A quality detection method for pseudoginsenoside F11 in Wuji Baifeng pills

Abstract

The present invention discloses a quality detection method for pseudoginsenoside F11 in Wuji Baifeng Pills. The present invention performs alcohol extraction on Wuji Baifeng Pills samples, performs detection according to high performance liquid chromatography and evaporative light detection, and judges the quality of Wuji Baifeng Pills by pseudoginsenoside F11 in Wuji Baifeng Pills. The quality safety of the medicine is ensured and the quality detection of pseudoginsenoside F11 in Wuji Baifeng Pills is convenient. The present invention has the characteristics of accuracy, high sensitivity, good repeatability, reliable results, and can effectively control the quality of the medicinal material and ensure the clinical efficacy of the medicine.

Description

Technical Field

[0001] The present invention relates to the quality control of Wuji Baifeng Pills, and particularly to a method for detecting the quality of pseudo-ginsenoside F11 in Wuji Baifeng Pills. Background Art

[0002] Wuji Baifeng Pills are prepared by crushing Rehmanniae Radix Praeparata, Rehmanniae Radix, Rhizoma Chuanxiong, Cornu Cervi Degelatinatum, Radix Stellariae, Gorgonopsis (fried), Rhizoma Dioscoreae, Radix Salviae Miltiorrhizae into coarse powder, and separately cutting into pieces the remaining twelve herbs including Gallus domesticus (with feathers, claws and intestines removed), Colla Corii Cervi, Carapax Trionycis Vinegar, Concha Ostreae Calcined, Ootheca Mantidis, Radix Ginseng, Radix Astragali seu Hedysari, Radix Angelicae Sinensis, Radix Paeoniae Alba, Rhizoma Cyperi Vinegar, Radix Asparagi, Radix Glycyrrhizae, etc. Then put them in a pot, add 1500 g of yellow rice wine, seal with a lid, stew over water until the wine is exhausted, take out, mix with the above-mentioned coarse powder, dry at low temperature, then crush into fine powder, sieve, and mix evenly. Add 30 - 40 g of refined honey and appropriate amount of water to every 100 g of the powder to make pills, and dry to make water-honey pills.

[0003] This product is water-honey pills in dark brown to black color; sweet and slightly bitter in taste; functions and indications: replenishing qi and nourishing blood, regulating menstruation and arresting leukorrhea. It is used for deficiency of both qi and blood, emaciation, weakness of waist and knees, irregular menstruation, metrorrhagia and leukorrhagia. At present, there has been no report on the quality inspection method of pseudo-ginsenoside F11 in Wuji Baifeng Pills. In order to better control the quality of Wuji Baifeng Pills and ensure the clinical efficacy, the present invention provides a method for detecting the quality of pseudo-ginsenoside F11 in Wuji Baifeng Pills. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for detecting the quality of pseudo-ginsenoside F11 in Wuji Baifeng Pills. The present invention has the characteristics of accuracy, high sensitivity, good repeatability, reliable results, and can effectively control the quality of the product and ensure the clinical efficacy of the drug.

[0005] The technical solution of the present invention:

[0006] A method for detecting the quality of pseudo-ginsenoside F11 in Wuji Baifeng Pills, the Wuji Baifeng Pills are prepared from 64 parts by weight of Gallus domesticus, 12.8 parts of Colla Corii Cervi, 6.4 parts of Carapax Trionycis Vinegar, 4.8 parts of Concha Ostreae Calcined, 4.8 parts of Ootheca Mantidis, 12.8 parts of Radix Ginseng, 3.2 parts of Radix Astragali seu Hedysari, 14.4 parts of Radix Angelicae Sinensis, 12.8 parts of Radix Paeoniae Alba, 12.8 parts of Rhizoma Cyperi Vinegar, 6.4 parts of Radix Asparagi, 3.2 parts of Radix Glycyrrhizae, 25.6 parts of Rehmanniae Radix Praeparata, 25.6 parts of Rehmanniae Radix, 6.4 parts of Rhizoma Chuanxiong, 4.8 parts of Cornu Cervi Degelatinatum, 2.6 parts of Radix Stellariae, 6.4 parts of Gorgonopsis (fried), 12.8 parts of Rhizoma Dioscoreae, 12.8 parts of Radix Salviae Miltiorrhizae. It is characterized in that the detection method:

[0007] (1) Preparation of reference solution: Take an appropriate amount of pseudo-ginsenoside F11 reference substance, accurately weigh it, and add methanol to make a solution containing 100 μg per 1 ml, thus obtaining.

[0008] (2) Preparation of the test solution: Take the aqueous honeyed pills of Wuji Baifeng Pills, grind them, take 6 g, and weigh accurately; place them in a stoppered conical flask, accurately add 40 - 160 ml of 40 - 80% methanol solution, stopper tightly, weigh, heat under reflux for 0.5 - 3 hours, cool, weigh again, make up the lost weight with 40 - 80% methanol solution, shake well, filter, accurately measure 50 ml of the filtrate, place it in an evaporating dish, evaporate to dryness, add 10 - 40 ml of water to dissolve, shake and extract with water-saturated n-butanol for 3 - 5 times, 10 - 50 ml each time, combine the n-butanol extracts, wash with ammonia test solution for 1 - 3 times, 20 - 110 ml each time, then wash with 20 - 110 ml of n-butanol-saturated water, separate the n-butanol layer, concentrate under reduced pressure, recover the solvent to dryness, add an appropriate amount of methanol to dissolve, transfer to a 1 - 10 ml volumetric flask, add methanol to the scale, shake well, filter, and take the subsequent filtrate as the test solution;

[0009] (3) Chromatographic conditions: The filler is octadecylsilane chemically bonded silica gel; the mobile phase is acetonitrile: aqueous solution = 20 - 40:60 - 80; the column temperature is 20 - 40 °C; the flow rate is 0.6 - 1.2 ml / min; evaporative light scattering detector: gain: 10 - 55, drift tube temperature: 30 - 90 °C, nebulizer temperature: 30 - 80 °C, gas pressure: 15 - 45 psi. The number of theoretical plates calculated by pseudo-ginsenoside F11 should be not less than 3000;

[0010] (4) Accurately吸取 5 μl, 20 μl of the reference solution and 10 μl of the test solution respectively, inject them into the liquid chromatograph, and determine according to the chromatographic conditions in step (3), calculate the content of pseudo-ginsenoside F11, and you will get it.

[0011] The method for detecting impurities of pseudo-ginsenoside F11 in the aforementioned Wuji Baifeng Pills, in step (1): Preparation of the reference solution: Take the reference substance of pseudo-ginsenoside F11, weigh accurately, and make a solution containing 100 μg per 1 ml with methanol, and you will get it.

[0012] The method for detecting impurities of pseudo-ginsenoside F11 in the aforementioned Wuji Baifeng Pills. In step (2): Preparation of the test solution: Take the water-honey pills of Wuji Baifeng Pills, grind them, take 6 g, and weigh accurately; place them in a stoppered conical flask, accurately add 50 - 70% methanol solution 50 - 150 ml, stopper tightly, weigh, heat under reflux for 0.5 - 2 hours, cool, weigh again, make up the lost weight with 50 - 70% methanol solution, shake well, filter, accurately measure 50 ml of the filtrate, place it in an evaporating dish, evaporate to dryness, add 20 - 30 ml of water to dissolve, shake and extract with water-saturated n-butanol 3 - 5 times, each time 20 - 40 ml, combine the n-butanol extraction solutions, wash with ammonia test solution 1 - 3 times, each time 25 - 100 ml, then wash with n-butanol-saturated water 30 - 100 ml, separate the n-butanol layer, concentrate under reduced pressure, recover the solvent to dryness, add an appropriate amount of methanol to dissolve, transfer to a 2 - 5 ml volumetric flask, add methanol to the scale, shake well, filter, and take the subsequent filtrate as the test solution.

