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.
SPE Procedure1. 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.
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.