GB/T 46279-2025Determination of amprolium hydrochloride, ethopabate and sulfaquinoxaline in feeds (English PDF)

4.1 Principle

The analyte in the sample was extracted with methanol solution, defatted with n-hexane, purified by hydrophilic-lipophilic equilibrium solid-phase extraction column, and then separated by high performance liquid chromatography.

The sample was measured using a UV detector in series with a fluorescence detector, and quantified using the external standard method.

4.2 Reagents or Materials

Unless otherwise specified, use only analytical grade reagents.

4.2.1 Water. GB/T 6682, Grade I.

4.2.2 Methanol. chromatographic grade. 4.2.3 n-Hexane.

4.2.4 Extraction solution. a mixture of methanol (4.2.2) and water, V(methanol) V(water) = 4 1.

4.2.5 Phosphate buffer. Weigh 0.2g potassium dihydrogen phosphate and 13.68g dipotassium hydrogen phosphate trihydrate, dissolve in water and dilute to a final volume. Mix 1000mL thoroughly.

4.2.6 Eluent. Transfer 10 mL of methanol (4.2.2), dilute with water and bring the volume to 100 mL, and mix well.

4.2.7 Sodium heptanesulfonate solution (0.005 mol/L). Weigh 1 g of sodium heptanesulfonate into 800 mL of water, stir to dissolve, and add 6 mL of trichloroethylene (TCM) sodium sulfate solution.

Ethylamine and 24 mL of glacial acetic acid were diluted with water to a final volume of 1000 mL and mixed thoroughly.

4.2.8 Reconstituted solution. a mixture of sodium heptanesulfonate solution (4.2.7) and methanol (4.2.2), V(sodium heptanesulfonate solution) V(methanol) = 1 1.

4.2.9 Standard stock solution (0.5 mg/mL). Accurately weigh amprolium hydrochloride (CAS No.. 137-88-2, purity not less than 98.0%), ethoxysulfate...

Methylparaben (CAS No.. 59-06-3, purity not less than 98.0%) and sulfaquinoline (CAS No.. 59-40-5, purity not less than 98.0%).

50 mg of each solution (accurate to 0.01 mg) was placed in separate 100 mL volumetric flasks, dissolved and diluted to volume with methanol (4.2.2), and mixed well. The solution was then stored at -18°C.

Stored at the following conditions, the shelf life of the standard stock solutions of amprolium hydrochloride and ethoxybenzoate is 9 months. The standard stock solution of sulfaquinoline is...

The stock solution has a shelf life of 6 months.

4.2.10 Standard intermediate solution (100 µg/mL). Accurately transfer 10 mL of each of the standard stock solutions (4.2.9) into separate 50 mL volumetric flasks.

Dilute with methanol (4.2.2) and bring to volume, then mix well. Store below -18°C; shelf life is 3 months. Ethoxybenzoate Standard Intermediate The solution was diluted 20 times with methanol (4.2.2) (5 µg/mL) before use.

4.2.11 Mixed Standard Series Solutions. Accurately transfer an appropriate amount of the intermediate standard solution (4.2.10) and prepare ampicillin hydrochloride using the reconstitution solution (4.2.8).

The mass concentrations of porphyrin were 1 µg/mL, 2 µg/mL, 5 µg/mL, 10 µg/mL, 20 µg/mL and 50 µg/mL, and the mass of ethoxyamide benzyl ester was...

The concentrations of sulfaquinoline were 0.05 µg/mL, 0.1 µg/mL, 0.25 µg/mL, 0.5 µg/mL, 1.0 µg/mL, and 2.5 µg/mL.

A series of mixed standard solutions with concentrations of 0.6 µg/mL, 1.2 µg/mL, 3 µg/mL, 6 µg/mL, 12 µg/mL, and 30 µg/mL. (Prepared immediately before use) Prepared on the spot.

4.2.12 Solid phase extraction column. hydrophilic-lipophilic balanced column, 60mg/3mL, or equivalent.

4.2.13 Microporous filter membrane. 0.45µm, organic system.

4.3 Instruments and Equipment

4.3.1 Liquid Chromatograph. Equipped with an ultraviolet detector (or diode array detector) and a fluorescence detector, with the fluorescence detector connected in series with the ultraviolet detector. After the measuring device.