[0013] Specifically, for the method for detecting impurities of pseudo-ginsenoside F11 in the aforementioned Wuji Baifeng Pills, in step (2): Preparation of the test solution: Take the water-honey pills of Wuji Baifeng Pills, grind them, take 6 g, and weigh accurately; place them in a stoppered conical flask, accurately add 60% methanol solution 100 ml, stopper tightly, weigh, heat under reflux for 1.5 hours, cool, weigh again, make up the lost weight with 60% methanol solution, shake well, filter, accurately measure 50 ml of the filtrate, place it in an evaporating dish, evaporate to dryness, add 25 ml of water to dissolve, shake and extract with water-saturated n-butanol 4 times, each time 30 ml, combine the n-butanol extraction solutions, wash with ammonia test solution 2 times, each time 50 ml, then wash with n-butanol-saturated water 50 ml, separate the n-butanol layer, concentrate under reduced pressure, recover the solvent to dryness, add an appropriate amount of methanol to dissolve, transfer to a 2 ml volumetric flask, add methanol to the scale, shake well, filter, and take the subsequent filtrate as the test solution.

[0014] The method for detecting impurities of pseudo-ginsenoside F11 in the aforementioned Wuji Baifeng Pills. In step (3): Chromatographic conditions: Using octadecylsilane chemically bonded silica gel as the filler; the mobile phase is acetonitrile: aqueous solution = 25 - 35:65 - 75; column temperature is 25 - 35 °C; flow rate: 0.8 - 1 ml / min; evaporative light scattering detector: gain: 20 - 50, drift tube temperature: 40 - 80 °C, nebulizer temperature: 35 - 75 °C, gas pressure: 20 - 40 psi. The number of theoretical plates calculated based on pseudo-ginsenoside F11 should be not less than 3000.

[0015] Specifically, for the method for detecting impurities of pseudo-ginsenoside F11 in Wuji Baifeng Pills described above, in step (3): Chromatographic conditions: using octadecylsilane-bonded silica gel as the filler; the mobile phase is acetonitrile: aqueous solution = 35:65; column temperature is 35°C; flow rate: 0.9 ml / min; evaporative light scattering detector: gain: 30, drift tube temperature: 45°C, nebulizer temperature: 40°C, gas pressure: 30 psi. The theoretical plate number calculated based on pseudo-ginsenoside F11 should not be less than 3000.

[0016] For the method for detecting impurities of pseudo-ginsenoside F11 in Wuji Baifeng Pills described above, in step (4): Precisely pipette 5 μl, 20 μl of the reference solution and 10 μl of the test solution respectively, inject them into the liquid chromatograph, and perform the determination according to the chromatographic conditions in step (3). Calculate the content of pseudo-ginsenoside F11 using the external standard two-point logarithmic equation to obtain the result.

[0017] Advantages of the present invention:

[0018] This method ensures the quality and safety of drug use by detecting pseudo-ginsenoside F11 in Wuji Baifeng Pills and facilitates the quality detection of pseudo-ginsenoside F11 in Wuji Baifeng Pills. The present invention has the characteristics of accuracy, high sensitivity, good repeatability, reliable results, being able to effectively control the quality of this medicinal material, and ensuring the clinical efficacy of the drug.

[0019] The inventor conducted the following experiments to verify the effect of the present invention:

[0020] Experimental example:

[0021] 1 Drug source

[0022] Wuji Baifeng Pills (14521001, 14521002A, 14521002B), the negative sample without ginseng, and the Wuji Baifeng Pills sample prepared by replacing ginseng in the prescription of Wuji Baifeng Pills with American ginseng were all prepared according to the method described in the present invention.

[0023] 2 Appearance

[0024] This product is a black-brown to black aqueous honey pill; it tastes sweet and slightly bitter.

[0025] 3 Content determination

[0026] This product is composed of twenty kinds of medicinal materials and has the effects of replenishing qi and nourishing blood, regulating menstruation and arresting leucorrhea. Ginseng is the monarch drug in the prescription and is included in the first volume of the Chinese Pharmacopoeia 2020 Edition. It has the effects of greatly replenishing primordial qi, restoring pulse and arresting collapse, tonifying the spleen and lungs, promoting fluid production and nourishing blood, calming the mind and improving intelligence, and is the main component for the prescription to exert its effects. In order to better control the quality of this pill, the content of pseudo-ginsenoside F11 was determined by high performance liquid chromatography and evaporative light scattering, and a methodology study was carried out.

[0027] 3.1 Instruments

[0028] Shimadzu LC-2030 high-performance liquid chromatograph; LCsolution chromatography workstation; Waters liquid chromatograph (USA), including: 2424 evaporative light scattering detector, column oven, GA-10B low-noise air pump, Empower chromatography workstation; ultrasonic herbal extractor (model: KS-500ZT, ultrasonic power: 500W, ultrasonic frequency: 28 / 33 / 40 / 42 / 50 / 59KHz; Kunshan Jielimei Ultrasonic Instrument Co., Ltd.).

[0029] 3.2 Drug Test

[0030] Acetonitrile was of chromatographic grade; n-butanol, methanol, and ammonia were of analytical grade; water was Wahaha purified water; ginsenoside F11 reference standard (Chengdu Purechen-standard co., Ltd.; batch number: 200926; for content determination: 98%)

[0031] 3.3 Chromatographic conditions

[0032] Chromatographic columns: Waters C18 column (250×4.6mm, 5μm), Agilent XDB-C18 column (250×4.6mm, 5μm), Shimadzu ODS-3 column (250×4.6mm, 5μm); Mobile phase: Acetonitrile-water solution (35:65); Column temperature: 35℃; Flow rate: 0.9ml / min; Evaporative light detector: Gain: 30, Drift tube temperature: 45℃, Nebulizer temperature: 40℃, Gas pressure: 30psi. The theoretical plate number, calculated based on ginsenoside F11, should be no less than 3000.

[0033] 3.4 Preparation of reference stock solution

[0034] Accurately weigh an appropriate amount of ginsenoside F11 reference standard, dissolve it in methanol, and prepare a ginsenoside F11 reference standard solution with a concentration of 0.09947 mg / ml.

[0035] 3.5 Preparation of the test solution

[0036] Take Wuji Baifeng Pills (water-honey pills), crush them, take 6 g, and weigh accurately; place them in a stoppered conical flask, accurately add 100 ml of 60% methanol solution, stopper tightly, weigh, heat under reflux for 1.5 hours, cool, weigh again, add 60% methanol solution to make up for the lost weight, shake well, filter, accurately measure 50 ml of the filtrate, place it in an evaporating dish, evaporate to dryness, add 25 ml of water to dissolve, shake and extract with water-saturated n-butanol 4 times, each time 30 ml, combine the n-butanol extracts, wash with ammonia test solution 2 times, each time 50 ml, then wash with 50 ml of n-butanol-saturated water, separate the n-butanol layer, concentrate under reduced pressure, recover the solvent to dryness, add appropriate amount of methanol to dissolve, transfer to a 2-ml volumetric flask, add methanol to the scale, shake well, filter, and take the consecutive filtrate to obtain the solution.

