DEVELOPMENT AND VALIDATION OF A LIQUID CHROMATOGRAPHIC METHOD FOR ANALYSIS OF FOOD COLOURS IN MULTIPLE FOOD MATRICES

INTRODUCTION

Food colours are added to different types of food commodities to increase their visual appearance often to help consumer to find them more appealing. The use of these additives is strictly regulated in many countries worldwide. There is a growing concern about the safety of some commonly used legal food colorants and there is a trend to replace the synthetic forms with natural products. Additionally, several dyes with known or suspected carcinogenic properties have been shown to be added illegally to foods. The most important food safety concerns in the field of food colours are lack of uniform regulation concerning legal food colours worldwide, possible link of artificial colours to hyperactive behaviour, replacement of synthetic colours with natural ones, and the presence of harmful illegal dyes—both known but also new, emerging ones in food. To detect these kinds of synthetic colour, a method has been developed by using High Performance Liquid Chromatograph coupled with UV detector.

 

PRINCIPLE

The colouring matter in food can be classified into two categories namely water soluble and fat soluble. There are altogether 8 permitted coal-tar food colours which can be used in certain food products under the provisions of Prevention of Food Adulteration Act, 1954. They include 3 red shades, namely Carmoisine, Ponceau 4R, Erythrosine, 2 yellow shades namely Tartrazine, Sunset Yellow FCF, 2 blue shades i.e. Brilliant Blue FCF, Indigo Carmine and 1 green shade i.e. Fast Green FCF.

The general scheme for identifying synthetic food colour in foods normally involves extraction of the colour from the prepared solutions of the food, separation of colours and identification and quantification of the separated colour.

In present study, a High Performance Liquid chromatographic method was developed for analysis of 7 water soluble synthetic food dyes namely Carmoisine, Ponceau 4R, Erythrosine, Tartrazine, Sunset Yellow FCF, Brilliant Blue FCF and Indigo Carmine in food products like sugar boiled confectionary, Jelly, Jams and fruit juices.

 

MATERIAL & METHODS

Standard preparation

Stock solution of about 1000 mg/L of each reference standard in water was prepared individually. The standards were labelled properly and stored at 2-8°C. From these standards intermediate and working mixture standards were prepared by serial dilution. The mixture standards were used for method validation.

Preparation of mobile phase

Mobile phase A (10 mM Ammonium acetate buffer): 0.7708 gm of ammonium acetate was taken and finally volume made up to 1000mL with double distilled water. The solution was then filtered through 0.45 µm membrane filter. Furthermore the solution was sonicated for 15 minutes in an ultrasonic bath.

Mobile phase B: HPLC grade methanol was used as Mobile phase B.

Preparation of calibration curve from mixtures of standard

A mixture stock solution (concentration of 100 mg/L) of 7 water soluble colours was first prepared. Then the following dilutions (Table-1) ranging from 1 mg/L to 20mg/L was prepared with double distilled water as diluent.

Table-1. Details for preparation of Working mixture standards

Stock Conc.

(mg/L)

Volume of Stock

(mL)

Volume of Diluent

(mL)

Final Volume

(mL)

Final Conc.

(mg/L)

Name
10 0.100 0.900 1 1 CC-1
10 0.200 0.800 1 2 CC-2
10 0.500 0.500 1 5 CC-3
100 0.100 0.900 1 10 CC-4
100 0.200 0.800 1 20 CC-5

 

Sample preparation

About 5 gm of sample was taken in a 50 mL centrifuge tube and 10 mL of water was added to it. The tube was then degassed in an ultrasonic bath for 15 minutes. After that volume of the solution was made up to 25 mL. The mixture was filtered through 0.45 µm membrane filter and aqueous extract was injected into HPLC system for determination.

 

Instrumental Condition

LC system: Shimadzu LC-2030
Column: Shim-pack GWS C-18, 5 µm, 250X4.6 mm
Injection Volume: 10 µL
Absorbance wavelength: 420nm, 510nm
Flow rate: 1mL/minute
Column temperature: 35 ºC

Gradient Programming:

Time (minutes) % of A % of B
0.01 90 10
2.00 90 10
4.00 70 30
6.00 50 50
8.00 30 70
10.00 10 90
12.00 30 70
14.00 50 50
16.00 90 10

 

RESULTS AND DISCUSSION

All the 7 food colours viz. Carmoisine, Ponceau 4R, Erythrosine, Tartrazine, Sunset Yellow FCF, Brilliant Blue FCF and Indigo Carmine were validated in various matrices like sugar boiled confectionary, Jelly, Jams and fruit juices at two different spiking levels. Various Performance characteristics like Specificity, Limit of Detection, Linearity, Matrix Effect, Accuracy, Precision, Limit of detection, Limit of Quantification and Robustness were assessed. The method was found to be specific for the analytes and matrix combinations. The response of the detector was found to be linear within 1-20 mg/L (Figures. 1-7). Spiked recoveries were performed by two analysts on two different days at two different concentration levels to evaluate the accuracy and precision of the method. A typical chromatogram of Reference Standard mixture, Chromatogram of Blank Control Sample and Control Sample fortified with Reference Standards are presented in Figure. 8, 9 and 10. At LOQ level (2 mg/Kg) the mean recoveries obtained were between 93.92 – 101.59 % with relative standard deviation ranging between 1.65 – 10.35 % whereas at the higher fortification level (10 mg/kg) the recovery ranged between a 90.81 – 102.90 % with relative standard deviation of  up to 3.21%.  LOQ was fixed at the level of 2 mg/kg.

CONCLUSIONS

The analytical method described in this study was optimised for determination of 7 water soluble synthetic food dyes namely Carmoisine, Ponceau 4R, Erythrosine, Tartrazine, Sunset Yellow FCF, Brilliant Blue FCF and Indigo Carmine in food products like sugar boiled confectionary, Jelly, Jams and fruit juices by HPLC. The UV detection and quantification after the separation of the colours through HPLC method provides the specificity of the analysis. This method can be used to determine the analytes in food products as mentioned above at extremely low concentration (~ 1.6 mg/Kg). The Limit of Quantification of the method is found to fall within the range of 1.61 mg/Kg to 9.72 mg/Kg. The calibration curve was found to be linear. This was apparent from the square of the correlation coefficient (r2) value which ranged between 0.9996 to 0.9999. All the method validation parameters were within acceptable limits. This method found to be cheap, easy, effective and quite rugged for the analytes and matrix combination. In conclusion the method was found to be fit for its intended purpose.

Figure 1 CC for Indigo carmine
Figure 1: CC for Indigo carmine
Figure 2 CC for Ponceau 4R
Figure 2: CC for Ponceau 4R

Figure 3: CC for Sunset yellow

Figure 4: CC for Carmoisine

Figure 5: CC for Erythrosine

Figure 6: CC for Tartrazine
Figure 7 CC for Brilliant blue
Figure 7: CC for Brilliant blue

Figure 8 Chromatogram of Reference Standard mixture
Figure 8: Chromatogram of Reference Standard mixture

Figure 9 Chromatogram of Blank Control Sample
Figure 9: Chromatogram of Blank Control Sample

Figure 10 Chromatogram of Control Sample fortified with Reference Standards
Figure 10: Chromatogram of Control Sample fortified with Reference Standards

Conducted by  Rituparna Mukherjee.

Leave a Reply

Your email address will not be published. Required fields are marked *