Triphenylmethane dyes (TPMs) are synthetic dyes used for a wide range of industrial applications. They are also used in aquaculture production for their anti-bacterial, anti-fungal, and anti-parasitic properties. However, because of their toxicity to humans TPMs are banned in the US and EU. As such, the FDA and EU Commission have established very low detection limits for these compounds in seafood products (1 μg/kg [US] and 2 μg/kg [EU]). As the parent dyes are rapidly metabolized to form leuco- metabolites that predominate and persist in fish tissue, detection limits are expressed as the sum of the parent drug and corresponding leuco-form.
TPMs are difficult compounds to analyze. This application note describes a simple SPE method that uses triethylammonium formate as the elution solvent (methanol containing triethylamine [TEA] and formic acid). The positively-charged TEA acts as a counter-ion to release the dyes from the ion-exchange sorbent. No evaporation step is included, which avoids any potential loss during this step. The eluted extracts can be analyzed directly by LC–MS–MS as TEA is MS-compatible and has no ion-pairing effect on the dyes. Ascorbic acid is included as an antioxidant. This simple method allows for the rapid analysis of nine dyes in seafood while achieving good accuracy, precision, and low sensitivity.
1. Sample Extraction: Weigh 2 g of sample into a 15 mL polypropylene centrifuge tube. Add 10 mL of 1% formic acid in acetonitrile and 1 mL of 1 M ascorbic acid. Shake for 15 min to extract. Centrifuge the samples for 10 min at ≥3000 rcf and 4 °C. Transfer the supernatant to a 50 mL polypropylene centrifuge tube and add 20 mL of McIlvaine's buffer (0.1 M, pH 3.5)*. Vortex the samples briefly (1 min) to mix and centrifuge for 5 min at ≥3000 rcf and 4 °C.
2. Condition SPE Cartridge: Add 3 mL methanol to the CSDAU206 cartridge. Add 3 mL of ultrapure water. Add 1 mL of McIlvaine's buffer (0.1 M, pH 3.5).
3. SPE Extraction: Load supernatant from step 1. Adjust vacuum for a flow of 1–3 mL per min.
4. Wash Cartridge: Add 3 mL of 0.1% formic acid and slowly draw through. Add 3 mL of 0.1% formic acid in MeOH and slowly draw through. Dry under vacuum for 1 min to remove excess solvent.
5. Elute Cartridge: Elute the dyes from the SPE cartridge using 3 mL elution solvent (1% TEA + 0.5% formic acid in MeOH). Vortex the samples for 2 min and transfer a 1 mL aliquot to an autosampler vial for analysis.
*0.1 M McIlvaines buffer pH 3.5 — mix 500 mL of 0.1 M citric acid with 400 mL 0.1 M disodium hydrogen phosphate. Adjust pH to the correct value (3.5) using the citrate or phosphate solution.
**Additional experimental parameters are available upon request or on the UCT website.
Table 1: Accuracy and Precision Data for Nine Dyes Spiked in Salmon Tissue.
This simple, fast, and cost-effective method allows for the rapid analysis of dyes in seafood by LC–MS–MS. The method achieves good accuracy and precision and low sensitivity with an average recovery of 91% and average relative standard deviation (RSD) of 4.3% at 1 ppb spike levels.
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