[0037] 3.6 Preparation of negative control sample solution

[0038] Crush Rehmanniae Radix Praeparata, Rehmanniae Radix, Chuanxiong Rhizoma, Cornu Cervi Degelatinatum, Radix Stellariae, Gorgonians (Fried), Dioscoreae Rhizoma, Salviae Miltiorrhizae Rhizoma et Radix into coarse powder. Except for Radix Ginseng, the remaining eleven herbs, namely, black-bone chicken (defeathered, clawless, and gutted), Colla Cornus Cervi, Carapax Trionycis (Vinegar-processed), Concha Ostreae (Calcined), Mantis Eggcases, Radix Astragali seu Hedysari, Radix Angelicae Sinensis, Radix Paeoniae Alba, Cyperi Rhizoma (Vinegar-processed), Radix Asparagi, and Radix Glycyrrhizae, are respectively broken into pieces as appropriate, placed in a pot, add 1500 g of yellow rice wine, cover and seal, stew over water until the wine is exhausted, take out, mix with the above-mentioned coarse powder, dry at low temperature, then crush into fine powder, sieve, and mix well. Add 30 - 40 g of refined honey and appropriate amount of water to every 100 g of the powder to make pills, dry, and make water-honey pills to obtain the negative sample without Radix Ginseng. Take the water-honey pills of the negative sample without Radix Ginseng, crush them, take 6 g, and weigh accurately; place them in a stoppered conical flask, accurately add 100 ml of 60% methanol solution, stopper tightly, weigh, heat under reflux for 1.5 hours, cool, weigh again, add 60% methanol solution to make up for the lost weight, shake well, filter, accurately measure 50 ml of the filtrate, place it in an evaporating dish, evaporate to dryness, add 25 ml of water to dissolve, shake and extract with water-saturated n-butanol 4 times, each time 30 ml, combine the n-butanol extracts, wash with ammonia test solution 2 times, each time 50 ml, then wash with 50 ml of n-butanol-saturated water, separate the n-butanol layer, concentrate under reduced pressure, recover the solvent to dryness, add appropriate amount of methanol to dissolve, transfer to a 2-ml volumetric flask, add methanol to the scale, shake well, filter, and take the consecutive filtrate to obtain the solution.

[0039] 3.7 Preparation of test solution by replacing Radix Ginseng in the prescription with Radix Panacis Quinquefolii

[0040] By pulverizing eight herbs, namely Rehmanniae Radix Praeparata, Rehmanniae Radix, Chuanxiong Rhizoma, Cornu Cervi Degelatinatum, Stellariae Radix, Euryales Semen (parched), Dioscoreae Rhizoma, and Salviae Miltiorrhizae Radix, into coarse powder, and separately breaking up the remaining twelve herbs, namely Gallus domesticus (with feathers, claws, and intestines removed), Colla Corii Cervi, Amydae Carapax (vinegar processed), Panacis Quinquefolii Radix, Concha Ostreae (calcined), Mantidis Ootheca, Astragali Radix, Angelicae Sinensis Radix, Paeoniae Radix Alba, Cyperi Rhizoma (vinegar processed), Asparagi Radix, and Glycyrrhizae Radix, into pieces as appropriate, placing them in a jar, adding another 1500 g of yellow rice wine, covering and sealing it, stewing it over water until the wine is exhausted, taking it out, mixing it with the above-mentioned coarse powder, drying it at low temperature, pulverizing it into fine powder again, sieving it, and mixing it evenly. Add 30 - 40 g of refined honey and an appropriate amount of water to every 100 g of the powder to make pills, drying them to obtain the Wuji Baifeng Pills sample with Panacis Quinquefolii Radix replacing Ginseng Radix in the prescription. Take the water-honey pills of the Wuji Baifeng Pills sample with Panacis Quinquefolii Radix replacing Ginseng Radix in the prescription, crush them, take 6 g, and weigh it precisely; place it in a stoppered conical flask, precisely add 100 ml of 60% methanol solution, stopper it tightly, weigh it, heat under reflux for 1.5 hours, cool it, weigh it again, make up the lost weight with 60% methanol solution, shake it well, filter it, precisely measure 50 ml of the filtrate, place it in an evaporating dish, evaporate it to dryness, add 25 ml of water to dissolve it, shake and extract it 4 times with n-butanol saturated with water, 30 ml each time, combine the n-butanol extracts, wash it 2 times with ammonia test solution, 50 ml each time, then wash it with 50 ml of water saturated with n-butanol, separate the n-butanol layer, concentrate it under reduced pressure, recover the solvent to dryness, add an appropriate amount of methanol to dissolve it, transfer it to a 2-ml volumetric flask, add methanol to the scale, shake it well, filter it, and take the continued filtrate to obtain the solution.

[0041] 3.8 Preparation of Blank Solution

[0042] Precisely measure 100 ml of 60% methanol solution, place it in a stoppered conical flask, stopper it tightly, weigh it, heat under reflux for 1.5 hours, cool it, weigh it again, make up the lost weight with 60% methanol solution, shake it well, filter it, precisely measure 50 ml of the filtrate, place it in an evaporating dish, evaporate it to dryness, add 25 ml of water to dissolve it, shake and extract it 4 times with n-butanol saturated with water, 30 ml each time, combine the n-butanol extracts, wash it 2 times with ammonia test solution, 50 ml each time, then wash it with 50 ml of water saturated with n-butanol, separate the n-butanol layer, concentrate it under reduced pressure, recover the solvent to dryness, add an appropriate amount of methanol to dissolve it, transfer it to a 2-ml volumetric flask, add methanol to the scale, shake it well, filter it, and take the continued filtrate to obtain the solution.

[0043] 3.9 Specificity Experiment

[0044] Respectively take the pseudo-ginseng reference substance solution, the test solution (batch numbers: 14521001, 14521002A, 14521002B), the ginseng-free negative test solution, the test solution of Wuji Baifeng Pills with Panacis Quinquefolii Radix replacing Ginseng Radix in the prescription, and the blank solution, and inject them into the liquid chromatograph according to the quality inspection method determination method respectively, and record the chromatograms. From Figures 1 to 8As can be seen in the chromatogram of the reference standard, ginsenoside F11 elutes at 14.1 min. Negative and blank samples do not show a peak at this point. The sample of Wuji Baifeng Wan, which uses American ginseng instead of ginseng in the prescription, shows a peak at this point. That is, under the conditions of this test, ginsenoside F11 should be completely separated from other components with a resolution greater than 1.5 and a theoretical plate number based on ginsenoside F11 not less than 3000.

[0045] 3.10 Accuracy Experiment

[0046] Accurately weigh six portions of the tested sample of Wuji Baifeng Wan (batch number: 14521002B, content of ginsenoside F11: 0.0021%), accurately add 1 ml of ginsenoside F11 reference solution, and process the samples according to the preparation method of the tested sample solution. Inject 10 μl of each sample and record the chromatogram. Calculate the content of ginsenoside F11, subtract the known content of ginsenoside F11 in the tested sample, and calculate the recovery rate by the ratio of the amount of ginsenoside F11 accurately added to the amount of ginsenoside F11 reference solution. The results are shown in Table 1 below. Figures 9 to 21 .

[0047] Table 1. Results of Recovery Rate Experiment

[0048]

[0049] The results showed that the method had a good recovery rate.

[0050] 3.11 Precision Test

[0051] (1) Repeatability

[0052] Accurately weigh a sample of Wuji Baifeng Wan (a traditional Chinese medicine formula) containing American ginseng instead of ginseng. Prepare six test solutions according to the method for preparing test solutions. Inject 10 μl of each solution into the chromatograph and record the chromatograms. Calculate the content of ginsenoside F11 impurity using the external standard logarithmic two-point method. Perform statistical comparison of the data. The results are shown in Table 2 below. Figures 22 to 34 .

[0053] Table 2 Results of Repeatability Experiments

[0054]

[0055] The results showed good repeatability.

