The contamination of melamine in food is a hot topic recently. Since melamine was detected in pet feed exported to the United States last year, dozens of brands of dairy products in China have now been contaminated with melamine. All of this caused people to worry about food safety, and it also triggered a wave of development and research on melamine detection methods.
Melamine is an important nitrogen heterocyclic organic chemical raw material. The aqueous solution is weakly alkaline, soluble in methanol, acetic acid, etc., slightly soluble in water and ethanol, insoluble in ether, benzene, etc., and poorly retained on traditional C18 columns. . Using this feature, people generally use ion-pair reagent liquid chromatography and liquid-mass spectrometry to detect. However, due to the complexity of the food matrix, sample preparation is critical. We use trichloroacetic acid extraction, MCX solid phase extraction small column purification, supplemented by J2 pressurized solid phase extraction instrument operation, to establish a set of fast, accurate, and reproducible sample external processing methods.
1. Materials and Methods
1.1 Main instruments and reagents: Agilent HP1100 high-performance liquid chromatograph, equipped with quaternary gradient pump, online vacuum degasser, automatic sampler, column thermostat, diode array detector, J2SCIENTIFIC (Green Cotton Giant Trade) company pressurization Solid phase extraction instrument, ultrasonic cleaner, centrifuge, solid phase extraction cartridge: OASISMCX, 6mL, 500mg. Melamine standard product, purity 99%.
1.2 Sample processing
1.2.1 Extraction Accurately weigh 5.0g of the sample after crushing and mixing, add 50mL of 1% trichloroacetic acid solution and 2mL of 2% lead acetate solution, mix and ultrasonically extract for 30min, and take the supernatant for purification after high speed centrifugation.
1.2.2 Purification Place the MCX solid phase extraction cartridge (6 mL, 500 mg) on ​​the sample holder of the J2 solid phase extraction instrument, add 5 mL of methanol and 5 mL of water respectively to activate, and then accurately transfer 5.0 mL of centrifugal fluid to the column. During activation and loading, raise the sample rack to the top and adjust the size of the N2 gas flow by adjusting the panel knob. The flow rate should not exceed 1mL / min. Then wash the SPE column with 5mL water and 5mL methanol again, discard the eluent, raise the column to the top and adjust the air valve knob to automatically dry the column under pressure, and elute with 5mL of 5% ammonia methanol solution, collect the eluent Blow dry with a nitrogen blower, dilute to 1.0mL with methanol-water (2: 8, V / V), pass a 0.45μm filter membrane, and measure on the machine.
1.3 Determination by liquid chromatography: slightly
2. Results and discussion
2.1 Sample extraction and purification Trichloroacetic acid has the function of precipitating protein in milk and its products, and helps to dissolve melamine. Because melamine is weakly alkaline. (Weak cation compounds), using cation exchange column for purification can achieve the effect of removing impurities, improving detection sensitivity, reproducibility and recovery rate. Figure 1 is a chromatogram obtained after direct extraction of a milk powder sample, and there are many impurity peaks. Figure 2 is a chromatogram after extraction and purification by a solid phase extraction cartridge. The interference of impurity peaks is basically removed.
Figure 1: The chromatogram of direct extraction and determination of milk powder samples
Figure 2: Chromatogram of milk powder sample extraction and purification by solid phase extraction column
2.2 Application of J2 Lumian Jumao Company's Pressurized Solid Phase Extractor
J2 Lumian Jumao's pressurized solid phase extraction instrument has the characteristics of high efficiency and simplicity. It can control the flow rate of the small column through the air flow adjustment valve in the device, and can directly dry the small column to ensure that each small column has good reproducibility during the activation and elution process. And can be used with different specifications of solid phase extraction cartridges (such as 1mL, 3mL, 6mL, etc.), one operation can process 48 samples at the same time. We added (2.0-50.0) ug / mL melamine with different concentrations to the milk powder to conduct the spike recovery and reproducibility experiments, and the recovery rate was between 95-99%. The relative standard deviations are all less than 3%.
3. Conclusion:
The pressurization operation of the J2 solid phase extraction instrument is achieved by applying a certain pressure of air or N2 above the solid phase extraction cartridge. This can accelerate the filtration speed, control the flow rate of the entire process of column activation, leaching, and elution, make the solution easy to enter the pores of the stationary phase, and facilitate the closer contact between the sample liquid and the stationary phase, thereby improving the extraction effect. It overcomes the problems of conventional manual solid phase extraction operations that are time-consuming, unable to ensure stable flow rates, and have large deviations in the results of different personnel operations. It eliminates errors in human operations through simple control, ensuring that dozens of samples are processed simultaneously in a short time and has Good reproducibility.
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