Determination of Soybean Yellow Roll Content by High Performance Liquid Chromatography (New Variety)

Determination of Soybean Yellow Roll Content by High Performance Liquid Chromatography

Soybean yellow roll (new variety)

Dadou Huangjuan

SEMEN SOJAE GERMINATUM

This product is obtained by germination and drying of mature seeds of leguminous plant soybean Glycine max (L.) Merr. The soybean is soaked in water to swell, the water is discharged, covered with a damp cloth, rinsed once a day with water or sprayed twice, and when the bud length is 0.5 cm to 1 cm, it is taken out and dried.

【Properties】 This product is slightly kidney-shaped, about 8mm long and about 6mm wide. The surface is yellow or yellowish brown, slightly shrunken, with a distinct umbilical point on one side and a curved radicle on one end. The outer cortex is brittle and often ruptured or detached. 2 cotyledons, yellow. The air is slight, the taste is light, and the chewing has a bean flavor. It is better to have large and full.

[Identification] (1) Take 1g of this product powder, add 30ml of diluted ethanol, sonicate for 30 minutes, centrifuge (3000 rev / min) for 10 minutes, filter, and evaporate the filtrate, add 1 ml of diluted ethanol to dissolve, and take the supernatant. Liquid, as a test solution. Another leucine reference substance was added, and diluted ethanol was added to prepare a solution containing 0.5 mg per 1 ml as a reference solution. According to the thin layer chromatography (Appendix VI B) test, take 5μl of each of the above two solutions, respectively, on the same silica gel G thin-layer plate, with n-butanol-glacial acetic acid-water (19:5:5) as the developing agent , unroll, remove, dry, spray with ninhydrin test solution, heat at 105 ° C until the spots are clear. In the chromatogram of the test sample, spots of the same color are displayed at positions corresponding to the chromatogram of the reference substance.

(2) Take 2 g of this product powder, add 30 ml of 80% ethanol, sonicate for 30 minutes, filter, and evaporate the filtrate, add 100 ml of ethanol to the residue to dissolve, and take the supernatant as the test solution. Another genus lignin reference substance was added, and 80% ethanol was added to make a solution containing 1 mg per 1 ml as a reference solution. According to the thin layer chromatography (Appendix VI B) test, draw 5~10μl of the test solution and 2μl of the reference solution, respectively, on the same silica gel G thin layer plate, with ethyl acetate-methanol-water (10:1.7: 1.3) As a developing agent, the cylinder was pre-saturated for 30 minutes, unrolled, taken out, air-dried, sprayed with 2% aluminum trichloride ethanol solution, heated at 105 ° C for several minutes, and placed under ultraviolet light (365 nm) for inspection. In the chromatogram of the test sample, fluorescent spots of the same color are displayed at positions corresponding to the chromatogram of the reference substance.

[Check] Moisture must not exceed 11.0% (Appendix IX H).

Total ash should not exceed 7.0% (Appendix IX K)

[Content determination] Determined by high performance liquid chromatography (Appendix VI D).

Chromatographic conditions and system suitability test with octadecylsilane bonded silica as a filler; methanol as mobile phase A, with 1% acetic acid solution as mobile phase B, gradient elution according to the following table; column temperature 35 ° C; The detection wavelength was 260 nm. The number of theoretical plates shall not be less than 5,000 according to the peaks of daidzin and genistein.

Preparation of the reference solution Take about 10mg of daidzin and genistin, which are accurately weighed, placed in a 50ml volumetric flask, add 70% methanol to the mark, shake well; accurately measure 1ml, set 10ml volumetric flask, add Methanol to the mark, shake well, that is.

Preparation of the test solution Take about 1.0g of this product powder (through the No. 4 sieve), accurately weighed, placed in a conical flask, precision added 70% methanol 25ml, weighed, heated to reflux for 2 hours, let cool Then weigh the weight, make up the lost weight with 70% methanol, shake, centrifuge (2000 rev / min) for 10 minutes, take the supernatant, filter, and take the filtrate, that is.

The measurement method accurately draws 10 μl of each of the reference solution and the test solution, and injects into a liquid chromatograph, and the measurement is obtained.

This product is calculated as dry product, and the total amount of daidzein (C ₂₁ H ₂₀ O ₉ ) and genistein (C ₂₁ H ₂₀ O ₁₀ ) should not be less than 0.08%.

[Processing] Take the rush and light bamboo leaves in the pot, add the appropriate amount of water to cook twice, the first 60 minutes, the second 30 minutes, combine the decoction, filtered. The filtrate and the soy yellow roll are placed together in a pot, and the solution is sucked up, taken out, and dried.

1kg of rush and 2kg of light bamboo leaves per 100kg of net medicine.

Syltech500 High Performance Liquid Chromatograph (single pump configuration / gradient)
1 pump P501 constant current high pressure infusion pump
1.1 Principle of liquid transport: double plunger series reciprocating mode, automatic pulse suppression system, and solvent compression factor program, can automatically compensate flow, liquid delivery is more accurate
1.2 Maximum conveying pressure: 6000PSI or more (about 42MPa or more)
1.3 Flow rate accuracy: 0.1%
1.4 Flow rate range: (0.001-10.000) mL / min minimum increment 0.001mL / min
1.5 Plunger rod cleaning: automatic plunger rod cleaning without additional equipment to protect the plunger rod
1.6 Security: equipped with power-on self-test, high and low voltage alarm and protection
1.7 Gradient System: Binary Gradient Mixing System / Single Pump System
1.7.1 Mixing range: 0 to 100%
1.7.2 Mixing principle: high pressure mixing
1.7.3 Mixed liquid number: greater than or equal to 2 yuan
1.7.4 Degassing device: both internal and external degassing
2 UV detector UV501 UV variable wavelength detector
2.1 Light source: Xenon lamp (optional tungsten lamp) The lamp life is 2000 hours, and the service life is more than 1000 hours.
2.2 Wavelength range: 190 to 720 nm (step increments of 1 nm)
2.3 Wavelength accuracy: <±1nm
2.4 Wavelength reproducibility: <±0.5nm
2.5 Bandwidth: less than 5nm
2.6 Noise: <±2.5*10-5AU (under certain conditions)
2.7 Drift: <±2.5*10-4AU (under certain conditions)
2.8 Flow cell volume: 10ul volume, optical path 10mm
2.9 Absorption range: 0.01AU/V-10AU/V (10 steps total) injection signal control (event), automatic zero adjustment
2.10 Function: With fixed-point wavelength measurement, data processing hardware embedded detector, digital signal direct acquisition and processing function
3 Column: VertexTMXB-C18 4.6*250mm*5um LC column with guard column
4 column oven: 10 degrees to 100 degrees (0.1 degree increments) at room temperature
5 N2010 Pharmacopoeia chromatography workstation: chromatography workstation (to meet the 2010 edition of the pharmacopoeia requirements, the United States made) Chinese and English operating software, support WIN9X/XP/2000/2003 and other operating systems, hardware embedded detector, digital signal direct acquisition and processing
6 Vertex7825i manual injection valve: Vertex7825i manual injection valve (with remote trigger switch), comes with 20UL quantitative, pressure 8000psi, injection times more than 10,000 times