SIMULTANEOUS DETERMINATION OF MALACHITE GREEN AND ITS METABOLITE LEUCOMALACHITE GREEN IN EEL PLASMA USING POSTCOLUMN OXIDATION

A rapid HPLC method with solid-phase extraction (SPE) clean-up for malachite green (MG) and leucomalachite green (LMG) in eel plasma was developed. MG and LMG were extracted with a buffered methanolic solution. The extract was subjected to aromatic sulphonic acid SPE. MG and LMG were eluted from the SPE column with methanol after a treatment with ammonia gas. The reconstituted eluate was analyzed on a Chromspher B column with acetonitrile-ion-pair buffer (pH 4.0) (6:4, v/v) as the mobile phase and detection at 610 nm after post-column oxidation with PbO2. The average recoveries for MG and LMG over the linear range of applicability (20-2500 ng/ml) were 82 +/- 1% and 83 +/- 1%, respectively. The limits of quantification were 5.0 mu g/l for MG and 0.9 mu g/l for LMG.