Abstract:

A UPLC-ESI-MS/MS method was established for rapid determination of malachite green (MG) and its metabolite
leucomalachite green (LMG) in aquatic fingerlings with quantification by isotope-labeled internal standard method. UPLC
and MS/MS analytical conditions were examined and optimized by a series of experiments. Samples were extracted with
acetonitrile, purified on a neutral Al2O3 solid phase extraction cartridge. All eluates were collected and blown to dryness under a
nitrogen stream. The residue was re-dissolved in acetonitrile and ammonium acetate (1+1), sonficated and filtered prior to UPLC
analysis. The mobile phase was composed of A (acetonitrile) and B (5 mmol/L of ammonium acetate containing 0.1% formic
acid) at a flow rate of 0.25 mL/min. The analytes were finally detected and quantified using a triple Quadrapole MS/MS system
in positive polarity multiple reaction mode. Malachite green and leucomalachite green showed good linearity over the range of
0.5 to 20.0 ng/ mL with correlation coefficient of 0.9991 and 0.9999, respectively. The limits of detection of this method were
0.03 μg/kg for malachite green and leucomalachite green, and limits of quantification were 0.10 μg/kg. The average recoveries
at three spiked levels (0.10, 1.0 μg/kg and 2.0 μg/kg) ranged from 84.3% to 100.7% with relative standard deviations from
4.12% to 9.18%. In addition, the method showed advantages of simplicity, sensitivity and accuracy, and could be applied for
simultaneous determination of malachite green and leucomalachite green in aquatic fingerlings.

Key words: aquatic fingerlings, malachite green, leucomalachite green, ultra performance liquid chromatography tandem mass spectrometry, metabolite