A method for detecting the content of multiple components in traditional Chinese medicine for promoting blood circulation and lowering blood sugar

Abstract

This invention provides a method for detecting the content of multiple components in traditional Chinese medicine for promoting blood circulation and lowering blood sugar. This method has the advantages of high stability, accuracy, and reproducibility. The content determination of 15 batches of traditional Chinese medicine for promoting blood circulation and lowering blood sugar showed that the content of 3'-OH puerarin was 0.0923%–0.2317%, the content of puerarin was 0.5320%–0.6843%, the content of puerarin-6-O xyloside was 0.0320%–0.0412%, the content of 3'-methoxypuerarin was 0.0972%–0.1615%, the content of puerarin apigenin was 0.1374%–0.1712%, the content of rutin was 0.1490%–0.3698%, and the content of salvianolic acid was 0.3849%–0.8152%. The multi-index content determination method of this invention can also be used for the internal quality control standards of Tongmai Jiangtang Capsules, further ensuring the stability of the product's intrinsic quality and clinical efficacy.

Description

Technical Field

[0001] This invention belongs to the field of chemical component analysis and detection of traditional Chinese medicine, and in particular relates to a method for detecting the content of multiple components in traditional Chinese medicine for promoting blood circulation and lowering blood sugar. Background Technology

[0002] The blood-activating and blood-regulating capsules described in this invention have the national drug approval number Z20026853. This medicine uses *Codonopsis pilosula* as the principal ingredient, *Salvia miltiorrhiza*, *Coptis chinensis*, and *Astragalus membranaceus* as assistant ingredients, and *Gynostemma pentaphyllum*, *Dioscorea opposita*, *Atractylodes lancea*, *Scrophularia ningpoensis*, *Malva verticillata* fruit, and leeches as adjuvant ingredients. The guiding ingredient is *Pueraria lobata*. The combination of these herbs has the effects of nourishing yin and clearing heat, activating blood circulation and unblocking collaterals, and is used for diabetes caused by deficiency of both qi and yin and obstruction of the blood vessels.

[0003] According to the prior art search and analysis conducted by the applicant, for example, Wu Yujia, Wan Haofang, et al., "Analysis of the components and fingerprint spectrum of Tongmai Jiangtang capsules based on HPLC-Q-Exactive liquid chromatography-mass spectrometry and HPLC", Chinese Traditional and Herbal Drugs, 2022-10-24, published an article that used 0.5% formic acid aqueous solution-0.5% formic acid acetonitrile solution as the mobile phase for gradient elution, column temperature 40℃, and flow rate 0.4 mL / min to detect 15 batches of Tongmai Jiangtang samples and determine the contents of protocatechuic acid, protocatechuic aldehyde, puerarin, 3-methoxypuerarin, puerarin apigenin, daidzein, salvianolic acid B, and tanshinone IIA. Yang Bin, Wang Haile, *Simultaneous Determination of Seven Components in Tongmai Jiangtang Capsules by HPLC Gradient Elution*, *China Modern Chinese Medicine*, 2020-05-14. This paper describes a method using acetonitrile-0.3% formic acid aqueous solution as the mobile phase, with gradient elution and detection wavelengths of 260 nm for the detection of verrucoside, gentianin, and gentianin; and 203 nm for the detection of codonopsis pilosula cyclic peptide B, gypenosides XLIX, gypenosides A, and gypenosides XVII. However, this method has several drawbacks. HPLC technology lacks versatility in production practice and suffers from low reproducibility. Therefore, developing an efficient and practical detection method is urgently needed. Summary of the Invention

[0004] This invention provides a method for detecting the content of multiple indicators of traditional Chinese medicine for promoting blood circulation and lowering blood sugar. This method has the advantages of high stability, accuracy, and reproducibility. The method determines the content of seven pharmacodynamic indicators closely related to clinical efficacy: 3'-hydroxypuerarin, puerarin, puerarin-6-O xyloside, 3'-methoxypuerarin, puerarin apigenin, rutin, and salvianolic acid B, and obtains the optimal chromatographic mobile phase conditions.

