Determination of Food Colorants (Ponceau 4R and Brilliant black) by Derivative Spectrophotometry

 

Saad Antakli, Leon Nejem, Rahaf Taftanaze

Department of Chemistry, Faculty of Science, University of Aleppo, Syria.

 

ABSTRACT:

Spectrophotometric method was developed and applied for simultaneous determination of two colorants: Ponceau 4R and Brilliant black in Syrian foodstuffs. This study was applied on several Syrian foodstuffs as: Royal Jelly, Archi jelly, Columbus drink (strawberry), Columbus drink (blueberry), Aquaz drink (blueberry). Derivative spectrophotometric method (DS) was applied for the determination of Ponceau 4R and Brilliant black, respectively. Ponceau 4R was determined by first derivative spectrophotometry (¹D₄₅₄), and Brilliant black was determined by first derivative spectrophotometry (¹D₆₂₄). Linearity ranges were 3 - 40 𝜇g/mL for Ponceau 4R and 2 - 35 𝜇g/mL for Brilliant black. Regression analysis showed a good correlation coefficients R² = 1 and R² = 0.9999 for Ponceau 4R and Brilliant black, respectively. The limit of detection (LOD) and limit of quantification (LOQ) were to be 0.825 𝜇g/mL and 2.50 𝜇g/mL for Ponceau 4R and 0.328 𝜇g/mL and 0.99 𝜇g/mL for Brilliant black, respectively. The proposed derivative method was successfully applied to analysis individual or mixture of Ponceau 4R and Brilliant black in foodstuffs. All studied samples showed dye levels conformity with Syrian legislation.

 

 

1. INTRODUCTION:

Food additives in general and colors in particular have increasingly under evaluation of their safety in use these days in many developed countries. Only about ten dyes are permitted to be used as food coloring agents, many others having been banned in the last two decades because of toxicity and carcinogenity¹. Research results have shown that Ponceau 4R, when consumed too excess, can cause adverse health effects such as reproductive toxicity, neurobehavioral effects and cancer².

 

Thus, various methods have been proposed to control the amount of colorants in food such as capillary zone electrophoresis (CZE)³, differential pulse polarography^4-5 and voltammetric methods as adsorptive voltammetry⁶.

 

Chromatographic methods have been used for colorants analysis in foods, high performance liquid chromatography (HPLC)⁷, high-performance ion chromatography⁸, reversed phase thin-layer plates⁹, reverse phase high performance liquid chromatographic (RP-HPLC) method^10-11 and micellar electro kinetic capillary chromatography (MEKC)¹², spectrophotometric^13-14 and derivative spectrophotometric methods¹⁵, high-performance liquid chromatography using a short column with photodiode array detection¹⁶, are successfully applied to determine other synthetic dyes.

 

The aim of this work is to develop a simple and accurate spectrophotometric method for simultaneous determination of Ponceau 4R (E124), and Brilliant black (E151), and their mixtures in different foodstuffs without prior treatment by UV/Vis spectrophotometry and derivative spectrophotometry (DS), where their mixtures give strawberry shades to products.

 

2. MATERIALS AND METHODS:

2.1. Apparatus:

All spectral measurements were carried out using a UV/Vis. spectrophotometer 80DV instrument Ltd (UK) connected to computer, quartz cells 1 cm, and centrifuge Centurion Scientific Ltd (UK).

 

2.2. Chemical regents:

Distilled water was used to prepare the solutions. The standard synthetic colorants were Ponceau 4R (CI 48440, CAS 2611-82-7) purity 85% and Brilliant black (CI 2519, CAS 2519-30-4) purity 83%. Both colorants were obtained from Sineset (France).

 

2.3. Standard preparation:

Stock solutions (100𝜇g/mL) of Ponceau 4R and Brilliant black were prepared by dissolving appropriate weights of standard colorants (after taking the purity of the colorants in consideration) in distilled water. The working standard solutions of each colorant were prepared by appropriate dilutions of stock solutions with distilled water to give concentrations between 0.9 – 50 𝜇g/mL of Ponceau 4R and 0.9 – 50𝜇g/mL of Brilliant black.

