.18

2.40

2.27

Rhodamine B

0.107

0.500

0.519

0.34

1.59

1.65

Malachite green

0.195

0.695

0.660

0.62

2.21

2.10

Colors

80 + 20 (v/v) acetonitrile–water, and showed a broad peak
with merged retention time (RT). An attempt was made
to test different combinations of acetonitrile with sodium
acetate buffer as the mobile phase to achieve optimal
resolution and peak symmetry. The addition of sodium
acetate reduced the polarity and resulted in much faster
(within 10 min) elution of all three dyes. Other mobile
phases, such as acetonitrile–sodium perchlorate (0.1 M,
pH 3.0, 50 + 50, v/v, to 70 + 30, v/v, linear gradient; 15),
ammonium acetate (0.1 M pH, 4.0): acetonitrile
(60 + 40,  v/v; 36), acetonitrile–acetate buffer (0.05 M,
pH 4.5; 60 + 40, v/v; 27), ammonium acetate (5 mM)
in 0.1% formic acid–0.1% formic acid in acetonitrile
(80 + 20, v/v, to 20 + 80, v/v, linear gradient; 17) were
also attempted. The isocratic mixture of acetonitrile–
sodium acetate (20 mM; 80 + 20) at pH 4.0 with a flow
rate of 1.5 mL/min was found to be the best. Auramine
separated at RT 4.08 min, Rhodamine B at 6.00 min, and
malachite green at 8.44 min (Figure 2).
Validation of the Method
The validation analytical method, including linearity,
sensitivity, LOD, LOQ, method precision, and recovery
experiment, was carried out. The linearity of the assay
was checked by running a duplicate set of each dye, and
the calibration graph was obtained by plotting the peak
area versus concentration. Auramine and Rhodamine  B

showed linearity at the concentrations of 0.2–25 and
0.2–50  mg/L, respectively, while malachite green gave
linearity at 0.1–50 mg/L. The regression coefficient of
the three dyes varied from 0.9983 to 0.9995 (Figure 3).

The LOD and the LOQ were determined by the U.S.
Environmental Protection Agency method (38). Seven
replicates of each dye at a concentration of 5  mg/L in
sugar, fatty, and starch-based food matrixes were spiked
and analyzed. The LOD and LOQ of the three studied
colors ranged from 0.107 to 0.371 and from 0.34 to
1.18 mg/L, respectively, in sugar-based matrixes. In the
case of fatty foods, the LOD and LOQ ranged from 0.519
to 0.713 and 1.65 to 2.27 mg/L. In starch-based matrixes
the ranges were 0.500–0.754 and 1.59–2.40  mg/L,
respectively, which were found to be higher than in
sugar-based matrixes (Table 4).
Method efficiency was tested in terms of RSDs for both
intraday and interday precision. The intraday precision (as
RSDr) for Rhodamine B varied from 0.84% at 5.0 mg/L to
4.30% at 10.00 mg/L. For auramine and malachite green
at these two concentrations, however, the % RSDrs were
close and in the range of 2.3–3.5. The interday precision
(as RSDR) of the three dyes at the two concentrations
ranged from 1.24 to 4.40% for Rhodamine B (Table 5).
The trueness of the method was evaluated in terms
of recovery experiments by spiking standard colors in
fatty, starchy, and sugar-based food samples at three
concentrations of 50, 100, and 150 mg/kg or mg/L. The
values showed a recovery of 75.0–88% for the three

colors from fatty and starchy foods, while sugar-based
water-soluble matrixes offered a higher recovery in the
range of 87.5–96.5% (Table 6). Among the colors, the
recovery was least in case of the malachite green (75%)
and maximum in the case of Rhodamine B (97.5%).

Table  5.  Intraday and interday precision for estimation of three basic dyes studied (n = 3)
Intraday precision
Colors
Auramine
Rhodamine B
Malachite green

Interday precision

Amt, mg/L

Avg. peak area

% RSDr

SE

Avg. peak area

% RSDR

SE

5.0

270159

3.18

4963

265908

1.89

2907

10.0

551768

2.27

7225

564047

1.24

4046

5.0

268211

0.84

1303

262008

3.31

5009

10.0

428361

4.30

10653

427092

4.40

10870

5.0

357741

3.21

6641

359453

2.92

6075

10.0

762783

3.54

15614

733417

1.78

7525


Dixit et al.: Journal of AOAC International Vol. 94, No. 6, 2011  1879



Table  6.  Recovery of individual dyes spiked in different food matrixesa
Sugar-based matrixes