[0005] The present invention also determined the content of 15 batches of Tongmai Jiangtang capsules, showing that the content of 3'-OH puerarin was 0.0923%–0.2317%, the content of puerarin was 0.5320%–0.6843%, the content of puerarin-6-O xyloside was 0.0320%–0.0412%, the content of 3'-methoxypuerarin was 0.0972%–0.1615%, the content of puerarin apigenin was 0.1374%–0.1712%, the content of rutin was 0.1490%–0.3698%, and the content of salvianolic acid was 0.3849%–0.8152%. The multi-index content determination method of the present invention can also be used for the internal quality control standards of Tongmai Jiangtang capsules, further ensuring the stability of the product's intrinsic quality and clinical efficacy.

[0006] The technical solution of this invention patent application is as follows:

[0007] A method for detecting the content of multiple components in a traditional Chinese medicine for promoting blood circulation and lowering blood sugar, the method comprising the following steps:

[0008] (1) Preparation of the test solution: Take the Chinese herbal medicine of the present invention, weigh it, add methanol, soak it, sonicate it, cool it, weigh it again, make up the weight loss with methanol, shake it well, filter it, and take the filtrate to obtain the test solution.

[0009] (2) Preparation of reference solution: Weigh 3'-hydroxypuerarin, puerarin, puerarin-6-O xyloside, 3'-methoxypuerarin, puerarin apigenin, rutin, and salvianolic acid B, and prepare a reference solution with methanol.

[0010] (3) Chromatographic conditions and system suitability test:

[0011] Chromatographic column; acetonitrile as mobile phase A, 0.02–0.04% phosphoric acid solution as mobile phase B, gradient elution ratio: 0–10 min, mobile phase A 3% → 10%, mobile phase B 97% → 90%; 10–14 min, mobile phase A 10% → 12%, mobile phase B 90% → 88%; 14–34 min, mobile phase A 12% → 12.5%, mobile phase B 88% → 87.5%; 34–44 min, mobile phase A 12.5% ​​→ 20.9%. Mobile phase B: 87.5% → 79.1%, 44–60 min; Mobile phase A: 20.9% → 23.6%; Mobile phase B: 79.1% → 76.4%, 60–74 min; Mobile phase A: 23.6% → 60%; Mobile phase B: 76.4% → 40%, 74–75 min; Mobile phase A: 60% → 3%; Mobile phase B: 40% → 97%, 75–80 min; Mobile phase A: 3%; Mobile phase B: 97%; Detection wavelengths: 210 nm and 250 nm.

[0012] (4) Content determination: inject the reference solution and the test solution into the liquid chromatograph, measure the content, and perform the corresponding conversion to obtain the content.

[0013] Preferably, the concentration of methanol in steps (1) and (2) of the detection method is 40% to 60%.

[0014] Preferably, the concentration of methanol in steps (1) and (2) of the detection method is 50%.

[0015] Preferably, in step (1) of the detection method, the ultrasonic processing power is 400-600W, the ultrasonic frequency is 30-50kHz, and the ultrasonic time is 40-50 minutes.

[0016] Preferably, in step (1) of the detection method, the ultrasonic processing power is 500W, the ultrasonic frequency is 40kHz, and the ultrasonic time is 45 minutes.

[0017] Preferably, in step (2) of the detection method, the concentrations of 3'-hydroxypuerarin in the reference solution are 0.0039–0.0774 mg / ml, puerarin concentration is 0.0242–0.4834 mg / ml, puerarin-6-O xyloside concentration is 0.0015–0.0306 mg / ml, 3'-methoxypuerarin concentration is 0.0044–0.0873 mg / ml, puerarin apigenin concentration is 0.0065–0.1307 mg / ml, rutin concentration is 0.0041–0.0819 mg / ml, and salvianolic acid B concentration is 0.0188–0.3764 mg / ml.

