(1) Reagents and materials Unless otherwise specified, the reagents used are of analytical grade and the water is deionized water, which meets the requirements of GB/T582-1992 secondary water.
1Methanol: Chromatographically pure 2 acetonitrile: chromatographically pure 3 solid phase extraction column: Sep-pak Alumin B (500mg/6ml)
4N-methylimidazole 5 trifluoroacetic acid 6 ivermectin standard: purity ≥ 95%
7 standard stock solution: Accurately weigh 100mg of ivermectin standard, dissolve it in 100ml volumetric flask with acetonitrile, make 1mg/m1 standard stock solution, save it in a 20~C refrigerator, valid for 3 months.
8 standard working solution: The standard stock solution of lmg/ml is diluted with acetonitrile into a series of standard working solutions.
9N-methylimidazole-acetonitrile solution: N-methylimidazole-acetonitrile (1:1).
10 Trifluoroacetic anhydride - acetonitrile solution: trifluoroacetic anhydride - acetonitrile (1; 2).
(2) Determination step 1 accurately extract the tissue sample homogenate (10000r / min homogenate 1min) (2.00 + 0.05) g, placed in a 50ml polypropylene centrifuge tube, add acetonitrile 6.0ml, vortex Spinning for 1 min, centrifugation at 7000 r/min for 5 min, the supernatant is the sample extract.
2 Purification After pre-washing the solid phase extraction column with 4 ml of acetonitrile, the sample extract was taken through the column, naturally flowed out, collected in a l0 ml stoppered test tube, and then washed with a solid phase extraction column of 3 ml of acetonitrile, and collected in a l0 ml stoppered test tube. .
3 Fluorescence Derivatization The solution in the test tube was blown dry with nitrogen at 500 c, and the remaining trace moisture was baked in a 500 c oven for 15-20 min. Add N-methylimidazole-acetonitrile solution 100u1 to the test tube, vortex for 30s, then add 150uL of trifluoroacetic anhydride-acetonitrile solution. Immediately appear white mist in the test tube and release heat. Immediately plug the test tube and gently shake it for 30s. After adding 750 uL of methanol, after standing for 15 min, the solution was transferred to a 1 ml injection bottle for analysis.
4 Standard curve drawing accurately transfer a certain concentration of ivermectin standard working solution, according to the above method for fluorescence derivatization, on the machine.
5 determination
a. Chromatographic conditions column: C18 column mobile phase: methanol: water = 92; 8
Column temperature: 25~C
Flow rate: 1.0ml/min
Injection volume: 50ul.
Excitation wavelength: 365nm
Emission wavelength: 475nm
b. Chromatographic determination Take an appropriate amount of sample solution and the corresponding standard working solution, do single or multi-point calibration, quantify the integral value of the chromatographic peak area. The response of ivermectin in the standard working fluid and the sample solution should be within the linear range of the instrument detection.
6 The blank test was carried out in accordance with the above measurement procedure except that no sample was added.
(3) Sensitivity and recovery rate The detection limit of ivermectin in muscle, liver, kidney and adipose tissue of pigs and chickens is lug/kg.
At a concentration level of 10 to 50 ug/kg, the recovery range is 70% to 90%.
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