[0056] (2) Intermediate precision

[0057] Another staff member precisely weighed a sample of Wuji Baifeng Wan (a traditional Chinese medicine formula) containing American ginseng instead of ginseng. The sample was prepared using the same method and conditions as described above, and the chromatogram was recorded. The content of ginsenoside F11 was calculated using the external standard logarithmic two-point method. Statistical comparisons were performed, and the results are shown in Table 3 and Figure 4. Figures 35 to 52.

[0058] Table 3 Results of intermediate precision experiments

[0059]

[0060] The results showed that the results were basically consistent with the repeatability results, indicating that different analysts and different instruments did not affect the measurement results of this method.

[0061] 4 detection limits

[0062] Accurately pipette 1 ml of the reference solution into a 2 ml volumetric flask, dilute with methanol, and bring to volume. Mix well. This concentration is used as the injection reference concentration (10.15 × 98% ÷ 100 × 1 ÷ 2 mg / ml). The signal-to-noise ratio was 3:1 at an injection volume of 5 μl. Therefore, the limit of detection for ginsenoside F11 was calculated to be 0.268475 μg. The results are shown in Table 4 below. Figure 53 , Figure 54 .

[0063] Table 4. Detection Limit Assessment Table

[0064] project concentration Peak area S / N Average S / N Detection limit 10.15×98％÷100×1÷2 5102 2.812824 2.812824

[0065] 4.1 Limit of Quantification

[0066] Accurately weigh 10.24 mg of the reference standard and place it in a 25 ml volumetric flask. Dilute with methanol and bring to the mark. Shake well. Accurately pipette 15 ml of the reference standard solution and place it in a 50 ml volumetric flask. Dilute with methanol and bring to the mark. Shake well. This concentration is used as the injection concentration of the reference standard (concentration: 10.24 × 98% ÷ 25 × 15 ÷ 50 mg / ml). The result shows that the signal-to-noise ratio is 10:1 at an injection volume of 5 μl. Therefore, the limit of quantification for ginsenoside F11 is calculated to be 0.602112 μg. The results are shown in Table 5 below. Figures 55 to 61 .

[0067] Table 5 Quantitative Limits Table

[0068]

[0069] 4.2 Examination of Linear Relationships

[0070] Under the chromatographic conditions described above, 4, 6, 8, 12, 16, 20, and 25 μl of the reference solution (concentration 0.09947 mg / ml) were accurately injected into the liquid chromatograph, and the peak areas were measured and the chromatograms were recorded. A linear regression calculation was performed using peak area A and injection volume C, yielding the following linear regression equation: y = 411378x - 12769, R0 2=0.9916, the results are shown in Table 6 below. It can be seen that the injection amount of the reference standard for ginsenoside F11 in the range of 0.39788μg-2.48675μg has a good linear relationship with the peak area. The results are shown in Table 6 below. Figures 62 to 68 .

[0071] Table 6. Linearity of Ginsenoside F11

[0072] 1 2 3 4 5 6 7 Injection volume (μg) 0.39788 0.59628 0.79576 1.19364 1.59152 1.9894 2.48675 Peak area A 7910 12388 18637 33087 50509 69078 93548

[0073] 4.3 Scope

[0074] Accurately weigh ginsenoside F11 reference standard and prepare reference standard solutions at concentrations of 80%, 100%, 120%, and 150%, respectively. Inject 10 μl of each solution into a high-performance liquid chromatograph (HPLC), determine the peak area, and record the chromatograms. The results are shown in Table 7 below. Figures 69 to 72 .

[0075] Table 7. Scope of Ginsenoside F11

[0076]

[0077]

[0078] The results showed that all four concentration points met the linearity requirement.

[0079] 4.4 Durability

[0080] Investigation of different chromatographic columns: Take Wuji Baifeng Wan (14521002A), and prepare the test solution according to the above method. Using the same test solution, but with other chromatographic conditions unchanged, use different manufacturers to investigate the applicability of the method. The results are shown in Table 8 below.

[0081] Condition 1: Agilent XDB-C18, 250×4.6mm, 5μm

[0082] Condition 2: Waters XDB-C18, 250×4.6mm, 5μm

[0083] Condition 3: Shimadzu ODS-3, 250×4.6mm, 5μm

[0084] Table 8. Investigation of different chromatographic columns

[0085] condition 1 2 3 Content (μg / g) 18 Not detected Not detected Retention time (min) 10.7 14.4 17.2

[0086] The results show that there are differences in detection results between different chromatographic columns. When actually testing samples, it is recommended to use the Agilent XDB-C18 (250×4.6mm, 5μm) chromatographic column. Attached Figure Description

[0087] Figure 1 : Chromatogram of the negative test sample solution of Wuji Baifeng Pills lacking ginseng in the specificity test;

[0088] Figure 2 : Chromatogram of the test sample solution of Wuji Baifeng Pills with American ginseng replacing ginseng in the prescription in the specificity test;

[0089] Figure 3 : Chromatogram of the test sample solution 14521001 in the specificity test;

[0090] Figure 4 : Chromatogram of the test sample solution 14521002A in the specificity test;

[0091] Figure 5 : Chromatogram of the test sample solution 14521002B in the specificity test;

[0092] Figure 6 : Chromatogram of the pseudo-ginseng reference substance solution (injection volume of 20 μl) in the specificity test;

[0093] Figure 7 : Chromatogram of the pseudo-ginseng reference substance solution (injection volume of 5 μl) in the specificity test;

[0094] Figure 8 : Chromatogram of the blank solution in the specificity test;

[0095] Figure 9 : Chromatogram of the test sample 1 of Wuji Baifeng Pills spiked with pseudo-ginsenoside F11 in the accuracy test;

[0096] Figure 10 : Chromatogram of the test sample 2 of Wuji Baifeng Pills spiked with pseudo-ginsenoside F11 in the accuracy test;

[0097] Figure 11 : Chromatogram of the test sample 3 of Wuji Baifeng Pills spiked with pseudo-ginsenoside F11 in the accuracy test;

[0098] Figure 12 : Chromatogram of the test sample 4 of Wuji Baifeng Pills spiked with pseudo-ginsenoside F11 in the accuracy test;

[0099] Figure 13 : Chromatogram of the test sample 5 of Wuji Baifeng Pills spiked with pseudo-ginsenoside F11 in the accuracy test;

[0100] Figure 14 : Chromatogram of the test sample 6 of Wuji Baifeng Pills spiked with pseudo-ginsenoside F11 in the accuracy test;

[0101] Figure 15 : Chromatogram of the pseudo-ginsenoside F11 reference substance solution Figure 1 (injection of 20 μl);

[0102] Figure 16 Accuracy test of ginsenoside F11 reference solution chromatogram Figure 2 (20ul injection);

[0103] Figure 17 Accuracy test of ginsenoside F11 reference solution chromatogram Figure 1 (5ul injection);

[0104] Figure 18 Accuracy test of ginsenoside F11 reference solution chromatogram Figure 2 (5ul injection);

[0105] Figure 19 Accuracy test of ginsenoside F11 reference solution chromatogram Figure 3 (5ul injection);

[0106] Figure 20 Accuracy test of ginsenoside F11 reference solution chromatogram Figure 4 (5ul injection);

[0107] Figure 21 Accuracy test of ginsenoside F11 reference solution chromatogram Figure 5 (5ul injection);

[0108] Figure 22 Chromatogram of sample 1 in the prescription for replacing ginseng with American ginseng in Wuji Baifeng Wan (a traditional Chinese medicine formula) in a repeatability experiment;

[0109] Figure 23 Chromatogram of sample 2 in the prescription of replacing ginseng with American ginseng in reproducibility test of Wuji Baifeng Wan;

