Determination and identification, according to European Union Decision 2002/657/EC, of malachite green and its metabolite in fish by liquid chromatography-tandem mass spectrometry using an optimized extraction procedure and three-way calibration.

This paper reports a multiresponse optimization of an extraction procedure in the simultaneous determination of malachite green (MG) and its metabolite (leucomalachite green, LMG) in fish by liquid chromatography with triple quadrupole mass spectrometry (LC-MS/MS). Prior to optimization, the active factors of the extraction procedure were determined by a screening experimental design. Then, in the optimal experimental conditions of the extraction, MG and LMG have been determined by using a three-way calibration model based on parallel factor analysis (PARAFAC). The procedure fulfils the performance requirements for a confirmatory method established by the European Union Decision 2002/657/EC. This norm establishes maximum permitted tolerances for relative abundance of the precursor/product ion pairs. There is a reported contradiction in the literature related to the fact that there are standard samples whose concentration is greater than CCalpha but the maximum permitted tolerances are not fulfilled in the identification of the analytes. In this work, it is shown that with the information provided by PARAFAC this contradiction is avoided. The figures of merit for PARAFAC and univariate calibration procedures were evaluated under optimal conditions in the extraction step. The figures of merit obtained were in the range of 0.13-0.23 microg kg(-1) for the decision limit, CCalpha, (alpha=0.01) and 0.22-0.39 microg kg(-1) for the detection capability, CCbeta, (beta=0.05), whereas mean relative errors in absolute value were in the range of 2.8-4.6% for MG and LMG with PARAFAC calibration. The proposed optimized extraction procedure using a PARAFAC calibration was also applied in the determination of MG and LMG in gilthead bream samples: the decision limit was in the range of 0.45-0.55 microg kg(-1), the detection capability was in the range of 0.76-0.92 microg kg(-1) for MG and LMG. Trueness was likewise confirmed and the mean of the absolute values of relative errors were between 4.2% and 7.2%.