Malachite Green in Fish (HPLC/UV)

This application brief describes how to determine malachite green in fish by HPLC/UV.

Malachite Green (MG) is an effective and inexpensive fungicide used in aquaculture. During metabolism MG reduces to Leucomalachite Green (LMG), which has been shown to accumulate in fatty fish tissues. Both MG and LMG have demonstrated putative carcinogenic activity, and thus, have been banned for use in aquaculture by both the United States Food and Drug Administration (US FDA) and European Union (EU).

Pretreatment

1. Weigh 1 g sample into a 30 mL centrifuge tube.
   
2. Add 50 μL TMPD* solution (1 mg/mL).
   
3. Add standard solutions (MG, LMG, 0.1 μg/mL) and internal standard, leave to stand for 10 minutes.
   
4. Add 10 mL McIlvaines Buffer (pH 2.6) /methanol (50:50, v/v) solution; homogenize for 45 seconds.
   
5. Centrifuge at 5000 rpm for 20 minutes, transfer supernatant into centrifuge tube.
   
6. Repeat steps 4 and 5, combine the two portions of supernatant. A 20 mL aliquot will be used for SPE.

*TMPD= N, N, N’, M’- Tetramethyl-1,4-phenylenediamine dihydrochloride

Solutions

McIlvaines Buffer (pH 2.6) :

1. 0.1 M citric acid monohydrate (A) - Dissolve citric acid monohydrate (10.5 g) in water (500 mL).

2. 0.2 M disodium hydrogen phosphate dihydrate (B) - Dissolve disodium hydrogen phosphate dihydrate (14.2 g) in water (500 mL).

3. Mix A (445.5 mL) and B (54.5 mL).

McIlvaines Buffer (pH 2.6): methanol (50:50 v/v):

Mix McIlvaines Buffer (pH 2.6) (250 mL) with methanol (250 mL).