Determination of the contents of astragaloside I and astragaloside I in Beiqi Yifei tablets by HPLC

Abstract

The invention discloses a method for determining the content of flavonoids A and astragaloside A in Beiqi Yifei tablets by high performance liquid chromatography, belongs to the field of drug analysis technology, and comprises the following steps: S1, preparation of reference solution; S2, preparation of test solution; S3, measuring 10 μl of reference stock solution I, reference stock solution II and test solution in sequence, and injecting into high performance liquid chromatograph for determination. The method for determining the content of flavonoids A and astragaloside A in Beiqi Yifei tablets by high performance liquid chromatography of the present invention is simple and fast to operate, replaces the existing standard thin layer chromatography method for determining the content of astragaloside A, and adds the content determination item of alcohol extraction medicinal flavor flavonoids A, and one operation can complete two content determinations at the same time, the method is simple and fast, and the content of flavonoids A and astragaloside A is determined accurately, with high sensitivity, and the method for evaluating Beiqi Yifei tablets is more comprehensive, further ensuring the product quality and efficacy, and meeting the requirements of drug quality control.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis, and in particular to a method for determining the contents of astragaloside I and astragaloside I in Beiqi Yifei tablets by using a high performance liquid chromatography method. Background Art

[0002] Beiqi Yifei Tablets are derived from the prescription of Mr. Chen Qiuling in Linyi, Shandong. This prescription has been used to treat tuberculosis in Shandong and Northeast China for nearly a hundred years. It was later passed on to his eldest son, Mr. Chen Zuoen. During his medical practice, Mr. Chen Zuoen combined the principles of traditional Chinese medicine treatment and the characteristics of tuberculosis at that time, and continuously improved and perfected the family prescription powder, gradually forming a fixed formula and making it into Chinese patent medicine honey pills for clinical use. In 2002, it was introduced by Tonghua Tuberculosis Hospital with a definite effect. In 2003, it was awarded the Jilin Provincial Hospital Preparation Number (Jilin Medicine Preparation Z03A0004). After treatment and observation of different types of tuberculosis patients, this variety of medicine alone can significantly improve the patient's physical condition and clinical symptoms, promote lesion absorption and cavity closure; combined with chemotherapy drugs can reduce their toxic side effects, enable patients to successfully complete conventional chemotherapy, and accelerate sputum bacteria negative conversion and lesion absorption.

[0003] In the prescription, Astragalus membranaceus greatly nourishes the spleen and lung qi, nourishes the lung yin, nourishes the spleen and helps the lung to help the source of biochemistry, and can also consolidate the surface and stop sweating as the main medicine, Ophiopogon japonicus nourishes yin and nourishes the blood, and Lilium obtains the qi of earth and metal to moisten the lungs and stop coughing, and can also clear the deficiency heat in the lungs, which are all the minister medicines, assisted by Fritillaria thunbergii to moisten the lungs and stop coughing, Stemona tuber and Bletilla striata to nourish the lungs and kill insects, Panax notoginseng to resolve blood stasis and stop bleeding, Platycodon grandiflorum to open and spread lung qi, and remove phlegm and discharge pus; Panax notoginseng is an important hemostatic medicine, which stops bleeding without leaving blood stasis. It is used together with Fritillaria thunbergii and Bletilla striata to strengthen the lungs, seal the damage, remove dead muscles, and disperse blood stasis, which has the wonderful effect of supporting the old and creating the new. Houttuynia cordata, Picrorhizon, and Cat's Claw are used to clear heat and disperse knots, resolve phlegm and stop coughing. The above medicines work together to play the role of nourishing qi and yin, moistening the lungs and stopping coughing, and promoting muscle growth and stopping bleeding. Attack and supplement are used at the same time, and all symptoms disappear.

[0004] Modern research shows that astragalus contains a variety of chemical components, such as saponins, flavonoids, lignans, amino acids, polysaccharides and various trace elements. The triterpenoid saponin compound it contains, astragaloside IV, is the main active ingredient of astragalus and the main active ingredient of Beiqi Yifei Tablets. Therefore, as a means of evaluating the quality standard of Beiqi Yifei Tablets, the accurate determination of astragaloside IV content is the key technology to ensure the efficacy of the product.

[0005] However, the current quality standard of Beiqi Yifei Tablets uses thin layer chromatography scanning to determine the content of astragaloside IV. However, the thin layer chromatography scanning method is cumbersome to operate, and there are many factors that affect it, and the results are not accurate enough. For example, during thin layer chromatography, the development temperature, the saturation of the development system, the size of the silica gel plate particles used, the tightness of the silica gel plate, etc. will affect the determination of the content of astragaloside IV. Although the co-thin layer method eliminates some errors, the reproducibility between experiments is poor. In addition, in order to eliminate the interference of other components and background interference, multiple steps of separation and purification must be performed, and the loss of astragaloside IV is also high during the purification process. At the same time, the current quality standard only contains one item of astragaloside IV for content determination, and the method for evaluating Beiqi Yifei Tablets is not comprehensive enough to ensure product quality and efficacy. Summary of the invention

[0006] In view of the above technical problems, the present invention provides a method for determining the contents of astragaloside I and astragaloside I in Beiqi Yifei tablets by high performance liquid chromatography.