[0018] As a further preferred embodiment of the present invention, in step (2) of the detection method, the concentrations of 3'-hydroxypuerarin in the reference solution are 0.03 mg / ml, puerarin concentration is 0.10 mg / ml, puerarin-6-O xyloside concentration is 0.10 mg / ml, 3'-methoxypuerarin concentration is 0.05 mg / ml, puerarin apigenin concentration is 0.07 mg / ml, rutin concentration is 0.04 mg / ml, and salvianolic acid B concentration is 0.2 mg / ml.

[0019] Preferably, the chromatographic column used in step (3) of the detection method is: Agilent XDB-C18, 4.6×250mm, 5μm.

[0020] Preferably, the concentration of the mobile phase B phosphoric acid solution in step (3) of the detection method is 0.03%.

[0021] Preferably, the content determination of the traditional Chinese medicine of the present invention is carried out according to the preparation of the test solution in step (1), the preparation of the reference solution in step (2), and the chromatographic conditions in step (3), wherein the content of 3'-OH puerarin is 0.0923%–0.2317%, the content of puerarin is 0.5320%–0.6843%, the content of puerarin-6-O xyloside is 0.0320%–0.0412%, the content of 3'-methoxypuerarin is 0.0972%–0.1615%, the content of puerarin apigenin is 0.1374%–0.1712%, the content of rutin is 0.1490%–0.3698%, and the content of salvianolic acid is 0.3849%–0.8152%.

[0022] To further illustrate the inventiveness of the liquid-phase multi-index pharmacodynamic component detection method of the present invention, the optimized experimental contents of the technical solution of the present invention are summarized as follows.

[0023] 1.1 Experimental pre-design and chromatographic determination results

[0024] 1.1.1 Chromatographic conditions and system suitability test

[0025] Column (Agilent XDB-C) 18 (4.6×250mm, 5μm); using acetonitrile as mobile phase A and 0.03% phosphoric acid solution as mobile phase B, gradient elution was performed according to the specifications in Table 1; the detection wavelengths were 210nm and 250nm; the injection volume was 10μl; and the mobile phase flow rate was 1ml / min.

[0026] Table 1 Elution gradient

[0027]

[0028]

[0029] 1.1.2 Preparation of the test solution

[0030] Take about 0.5g of the powder (passed through a No. 3 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 50ml of 50% methanol, seal tightly, weigh it, soak for 1 hour, sonicate (power 500W, frequency 40kHz) for 45 minutes, cool, weigh it again, replenish the lost weight with 50% methanol, shake well, filter, and collect the filtrate to obtain the product.

[0031] 1.1.3 Determination Method

[0032] Accurately pipette 10 μl each of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0033] 1.1.4 Results

[0034] The chromatographic peaks were not effectively separated, and the gradient elution conditions need to be optimized.

[0035] 1.2 Optimization of the mobile phase

[0036] 1.2.1 Chromatographic conditions and system suitability test

[0037] The chromatographic column (Agilent XDB-C18, 4.6×250mm, 5μm) was used; acetonitrile was used as mobile phase A, and mobile phase B consisted of 0.1% phosphoric acid solution, 0.05% phosphoric acid solution, and 0.11% phosphoric acid solution, respectively, with gradient elution performed according to the specifications in Table 2; the detection wavelengths were 250nm and 210nm; the injection volume was 10μl; and the mobile phase flow rate was 1ml / min.

[0038] Table 2 Elution gradient

[0039]

[0040] 1.2.2 Preparation of the test solution

[0041] Prepare the test solution using the method described in section 1.1.2.

