On World Chocolate Day: Analyzing Disodium EDTA in Chocolate by HPLC
Introduction
Chocolate is one of the most widely consumed sweet confections on Earth, enjoyed across diverse demographics and cultures for its unique palatability and sensory characteristics. From premium dark chocolate to milk chocolate and filled confectionery, its global popularity continues to grow alongside increasing consumer expectations for product quality, safety, and regulatory compliance.
Characterizing the specific constituent content in chocolate is of paramount importance due to the inherent complexity of the food matrix. Chocolate is a dense, multi-phase system comprised of lipids, carbohydrates, polyphenols, and proteins, which severely interfere with direct analytical measurements.
In this article, we present an HPLC method to determine Disodium EDTA (Na2EDTA) in chocolate. Disodium EDTA is an additive used as a chelating agent to sequester trace metal ions, thereby preventing lipid oxidation and extending shelf life. Because excessive intake of EDTA can disrupt essential mineral absorption in humans, the maximum permissible limits for its inclusion in food products is strictly controlled by regulatory bodies.
Why HPLC?
Quantifying trace levels of disodium EDTA within the highly interfering, fat-rich matrix of chocolate is significantly challenging due to the interference of chocolate's content with the analysis. To overcome this challenge, standard methods would require solid-phase extraction (SPE) to help with removing matrix interferences and enriching the target analyte.
However, in the practice, after the samples passed through Welchrom P-SAX SPE cartridge, the target analyte was not adsorbed, limiting the recovery rate. Therefore, we used trichloromethane (CHCl3) to extract instead, followed by HPLC to analyze the extracted sample directly.
The Method
Pretreatment
Weigh 5 ± 0.01 g of the chocolate sample, place in a 50 mL centrifuge tube, add 25 mL water, vortex to mix, sonicate for 20 minutes, and centrifuge at 7500 r/min for 5 minutes. Transfer the supernatant to another 50 mL centrifuge tube, and repeat the extraction with the remaining residue. Combine both supernatants, and dilute to 50 mL by water. Pipette 10 mL, add 5 mL of CHCl3, mix well, sonicate for 20 minutes, and centrifuge at 7500 r/min for 5 minutes. Transfer 5 mL of the supernatant, add 0.5 mL of iron trichloride (FeCl3), sonicate for 20 minutes, and filter through membrane.
Chromatographic Conditions
- Column: Ultisil Plus C18 (4.6×250 mm, 5 µm)
- Mobile Phase: Methanol / TBAB-CH3COONa = 15 / 85
- Column Temperature: 35 °C
- Flow Rate: 0.8 mL/min
- Injection Volume: 10 µL
- Mobile Phase Preparation:
- TBAB-CH3COONa: Weigh 6.45 g of tetrabutylammonium bromide (TBAB) and 2.46 g of sodium acetate, add 1000 mL of water, and sonicate to dissolve. Then adjust the pH to 4.0 by phosphoric acid.
- Methanol: HPLC-grade.
Chromatogram and Data
Result: After extraction by trichloromethane and separation by Ultisil Plus C18 column, the recovery rate of the target chocolate sample reaches 88.2%.