GB/T 47386-2026Determination of brassinolide in fertilizers - High performance liquid chromatography (English PDF)

5.1.2 Reagents or materials

5.1.2.1 Unless otherwise specified, only reagents confirmed to be of analytical grade and Grade I water as specified in GB/T 6682 shall be used in the analysis.

5.1.2.2 Methanol. chromatographic grade.

5.1.2.3 Acetonitrile. chromatographic grade.

5.1.2.4 Benzylboronic acid. purity >= 98.0%.

5.1.2.5 Phenylboronic acid methanol solution (

1.0 g/L). Accurately weigh

1.0 g of phenylboronic acid into a 1 L volumetric flask, dissolve it in methanol and dilute to the mark. 5.1.2.6 24-Epibrassinolide Standard (CAS No.. 78821-43-9). Purity >= 95.0%. 5.1.2.7 28-Homobrassinolide Standard (CAS No.. 82373-95-3). Purity >= 96.0%. 5.1.2.8 28-Epoxybrassinolide Standard (CAS No.. 80483-89-2). Purity >= 95.0%.

5.1.2.9 Mixed Standard Stock Solution (1000 µg/mL). Accurately weigh 24-epibrassinolide, 28-homobrassinolide, and 28-epibrassinolide... Dissolve

0.01 g (accurate to 0.0001

g) of each lactone standard in a 10 mL volumetric flask, dilute to the mark with methanol, shake well, and let stand. Store at -18°C away from light. Shelf life is 30 days.

5.1.2.10 Mixed Standard Solution (100 µg/mL). Transfer 1 mL of 1000 µg/mL mixed standard stock solution (5.1.2.9) into a 10 mL... In a volumetric flask, dilute to the mark with methanol and shake well. Prepare fresh before use.

5.1.3 Instruments and Equipment

5.1.3.1 Commonly used laboratory instruments and equipment.

5.1.3.2 High performance liquid chromatograph, equipped with an ultraviolet detector or a diode array detector.

5.1.3.3 Electronic balance, accuracy 0.0001g.

5.1.3.4 Ultrasonic cleaner.

5.1.3.5 Vortex mixer.

5.1.3.6 Organic phase microporous filter membrane, pore size 0.45µm, nylon microporous filter membrane or equivalent.

5.1.4 Test Procedure

5.1.4.1 Preparation of test solution Weigh 0.5g to 2.0g (accurate to 0.0001g) of the sample into a 50mL plastic centrifuge tube, and accurately add 10mL of phenylboronic acid methanol solution. Add 10 mL of liquid to 10 mL of methanol, vortex for 1 min, sonicate at room temperature for 10 min, allow to stand for 30 min, transfer to a 25 mL volumetric flask, and dilute with methanol. Volume up to the mark, shake well, take 1 mL of supernatant and filter through a 0.45 µm organic phase microporous membrane, ready for analysis.

5.1.4.2 Measurement 5.1.4.2.1 Chromatographic Reference Conditions The chromatographic reference conditions are as follows:

a) Chromatographic column. 100mm × 4.6mm (inner diameter), stainless steel column, packed with 2.7µm C18 material. Or a chromatographic column with equivalent performance. Spectral column.

b) Mobile phase. V(acetonitrile).V(water) = 85.15.

0.8 mL/min.

d) Column temperature. 30°C.

e) Detection wavelength. 222nm.

f) Injection volume. 5 µL. 5.1.4.2.2 Plotting the Standard Curve Transfer

0.25 mL,

0.50 mL,

5.00 mL of the mixed standard solution (5.1.2.10) into 25 mL volumetric flasks, respectively. Add 10 mL of phenylboronic acid methanol solution to the solution, dilute to volume with methanol, shake well, and allow to stand for 30 min to prepare a solution with a mass concentration of 1.0 µg/mL. Standard working solutions of 2.0 µg/mL, 5.0 µg/mL, 10.0 µg/mL and 20.0 µg/mL were analyzed under the chromatographic conditions described in 5.1.4.2.1. A standard working curve was plotted with the peak area of the chromatographic solution as the ordinate and the corresponding concentration of the standard working solution as the abscissa. (Brassinolide standard) A typical chromatogram of the quasi-working solution obtained by high performance liquid chromatography-ultraviolet detection is shown in Figure A.1 of Appendix A. 5.1.4.2.3 Measurement Take the sample solution obtained in 5.1.4.1, and perform the determination under the chromatographic conditions in 5.1.4.2.1.Record the retention time and peak surface of the chromatographic peak. The product was quantified using the external standard method, and parallel experiments were conducted simultaneously. The response values of the analyte in the sample solution should all be within the linear range of the standard curve. If the sample solution exceeds the linear range, it can be diluted with methanol before measurement.