[0042] 1.2.3 Results

[0043] If the separation with 0.03% phosphoric acid water does not significantly change the chromatographic peak shape, while 0.1% phosphoric acid water and 0.11% phosphoric acid water improve the chromatographic peak shape, and the difference between 0.1% phosphoric acid water and 0.11% phosphoric acid water is not significant, so 0.1% phosphoric acid water is selected. It is necessary to optimize the gradient elution conditions of the mobile phase.

[0044] 1.3 Optimization of Gradient Elution Conditions

[0045] 1.3.1 Chromatographic conditions and system suitability test

[0046] The chromatographic column (Agilent XDB-C18, 4.6 × 250 mm, 5 μm) was used; acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B, with gradient elution performed according to the specifications in Table 3; the detection wavelengths were 210 nm and 250 nm; the injection volume was 10 μl, and the mobile phase flow rate was 1 ml / min. The elution conditions were optimized 18 times, as follows, to obtain the optimal gradient elution conditions.

[0047] Table 3 Elution gradient optimization

[0048]

[0049]

[0050]

[0051]

[0052]

[0053]

[0054]

[0055] As can be seen from the experimental results in Table 3, Method 18 is the better gradient elution condition obtained through optimized elution of the gradient process.

[0056] 1.4 Optimization of the preparation method of the test solution

[0057] 1.4.1 Preparation of the test solution

[0058] Take approximately 0.5g of the powder (passed through a No. 3 sieve), accurately weigh it, divide it into 3 parallel portions, place each portion in a stoppered conical flask, accurately add 25ml of methanol, 50% methanol, and 70% methanol respectively, seal tightly, weigh, sonicate (power 500W, frequency 40kHz) for 30 minutes, cool, weigh again, replenish the lost weight with solvent, shake well, filter, and collect the filtrate to obtain the final product.

[0059] 1.4.2 Determination Method

[0060] Accurately pipette 10 μl each of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0061] 1.4.3 Results

[0062] When methanol was used as the extraction solvent, the chromatographic peaks between 19 and 22 min could not be effectively separated. Further optimization of the elution gradient did not solve the problem. The sample content extracted with 50% methanol was slightly higher than that extracted with 70% methanol; therefore, 50% methanol was chosen as the extraction solvent.

[0063] 1.5 Investigation into the amount of extraction solvent used

[0064] 1.5.1 Chromatographic conditions and system suitability test: Chromatographic column (Agilent XDB-C18, 4.6×250mm, 5μm); acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution was performed according to the optimized gradient elution conditions, see Table 4; detection wavelengths were 210nm and 250nm; injection volume was 10μl; mobile phase flow rate was 1ml / min.

[0065] 1.5.2 Preparation of the test solution

[0066] Take approximately 0.5g of the powder (passed through a No. 3 sieve), accurately weigh it, divide it into 3 parallel portions, place them in stoppered conical flasks, accurately add 20ml, 25ml and 30ml of 50% methanol respectively, seal tightly, weigh, sonicate (power 500W, frequency 40kHz) for 30 minutes, cool, weigh again, make up the weight loss with solvent, shake well, filter, and collect the filtrate to obtain the product.

[0067] 1.5.3 Determination Method

[0068] Accurately pipette 10 μl each of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0069] 1.5.4 Results

[0070] The sample content is high when using 25 ml of extraction solvent, so 25 ml of 50% methanol is selected as the extraction solvent.

[0071] 1.6 Selection of Measurement Wavelength

[0072] The peak area measured at a wavelength of 250 nm is greater than that measured at a wavelength of 210 nm, so 250 nm was chosen as the measurement wavelength.

[0073] 1.7 Identification of Chemical Components

[0074] Using reference solutions and spiked recovery experiments, nine components were identified, including 3'-hydroxypuerarin, puerarin-6-O xyloside, 3'-methoxypuerarin, puerarin apigenin, rutin, salvianolic acid B, daidzein, and daidzeinogen.