[0110] Figure 24 Chromatogram of sample 3 in the prescription for replacing ginseng with American ginseng in repeatability test;

[0111] Figure 25 Chromatogram of sample 4 in the prescription for replacing ginseng with American ginseng in the repeatability experiment;

[0112] Figure 26 Chromatogram of sample 5 of American ginseng in the prescription for replacing ginseng in Wuji Baifeng Wan (a traditional Chinese medicine formula) in a repeatability experiment;

[0113] Figure 27 Chromatogram of sample 6 in the prescription for replacing ginseng with American ginseng in the repeatability experiment;

[0114] Figure 28 Chromatography of reproducible ginsenoside F11 reference solution Figure 1 (20ul injection);

[0115] Figure 29Chromatography of reproducible ginsenoside F11 reference solution Figure 2 (20ul injection);

[0116] Figure 30 Chromatography of reproducible ginsenoside F11 reference solution Figure 1 (5ul injection);

[0117] Figure 31 Chromatography of reproducible ginsenoside F11 reference solution Figure 2 (5ul injection);

[0118] Figure 32 Chromatography of reproducible ginsenoside F11 reference solution Figure 3 (5ul injection);

[0119] Figure 33 Chromatography of reproducible ginsenoside F11 reference solution Figure 4 (5ul injection);

[0120] Figure 34 Chromatography of reproducible ginsenoside F11 reference solution Figure 4 (5ul injection);

[0121] Figure 35 Intermediate precision experiment: Chromatogram of ginseng substitute for ginseng in the prescription of Wuji Baifeng Wan (Experimenter 1);

[0122] Figure 36 Intermediate precision experiment: Chromatogram of sample 2 (experimenter 1) of American ginseng replacing ginseng in the prescription of Wuji Baifeng Wan;

[0123] Figure 37 Intermediate precision test: Chromatography of ginsenoside F11 reference solution Figure 1 (Experimenter 1, 20ul injection);

[0124] Figure 38 Intermediate precision test: Chromatography of ginsenoside F11 reference solution Figure 2 (Experimenter 1, 20ul injection);

[0125] Figure 39 Intermediate precision test: Chromatography of ginsenoside F11 reference solution Figure 1 (Experimenter 1, 5ul injection);

[0126] Figure 40 Intermediate precision test: Chromatography of ginsenoside F11 reference solution Figure 2 (Experimenter 1, 5ul injection);

[0127] Figure 41Intermediate precision test: Chromatography of ginsenoside F11 reference solution Figure 3 (Experimenter 1, 5ul injection);

[0128] Figure 42 Intermediate precision test: Chromatography of ginsenoside F11 reference solution Figure 4 (Experimenter 1, 5ul injection);

[0129] Figure 43 Intermediate precision test: Chromatography of ginsenoside F11 reference solution Figure 5 (Experimenter 1, 5ul injection);

[0130] Figure 44 Intermediate precision experiment: Chromatogram of sample 1 (experimenter 2) of American ginseng replacing ginseng in the prescription of Wuji Baifeng Wan;

[0131] Figure 45 Intermediate precision experiment: Chromatogram of sample 2 (experimenter 2) of American ginseng replacing ginseng in the prescription of Wuji Baifeng Wan;

[0132] Figure 46 Intermediate precision test: Chromatography of ginsenoside F11 reference solution Figure 1 (Experimenter 2, 20ul injection);

[0133] Figure 47 Intermediate precision test: Chromatography of ginsenoside F11 reference solution Figure 2 (Experimenter 2, 20ul injection);

[0134] Figure 48 Intermediate precision test: Chromatography of ginsenoside F11 reference solution Figure 1 (Experimenter injects 2.5 μL of sample);

[0135] Figure 49 Intermediate precision test: Chromatography of ginsenoside F11 reference solution Figure 2 (Experimenter injects 2.5 μL of sample);

[0136] Figure 50 Intermediate precision test: Chromatography of ginsenoside F11 reference solution Figure 3 (Experimenter injects 2.5 μL of sample);

[0137] Figure 51 Intermediate precision test: Chromatography of ginsenoside F11 reference solution Figure 4 (Experimenter injects 2.5 μL of sample);

[0138] Figure 52 Intermediate precision test: Chromatography of ginsenoside F11 reference solution Figure 5 (Experimenter injects 2.5 μL of sample);

[0139] Figure 53 Chromatogram of blank solution for detection limit experiment;

[0140] Figure 54 Chromatogram of ginsenoside F11 reference solution for detection limit experiment;

[0141] Figure 55 Chromatogram of blank solution in the limit of quantitation test;

[0142] Figure 56 Quantitation limit test of ginsenoside F11 reference solution chromatogram Figure 1 ;

[0143] Figure 57 Quantitation limit test of ginsenoside F11 reference solution chromatogram Figure 2 ;

[0144] Figure 58 Quantitation limit test of ginsenoside F11 reference solution chromatogram Figure 3 ;

[0145] Figure 59 Quantitation limit test of ginsenoside F11 reference solution chromatogram Figure 4 ;

[0146] Figure 60 Quantitation limit test of ginsenoside F11 reference solution chromatogram Figure 5 ;

[0147] Figure 61 Quantitation limit test of ginsenoside F11 reference solution chromatogram Figure 6 ;

[0148] Figure 62 : Chromatogram of ginsenoside F11 reference solution (injection volume 25 μL) in linearity assessment experiment;

[0149] Figure 63 : Chromatogram of ginsenoside F11 reference solution (injection volume 20 μL) in linearity assessment experiment;

[0150] Figure 64 : Chromatogram of ginsenoside F11 reference solution (injection volume 16 μL) in linearity assessment experiment;

[0151] Figure 65 : Chromatogram of ginsenoside F11 reference solution (injection volume 12ul) in linear relationship investigation experiment;

[0152] Figure 66 : Chromatogram of ginsenoside F11 reference solution (injection volume 8 μL) in linear relationship investigation experiment;

[0153] Figure 67 : Chromatogram of ginsenoside F11 reference solution (injection volume 6 μL) in linearity assessment experiment;

[0154] Figure 68 : Chromatogram of ginsenoside F11 reference solution (injection volume 4 μL) in linear relationship investigation experiment;

[0155] Figure 69 Linearity range investigation experiment: Chromatography of ginsenoside F11 reference solution Figure 1 ;

[0156] Figure 70 Linearity range investigation experiment: Chromatography of ginsenoside F11 reference solution Figure 2 ;

[0157] Figure 71 Linearity range investigation experiment: Chromatography of ginsenoside F11 reference solution Figure 3 ;

[0158] Figure 72 Linearity range investigation experiment: Chromatography of ginsenoside F11 reference solution Figure 4 ;

[0159] Figure 73 Durability test: Chromatography of ginsenoside F11 reference solution Figure 1 (5ul injection, Waters XDB-C18 column, 250×4.6mm, 5μm);

[0160] Figure 74 Durability test: Chromatography of ginsenoside F11 reference solution Figure 2 (5ul injection, Waters XDB-C18 column, 250×4.6mm, 5μm);

[0161] Figure 75 Durability test: Chromatography of ginsenoside F11 reference solution Figure 3 (5ul injection, Waters XDB-C18 column, 250×4.6mm, 5μm);

[0162] Figure 76 Durability test: Chromatography of ginsenoside F11 reference solution Figure 4 (5ul injection, Waters XDB-C18 column, 250×4.6mm, 5μm);

[0163] Figure 77 Durability test: Chromatography of ginsenoside F11 reference solution Figure 5 (5ul injection, Waters XDB-C18 column, 250×4.6mm, 5μm);

[0164] Figure 78 Durability test: Chromatography of ginsenoside F11 reference solution Figure 1 (20 μL injection, Waters XDB-C18 column, 250 × 4.6 mm, 5 μm);