4.3.2 Analytical balance. accuracy 0.1mg, 0.01mg. 4.3.3 Vortex mixer. 4.3.4 Ultrasonic cleaner.

4.3.5 Centrifuge. The rotation speed shall not be less than 5000 r/min.

4.3.6 Solid phase extraction apparatus.

4.3.7 Nitrogen blowing device.

4.4 Samples

Prepare samples according to GB/T 20195, at least.200g, crush them so that they all pass through a test sieve with a 0.425mm aperture, mix thoroughly, and load into...

Store in a ground glass bottle for later use.

4.5 Test Procedure

4.5.1 Extraction

Perform two parallel experiments. Weigh 2 g (m, accurate to 0.1 mg) of the sample into a 50 mL centrifuge tube and accurately add 25 mL of extraction solution.

(4.2.4) Mix well, extract by sonication for 30 min (shaking once every 10 min during this period), centrifuge at 5000 r/min for 5 min, collect the supernatant and residue.

Repeat the extraction once, combine the supernatants (V), and set aside.

4.5.2 Purification

4.5.2.1 Degreasing

Transfer 8 mL of the stock solution (4.5.1), add 5 mL of n-hexane (4.2.3), vortex for 1 min, and centrifuge at 5000 rpm for 5 min. Accurately transfer...

Take 5 mL (V1) of the lower layer solution (1 mL for additive premixed feed or mixed feed additive), and purge with nitrogen at 40°C until nearly Dry the sample, dissolve it in 3 mL of phosphate buffer (4.2.5) by vortexing, mix well, centrifuge at 5000 r/min for 5 min, and take all the supernatant for later use.

4.5.2.2 Solid-phase extraction

The solid-phase extraction column (4.2.12) was activated sequentially with 3 mL of methanol (4.2.2), 3 mL of water, and 3 mL of phosphate buffer (4.2.5). The prepared solution...

(4.5.2.1) Pass the entire column through the column, elute sequentially with 3 mL of water and 3 mL of eluent (4.2.6), dry under vacuum, elute with 3 mL of methanol (4.2.2), and collect.

The eluent was dried nearly to dryness under nitrogen at 40°C. 1 mL (V2) of the reconstitution solution (4.2.8) was accurately added to dissolve the eluent, and the mixture was vortexed to mix. Microporous membrane.

(4.2.13) Filtering, to be tested.

4.5.3 Measurement

4.5.3.1 Reference conditions for high performance liquid chromatography

The reference conditions for high performance liquid chromatography are as follows.

a) Chromatographic column. C18 column, 250 mm in length, 4.6 mm in inner diameter, 5 µm in particle size, or equivalent;

b) Column temperature. 30°C;

c) Mobile phase. Phase A is sodium heptanesulfonate solution (4.2.7), Phase B is methanol (4.2.2), and the gradient elution program is shown in Table 1;

d) Flow rate. 1 mL/min;

e) Injection volume. 20 µL;

f) Ultraviolet detection wavelength. 267nm;

g) Fluorescence detector. excitation wavelength 306nm, emission wavelength 350nm.

Table 1 Gradient elution program time min Phase A (volume fraction) Phase B (volume fraction) 0.0 70 30 30.0 52 48 32.0 10 90 35.0 10 90 35.1 70 30 40.0 70 30

4.5.3.2 Determination of mixed standard series solutions and sample solutions

Under optimal instrument conditions, the mixed standard series solution (4.2.11) and the sample solution (4.5.2.2) were respectively tested using ultraviolet light detection.

A fluorescence detector (or diode array detector) was used to determine aminopropyl hydrochloride and sulfaquinoline, while a fluorescence detector was used to determine ethoxybenzoate. Mixed standards.

The high-performance liquid chromatogram of the solution is shown in Appendix A.

4.5.3.3 Qualitative

Qualitative analysis is based on retention time. The retention times of the analyte in the sample solution and the mixed standard series solutions (with equivalent mass concentrations) are consistent, indicating their relative...

The deviation is within ±2.5%.

4.5.3.4 Quantitative analysis

A standard curve should be plotted with the mass concentration of the analyte in the standard solution on the x-axis and the peak area on the y-axis. The correlation coefficient should not be lower than that of the standard curve.