[0075] The beneficial effects of the technical solution of this invention are as follows:

[0076] (1) Through extensive experimentation and trial, the optimal liquid chromatography conditions were finally found: using 0.02–0.04% phosphoric acid solution as mobile phase B, the gradient elution ratios are as follows: 0–10 min, mobile phase A 3% → 10%, mobile phase B 97% → 90%; 10–14 min, mobile phase A 10% → 12%, mobile phase B 90% → 88%; 14–34 min, mobile phase A 12% → 12.5%, mobile phase B 88% → 87.5%; 34–44 min, mobile phase A 12% → 12.5%. 5%→20.9%, mobile phase B is 87.5%→79.1%, 44~60min, mobile phase A is 20.9%→23.6%, mobile phase B is 79.1%→76.4%, 60~74min, mobile phase A is 23.6%→60%, mobile phase B is 76.4%→40%, 74~75min, mobile phase A is 60%→3%, mobile phase B is 40%→97%, 75~80min, mobile phase A is 3%, mobile phase B is 97%; detection wavelengths are 210nm and 250nm.

[0077] (2) Based on the methodological investigation of the liquid chromatography of this invention, the precision test results of the test solution are as follows: 3'-hydroxypuerarin RSD≤0.89%, puerarin RSD≤0.52%, puerarin-6-O xyloside RSD≤1.39%, 3'-methoxypuerarin RSD≤0.71%, puerarin apigenin RSD≤0.69%, rutin RSD≤1.07%, and salvianolic acid B RSD≤1.38%. The stability test results of the test solution are as follows: 3'-hydroxypuerarin RSD≤0.77%, puerarin RSD≤0.56%, puerarin-6-O xyloside RSD≤0.75%, 3'-methoxypuerarin RSD≤0.39%, puerarin apigenin RSD≤0.16%, rutin RSD≤2.20%, and salvianolic acid B RSD≤1.51%. Accuracy test results: 3'-hydroxypuerarin RSD≤2.52%, puerarin RSD≤2.70%, puerarin-6-O xyloside RSD≤2.49%, 3'-methoxypuerarin RSD≤2.22%, puerarin apigenin RSD≤3.45%, rutin RSD≤2.38%, and salvianolic acid B RSD≤2.51%. Therefore, the multi-index content determination method of this invention has the advantages of high stability, accuracy, and reproducibility.

[0078] (3) Analysis of the content of 15 batches of Tongmai Jiangtang capsules revealed the following: 3'-OH puerarin content ranged from 0.0923% to 0.2317%, puerarin content ranged from 0.5320% to 0.6843%, puerarin-6-O xyloside content ranged from 0.0320% to 0.0412%, 3'-methoxypuerarin content ranged from 0.0972% to 0.1615%, puerarin apigenin content ranged from 0.1374% to 0.1712%, rutin content ranged from 0.1490% to 0.3698%, and salvianolic acid content ranged from 0.3849% to 0.8152%. This multi-index content determination method can also be used for the internal quality control standards of Tongmai Jiangtang capsules, further ensuring the stability of the product's intrinsic quality and clinical efficacy. Attached Figure Description

[0079] Figure 1 chromatogram of negative tanshinone;

[0080] Figure 2 Negative chromatogram of kudzu root;

[0081] Figure 3 The chromatogram of the mixed standard is shown below. Among them, chromatographic peak 1 is 3'-hydroxypuerarin; chromatographic peak 2 is puerarin; chromatographic peak 3 is puerarin-6'-O-xyloside; chromatographic peak 4 is 3'-methoxypuerarin; chromatographic peak 5 is puerarin apigenin; chromatographic peak 6 is rutin; and chromatographic peak 7 is salvianolic acid B.