[0007] In order to achieve the above object, the technical solution provided by the present invention is as follows:

[0008] A method for determining the content of astragaloside I in Beiqi Yifei tablets by high performance liquid chromatography comprises the following steps:

[0009] S1. Preparation of reference solution: weigh 10 mg of each of the reference substances of Fritillaria sutchuenensis and Astragaloside IV, place in different 10 mL volumetric flasks, add methanol to dissolve and dilute to scale, shake well, and obtain reference substance stock solution I; measure appropriate amount of each reference substance stock solution I, mix well, and dilute with methanol to obtain reference substance stock solution II containing 50 μg / mL of Fritillaria sutchuenensis and 100 μg / mL of Astragaloside IV;

[0010] S2. Preparation of test solution: weigh 0.5000 g of sample, add methanol for Soxhlet extraction for 4 hours, concentrate under reduced pressure to 10 mL, and filter to obtain the solution;

[0011] S3, measure 10μl of reference substance stock solution I, reference substance stock solution II and test solution, inject into high performance liquid chromatography for determination, calculate with external standard two-point method logarithmic equation, and get the determination result. Each tablet of this product contains astragaloside C 41 H 68 O 14 The amount should not be less than 0.04mg. Each tablet of this product contains Fritillaria cirrhosae and Fritillaria acetoin A C. 41 H 68 O 14 It shall not be less than 0.05mg.

[0012] Wherein, the Soxhlet extraction temperature in step S2 is 110°C.

[0013] In step S3, the detector of the high performance liquid chromatograph is an evaporative light scattering detector.

[0014] The HPLC conditions in step S3 are: chromatographic column: C18; mobile phase A: 0.1% formic acid + 0.03% triethylamine aqueous solution; mobile phase B: acetonitrile; column temperature: 35°C; flow rate: 0.8 mL / min; evaporative light scattering detector conditions: drift tube temperature: 75°C; air flow rate: 2.5 mL / min.

[0015] Compared with the prior art, the technical effects of the present invention are:

[0016] The method for determining the contents of Fritillaria sudoriferum and Astragaloside I in Beiqi Yifei tablets by high performance liquid chromatography provided by the present invention is simple and rapid to operate, and replaces the existing standard thin layer chromatography method for determining the content of Astragaloside I, and adds a content determination item of Fritillaria sudoriferum A, an alcohol-extracted medicinal flavor, so that two content determinations can be completed simultaneously in one operation. The method is simple and rapid, and the contents of Fritillaria sudoriferum and Astragaloside I are determined accurately with high sensitivity. The method for evaluating Beiqi Yifei tablets is more comprehensive, and the product quality and efficacy are further guaranteed, and the requirements for drug quality control are met. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For ordinary technicians in this field, other drawings can also be obtained based on these drawings.

[0018] Figure 1 The standard curve of Fritillaria cirrhosae A provided in the embodiment of the present invention;

[0019] Figure 2 The standard curve of Huangshi glycoside provided in the embodiment of the present invention. DETAILED DESCRIPTION

[0020] In order to make those skilled in the art better understand the technical solution of the present invention, the present invention will be further described in detail below in conjunction with the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. The experimental methods in the following embodiments are conventional methods unless otherwise specified. The test materials used in the following embodiments are purchased from conventional biochemical reagent stores unless otherwise specified.

[0021] The sample used was Beiqi Yifei Tablets produced by Tonghua Jitong Pharmaceutical Co., Ltd. (formerly Tonghua Yongcang Pharmaceutical Co., Ltd.).

[0022] [Prescription] Astragalus 200g, Lily 100g, Ophiopogon 100g, Fritillaria 80g

[0023] Stemona 150g Platycodon 100g Picrorhizoma 150g Cat's Claw 120g

[0024] Houttuynia cordata 150g Bletilla striata 100g Panax notoginseng 50g

[0025] [Preparation method] The above eleven herbs, Panax notoginseng and Bletilla striata are crushed into fine powder; Fritillaria thunbergii, Stemona tuberosa and Houttuynia cordata are extracted twice with 70% ethanol, the first time for 2 hours and the second time for 1 hour, the extracts are combined, the ethanol is recovered and concentrated into a clear paste; the residue and the other six herbs including Astragalus membranaceus are decocted with water twice, each time for 1.0 hour, the decoctions are combined, filtered, the filtrate is combined with the above clear paste, concentrated to a thick paste with a relative density of 1.30-1.32 (50℃), Panax notoginseng and Bletilla striata fine powder are added, mixed, dried, crushed into fine powder, added with appropriate amount of starch, made into granules, dried, pressed into 1000 tablets, and film-coated.

