Melamine is an important triazine nitrogen-containing heterocycle organic chemical raw material, mainly used for the production of melamine-formaldehyde resin, widely used in wood processing, plastics, coatings, paper, textiles, leather and other industries, is a white crystal . The Sanlu milk powder incident has caused widespread concern over the detection of melamine. The General Administration of Quality Supervision, Inspection and Quarantine and the National Standardization Administration have successively issued the “Guidelines for the Detection of Melamine in Raw Milk and Dairy Products†(GB/T22388-2008) and “Ingredients in Milk The national standard for rapid detection of melamine by liquid chromatography (GB/T22400-2008). This article refers to the above criteria and optimizes the preparation conditions. A liquid chromatographic method is established. The pretreatment is simple. The detection limit, precision, reproducibility, and recovery rate are all in line with national standards.
1 Experimental section
1.1 Instruments and Reagents
High Performance Liquid Chromatography (includes UV1620 UV-Vis Detector 1; P1620 High Pressure Constant Flow Pump 1; AT-330 Column Thermostat; FA2004 Analytical Balance; TGL-16G-A Centrifuge; Melamine Standards >99% Sodium octane sulfonate (chromatographically pure); phosphoric acid (analytically pure), acetonitrile (chromatographically pure), purified water.
1.2 Standard solution preparation
The melamine standard was accurately weighed, dissolved in 50% aqueous methanol, and a standard stock solution of 1.422 mg/ml was prepared and stored at 4°C in the dark. According to the experimental requirements, the mobile phase is gradually diluted to the appropriate concentration of the standard working solution.
1.3 Sample Preparation
Weigh 2g of yoghurt sample and 50ml with a centrifuge tube, add acetonitrile: water = 50:50 mixed solution 15ml, fully mixed and ultrasonic extraction 15min. Take 250ul extract, add 0.1mol/l hydrochloric acid 750ul, mix, centrifuge at 12000r/min for 5min, take the supernatant, 0.22um filter membrane, as HPLC determination solution.
1.4 chromatographic conditions:
Column: Globalsil C18 5μm (ID4.6mm×250mm)
Mobile phase: acetonitrile/buffer salt=15/85 (buffer salt: 10 mM sodium octane sulfonate solution containing 0.1% phosphoric acid)
Flow rate: 1.0ml/min
Wavelength: 240nm
Temperature: 40°C
Injection volume: 20μl
2 experimental results
2.1 Precision Experiment
Take the concentration of 0.569μg/ml melamine standard working solution, according to the above chromatographic conditions, continuous injection 5 times, calculate the RSD (%) with the peak area of ​​each component, the results are shown in Table 1: Relative standard deviation of retention time (RSD) It is 0.23% and the peak area RSD is 0.57%.
Table 1 Precision Experiment
NO.
Retention time(min)
Peak High
Peak Area
1
11.933
289
4216.8
2
11.990
290
4264.2
3
11.928
287
4246.0
4
11.927
287
4242.8
5
11.923
284
4218.6
RSD(%)
0.23
0.82
0.57
Figure 1 Melamine Standards
2.2 Standard curve
The melamine standard working solutions with the concentrations of 0.142, 0.284, 0.853, 1.422, 7.110, and 14.220 μg/mL were respectively prepared. The above six standard working solutions were injected from low to high concentrations under the above chromatographic conditions. The standard curve was obtained by linear regression of the peak area (y-axis of the ordinate) with the reference substance concentration (abscissa X-axis, μg/ml) using the least squares method.
2.3 Sample Recovery and Reproducibility Experiments
Take yoghurt sample with known melamine content, add a certain amount of standard stock solution, and follow the “sample pretreatment†operation. Prepare 3 parts in parallel and obtain the sample solution and then analyze according to the above chromatographic conditions to obtain sample recovery rate and reproduction. Sexual results, see Table 2.
Table 2 Sample Recovery and Reproducibility (n=3)
Spiked(mg/kg)
Recovery(%)
RSD(%)
35.0
87.2
1.37
Figure 2 blank yogurt sample
Figure 3 Supplemented yogurt sample
2.4 Limit of detection and limit of quantification
According to the noise value, according to 3 times the signal to noise ratio, calculate the theoretical detection limit of 0.221 mg/kg; according to 10 times the signal to noise ratio, calculate the limit of quantification, it is 0.738 mg/kg.
3 Conclusion
This method optimizes the HPLC method for the determination of melamine in dairy products. This method simplifies the pretreatment steps, is easy to operate, has a good linear relationship, has good precision and reproducibility, and the recovery rate meets the requirements of national standards. , Detection limit and limit of quantification also meet the current melamine detection requirements.