 

2.4. Calibration Curve:

To construct the calibration curve, five standard solutions for each concentration were prepared and the absorbance was measured of each solution five times.

 

2.5. Sample preparation:

Ponceau 4R was studied in three products: Royal Jelly (cherries), Archi jelly (cherries), Columbus drink (strawberry). Ponceau 4R and Brilliant black were also studied in two products: Aquaz drink (blueberry) Columbus drink (blueberry).

·       0.6 g of Royal Jelly (cherries) sample was accurately weighted and dissolved in distilled water at 70 °C, then solution was transferred into a 10 mL volumetric flask and adjusted to volume with distilled water.

·       0.3 g of Archi jelly (cherries) sample was accurately weighted and dissolved in distilled water at      70 °C, then solution was transferred into a 10 mL volumetric flask and adjusted to volume with distilled water.

·       0.1 g of Columbus drink (strawberry) sample was accurately weighted and dissolved in distilled water, then solution was centrifuged during 5 min. at 6000, and then solution was transferred into   a 10 mL volumetric flask and adjusted to volume with distilled water.

·       0.2 g of Columbus drink (blueberry) sample was accurately weighted and dissolved in distilled water, then solution was centrifuged during 5 min. at 6000, and then solution was transferred into   a 10 mL volumetric flask and adjusted to volume with distilled water.

·       0.1 g of Aquaz drink (blueberry) sample was accurately weighted and dissolved in distilled water, then solution was centrifuged during 5 min. at 6000, and then solution was transferred into a 10 mL volumetric flask and adjusted to volume with distilled water.

 

3. RESULTS AND DISCUSSION:

Absorption spectra of the standard colorants 10𝜇g/mL Ponceau 4R and 10𝜇g/mL Brilliant black solutions and 10𝜇g/mL of each of them as a mixture solution were recorded within a wave length range of 400 – 700nm against distilled water, Fig. 1. As can be seen, Ponceau 4R cannot be determined by direct measurement of absorbance at 508nm, and Brilliant black cannot too at 570nm, because of the overlapped spectra. On the other hand, derivative spectrophotometry showed more resolution. Where it made the determination of Ponceau 4R and Brilliant black mixture possible without pretreatment. The first derivative spectrum at zero-crossing point was used to determine Ponceau 4R in the presence of Brilliant black at 454 nm (Fig. 2, a). The first derivative spectrum at zero-crossing point was used to determine Brilliant black in the presence of Ponceau 4R at 624 nm (Fig.2, b).

 

 

Fig. 1. Zero-order spectra of:

a- Ponceau 4R, b- Brilliant black.

c- Mixture of Ponceau 4R and Brilliant black.

 

 

Fig. 2. First derivative spectra of:

a- Ponceau 4R, b- Brilliant black.

c- Mixture of Ponceau 4R and Brilliant black.

3.1. Stability of the coloration:

Time effect on the stability of colors in light and darkness, was studied in two different concentrations 8 and 15µg/mL of both colorant. We did not notice any significant changes during the absorption measurement within minimum 12 weeks in both cases light and darkness. 

4. METHOD VALIDATION:

The validity of the proposed method was assessed by linearity (evaluated by regression equation), limit of detection (LOD) and limit of quantification (LOQ), accuracy (reported as recovery or percentage) and precision (reported as RSD %).

 

4.1. Linearity:

The concentration linearity of Ponceau 4R was in the range 3 - 40𝜇g/mL at 454 nm by ¹D_454, Figs. 3 & 4 and the concentration linearity of Brilliant black was in the range 2- 35µg/mL at 624 nm by ¹D_624, Figs. 7& 8.

 

 

Fig. 3. First derivative spectra of Ponceau 4R:

C₁: 3 𝜇g/mL, C₂: 5 𝜇g/mL, C₃: 10 𝜇g/mL, 

C₄: 15 𝜇g/mL, C₅: 20 𝜇g/mL, C₆: 25 𝜇g/mL,

C₇: 30 𝜇g/mL, C₈: 35 𝜇g/mL, C₉: 40 𝜇g/mL.

