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

A Simple Method for Simultaneous Determination of Basic Dyes  Encountered in Food Preparations by ReversedPhase HPLC Một phương pháp đơn giản để xác định đồng thời cơ bản nhuộm Gặp phải trong chế phẩm thực phẩm bằng cách đảo ngược pha HPLC

A Simple Method for Simultaneous Determination of Basic Dyes  Encountered in Food Preparations by Reversed-Phase HPLC

S umita D ixit , S ubhaSh K K hanna , and m uKul D aS 1

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

Received November 16, 2010 Accepted by SG January 12, 2011.

1 Corresponding author’s e-mail: mditrc@rediffmail.com

DOI: 10.5740/jaoacint.10-450

FOOD COMPOSITION AND ADDITIVES

The present method utilizes a simple

pretreatment step, cleanup on polyamide SPE

cartridges, and HPLC resolution on

reversed-phase 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

λ max 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.

Colors 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

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

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

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

Figure 1 Structures of the three basic nonpermitted

colors.

Table 1 Common names, Color Index (CI)

names, CI numbers, and λ max of three basic dyes

encountered in foods

Common name CI name CI No λ max , nm

Auramine Basic Yellow 2 41000 430

Rhodamine B Basic Violet 10 45170 525

Malachite green Basic Green 4 42000 618

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

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

Table 2 Adsorption of colors on polyamide

cartridges at different pH concentrations

pH Concentration Amount of color adsorbed, µg

a Pure Millipore water has a pH of 7.7; therefore, 8.0 pH was

chosen as the starting concentration.

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

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

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/Lto 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 4 LOD and LOQ of three basic dyes in different food commodity groups

LOD, mg/L LOQ, mg/L

Colors 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

Table 5 Intraday and interday precision for estimation of three basic dyes studied (n = 3)

Intraday precision Interday precision Colors Amt, mg/L Avg peak area % RSD r SE Avg peak area % RSD R SE Auramine 5.0 270159 3.18 4963 265908 1.89 2907

10.0 551768 2.27 7225 564047 1.24 4046 Rhodamine B 5.0 268211 0.84 1303 262008 3.31 5009

10.0 428361 4.30 10653 427092 4.40 10870 Malachite green 5.0 357741 3.21 6641 359453 2.92 6075

10.0 762783 3.54 15614 733417 1.78 7525

Table 6 Recovery of individual dyes spiked in different food matrixesa

Sugar-based matrixes Starch-based matrixes Fatty food matrixes

Dyes Concn, mg/L or mg/kg Recovery, % RSD, % Recovery, % RSD, % Recovery, % RSD, % Auramine 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 Rhodamine B 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 Malachite green 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

a 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, %

Sugar-based matrixes

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 116.44 1.23 Auramine 46.28 1.88 Sugar-coated colored fennel Rhodamine B 103.43 1.34

Starch-based matrixes

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 Fatty food matrixes

Cream biscuit Rhodamine B 26.16 4.03

Cereal/pulse-based sweets Rhodamine B 29.38 3.59

Auramine 24.40 4.35 Milk-based sweets Rhodamine B 24.31 4.33

a Data represent mean of duplicate values for each analyzed sample.

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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