GB/T 23788-2026Determination of soy isoflavones in health foods (English PDF)

5.1 Reagents

5.1.1 Water. Grade I water as specified in GB/T 6682.

5.1.2 Acetonitrile (CH3CN). chromatographic grade.

5.1.3 Methanol (CH3OH). chromatographic grade.

5.1.4 Dimethyl sulfoxide (C2H6OS). chromatographic grade.

5.2 Reagent Preparation 5.2.1 80% methanol solution. Measure 800 mL of methanol (5.1.3), dilute with water to 1000 mL, and mix well. 5.2.2 0.01% phosphoric acid aqueous solution. Take

0.1 mL of phosphoric acid, dilute with water to 1000 mL, and mix well. 5.2.3 50% methanol solution. Measure 50 mL of methanol (5.1.3), dilute with water to 100 mL, and mix well.

5.3 Standard Samples/Standard Substances

5.3.1 Daidzin (C21H20O9, CAS No.. 552-66-9). Purity >= 98%, or conforming to nationally certified standards. Sample/standard substance.

5.3.2 Glycitin (C22H22O10, CAS No.. 40246-10-4). Purity >=98%, or certified by the state and granted a certificate. Standard samples/standard substances.

5.3.3 Genistin (C21H20O10, CAS No.. 529-59-9). Purity >= 98%, or certified by the state and granted a certificate. Standard samples/standard substances.

5.3.4 Daidzein (C15H10O4, CAS No.. 486-66-8). Purity >= 98%, or certified by the state and granted a certificate. Standard samples/standard substances.

5.3.5 Glycitein (C16H12O5, CAS No.. 40957-83-3). Purity >= 98%, or certified by the state and granted a certificate. Standard samples/standard substances.

5.3.6 Genistein (C15H10O5, CAS No.. 446-72-0). Purity >= 98%, or certified by the state and granted a certificate. Standard samples/standard substances.

5.4 Preparation of Standard Solutions

5.4.1 Soy isoflavone standard stock solution (1000 µg/mL) Weigh 25 mg each of the standard samples (5.3) of daidzein, genistein, daidzein, daidzein, genistein, and genistein (accurate to 100 mg/mg).

0.1 mg (calculated according to the purity of the standard sample), added to 10 mL of dimethyl sulfoxide (5.1.4), sonicated until fully dissolved, transferred to 25 mL of brown... In a volumetric flask, dilute to the mark with methanol (5.1.3) and mix well. Store in a refrigerator at 4°C; shelf life is 3 months.

5.4.2 Intermediate Standard Solution of Soy Isoflavones Transfer

2.5 mL each of the three standard stock solutions (5.4.1) of daidzein, genistein, and genistein, and transfer

2.5 mL of each of the stock solutions of daidzein, genistein, and genistein. Take

0.5 mL of each of the three standard stock solutions of genistein (5.4.1) and place them in a 10 mL volumetric flask. Dilute to the mark with methanol (5.1.3) and mix well. The mixed standard solution of soybean isoflavones contained 250 µg/mL of daidzein, genistein, and genistein, and daidzein were also present. The concentrations of genistein and famigrin are 50 µg/mL. Store at 4°C. Shelf life is 3 months.

5.4.3 Standard working solutions of soybean isoflavones Transfer

0.02 mL,

0.2 mL,

0.4 mL,

2.0 mL of the mixed standard intermediate solution (5.4.2) respectively, and dilute with 50% methanol solution. (5.2.3) Adjust the volume to 5 mL, mix well, and prepare a solution with mass concentrations of 1.0 µg/mL, 10.0 µg/mL, and 10.0 µg/mL for daidzein, genistein, and genistein, respectively. The concentrations of daidzein, genistein, and genistein were 20.0 µg/mL, 50.0 µg/mL, and 100.0 µg/mL, respectively, and the mass concentrations were 0.2 µg/mL, 0.0... 2.0 µg/mL, 4.0 µg/mL, 10.0 µg/mL, 20.0 µg/mL. Prepare fresh before use.

5.5 Materials

5.5.1 Filter membrane. 0.45µm, organic system.

6.3 Analytical balance. accuracy

0.1 mg and

0.001 g.

6.4 Centrifuge. The speed shall not be less than 8000 r/min.

6.5 High-speed pulverizer.

7.2 Sample Preparation

7.2.1 Solid samples (hard capsules, tablets), semi-solid samples (soft capsules) Accurately weigh

0.10 g to

0.50 g of the sample (accurate to

0.001 g), place it in a 50 mL stoppered centrifuge tube, and add 40 mL of 80% methanol. After mixing the solution (5.2.1) thoroughly, shake for 10 min, sonicate for 20 min, cool to room temperature, and transfer the sample solution to a 50 mL volumetric flask. Rinse the centrifuge tubes with 80% methanol solution (5.2.1), transfer all washings to a 50 mL volumetric flask, and rinse with 80% methanol solution. (5.2.1) Make up to volume and shake well. Take an appropriate amount of solution into a centrifuge tube, centrifuge at 8000 r/min for 5 min, take the supernatant and filter it through a 0.45 µm filter membrane. (5.5.1) Filter, discard 1 mL of the initial filtrate, and keep it for later use.

7.2.2 Liquid samples (oral liquids, alcoholic beverages, soft drinks) Accurately transfer

5.0 g of the well-mixed liquid sample into a 50 mL stoppered centrifuge tube, and add... Add 40 mL of 80% methanol solution (5.2.1), mix thoroughly, shake for 10 min, sonicate for 20 min, cool to room temperature, and transfer to a 50 mL volumetric immersion tank. In a volumetric flask. Rinse the centrifuge tube with 80% methanol solution (5.2.1), transfer all washings to a 50 mL volumetric flask, and rinse with 80% methanol solution. (5.2.1) Make up to volume and shake well. Take an appropriate amount of solution into a centrifuge tube, centrifuge at 8000 r/min for 5 min, take the supernatant and filter it through a 0.45 µm filter membrane. (5.5.1) Filter, discard 1 mL of the initial filtrate, and keep it for later use. Note

1.Samples such as alcoholic beverages that are completely miscible with 80% methanol solution (5.2.1) do not require shaking, ultrasonic extraction and centrifugation. Note

2.Adjust the dilution factor f appropriately according to the content of the component in the sample to ensure that the mass concentration of the component is within the range of the standard curve determination.

7.3 Chromatographic Reference Conditions The reference conditions for liquid chromatography are as follows:

a) Chromatographic column. AQC18 column (particle size 5.0µm, 4.6mm×250mm), or equivalent hydrophilic column.

b) Mobile phase. Phase A is acetonitrile (5.1.2), and Phase B is 0.01% phosphoric acid aqueous solution (5.2.2). Gradient elution conditions are shown in Table 1.

d) Column temperature. 30°C.

e) Injection volume. 10 µL.

f) Wavelength. 260nm.