[0165] Figure 79 Durability test: Chromatography of ginsenoside F11 reference solution Figure 2 (20 μL injection, Waters XDB-C18 column, 250 × 4.6 mm, 5 μm);

[0166] Figure 80 Durability test of Wuji Baifeng Wan test sample solution chromatogram Figure 1 (Batch number 14521002A, chromatographic column WatersXDB-C18, 250×4.6mm, 5μm);

[0167] Figure 81 Durability test of Wuji Baifeng Wan test sample solution chromatogram Figure 2 (Batch number 14521002A, chromatographic column WatersXDB-C18, 250×4.6mm, 5μm);

[0168] Figure 82 Durability test: Chromatography of ginsenoside F11 reference solution Figure 1 (5ul injection, Agilent XDB-C18 column, 250×4.6mm, 5μm);

[0169] Figure 83 Durability test: Chromatography of ginsenoside F11 reference solution Figure 2 (5ul injection, Agilent XDB-C18 column, 250×4.6mm, 5μm);

[0170] Figure 84 Durability test: Chromatography of ginsenoside F11 reference solution Figure 3 (5ul injection, Agilent XDB-C18 column, 250×4.6mm, 5μm);

[0171] Figure 85 Durability test: Chromatography of ginsenoside F11 reference solution Figure 4 (5ul injection, Agilent XDB-C18 column, 250×4.6mm, 5μm);

[0172] Figure 86 Durability test: Chromatography of ginsenoside F11 reference solution Figure 5 (5ul injection, Agilent XDB-C18 column, 250×4.6mm, 5μm);

[0173] Figure 87 Durability test: Chromatography of ginsenoside F11 reference solution Figure 1 (20 μL injection, Agilent XDB-C18 column, 250 × 4.6 mm, 5 μm);

[0174] Figure 88 Durability test: Chromatography of ginsenoside F11 reference solution Figure 2 (20 μL injection, Agilent XDB-C18 column, 250 × 4.6 mm, 5 μm);

[0175] Figure 89 Durability test of Wuji Baifeng Wan test sample solution chromatogram Figure 1 (Batch number 14521002A, Agilent XDB-C18 column, 250×4.6mm, 5μm);

[0176] Figure 90 Durability test of Wuji Baifeng Wan test sample solution chromatogram Figure 2 (Batch number 14521002A, Agilent XDB-C18 column, 250×4.6mm, 5μm);

[0177] Figure 91 Durability test of Wuji Baifeng Wan test sample solution chromatogram Figure 1 (Batch number 14521002A, chromatographic column Shimadzu ODS-3, 250×4.6mm, 5μm);

[0178] Figure 92 Durability test of Wuji Baifeng Wan test sample solution chromatogram Figure 2 (Batch number 14521002A, chromatographic column Shimadzu ODS-3, 250×4.6mm, 5μm);

[0179] Figure 93 Durability test: Chromatography of ginsenoside F11 reference solution Figure 1 (20 μL injection, Shimadzu ODS-3 column, 250 × 4.6 mm, 5 μm);

[0180] Figure 94 Durability test: Chromatography of ginsenoside F11 reference solution Figure 2 (20 μL injection, Shimadzu ODS-3 column, 250 × 4.6 mm, 5 μm);

[0181] Figure 95 Durability test: Chromatography of ginsenoside F11 reference solution Figure 1 (5ul injection, chromatographic column: Shimadzu ODS-3, 250×4.6mm, 5μm);

[0182] Figure 96 Durability test: Chromatography of ginsenoside F11 reference solution Figure 2 (5ul injection, chromatographic column: Shimadzu ODS-3, 250×4.6mm, 5μm);

[0183] Figure 97 Durability test: Chromatography of ginsenoside F11 reference solution Figure 3 (5ul injection, chromatographic column: Shimadzu ODS-3, 250×4.6mm, 5μm);

[0184] Figure 98 Durability test: Chromatography of ginsenoside F11 reference solution Figure 4 (5ul injection, chromatographic column: Shimadzu ODS-3, 250×4.6mm, 5μm);

[0185] Figure 99 Durability test: Chromatography of ginsenoside F11 reference solution Figure 5 (5ul injection, chromatographic column: Shimadzu ODS-3, 250×4.6mm, 5μm); Detailed Implementation

[0186] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0187] Example 1:

[0188] Formula: Wuji Baifeng Wan (Black Chicken White Phoenix Pill), made from 640g of black chicken (with feathers, claws, and intestines removed), 128g of deer antler glue, 64g of vinegar-processed turtle shell, 48g of calcined oyster shell, 48g of mantis egg case, 128g of ginseng, 32g of astragalus, 144g of angelica, 128g of white peony root, 128g of vinegar-processed cyperus rhizome, 64g of asparagus root, 32g of licorice root, 256g of prepared rehmannia root, 256g of rehmannia root, 64g of chuanxiong rhizome, 48g of deer antler powder, 26g of stellaria root, 64g of stir-fried euryale seed, 128g of yam, and 128g of salvia miltiorrhiza.

[0189] Process: According to the formula, eight ingredients—Rehmannia glutinosa (processed), Rehmannia glutinosa, Ligusticum chuanxiong, deer antler powder, Stellaria dichotoma, Euryale ferox (fried), Dioscorea opposita, and Salvia miltiorrhiza—are coarsely ground. The remaining twelve ingredients—black-boned chicken (feathered, claws, and intestines removed), deer antler glue, vinegar-processed turtle shell, calcined oyster shell, mantis egg case, ginseng, Astragalus membranaceus, Angelica sinensis, Paeonia lactiflora, vinegar-processed Cyperus rotundus, Asparagus cochinchinensis, and Glycyrrhiza uralensis—are also chopped and placed in a jar. 1500g of rice wine is added, the jar is sealed, and the mixture is steamed until the wine is absorbed. The mixture is then removed, mixed with the coarse powder, dried at a low temperature, and further ground into a fine powder. The powder is sieved and mixed thoroughly. For every 100g of powder, 30-40g of refined honey and an appropriate amount of water are added to form pills. These pills are then dried to obtain the honey-water pills.

[0190] Quality inspection method: A quality inspection method for ginsenoside F11 in Wuji Baifeng Wan (a traditional Chinese medicine formula).

[0191] Characteristics: This product is a dark brown to black water-honey pill; it tastes sweet and slightly bitter.

[0192] Content determination: Determined by high performance liquid chromatography (General Rule 0512).

[0193] Chromatographic conditions: Octadecylsilane-bonded silica gel as the stationary phase; acetonitrile-water solution (35:65) as the mobile phase; column temperature 35℃; flow rate: 0.9 ml / min; evaporative light detector: gain 30; drift tube temperature: 45℃; nebulizer temperature: 40℃; gas pressure: 30 psi. The theoretical plate number, calculated based on ginsenoside F11, should be no less than 3000.

[0194] Preparation of reference solution: Take an appropriate amount of ginsenoside F11 reference standard, accurately weigh it, and add methanol to prepare a solution containing 100 μg per ml.

[0195] Preparation of the test solution: Take 6g of the water-honey pills of this product, crush them, and accurately weigh them; place them in a stoppered conical flask, accurately add 100ml of 60% methanol solution, stopper tightly, weigh, heat under reflux for 1.5 hours, cool, weigh again, replenish the lost weight with 60% methanol solution, shake well, filter, accurately measure 50ml of the filtrate, place it in an evaporating dish, evaporate to dryness, add 25ml of water to dissolve, add water-saturated n-butanol and shake to extract 4 times, 30ml each time, combine the n-butanol extracts, wash twice with ammonia solution, 50ml each time, then wash with 50ml of n-butanol-saturated water, separate the n-butanol solution, concentrate under reduced pressure, recover the solvent to dryness, add an appropriate amount of methanol to dissolve, transfer to a 2ml volumetric flask, add methanol to the mark, shake well, filter, and take the filtrate, which is the test solution.