Dyes

Concn, mg/L or
mg/kg
Recovery, %

Auramine

Rhodamine B

Malachite green

a

RSD, %

Starch-based matrixes
Recovery, %

RSD, %

Fatty food matrixes
Recovery, %

RSD, %

50

94.4

3.31

85.1

5.15

85.5

2.48

100

93.3

2.21

87.0

2.28

85.2

1.99

150

96.5

0.97

85.2

3.32

88.0

3.80

50

97.5

1.67

87.2

1.14

85.5

4.67

100

96.3

1.55

87.6

2.58

87.2

2.48

150

97.2

1.13

82.9

0.60

81.9

1.24

50

87.5

2.71

77.5

3.14

74.6

4.17

100

88.6

2.04

77.5

1.28

74.9

1.49

150

88.5

1.81

77.0

2.94

76.6

2.52

  Recovery of dyes was performed in duplicate; mean data shown.

Table  7.  Determination of dyes in food products collected from the local marketa
Sample

Dyes found

Concn, mg/kg

RSD, %

Rhodamine B

62.86

3.98

Rhodamine B

34.24

4.79

Rhodamine B

47.85

3.75

Rhodamine B

55.44

3.09

Rhodamine B

77.63

3.53

Rhodamine B

108.35

2.76

Rhodamine B

116.44

1.23

Auramine

46.28

1.88

Rhodamine B

103.43

1.34

Rhodamine B

49.89

4.90

Rhodamine B

204.74

2.51

Auramine

39.69

2.00

Auramine + Malachite green

65.56

3.59

Colored peas

Rhodamine B

20.52

4.55

Sweetened puffed rice

Rhodamine B

43.01

3.26

Rhodamine B

40.10

4.00

Rhodamine B

51.11

3.46

Sugar-based matrixes

Candy floss

Sugar toy

Sugar-coated colored fennel

Starch-based matrixes
Fryums

Fatty food matrixes
Cream biscuit

Rhodamine B

26.16

4.03

Fruit cake

Rhodamine B

34.54

3.46

Cereal/pulse-based sweets

Rhodamine B

29.38

3.59

Auramine

24.40

4.35

Rhodamine B

24.31

4.33

Milk-based sweets
a

  Data represent mean of duplicate values for each analyzed sample.


1880  Dixit et al.: Journal of AOAC International Vol. 94, No. 6, 2011

Application to Real Samples
Five samples each of pink-, yellow-, and green-colored
listed food commodities were analyzed; results revealed
that all samples of candy floss (mostly consumed by
children) contained Rhodamine B. One sample each
of pink-colored commodities, including cream biscuit,

fruit cake, cereal/pulse-based sweets, colored fried
peas, milk-based sweets, and sugar-coated fennel seeds,
showed the presence of Rhodamine B (Table 7). Two
pink samples of sugar-derived toys and starch-based
savories also showed Rhodamine B. One sample each
of yellow-colored commodities, including fryums,
sugar toys, and cereal/pulsed-based sweets, was found
to contain auramine. In the case of sweetened puffed
rice, three out of five samples had Rhodamine B and
one green-looking sample of fryum contained a blend
of auramine plus malachite green. The presence of
nonpermitted colors ranged from 20.5 to 204.7 mg/kg in
the foodstuffs (Table 7). The intake of nonpermitted basic
colors at such levels could be hazardous in view of their
toxic potential.
Conclusions
The present method utilizes a simple pretreatment
step, cleanup on polyamide SPE cartridges, and HPLC
resolution on a reversed-phase C18 to detect the three
basic nonpermitted dyes encountered in different food
matrixes. The recoveries of spiked dyes with this method
ranged from 75 to 96.5%. HPLC resolution was optimal
with an acetonitrile–sodium acetate buffer (20 mM), in
which all three dyes were completely separated within
10 min. The proposed method offers to reduce any
interference of starch- and fat-based food matrixes.
Monitoring of colors at their respective λmax gives high
sensitivity and scope to the testing of typical green color
blends in a broad variety of real market samples. The
study demonstrated that the use of a combination of a

simple SPE cleanup and HPLC resolution with UV-Vis
end point detection was useful in screening the presence
of these three basic nonpermitted dyes in a variety of food
matrixes.
Acknowledgments
We are grateful to the Director of the Indian Institute
of Toxicology Research (IITR) for his keen interest in
the present study. Author S.K. Khanna is a superannuated
scientist from IITR. Financial support from the Council
of Scientific and Industrial Research Network Project No.
17 is gratefully acknowledged. The manuscript is IITR
Communication No. 2889.

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