5.1.4.3 Blank Test Except for not weighing the sample, all measurements were performed according to the above steps.

5.1.5 Experimental Data Processing The content of the analyte Xi1 in the sample, expressed as a mass fraction (%), is calculated according to formula (1).

5.1.6 Precision In the same laboratory, by the same operator using the same equipment, following the same testing methods, and within a short period of time, the same test object is subjected to... The absolute difference between two independent test results obtained from mutually independent tests should not exceed 20% of the arithmetic mean of the two measurements. The condition is that the arithmetic mean of these two measurements is no more than 5% in cases where it is 20% of the total. In different laboratories, different operators use different equipment, follow the same testing methods, and independently test the same object. The absolute difference between two independent test results obtained from the test should not exceed 30% of the arithmetic mean of the two measurements. The premise is that the arithmetic mean of the measured values is 30% of the cases where it does not exceed 5%.

5.2.2 Reagents or materials

5.2.2.1 Unless otherwise specified, only reagents confirmed to be of analytical grade and Grade I water as specified in GB/T 6682 shall be used in the analysis.

5.2.2.2 Industrial nitrogen.

5.2.2.3 Methanol. chromatographic grade.

5.2.2.4 Acetonitrile. chromatographic grade. 5.2.2.5 24-Epibrassinolide standard. Same as 5.1.2.6. 5.2.2.6 28-Homobrassinolide standard. Same as 5.1.2.7. 5.2.2.7 28-Epoxy brassinolide standard. Same as 5.1.2.8.

5.2.2.8 Mixed standard stock solution. Same as 5.1.2.9.

5.2.2.9 Mixed standard solutions. Same as 5.1.2.10.

5.2.3 Instruments and Equipment

5.2.3.1 Commonly used laboratory instruments and equipment.

5.2.3.2 High performance liquid chromatograph with an electrical atomization detector.

5.2.3.3 Electronic balance, accuracy 0.0001g.

5.2.3.4 Ultrasonic cleaner.

5.2.3.5 Vortex mixer.

5.2.3.6 Organic phase microporous filter membrane, pore size 0.45µm, nylon microporous filter membrane or equivalent.

5.2.4 Test Procedure

5.2.4.1 Preparation of test solution Weigh 0.5g~2.0g (accurate to 0.0001g) of sample into a 50mL plastic centrifuge tube, add 20mL of methanol, vortex for 1min, and centrifuge at room temperature. Sonicate at room temperature for 10 min, transfer to a 25 mL volumetric flask, dilute to the mark with methanol, shake well, and filter 1 mL of the supernatant through a 0.45 µm organic phase filter. Microporous filter membrane, to be tested.

5.2.4.2 Measurement 5.2.4.2.1 Chromatographic Reference Conditions The chromatographic reference conditions are as follows:

a) Chromatographic column. 100mm × 4.6mm (inner diameter), stainless steel column, packed with 2.7µm C18 material. Or a chromatographic column with equivalent performance. Spectral column.

b) Mobile phase. V(acetonitrile).V(water) = 38.62.

0.6 mL/min.

d) Column temperature. 30°C.

e) Injection volume. 8 µL.

f) Acquisition frequency. 5Hz.

g) Atomization temperature. 50°C. 5.2.4.2.2 Plotting the Standard Curve Transfer

0.25 mL,

0.50 mL,

5.00 mL of the mixed standard solution (5.2.2.9) to 25 mL volumetric flasks, respectively. After diluting the methanol to a final volume, shake well to prepare standards with mass concentrations of 1.0 µg/mL, 2.0 µg/mL, 5.0 µg/mL, 10.0 µg/mL, and 20.0 µg/mL. The working solution was measured under the chromatographic conditions described in 5.2.4.2.1.The peak area was plotted on the ordinate, and the corresponding concentration of the standard working solution was plotted on the x-axis. Plot the standard working curve. A typical chromatogram of the brassinolide standard working solution obtained by high-performance liquid chromatography-electrospray ionization is shown in the appendix. Figure B.1 of B. 5.2.4.2.3 Test solution determination Take the sample solution obtained according to procedure 5.2.4.1, and perform the determination under the chromatographic conditions of 5.2.4.2.1.Record the retention time and color of the analyte. Peak area was determined, and quantification was performed using the external standard method, with parallel experiments conducted simultaneously. The response values of the analyte in the sample solution should all be within the linear range of the standard curve. If the sample solution is outside the linear range, it can be diluted with methanol before measurement.

5.2.4.3 Blank Test Except for not weighing the sample, all measurements were performed according to the above steps.

5.2.5 Experimental Data Processing The content of the analyte Xi2 in the sample, expressed as a mass fraction (%), is calculated according to formula (2).