Method for Detecting Malachite Green in Feed by HPLC
1. High performance liquid chromatography
High performance liquid chromatography for the detection of chloramphenicol is a sensitive method with high reliability. This method has good repeatability and few false positives, which can be quantitatively identified, but the detection limit is high, which is 5~. 10 μg/kg, the recovery rate is low. Because the composition of the animal is complex, some impurities interfere with the determination. When applying this method, careful precautions must be taken for sample pretreatment to improve the accuracy and sensitivity of the measurement.
2. Gas chromatography
At present, the more commonly used method for confirming chloramphenicol is gas chromatography. The method is characterized by high separation efficiency, high selectivity, high sensitivity, low detection limit, rapidity, wide application, and can also be used for preparing high purity materials. It separates components with very close partition coefficients, separating out extremely complex mixtures. The detection limit is generally in the order of μg/kg, which is often used for the detection of antibiotic drug residues. However, most veterinary drugs have a high polarity or boiling point, which requires cumbersome derivatization steps, thus limiting the application of gas chromatography.
The detection of chloramphenicol in feed is currently carried out by gas chromatography. The method first extracts the compound feed with ethyl acetate, and extracts a part of the extract with nitrogen to remove the ethyl acetate. The residue is dissolved in a methanol/sodium chloride solution, and the oil and fat are removed by liquid-liquid extraction with n-hexane. The pyridine was extracted with ethyl acetate, dried, and purified by a C18 cartridge. After derivatization with a derivatizing agent, it was detected by gas chromatography equipped with an electron capture detector. The gas chromatographic conditions are as follows.
Carrier gas: nitrogen 1.5 ml / min; make-up gas: nitrogen 25 ml / min; split ratio: 1:10. Temperature: 280 °C in the gasification chamber; 300 °C in the detector; 230 °C in the oven.
Under the above-mentioned instrument conditions, inject 1 μl of the standard working solution, adjust the volume of the sample to make the peak of chloramphenicol in the sample solution close to the peak of chloramphenicol in the standard working solution, and compare the retention time and peak value to the chloramphenicol The quantitative calculation is performed. Repeatability: The relative deviation of the results of two parallel measurements shall not exceed 5%. Recovery rate: When the dosage is 0.01 mg/kg, the recovery rate is 90%~105%. The detection limit of this method is 0.005 mg/kg.
In general, immunological assays based on antigen-antibody-specific reactions have high sensitivity, specificity, simple sample pretreatment, and short analysis time. In recent years, enzyme-linked immunoassay has been greatly developed, the false positive rate is significantly reduced, and it is widely adapted. At present, enzyme-linked immunosorbent assay is used in meat, fish, shrimp, egg, urine, serum, milk, honey and feed to screen for chloramphenicol detection.
The internationally recognized quantitative confirmation method is still the physical and chemical analysis method, mainly gas chromatography and liquid chromatography. These two methods have high sensitivity, accurate results, good repeatability and few false positives. The disadvantage is that the sample preparation process is complicated. The instrumentation is high and expensive, and the analysis speed is slow. Compared with the two, due to the high detection limit of HPLC, it has gradually failed to meet the requirements of the detection limit of detection methods in developed countries. After the emergence of the combined technology, LC-MS has a higher sensitivity, lower detection limit, and accurate and reliable results. It is gradually replacing a single chromatographic technique in the analysis of chloramphenicol residues.