| Recent studies have shown that malachite green (MG) and crystal violet (CV) are potential carcinogen, teratogen and mutagen. Therefore, MG and CV are highly restricted or not permitted for use as an aquaculture veterinary drug in many countries and areas including the European Union, United States, Canada, China and Japan, etc., due to its potential negative impacts on the environment and public health. However, MG and CV are still being used worldwide illegally in fish farming and agriculture because of its low cost, ready availability and high efficiency in delivering resistance to important protozoan and fungal infections. News has it that malachite green residue was detected in aquatic products exported from China. Not only was it a quite attack for China’s export industry, it also reflects that the environment, especially water and soil, is suffering or is going to suffer from the serious problem of MG and CV pollution. For sticking to the policy of Sustainable Economic Development, China has got to control the illegal use of MG and CV and develop reliable methods for detection. High performance liquid chromatography (HPLC), HPLC coupled with mass spectrometry (MS) and enzyme-linked immunosorbent assays are broadly used to determine MG and CV. However, the pretreatment of the sample and operation of the instruments are not convenient for in-situ detection nor is it economically efficient. Thus, it is urgent that sensitive, rapid and affordable methods available to be used for the detection of MG and CV. Highly sensitive, luminol chemiluminescence (CL)-based analytical techniques have received considerable attention from researchers in recent years due to the advantages of low detection limits, short analysis time, simplicity in measurement, and availability of large calibration ranges.In this study, the influences of MG and CV on the CL of the "Na2CO3-luminol-H2O2" system were described separately. Single factor experiments and orthogonal experiments were designed for optimization of the best reaction conditions. Calibration curves for MG and CV measured by the CL method were drawn under the optimal experimental conditions. Methods based on microplate and "Na2CO3-luminol-H2O2" for detection of MG and CV were established separately, which were then used in 4 spiked water samples. Molecularly imprinted polymers (MIPs) for MG and CV (MG-MIPs and CV-MIPs) were synthesized, then characterized by IR spectrum, SEM, isothermal adsorption experiments and Scatchard analysis and finally used as stuffing for solid phase extraction (MISPE) for pretreatment of spiked soil samples. Detection of residues in pretreated samples was conducted via the established method mentioned above.A rapid simple and high-throughput chemiluminescence method for the determination of MG and CV has been successfully established, which was based on the use of microplate and the phenomenon that MG and CV greatly inhibited the CL signal of the "Na2CO3-luminol-H2O2" system. For MG, the decrement of the CL intensity was found to be proportional to the concentration of MG, and the calibration graph was linear in the range of 2 ng·mL-1 to 800 ng·mL-1 with a detection limit of 0.76 ng·mL-1 (3σ) and a regression coefficient of R2=0.9954. The recovery ratios for the 4 MG-spiked water samples were 90.4%-112.1% with R.S.D.2.7%～6.1%. For CV, the decrement of the CL intensity was found to be proportional to the concentration of CV, and the calibration graph was linear in the range of 2 ng·mL-1 to 800 ng·mL-1 with a detection limit of 0.59 ng·mL-1 (3a) and a regression coefficient of R2=0.9939. The recovery ratios for the 4 CV-spiked water samples were 88.8%～106.2% with R.S.D.3.5%-10.9%. The method has been applied to the determination of MG/CV in soil samples with pretreatment by MISPE, the recovery ratios for MG increased from 43.1%-57.9% to 71.5%-83.3% and the recovery ratios for CV increased from 44.1%-62.5% to 62.1%-84.0%, which indicated that MISPE was effective for the pretreatment of complex samples.In conclusion, detection methods for MG and CV based on the inhibition of CL of the "Na2CO3-luminol-H2O2" system have been established. Coupled with the usage of microplate, it becomes easy to operate parallel detection of single sample and multiple samples detection at one time. With the advantages of high degree of automation and simplified procedure, this method has helped to build a foundation for realizing of real-time monitoring in environmental protection. |
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