Abstract: Sulfaquinoxaline belongs to the class of sulfonamide antibiotics and has anti-coccidial effects. It is widely used in poultry industry. It is rapidly absorbed after oral administration, but it is slowly excreted and remains in tissues and eggs for a long time. Produce side effects such as drug resistance.
The World Health Organization (WHO) stipulates that the maximum residue limit (MRL) of animal tissues and milk is 100 μg / kg. Japan and other countries stipulate that broilers cannot be detected. Therefore, the use of this medicine should strictly control the medication period and dosage to ensure product safety and smooth foreign trade.
According to the British Veterinary Pharmacopoeia, the pure sulfaquinoxaline is a yellow powder, almost odorless, insoluble in water, soluble in methanol and ethanol, and easily soluble in alkaline solutions. At present, the detection method of sulfaquinoxaline in feed is the AOAC standard of the United States, which uses spectrophotometry, which requires two different determination methods for feeds containing and without assanic acid. However, the current domestic feed composition is more complicated, and the tester is unknown about the feed situation. It is difficult to determine which method to use; and this method requires a diazotization reagent to perform the coupling reaction, which is highly toxic and will cause operators and the environment. harm. Therefore, we have not adopted this method. In addition, there are reports in the literature that the determination by gas chromatography still requires diazotization. We ultimately chose high-performance liquid chromatography.
1. Determination method 1.1 Principle Extract sulfaquinoxaline in feed with methanol aqueous solution, centrifuge, filter, separate on HPLC instrument, and measure at 240nm with ultraviolet detector.
1.2 Reagents and solutions The reagents used below, unless otherwise specified, are analytically pure reagents; the water is distilled water, and the chromatographic water conforms to the first-grade water specified in GB / T6682.
1.2.1 Sulfaquinoxaline standard product: containing sulfaquinoxaline (C14H12N4O2S) 95.0%.
1.2.2 Methanol: chromatographically pure.
1.2.3 Phosphate solution: Take 3.40 g of potassium dihydrogen phosphate and 5.71 g of dipotassium hydrogen phosphate, add water to dissolve and dilute to 1000 mL.
1.2.4 Sulfaquinoxaline standard solution: Accurately weigh 50 mg of sulfaquinoxaline (4.1), dissolve in methanol and dilute to a stock solution of 0.1 mg / mL. Store in a refrigerator at 4 ° C, protected from light, and valid for 1 month. Before use, dilute this stock solution with water to a standard working solution of appropriate concentration.
1.2.5 Extraction solution: 100mL of methanol + 50mL of water 1.3 Instruments 1.3.1 Commonly used instruments and equipment in the laboratory 1.3.2 High-performance liquid chromatograph (with UV detector)
1.3.3 Analytical balance: Sensitivity is 0.0001g and 0.001g 1.3.4 Vortex Shaker 1.3.5 Centrifuge: 4000r / min 1.3.6 Syringe filter: prepare pore size of 0.45μm microporous membrane 1.4 Analysis step 1.4. 1 Extract and weigh 5g sample, accurate to 0.001g, add 50mL of extraction solution (4.5), mix with vortex oscillator, extract in ultrasonic water bath for 15min, take out and shake once in the middle, then centrifuge at 4000r / min for 5min, let stand, take The supernatant was passed through a 0.45μm filter membrane for liquid chromatography.
1.4.2 Preparation of standard curve Accurately absorb appropriate amount of stock solution, dilute with water or mobile phase to sulfaquinoxaline standard solutions with concentrations of 0.10, 0.50, 1.00, 2.00, and 10.0 μg / mL to make a standard curve.
1.4.3 Determination 1.4.3.1 Chromatographic conditions Chromatographic column: C18 column length 150mm, column internal diameter 4.6mm, particle size 5μm or equivalent performance.
Mobile phase: phosphate solution 75mL + methanol 25mL, pass 0.45μm filter membrane before use, and degas by ultrasound.
Flow rate: 1mL / min.
Detection wavelength: 240nm.
Injection volume: 10 ～ 20μL.
1.4.3.2 Qualitative and quantitative Qualitative according to the retention time of the standard, quantitative is calibrated by standard curve or single point.
1.4.4 Calculation of results The mass of sulfaquinoxaline in each kilogram of sample is calculated according to the following formula: m1
x = — × D mx——the mass of sulfaquinoxaline in each kilogram of sample (mg); m1——the mass of sulfaquinoxaline corresponding to the peak area of ​​the chromatogram (μg); D——the dilution factor; m—— The mass (g) of the sample.
The results of the parallel determination are expressed as an arithmetic mean value and are retained to one decimal place.
2. Results and discussion 2.1 Determination of chromatographic conditions Most sulfonamides often use phosphoric acid / acetonitrile as the mobile phase, and the peak time of sulfaquinoxaline under this system is 56 min, which affects the detection efficiency. Switch to phosphate / methanol system as mobile phase.
By changing the ratio of phosphate to methanol, the retention time of sulfaquinoxaline changed.
Phosphate: retention time of sulfaquinoxaline (min)
70: 30 4.86 75: 25 7.80 80: 20 12.32 The peak of early sulfaquinoxaline and the peak of impurities interfere with each other. The peak of late affects the detection efficiency and the peak shape becomes wider. A phosphate to methanol ratio of 75:25 is more suitable.
2.2 Method linearity and minimum detection limit For the added concentrations of 5.0, 10.0, 20.0, and 100.0 mg / kg, the above method was used.
Add concentration (mg / kg) 5.0 10.0 20.0 100.0 Peak area 35906 71684 122536 771414 The linear equation is: y = 7821.8 ～ 13601.6 The correlation coefficient is: r = 0.9992 The minimum detection concentration of the method is 5.0mg / kg. The dosage of sulfaquinoxaline compound chicken feed is 60g / t, which can detect concentrations less than 10 times the dosage, which can meet the testing needs.
2.3 The accuracy and precision of the method According to the allowable usage of sulfaquinoxaline in compound feed is 60 mg / kg, the compound feed is taken to make 5, 60, 100 mg / kg sulfaquinoxaline added samples, each Do 5 parallel concentrations, extract, measure on the machine, calculate the recovery rate and method coefficient of variation.
Coefficient of variation of recovery rate of measured value of added amount (mg / kg) (mg / kg) (%) (n = 5)
5 4.5 89.4 6.7 60 55.6 92.6 2.1 100 95.2 95.2 4.6 2.4 Scope of application Take additive pre-mixed feed and concentrated materials to make additional samples, extract, and appropriately dilute and measure on the machine.
Coefficient of variation of recovery rate of measured value of added amount (mg / kg) (mg / kg) (%) (n = 5)
Additive pre-mixed feed 200 190.2 95.1 5.4 Additive pre-mixed feed 1500 1480.6 98.7 2.1 Concentrated material 100 91.5 91.5 6.2 In short, the above experimental examination shows that this method is not only applicable to compound feed, but also applicable to concentrated and additive pre-mixed feed.
3. Conclusion Through the above experimental investigation, the extraction operation of this method is simple and fast. Under this chromatographic condition, the resolution is high and the interference is less. It is suitable for the determination of sulfaquinoxaline in compound feed, concentrate and additive premixed feed. The minimum detection concentration of the method meets the actual supervision and detection requirements, and has good accuracy and precision.
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