 

 

Fig. 4. calibration curve for ponceau 4R

C₁: 3 𝜇g/mL, C₂: 5 𝜇g/mL, C₃: 10 𝜇g/mL,

C₄: 15 𝜇g/mL, C₅: 20 𝜇g/mL, C₆: 25 𝜇g/mL,

C₇: 30 𝜇g/mL, C₈: 35 𝜇g/mL, C₉: 40 𝜇g/mL.

at 454 nm, n = 5 for each concentration.

 

 

Fig. 5. First derivative spectra of Brilliant black:

C₁: 2 𝜇g/mL, C₂: 6 𝜇g/mL, C₃:10 𝜇g/mL,

C₄: 14 𝜇g/mL, C₅: 18 𝜇g/mL, C₆: 22 𝜇g/ml,

C₇: 26 𝜇g/mL, C₈: 30 𝜇g/mL, C₉: 35 𝜇g/mL.

 

 

Fig. 6. Calibration curve for Brilliant black:

C₁: 2 𝜇g/mL, C₂: 6 𝜇g/mL, C₃:10 𝜇g/mL,

C₄: 14 𝜇g/mL, C₅: 18 𝜇g/mL, C₆: 22 𝜇g/ml,

C₇: 26 𝜇g/mL, C₈: 30 𝜇g/mL, C₉: 35 𝜇g/mL.

at 624 nm, n = 5 for each concentration.            

 

4.2. Limit of detection (LOD) and Limit of quantification (LOQ):

LOD and LOQ were calculated in Table 1 using the following equations:

 

             3.3 × SD                     10 × SD

LOD = ------------       LOQ = -------------

                   m                               m

 ^;

Where SD is the standard deviation of y intercepts of regression lines and m is the slope of the calibration curve.

 

 

 

 

Table 1: Statistical data for calibration graphs.

Method	Analyte	Selected wavelength (nm)	Linearity range 𝜇g/mL	Correlation coef. (R2)	LOD 𝜇g/mL	LOQ 𝜇g/mL
DS	Ponceau 4R	1D454	3 – 40	1,0000	0.825	2.50
DS	Brilliant black	1D624	2 – 35	0.9999	0.328	0.99

 

4.3. Accuracy:

To appoint the precision and accuracy of the proposed method, five replicate determinations were carried out on three different concentrations of standards Ponceau 4R and Brilliant black. The validation results are presented in Table 2.

 

 

 

 

 

4.4. Sensitivity Sandell’s:

Sensitivity of the proposed method for Ponceau 4R and Brilliant black was determined by calculating Sandell’s sensitivity (SS). It was to be SS = 0.0090 µg/cm² and 0.34 µg/cm², respectively.

 

4.5. Recovery:

The recovery was studied by three addition standards for every product. Table 3 presents the recoveries results for the five studied products:

 

Table 2: Precision and accuracy for the simultaneous determination of Ponceau 4R and Brilliant black for the proposed methods

Method	Colorant	Theoretical concentration (μg/mL)	*Observed concentration (μg/mL)	Precision RSD (%)	Accuracy (%)
DS	Ponceau 4R	5	4.95	2.26	99.00
		10	10.05	1.11	100.50
		15	15.10	0.91	100.67
DS	Brilliant black	5	4.98	2.98	99.60
		10	9.90	0.71	99.00
		15	15.20	0.72	101.33

Accuracy (%) = (observed concentration/theoretical concentration) _˟ 100. Precision (%) = (standard deviation/mean 

concentration) _˟100. * five separate determinations were performed and calculated the mean.

