National Standard of the People's Republic of China GB/T 18932. 26-2005
Method for determination of residual amount of nitrocellulose, nitroxanide and dimetridin in honey - Liquid chromatography
Method for the determination of metronidazole, ronidazole and
Dimetridazole residues in honey
Determination of the residual amount of metronidazole, lovonicidone, and dimetridin in honey. The liquid chromatography range GB/T 18 9 32 This section specifies the honey in the honey, strontium, strontium, A method for the determination of the residual amount of dimetridin. This section applies to the determination of the residual amount of metronidazole, lovonicidone, and dimetridinium in honey. The detection limits of this part of the method: metronidazole 4, nitrobacteria, and dimethoate. 010 mg / kg.
2 Normative references The following documents contain provisions which, through reference in this Part of GB/D 1 89 32, constitute provisions of this part. For dated references, all subsequent amendments (not including errata content) or revisions do not apply to this section. However, parties to agreements based on this section are encouraged to study whether they can use these documents. version. For undated references, the new version of Zui applies to this section. GB/T 6 37 9 Precision of test methods The repeatability and reproducibility of standard test methods are determined by interlaboratory tests (GB/T 63 79 -19 86, neq ISO 57 25; 1 98 1) GB/T 6 68 2 Analytical laboratory water specifications and test methods (GB/T 66 8 2-19 92, n eq I SO 369 6: 19 87)
3 Principle The nitromeridazole residue in the sample is extracted with ethyl acetate. The extract is evaporated to dryness, dissolved in water, purified by Oas is HLB solid phase extraction column and BAKERBOND Carboxylic Acid solid phase extraction column. Detector measurement, external standard method for quantification.
4 Reagents and materials Unless otherwise indicated, the reagents used were of analytical grade and the water was the first grade water specified in GB/T 6 68 2.
4 .1 Methanol: Chromatographically pure. 42 acetonitrile: pure chromatographic. 4 .3 ​​Acetate Acetate: Chromatographically pure. 4. 4 anhydrous sodium acetate: analytically pure. 4 .5 bing acetic acid: excellent grade pure. 4 ä¹‹é—´çš„ç¢³é…¸é’ é’ , 4。 6 methanol dehydration (5 +95): Measure 5 mL of methanol and 95 mL of water mixed. 4. 7 acetate buffer solution; 0.82 g / L. Weighed 0.82 g of anhydrous sodium acetate, Dissolve in 800 ml of water, adjust the pH with bing acetic acid until
4.3, dilute with water to 1 L, 4.8 Eluent: methanol + acetonitrile + acetate buffer solution (40 + 6 + 54). 4. 9 Oasis HLB solid phase extraction column or equivalent: 5 00 mg, 6 mL. Pre-treat with 5 mL of methanol and 10 mL of water before use to keep the column moist. 4. 1 0 BAKERBOND Carboxylic Acid solid phase extraction column or equivalent: 500 mg, 3 mL . Pre-treat with 5 mL of ethyl acetate before use to keep the column moist.
4.1 1 Metronidazole, lovonicidone, dimethoprim standard substance: purity) 9 8%. 4.1 2 nitrocellulose, lovonicidone, dimethoprim standard stock solution: 1. 0 Mg/mL. Accurately weigh the appropriate amount of standard solution of metronidazole, lovonicidone, and dimethoprim, and prepare a standard stock solution of 1.0 mg/mL with methanol. The stock solution can be stored in a refrigerator at a temperature below 4 ° C for two months. 4.1 3 Mixture standard working solution of metronidazole, naloxine and dimethoprim: Take appropriate amount of standard storage solution of metronidazole, lovonicidone, and dimetridin as needed, and dilute with mobile phase. Mix the standard working solution at the appropriate concentration. The mixed standard working solution should be used now.
5 instruments
5. 1 Liquid chromatograph: equipped with a UV detector. 5. 2 Analytical balance: Sensing. 1 mg and 0.01 g each. 5. 3 Liquid mixer. 5. 4 Oscillator. 5. 4 Solid phase extraction vacuum device 5. 5 Oscillator. 5. 6 stoppered glass centrifuge tube: 50 ml, . 5. 7 Vacuum pump: The vacuum should reach 80 kPa, 5. 8 centrifuge. 5.9 Rotary evaporator. 5. 1 0 sample tube: 5 mL.
5. 11 pH meter: Measurement accuracy ± 0.0 2. 5. 12 Pear bottle: 200 ml
6 Preparation and preservation of samples
3.1 Preparation of the sample For laboratory samples without crystallisation, stir them evenly. For crystallized samples, in a closed condition, warm in a water bath not exceeding 60 °C, oscillate, and after the samples are all melted, stir well and rapidly cool to room temperature. Split. 5 kg as a sample. The prepared sample is placed in a sample vial, sealed, and labeled.
