Determination of malachite green and leucomalachite green in carp muscle by liquid chromatography with visible and fluorescence detection.

A liquid chromatography-VIS/FLD method for the analysis of malachite green (MG) and its major metabolite, leucomalachite green (LMG) in carp muscle has been described. The method consists in an extraction with acetonitrile-buffer mixture followed by partioning with dichloromethane. Clean up and isolation were performed on SCX solid phase extraction (SPE) column. Chromatographic separation was achieved by using phenyl-hexyl column with an isocratic mobile phase consisting of acetonitrile and acetate buffer (0.05 M, pH 4.5) (60:40, v/v). Liquid chromatography with absorbance detector (lambda = 620 nm) was used for the determination of MG while LMG was detected by fluorescence detector (lambda(ex) = 265 nm and lambda(em) = 360 nm). The both detectors were connected on-line which allowed direct analysis of a sample extract for MG and LMG without the need for any post-column procedure. The whole method has been validated, according to the EU requirements (Commission Decision 2002/657/EC). Specificity, stability, decision limit (CCalpha), detection capability (CCbeta), accuracy and precision were determined. Average recoveries of MG and LMG from muscle fortified at three levels (0.5, 1 and 2 microg/kg) were 62% (range from 60.4 to 63.5%) and 90% (range from 89.0 to 91.5%), respectively. Relative standard deviations (RSD) of recoveries at all fortification levels were less than 10.9 and 8.6% for MG and LMG, respectively. The calculated CCalpha for MG and LMG were 0.15 and 0.13 microg/kg, and CCbeta were 0.37 and 0.32 microg/kg, complying with the minimum required performance limit (MRPL) of 2 microg/kg (sum of MG and LMG).