[0196] Assay: Accurately pipette 5 μl, 20 μl of reference solution and 10 μl of test solution into the liquid chromatograph, respectively, and determine the content of ginsenoside F11 by using the logarithmic equation of the external standard two-point method.

[0197] This product contains ginsenoside F11(C) 42 H 72 O 14 For water-honey pills, each 1g should not exceed 40μg.

[0198] Example 2:

[0199] Formula: Wuji Baifeng Pills, which are composed of 640 g of black-boned chicken (with feathers, claws and intestines removed), 128 g of deer horn glue, 64 g of stir-fried turtle shell with vinegar, 48 g of calcined oyster, 48 g of membranous wing of mantis, 128 g of ginseng, 32 g of astragalus membranaceus, 144 g of angelica sinensis, 128 g of white peony root, 128 g of cyperus rotundus with vinegar, 64 g of asparagus, 32 g of liquorice root, 256 g of prepared rehmannia root, 256 g of rehmannia root, 64 g of chuanxiong rhizome, 48 g of deer horn cream, 26 g of stellaria dichotoma, 64 g of gorgon fruit (stir-fried), 128 g of Chinese yam, and 128 g of salvia miltiorrhiza.

[0200] Process: According to the formula, first pulverize the eight herbs of prepared rehmannia root, rehmannia root, chuanxiong rhizome, deer horn cream, stellaria dichotoma, gorgon fruit (stir-fried), Chinese yam, and salvia miltiorrhiza into coarse powder. Then, cut the remaining twelve herbs of black-boned chicken (with feathers, claws and intestines removed), deer horn glue, stir-fried turtle shell with vinegar, calcined oyster, membranous wing of mantis, ginseng, astragalus membranaceus, angelica sinensis, white peony root, cyperus rotundus with vinegar, asparagus, and liquorice root into pieces as appropriate, place them in a pot, add 1500 g of yellow rice wine, seal it, stew over water until the wine is completely evaporated, take it out, mix it with the above-mentioned coarse powder, dry it at low temperature, then pulverize it into fine powder, sieve it, and mix it evenly. Add 30 - 40 g of refined honey and an appropriate amount of water to every 100 g of the powder to make pills, and dry them to obtain the water-honey pills.

[0201] Quality inspection method: A quality inspection method for pseudo-ginsenoside F11 in Wuji Baifeng Pills.

[0202] Appearance: This product is water-honey pills in blackish-brown to black color; it tastes sweet and slightly bitter.

[0203] Content determination: Determine according to the high performance liquid chromatography method (General Principles 0512):

[0204] Chromatographic conditions: Use octadecylsilane chemically bonded silica gel as the filler; use acetonitrile - aqueous solution (25:65) as the mobile phase; column temperature is 25 °C; flow rate: 0.8 ml / min; evaporative light scattering detector: gain: 20, drift tube temperature: 40 °C, nebulizer temperature: 35 °C, gas pressure: 20 psi. The theoretical plate number calculated by pseudo-ginsenoside F11 should be not less than 3000.

[0205] Preparation of test solution: Take the water-honey pills of Wuji Baifeng Pills, grind them, take 6 g, accurately weigh it; place it in a stoppered conical flask, accurately add 40 ml of 40% methanol solution, tightly stopper it, weigh it, heat under reflux for 0.5 hour, cool it, weigh it again, make up the lost weight with 40% methanol solution, shake it well, filter it, accurately measure 50 ml of the filtrate, place it in an evaporating dish, evaporate to dryness, add 25 ml of water to dissolve it, extract it with water-saturated n-butanol by shaking for 3 times, each time 10 ml, combine the n-butanol extraction solutions, wash it once with ammonia test solution, each time 20 ml, then wash it with 20 ml of n-butanol-saturated water, separate the n-butanol layer, concentrate it under reduced pressure, recover the solvent to dryness, add an appropriate amount of methanol to dissolve it, transfer it to a 1 ml volumetric flask, add methanol to the scale, shake it well, filter it, and take the continuous filtrate as the test solution.

[0206] This product contains ginsenoside F11(C) 42 H 72 O 14 For water-honey pills, each 1g should not exceed 40μg.

[0207] Example 3:

[0208] Formula: Wuji Baifeng Wan (Black Chicken White Phoenix Pill), made from 640g of black chicken (with feathers, claws, and intestines removed), 128g of deer antler glue, 64g of vinegar-processed turtle shell, 48g of calcined oyster shell, 48g of mantis egg case, 128g of ginseng, 32g of astragalus, 144g of angelica, 128g of white peony root, 128g of vinegar-processed cyperus rhizome, 64g of asparagus root, 32g of licorice root, 256g of prepared rehmannia root, 256g of rehmannia root, 64g of chuanxiong rhizome, 48g of deer antler powder, 26g of stellaria root, 64g of stir-fried euryale seed, 128g of yam, and 128g of salvia miltiorrhiza.

[0209] Process: According to the formula, eight ingredients—Rehmannia glutinosa (processed), Rehmannia glutinosa, Ligusticum chuanxiong, deer antler powder, Stellaria dichotoma, Euryale ferox (fried), Dioscorea opposita, and Salvia miltiorrhiza—are coarsely ground. The remaining twelve ingredients—black-boned chicken (feathered, claws, and intestines removed), deer antler glue, vinegar-processed turtle shell, calcined oyster shell, mantis egg case, ginseng, Astragalus membranaceus, Angelica sinensis, Paeonia lactiflora, vinegar-processed Cyperus rotundus, Asparagus cochinchinensis, and Glycyrrhiza uralensis—are also chopped and placed in a jar. 1500g of rice wine is added, the jar is sealed, and the mixture is steamed until the wine is absorbed. The mixture is then removed, mixed with the coarse powder, dried at a low temperature, and further ground into a fine powder. The powder is sieved and mixed thoroughly. For every 100g of powder, 30-40g of refined honey and an appropriate amount of water are added to form pills. These pills are then dried to obtain the honey-water pills.

[0210] Quality inspection method: A quality inspection method for ginsenoside F11 in Wuji Baifeng Wan (a traditional Chinese medicine formula).

[0211] Characteristics: This product is a dark brown to black water-honey pill; it tastes sweet and slightly bitter.

[0212] Content determination: Determined by high performance liquid chromatography (General Rule 0512).

[0213] Chromatographic conditions: Octadecylsilane-bonded silica gel as the stationary phase; acetonitrile-water solution (35:75) as the mobile phase; column temperature 35℃; flow rate 1.0 ml / min; evaporative light detector: gain 50; drift tube temperature: 75℃; nebulizer temperature: 60℃; gas pressure: 40 psi. The theoretical plate number, calculated based on ginsenoside F11, should be no less than 3000.

[0214] Preparation of reference solution: Take an appropriate amount of ginsenoside F11 reference standard, accurately weigh it, and add methanol to prepare a solution containing 100 μg per ml.

[0215] Preparation of reference solution: Take an appropriate amount of ginsenoside F11 reference standard, accurately weigh it, and add methanol to prepare a solution containing 100 μg per ml.