5.2 Sample storage Store the sample at room temperature.
7 Determination steps
7. 1 Extract and weigh 6g sample (accurate to 0.01 g). Place in a 50 MI glass-filled centrifuge tube, add 6 mI of water, mix on a liquid mixer, add 20 ml of acetic acid. Vinegar, shake 20 μ n on a shaker, centrifuge, and take the supernatant into a pear-shaped bottle. Then, it was extracted once with 20 ml of acetic acid, and the supernatant was combined, evaporated to dryness under reduced pressure on a rotary evaporator at 45 ° C, and dissolved in 5 ml of water, to be purified.
7.2 Purification Transfer the above 5 mL aqueous solution to the Oas is HLB column, pass the Oas is solid phase extraction column at a flow rate of 3 mL/min or less, and wash the pear-shaped bottle with 5 mL of water and transfer to the Oas is HLB column. Wash the column, wash the column with 5 mL of methanol + water (4.6), and discard all of the effluent. Under a negative pressure of 65 kPa, drain 20 μ n under reduced pressure, and place the Oa sis HLB column down to the Carboxylic Acid solid phase extraction column, and elute Oa sis with 5 MI acetic acid at a flow rate of 3 mL/min or less. HLB column and passed  Carboxylic Acid solid phase extraction column , discard the Oa sis HLB column, wash the Carb oxylic Acid solid phase extraction column with 5 ml of ethyl acetate and 5 mL of acetonitrile, respectively, and discard all the effluent. At a negative pressure of 65 kPa, drain 5 mM under reduced pressure, elute with 2 m working one eluent ((4.8), collect the eluent in a 5 mL sample tube, and dilute to the eluent to 2 mL, for liquid chromatography. 7.3 Determination 7. 3.1 Liquid chromatographic conditions a) Column: Di a mo ns il C, a , 5 pm, 250 mm X 4.6 mm (inside diameter) or equivalent; b) Mobile phase: acetonitrile + acetate buffer solution (1 0+9 0); c) flow rate: 1.0 mI ./mi n; d) column temperature: 250 C; e) injection volume: 5 0 KL; f) Detection wavelength: 315 nmo
7.3.2 Determination of liquid chromatography by using standard solutions of metronidazole, naloxine, and dimetridinium, respectively, with the peak area as the ordinate and the working solution concentration as the abscissa to draw the standard working curve. The working curve quantifies the sample. The response values ​​of metronidazole, naloxine, and dimetridin in the sample solution should be within the linear range determined by the instrument. See Figure A.1 for chromatograms of standard substances such as metronidazole, naloxine, and dimethoate. Under the above chromatographic conditions, the reference retention times of metronidazole, naloxine, and dimetridin are shown in Table 1. Table 1 Reference retention times of metronidazole, naloxine, and dimetridin Nitroimidate name} retention time / fli. } Two FP M r A qm mw A qE p AV * ut Two cattle method Addition recovery data see Appendix Bo Parallel Test According to the above steps, the same sample is tested in parallel. In the blank test, except for not taking the sample, the blank test was completed at the same time as above.
1 s. o
7.5
The result of the calculation is calculated according to formula (1):
1 0 0 0 1 0 0 0 (1)
V-m
C
One by one X
Where: X- The residual amount of the component to be tested in the sample, in milligrams per kilogram (mg/kg); the concentration of the tested component solution obtained from the standard working curve, in micrograms per milliliter (Rg/mL); The final volume of the sample solution is measured in milliliters (mL); m—the mass of the final sample represented by the sample solution, in grams (9).
Note: The calculation result should be deducted from the blank value.
9 Precision The precision data in this section is determined in accordance with the provisions of GB/T 6 37 9. The values ​​of repeatability and reproducibility are calculated with a reliability of 9 5%. 9.1 Repeatability Under repetitive conditions, the absolute difference between the two independent test results obtained does not exceed the repeatability limit (r), the content of metronidazole, lovonicidone, and dimetridinium in honey. And the repeatability equation is shown in Table 2.
Table 2 contains t range and repeatability and reproducibility equation name content range of eight mg / kg) repeatability limit r reproducibility R
Metronidazole wow 0. 010^-0. 100 r=0. 030 3 m+2. 167 3 R=0.080 7 m+1. 040 8
洛洛哒Wow 0. 010 0. 100 r =0.060 8 m+1. 872 7 R=0.062 0 m10. 719 0
Dimethoxymethane 0. 010^0. 100 - 0.062 7 m+1. 441 1 I g R=O. 709 9 I g m-0. 437 7
Note:. The arithmetic mean of the results of the two measurements.
If the difference exceeds the repeatability limit, the test results should be discarded and the determination of two individual tests repeated.
9. 2 Reproducibility Under reproducibility conditions, the absolute difference between the two independent test results obtained does not exceed the reproducibility limit (R), the content of metronidazole, lovonicidone, and dimetridin in honey. The range and reproducibility equations are shown in Table 20.
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