[0026] [Properties] This product is a film-coated tablet, which is brown-yellow in color after removing the coating; it tastes slightly bitter.

[0027] The instrument used was Huapu U6000 high performance liquid chromatograph and Tongwei 5800 evaporative light detector.

[0028] Chromatographic column: Welch Ultimate XB-C18 (4.6mm×250mm, 5μm)

[0029] Determined according to the high performance liquid chromatography method of "Chinese Pharmacopoeia" (General Chapter 0512).

[0030] Chromatographic conditions and system suitability test

[0031] Octadecylsilane bonded silica gel was used as filler; 0.1% formic acid + 0.03% triethylamine aqueous solution was used as mobile phase A, acetonitrile was used as mobile phase B, and gradient elution was performed as specified in the following table; evaporative light detector was used for detection; column temperature was 35°C; drift tube temperature was 75°C, flow rate was 0.8 mL·min -1 , air flow rate 2.5ml·min -1 . Perform gradient elution according to the table below:

[0032] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0min 90％ 10％ 5min 90％ 10％ 15min 60％ 40％ 20min 50％ 50％ 25min 30％ 70％ 30min 30％ 70％ 35min 90％ 10％ 45min 90％ 10％

[0033] Preparation of reference stock solution

[0034] Accurately weigh 10 mg each of the reference substances of astragaloside A and astragaloside I, place them in different 10 mL volumetric flasks, add methanol to dissolve and dilute to the scale, shake well, and obtain the reference substance stock solution I.

[0035] Accurately measure appropriate amounts of each reference substance stock solution I, mix well, and dilute with methanol to a concentration of 50 μg·mL of para-fritillary glycoside A. -1 , Astragaloside Ⅳ 100 μg·mL -1 Reference substance stock solution II.

[0036] Preparation of test solution

[0037] Take 15 samples, grind them into powder, weigh them accurately, accurately weigh 0.5000g (accurate to 0.01mg) of uniform sample, put it into the filter paper tube, put the filter paper tube into the extraction tube of Soxhlet extractor, connect it to the fat flask that has been dried to constant weight, add methanol from the upper end of the condenser of the extractor to 2 / 3 of the volume of the flask, pass condensed water, heat to 110℃, extract for 4 hours, concentrate under reduced pressure to 10mL, and filter to obtain.

[0038] Determination method

[0039] Accurately pipette 10 μl of reference substance reserve solution I, reference substance reserve solution II and test solution respectively, inject into liquid chromatograph and measure to obtain the result.

[0040] Each tablet of this product contains astragaloside I (C 41 H 68 O 14 ) shall not be less than 0.04mg.

[0041] Each tablet of this product contains Fritillaria cirrhosae acetoin (C 41 H 68 O 14 ) shall not be less than 0.05mg.

[0042] Example 1 Specificity Experiment

[0043] Take reference substance stock solution I, reference substance stock solution II, and test solution in turn, inject and test according to the chromatographic conditions in 3.2, and record the chromatogram. The results show (Table 1) that reference substance stock solution I, reference substance stock solution II, and test solution do not interfere with the determination.

[0044] Table 1 Specificity test results

[0045]

[0046] Example 2 Sample processing method determination experiment

[0047] Weigh three portions of 0.5000g of the test sample, add water to make the volume 10mL, and treat the test sample by ultrasonic treatment for 4h, heating reflux for 4h, and Soxhlet extraction for 4h. Take the filtrate under each treatment condition, inject and test according to the above chromatographic conditions, and record the chromatogram. The results show (Table 2) that the content of each component in the test sample is the highest under Soxhlet extraction conditions, and the extraction effect is the best.

[0048] Table 2 Experimental results determined by sample processing method

[0049]

[0050]

[0051] Example 3 Linear relationship experiment

[0052] Take the reference stock solution II and dilute it to the concentration of astragaloside Ⅳ of 0, 20, 40, 60, 80, 100 μg·mL -1 , the concentration of Fritillaria cirrhosae was 0, 10, 20, 30, 40, 50 μg·mL -1 The standard solution was injected and tested according to the above chromatographic conditions, the chromatogram was recorded, the peak area was determined, and the standard curve was drawn with the common logarithm of the product of the standard solution concentration and the injection volume as the abscissa and the common logarithm of the peak area as the ordinate. The results showed that within the injection content range of 0 to 10 μg, the linear equation of astragaloside IV was y = 1.5211x + 1.6968, and the correlation coefficient R 2 is 0.9997; in the range of 0-5 μg of injection content, the linear equation of Fritillaria cirrhosae A is y=1.6377x+2.2522, and the correlation coefficient R 2 is 0.9994; each component has a good linear relationship, see Figure 1 and Figure 2 .