 

Table 3: Recoveries of Ponceau 4R for Royal jelly (cherries), Archi jelly (cherries), Columbus (strawberry) and Ponceau 4R with Brilliant Black for Columbus (cherries), Aquaz drink (blueberry):

Product	Colorant	Sample µg/mL	Added µg/mL	Total* Found µg/mL	Rec. %	RSD %	SD µg/ml	Rec. %
Royal jelly (cherries)	Ponceau 4R	15.00	12.00	27.15	101.25	3.47	3.43	100.64
		15.00	15.00	30.10	100.67	3.02	3.00
		15.00	18.00	33.00	100.00	2.21	2.21
Archi jelly (cherries)	Ponceau 4R	9.25	7.40	16.60	99.32	3.02	3.04	98.93
		9.25	9.25	18.40	98.92	1.48	1.50
		9.25	11.11	20.20	98.56	1.91	1.94
Columbus (strawberry)	Ponceau 4R	18.20	14.56	32.80	100.27	3.30	3.29	99.91
		18.20	18.20	36.35	99.73	2.29	2.30
		18.20	21.84	39.98	99.73	1.13	1.33
Aquaz drink (blueberry)	Ponce 4R	20.30	16.24	36.80	101.60	3.14	3.09	100.07
		20.30	20.30	40.70	100.49	2.24	2.23
		20.30	24.36	44.20	98.11	1.34	1.37
	Brilliant Black	3.28	2.62	5.86	98.47	2.99	3.04	99.31
		3.28	3.28	6.55	99.70	3.43	3.44
		3.28	3.94	7.21	99.75	3.45	3.46

*n = 5.

 

5. ANALYTICAL APPLICATION:

The developed method was applied for quantitative determination of food colorants in six products foodstuffs from Syrian local markets. The samples were prepared as described in the section of samples preparation and analysed. Quantitative analysis was done by using calibration curves. The obtained results are summarized in Table 4 for five different batches of each studied Syrian trademarks products.

 

In general, the used amounts of Ponceau 4R were relatively higher than those of Brilliant black in all analysed products. However, the concentrations of the detected colorants were much lower than the Syrian limits¹⁷: 200 mg/kg for Ponceau 4R and 200 mg/kg for Brilliant black. The daily dose taken by each individual of the Ponceau 4R and Brilliant black was also calculated when taking only one serving (each sachet is sufficient to prepare four servings) and then compared with acceptable daily intake (ADI): 4 mg/kg body weight (or 240mg/60 kg, average of body weight) for Ponceau 4R and 5mg/kg body weight (or 300mg/60 Kg, average of  body weight) for Brilliant black.

 

The relative standard deviations RSD % (n = 5) of the quantitative results were in the range of          0.97 - 4.76 % and 0.94 - 4.31 % for Ponceau 4R and Brilliant black, respectively. Table 4, a-b present the determination results of colorants Ponceau 4R and Brilliant black, in four Syrian trademarks for five different batches for each: (Royal Jelly, Archi jelly, Columbus drink strawberry, Aquaz drink blueberry).

 

 

Table 4, a: Results of colorant Ponceau 4R:

Royal jelly product
No. of sample	1	2	3	4	5	Range of concentration
Concentration mg/sachet (80g)	24.5	22.5	28.0	29.1	22.7	22.5 – 29.1 mg/sachet
SD mg/sachet	0.38	0.44	0.60	1.02	0.86
RSD %	1.55	1.96	2.14	3.51	3.79
Concentration mg/bowl *	6.13	5.63	7.00	7.28	5.68
Archi jelly product
No. of sample	1	2	3	4	5	Range of concentration
Concentration mg/sachet (80g)	37.0	42.6	15.6	13.6	11.8	11.8 – 42.6 mg/sachet
SD mg/sachet	0.55	1.56	0.60	0.33	0.56
RSD %	1.49	3.66	3.85	2.43	4.75
Concentration mg/bowl *	9.25	10.65	3.90	3.40	2.95
Columbus drink product
No. of sample	1	2	3	4	5	Range of concentration
Concentration mg/sachet (40g in 1L)	72.7	71.7	74.5	67.0	62.3	62.3 – 74.5 mg/sachet
SD mg/sachet	1.05	1.72	2.00	0.84	1.06
RSD%	1.44	2.40	2.68	1.25	1.70
Concentration mg/bowl **	18.18	17.93	18.63	16.75	15.58

 n = 5, ^*(20 g in bowl 250 mL). ** (10 g in cup 250 mL).

