Trace Detection of Erythrosine B in Sugar

Erythrosine B (EB), also known as Red Dye #3, is a synthetic dye approved for use in candy in the US, and in pharmaceuticals and cosmetics in the EU and elsewhere. However, rodent studies suggest that ingestion of EB can promote thyroid tumor formation. EB may also be implicated as a dietary factor contributing to hyperkinesis in children. WHO recommends a daily intake of EB less than 0.1 mg/kg of body weight. While consumption of EB below this threshold is deemed acceptable, it is important to monitor the use of EB to ensure that dietary guidelines are both appropriate and adequately enforced.

With Misa (Metrohm Instant SERS Analyzer), the sensitive and selective detection of EB is demonstrated in a simple assay format easily adapted for on site surveillance testing. The ability to obtain fast results with a portable test platform recommends Misa as a competitive and cost-effective alternative to laboratory technologies (e.g., CE, HPLC) currently employed for detecting EB in foodstuffs.

Misa is a portable screening tool for detecting food additives, including chemical colorants. In this application note, a facile extraction and analysis procedure is used to detect EB in spiked sugar and colored decorating sugar.

Figure 1. Standard SERS reference spectrum of erythrosine B.

To establish a reference spectrum for EB, a pure standard in water (5 μg/mL) was analyzed using gold nanoparticles (Au NPs). The unique SERS spectrum shown in Figure 1 can be used to create a library entry for EB.

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To simulate testing for EB in a food product, solid EB was mixed thoroughly with pure sugar to prepare a concentration range of test samples: 100, 50, 25, and 10 μg/g. To extract EB, 100 mg of each dry sample was dissolved in 1 mL of ethanol and allowed to sit for 5 minutes. A test sample was prepared by pipetting 100 μL of the ethanolic extract into a glass vial containing 800 μL of Au NPs and 100 μL of 0.5 mol/L NaCl. The sample was shaken and placed into the Misa vial attachment for analysis.

To test for EB in Betty Crocker Pink™, a commercially available colored decorating sugar, samples were prepared as previously described.

**Table 1.** Experimental parameters
Instrument		Acquisition
Firmware	0.9.33	Laser Power	5
Software	Misa Cal V1.0.15	Int. Time	1 s
Misa Vial Attachment	6.07505.040	Averages	10
ID Kit - Au NP	6.07506.440	Raster	ON

Overlaid SERS spectra for ethanolic extracts of sugar spiked with EB demonstrate detection down to 10 μg/g.

Figure 2. Overlaid, baselined, background-subtracted SERS spectra of EB in sugar with Misa and Au NPs.

Based on peak comparison to reference spectra recorded for the spiked sugar samples, the presence of EB in pink sugar is confirmed. Note: Minor peak shifts, as evident in Figure 3, do not compromise library identification capabilities.

Figure 3. Overlaid, baselined, background-subtracted SERS spectra of EB in sugar with Misa and Au NPs.

Detection of erythrosine B in the field

Using the large end of the scoop, add 3–4 scoops of sample to a 2 mL vial. Add ethanol to the vial until halfway full. Cap and shake the vial gently to mix, then let sample rest for 5 minutes. Fill a clean vial halfway full with Au NPs. Using pipettes, add 2 drops each of sample solution and NaCl solution to Au NPs, then cap and shake the vial gently to mix. Insert into vial attachment on Misa for measurement.

**Table 2.** Requirements for field test protocol
ID Kit - Au NP	6.07506.440
includes:	Gold nanoparticles (Au NP)
	Scoop
	Disposable pipettes
	2 mL glass vials
Reagents
Ethanol
NaCl solution	3 g NaCl in 100 mL water
Test settings	Use ID Kit OP on MISA

Misa provides a robust solution for detecting EB in processed foods. This analysis requires minimal user training and consumables, thereby providing a facile analytical methodology for on-site chemical surveillance in both high- and low resource testing environments.

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