[0053] Example 4 Precision Experiment

[0054] Take the reference stock solution II of Fritillaria sutchuenensis and Astragaloside I, inject it continuously 6 times for detection according to the above chromatographic conditions, record the chromatogram, measure the peak area, calculate the corresponding concentration and relative standard deviation (RSD), see Table 3 for details. The results show that the precision of this method is good.

[0055] Table 3 Precision test results

[0056]

[0057]

[0058] Example 5 Reproducibility Experiment

[0059] Take 6 samples from the same batch to prepare the test solution, measure it according to the above chromatographic conditions, record the chromatogram, measure the peak area, and calculate its concentration and relative standard deviation (RSD), as shown in Table 4. The results show that this method has good reproducibility.

[0060] Table 4 Reproducibility test results

[0061]

[0062]

[0063] Example 6 Detection Limit and Quantification Limit

[0064] According to the requirements of the pharmaceutical industry, the detection limit is calculated by taking the ratio of three times the standard deviation to the sensitivity, that is, Dmin = (3Rn / S). The baseline noise is measured without adding samples under the same conditions as when analyzing the standard and samples. In this experiment, the minimum detection limit of each component is 2μg·mL based on three times the baseline noise. -1 The limit of quantification of each component was 5 μg·mL based on ten times the baseline noise. -1 The results are shown in Table 5.

[0065] Table 5 Detection limit and quantification limit

[0066] Detection limit Limit of Quantitation <![CDATA[2μg·mL -1 ]]> <![CDATA[5μg·mL -1 ]]>

[0067] Example 7 Recovery Experiment

[0068] Weigh three samples of 0.5000g in parallel, add 160μL, 200μL, and 240μL of the reference solution according to the low, medium, and high concentrations, respectively, prepare the test solution for the recovery experiment according to the test solution preparation method, take samples for detection, and record the chromatogram. The results are shown in Table 6, and the recovery rates are high at low, medium, and high concentrations.

[0069] Table 6 Recovery test results

[0070]

[0071]

[0072] Example 8 Sample Testing

[0073] Weigh three samples in parallel, prepare the test solution according to the test solution preparation method, take samples for detection, record the chromatogram, and calculate the content of each component in the sample according to the peak area using the external standard method. The results are listed in Table 7, and the results all meet the requirements.

[0074] Table 7 Sample test results

[0075]

[0076] The above description is only by way of illustration of certain exemplary embodiments of the present invention. It is undoubted that those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the above drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A method for determining the content of astragaloside I in Beiqi Yifei tablets by high performance liquid chromatography, characterized in that: The following steps are involved: S1. Preparation of reference solution: weigh 10 mg of each of the reference substances of Fritillaria sutchuenensis and Astragaloside IV, place in different 10 mL volumetric flasks, add methanol to dissolve and dilute to scale, shake well, and obtain reference substance stock solution I; measure appropriate amount of each reference substance stock solution I, mix well, and dilute with methanol to obtain reference substance stock solution II containing 50 μg / mL of Fritillaria sutchuenensis and 100 μg / mL of Astragaloside IV; S2. Preparation of test solution: weigh 0.5000 g of sample, add methanol for Soxhlet extraction for 4 hours, concentrate under reduced pressure to 10 mL, and filter to obtain the solution; S3, measure 10μl of reference substance stock solution I, reference substance stock solution II and test solution, inject into high performance liquid chromatography for determination, calculate with external standard two-point method logarithmic equation, and get the determination result. Each tablet of this product contains astragaloside C 41 H 68 O 14 The amount should not be less than 0.04mg. Each tablet of this product contains Fritillaria cirrhosae and Fritillaria acetoin A C. 41 H 68 O 14 The amount shall not be less than 0.05 mg; wherein, the high performance liquid chromatography conditions are as follows: chromatographic column: C18; Column temperature: 35°C; Mobile phase A: 0.1% formic acid + 0.03% triethylamine aqueous solution; Mobile phase B: acetonitrile; Flow rate: 0.8 mL / min; Gradient elution according to the following table: The detector of the high performance liquid chromatograph is an evaporative light scattering detector; the conditions of the evaporative light scattering detector are as follows: drift tube temperature: 75°C; air flow rate: 2.5 mL / min.

2. The method for determining the content of astragaloside I in Beiqi Yifei tablets by high performance liquid chromatography according to claim 1, characterized in that: The Soxhlet extraction temperature in step S2 is 110°C.

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