 

Table 4, b: Results of colorants Ponceau 4R and Brilliant black in Aquaz drink (blueberry).

No. of sample	Colorant	Concentration mg/sachet*	SD	RSD %	Concentration mg/cup**	Range of concentration
1	Ponceau 4R	16.0	0.59	3.69	4.00	15.0 – 19.4 mg/sachet for Ponceau 4R, 2.6 - 5.0 mg/sachet for Brilliant black
	Brilliant black	2.8	0.09	3.21	0.70
2	Ponceau 4R	16.2	0.59	3.64	4.05
	Brilliant black	2.6	0.09	3.46	0.65
3	Ponceau 4R	15.0	0.39	2.60	3.75
	Brilliant black	2.7	0.10	3.70	0.67
4	Ponceau 4R	16.5	0.16	0.97	4.13
	Brilliant black	3.3	0.13	3.94	0.83
5	Ponceau 4R	19.4	0.30	1.55	4.85
	Brilliant black	5.0	0.10	2.00	1.25

n = 5, *(sachet = 8 g in 1L), ** (2 g in cup 250 mL).

 

6. Method of standard additions:

This method was used to determine the concentrations of Ponceau 4R and brilliant black in Columbus (blueberry) product. A series with a standard solutions of Ponceau 4R and brilliant black to five flasks (10mL) containing specific weight of the sample were prepared by adding increasing standard concentrations (0, 5, 10, 15 and 20 µg/mL) of each colorant, completing the flasks with distilled water.

The graph curves are then plotted between the added concentrations of Ponceau 4R and brilliant black and the mean derivative absorption of five experiments. The negative intersection in absolute value which expresses the concentration of the colorants in the studied sample. Table 5 presents the concentrations of the both colorants Ponceau 4R and brilliant black in the Columbus drink (blueberry) product, for five different batches:

 

Table 5: Results of colorants Ponceau 4R and brilliant black in Columbus drink (blueberry).

No. of sample	Colorant	Concentration mg/sachet*	SD	RSD %	Concentration mg/cup**
1	Ponceau 4R	57.1	2.72	4.76	14.28
	Brilliant black	37.1	1.60	4.31	9.28
2	Ponceau 4R	71.9	1.89	2.63	17.98
	Brilliant black	58.1	2.04	3.51	15.53
3	Ponceau 4R	47.1	2.03	4.31	11.78
	Brilliant black	27.6	0.97	3.51	6.90
4	Ponceau 4R	51.4	2.18	4.24	12.85
	Brilliant black	43.8	0.41	0.94	10.95
5	Ponceau 4R	57.1	1.04	1.82	14.28
	Brilliant black	37.6	1.60	4.26	9.40
Range of concentration	47.1 – 71.9 mg/sachet for Ponceau, 27.6 – 58.1 mg/sachet for Brilliant black

n = 5, *(sachet = 40 g in 1L), ** (10g in cup 250mL).

 

 

 

7. CONCLUSION:

Ponceau 4R and Brilliant blacklevels were estimated in individual form and in their mixtures in different studied local Syrian foodstuffs by first derivative spectrophotometric method using zero-crossing point.

 

Colorants levels were lower than their maximum values which established by the Syrian legislation¹⁷. The proposed method of Ponceau 4R and Brilliant black determination are accurate, simple, sensitive, easy and directly applicable in quantitative analysis without previous chemical treatment in individual or binary mixture.

 

The proportions of mixture of Ponceau 4R and Brilliant black in the studied products were different from one to another. It was seen also that Ponceau 4R level in general higher than Brilliant black in all analyzed products. The food additives in the different studied products did not have any interference on the analysis of the Ponceau 4R and Brilliant black.

 

8. ACKNOWLEDGEMENT:

The Ministry of High Education in Syria financially and technically supported this work through department of Chemistry, Faculty of Science, and University of Aleppo, Syria.

 

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Received on 22.05.2019           Modified on 25.06.2019

Accepted on 30.07.2019         © RJPT All right reserved