[0216] Preparation of the test solution: Take 6g of the water-honey pills of this product, crush them, and accurately weigh them; place them in a stoppered conical flask, accurately add 100ml of 80% methanol solution, stopper tightly, weigh, heat under reflux for 3 hours, cool, weigh again, replenish the lost weight with 80% methanol solution, shake well, filter, accurately measure 50ml of the filtrate, place it in an evaporating dish, evaporate to dryness, add 40ml of water to dissolve, add water-saturated n-butanol and shake to extract 5 times, 50ml each time, combine the n-butanol extracts, wash 3 times with ammonia solution, 110ml each time, then wash with 110ml of n-butanol-saturated water, separate the n-butanol solution, concentrate under reduced pressure, recover the solvent to dryness, add an appropriate amount of methanol to dissolve, transfer to a 10ml volumetric flask, add methanol to the mark, shake well, filter, and take the filtrate, which is the test solution.

[0217] Assay: Accurately pipette 5 μl, 20 μl of reference solution and 10 μl of test solution into the liquid chromatograph, respectively, and determine the content of ginsenoside F11 by using the logarithmic equation of the external standard two-point method.

[0218] This product contains ginsenoside F11(C) 42 H 72 O 14 For water-honey pills, each 1g should not exceed 40μg.

Claims

1. A quality detection method for pseudoginsenoside F11 in Wuji Baifeng Pills, characterized in that: The Wuji Baifeng Pills are made of 64 parts of black chicken, 12.8 parts of antler glue, 6.4 parts of vinegar turtle shell, 4.8 parts of calcined oyster, 4.8 parts of silkworm cocoon, 12.8 parts of ginseng, 3.2 parts of astragalus, 14.4 parts of angelica, 12.8 parts of white peony root, 12.8 parts of vinegar cyperus, 6.4 parts of asparagus, 3.2 parts of liquorice, 25.6 parts of prepared rehmannia root, 25.6 parts of rehmannia root, 6.4 parts of chuanxiong, 4.8 parts of antler frost, 2.6 parts of bupleurum, 6.4 parts of stir-fried euryale, 12.8 parts of yam and 12.8 parts of salvia miltiorrhiza, characterized in that: the detection method: (1) Preparation of reference solution: Take an appropriate amount of pseudoginsenoside F11 reference substance, accurately weigh it, and add methanol to make a solution containing 100 μg per 1 ml. (2) Preparation of test solution: Take Wuji Baifeng Pills, grind them, take 6 g, and weigh accurately; put them into a stoppered conical flask, accurately add 40-160 ml of 40-80% methanol solution, stopper tightly, weigh the weight, heat and reflux for 0.5-3 hours, cool, weigh again, make up the lost weight with 40-80% methanol solution, shake well, filter, accurately measure 50 ml of the filtrate, put it into an evaporating dish, evaporate to dryness, and add 10-40 ml of water l to dissolve, add water-saturated n-butanol and shake to extract 3-5 times, 10-50ml each time, combine the n-butanol extracts, wash with ammonia test solution 1-3 times, 20-110ml each time, then wash with n-butanol saturated water 20-110ml, take the n-butanol solution, concentrate under reduced pressure, recover the solvent to dryness, add an appropriate amount of methanol to dissolve, transfer to a 1-10ml volumetric flask, add methanol to the scale, shake well, filter, and take the filtrate, which is the test solution; (3) Chromatographic conditions: octadecylsilane bonded silica gel as filler; mobile phase: acetonitrile: aqueous solution = 20-40:60-80; column temperature 20-40°C; flow rate: 0.6-1.2 ml / min; evaporative light detector: gain: 10-55, drift tube temperature: 30-90°C, nebulizer temperature: 30-80°C, gas pressure: 15-45 psi; the theoretical plate number calculated based on pseudoginsenoside F11 should be no less than 3000; (4) Accurately pipette 5 μl and 20 μl of the reference solution and 10 μl of the test solution respectively, inject them into the liquid chromatograph, and measure according to the chromatographic conditions of step (3) to calculate the content of pseudoginsenoside F11.

2. The quality detection method of pseudoginsenoside F11 in Wuji Baifeng Pills according to claim 1, characterized in that: In step (1): Preparation of reference solution: accurately weigh the reference substance of pseudoginsenoside F11, and add methanol to prepare a solution containing 100 μg per 1 ml.

3. The quality detection method of pseudoginsenoside F11 in Wuji Baifeng Pills according to claim 1, characterized in that: In step (2): Preparation of the test solution: Take Wuji Baifeng Pills, grind them, take 6 g, and weigh accurately; put them in a stoppered conical flask, accurately add 50-150 ml of 50-70% methanol solution, stopper tightly, weigh the weight, heat and reflux for 0.5-2 hours, cool, weigh again, make up the lost weight with 50-70% methanol solution, shake well, filter, accurately measure 50 ml of the filtrate, put it in an evaporating dish, evaporate to dryness, add water for 20-3 0ml to dissolve, add water-saturated n-butanol and shake to extract 3-5 times, 20-40ml each time, combine the n-butanol extracts, wash with ammonia test solution 1-3 times, 25-100ml each time, then wash with n-butanol saturated water 30-100ml, take the n-butanol solution, concentrate under reduced pressure, recover the solvent to dryness, add an appropriate amount of methanol to dissolve, transfer to a 2-5ml volumetric flask, add methanol to the scale, shake well, filter, and take the filtrate, which is the test solution.

4. The quality detection method of pseudoginsenoside F11 in Wuji Baifeng Pills according to claim 1 or 3, characterized in that: In step (2): Preparation of the test solution: Take Wuji Baifeng Pills, grind them, take 6 g, and weigh accurately; place in a stoppered conical flask, accurately add 100 ml of 60% methanol solution, stopper, weigh, heat and reflux for 1.5 hours, cool, weigh again, make up the lost weight with 60% methanol solution, shake well, filter, accurately measure 50 ml of the filtrate, place in an evaporating dish, evaporate to dryness, add 25 ml of water to dissolve, add saturated n-butanol with water and shake to extract 4 times, 30 ml each time, combine the n-butanol extracts, wash twice with ammonia test solution, 50 ml each time, then wash with 50 ml of n-butanol saturated water, take the n-butanol solution, concentrate under reduced pressure, recover the solvent to dryness, add an appropriate amount of methanol to dissolve, transfer to a 2 ml volumetric flask, add methanol to the mark, shake well, filter, and take the filtrate, which is the test solution.

5. The quality detection method of pseudoginsenoside F11 in Wuji Baifeng Pills according to claim 1, characterized in that: In step (3): chromatographic conditions: octadecylsilane bonded silica gel as filler; mobile phase of acetonitrile: aqueous solution = 25-35:65-75; column temperature 25-35°C; flow rate: 0.8-1 ml / min; evaporative light detector: gain: 20-50, drift tube temperature: 40-80°C, nebulizer temperature: 35-75°C, gas pressure: 20-40 psi; the theoretical plate number calculated based on pseudo-ginsenoside F11 should be no less than 3000.

6. The quality detection method of pseudoginsenoside F11 in Wuji Baifeng Pills according to claim 1 or 5, characterized in that: In step (3): Chromatographic conditions: octadecylsilane bonded silica gel as filler; mobile phase acetonitrile: aqueous solution = 35:65; column temperature 35°C; Flow rate: 0.9 ml / min; Evaporative light detector: gain: 30, drift tube temperature: 45 °C, nebulizer temperature: 40 °C, gas pressure: 30 psi; The theoretical plate number calculated based on pseudo-ginsenoside F11 should be no less than 3000.

7. The quality detection method of pseudoginsenoside F11 in Wuji Baifeng Pills according to claim 1, characterized in that: In step (4): 5 μl and 20 μl of the reference solution and 10 μl of the test solution are respectively accurately aspirated and injected into a liquid chromatograph, and the determination is performed according to the chromatographic conditions of step (3). The content of pseudoginsenoside F11 is calculated using an external standard two-point method logarithmic equation to obtain the result.

Images