Malachite Green and Leucomalachite Green Analysis
Malachite green (MG) is an effective and inexpensive fungicide with a long history of use, mainly in Asian countries. There is concern about illegal use of MG because of potential harm to humans. MG entering water cycles is easily absorbed by fish tissue and is reduced to leucomalachite green (LMG) during metabolism. LMG accumulates in, and is stored in, fat tissue. Based on European Commission decision 2002/657/EC, a test method must be able to detect 2 micrograms of MG + LMG per kilogram of fish muscle.
Law amendment to prohibit use of malachite green, news.gov.hk, August 19, 2005
General Chromatographic Methodology
Often reversed phase HPLC, typically on an octadecylsilane column with a guard column with the same stationary phase, is used to analyze for MG and LMG. Mobile phase composition is an organic and aqueous buffer mixture, often close to a 50:50 ratio, with acetonitrile being most common organic component. The aqueous buffer is adjusted to approximately pH 4.0 to 4.5.
The column typically is 100 mm or longer. The diameter is dictated by the detector: spectrometric instruments with large detector cells require larger diameter columns (4.6 mm) while spectrometric instruments with small detector cells, or with a mass spectrometer, a smaller diameter column is recommended, (2.1 mm or less).
Several detection methods can be employed, including UV-visible, fluorescence spectrophotometry, and mass spectrometry. MG is active at 620 nm and LMG at 265 nm, making it difficult to observe both in one spectrophotometric method. This can be circumvented by utilizing an oxidizing post column (e.g. lead oxide), in addition to the HPLC analytical column, to convert LMG to MG.
LMG converted to MG can be distinguished from MG by the different retention times. Alternatively a fluorescence detector can be added to the eluent flow path, to detect LMG by excitation at 265 nm and emission at 360nm. Mass spectrometry allows detection of both compounds without oxidation or use of two detectors.