[0082] Figure 4 High-performance liquid chromatogram of the Tongmai Jiangtang capsule sample. Detailed Implementation

[0083] Unless otherwise defined, the technical or scientific terms used in the specification and claims of this patent application shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0084] Example 1: Construction of the multi-index component content detection method of traditional Chinese medicine for promoting blood circulation and lowering blood sugar according to the present invention

[0085] 1. Experimental Instruments and Materials

[0086] 1.1 Instruments and Equipment

[0087] High-performance liquid chromatography-mass spectrometry (LC-2030CPLUS, Shimadzu, Japan); High-performance liquid chromatography-mass spectrometry (U3000, Thermo Fisher Scientific); High-power ultrasonic cleaner (KQ5200DE, Kunshan Ultrasonic Instrument Co., Ltd.); Electronic balance (TE124S, Sartorius); Electronic balance (SQP-SECURA225D-1CN, Sartorius).

[0088] 1.2 Experimental Materials

[0089] 3'-Hydroxypuerarin (Mansite, batch number MUST-24080119, 98.32%), Puerarin (Lemeitian Pharmaceutical Desite Biotechnology, batch number DSTDG000202, 98%), Puerarin-6-O-xyloside (Shanghai Yuanye Biotechnology, batch number P14D9S77617, 98%), 3'-Methoxypuerarin (Mansite, batch number MUST-24010418, purity 99.11%), Puerarin apigenin (Mansite, batch number MUST-24012503, purity 99.72%), Rutin (Mansite, batch number MUST-23111224, 99.04%), Tanshinone B (China National Institute for Food and Drug Control, batch number 111562-202318, 97.5%). Tongmai Jiangtang Capsules are supplied by Baoding Tianhao Pharmaceutical Co., Ltd.; acetonitrile and methanol are of chromatographic grade, and phosphoric acid and other components are of analytical grade. Tongmai Jiangtang Capsules, manufactured by Hebei Tianhao Pharmaceutical Co., Ltd., have batch numbers 240404, 240602, 240502, 240403, 240401, 240503, 231002, 240601, 231004, 240501, 231103, 231101, 231001, 231102, and 231003.

[0090] 2. Experimental methods and results

[0091] 2.1.1 Chromatographic conditions and system suitability test

[0092] Chromatographic column (Agilent XDB-C18, 4.6×250mm, 5μm); acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, elution gradient shown in Table 4; detection wavelength 250nm; injection volume 10μl; mobile phase flow rate 1ml / min. The theoretical plate number, calculated based on salvianolic acid B, should be no less than 6000. See chromatogram below. Figure 1-4 .

[0093] Table 4 Elution gradient

[0094]

[0095] 2.1.2 Preparation of the reference solution

[0096] Accurately weigh 3'-hydroxypuerarin, puerarin-6-O xyloside, 3'-methoxypuerarin, puerarin apigenin, rutin, and salvianolic acid B to prepare reference solutions with concentrations of 0.03, 0.10, 0.01, 0.05, 0.07, 0.04, and 0.2 mg / ml, respectively.

[0097] 2.1.3 Preparation of the test solution

[0098] Take about 0.5g of the powder (passed through a No. 3 sieve), accurately weigh it, divide it into 3 parallel portions, place each portion in a stoppered conical flask, accurately add 25ml of 50% methanol to each flask, seal tightly, weigh, sonicate (power 500W, frequency 40kHz) for 30 minutes, cool, weigh again, make up the lost weight with solvent, shake well, filter, and collect the filtrate to obtain the product.

[0099] 2.1.4 Determination Method: Accurately pipette 10 μl each of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0100] 2.1.5 Calculation Formula

[0101] In the formula: A 供试品 The peak area of ​​the test solution; A 对照品 c is the peak area of ​​the reference solution; 对照品 The concentration of the reference solution is mg / ml; W 供试品 Weigh the sample for testing.

[0102] 2.2 Verification Content

[0103] 2.2.1 System Adaptability Verification

[0104] 2.2.1.1 Solution Preparation

[0105] (1) Preparation of the test solution

[0106] Prepare one test solution according to the preparation method of the test solution.

[0107] (2) Preparation of reference solution

[0108] Accurately weigh two portions each of 3'-hydroxypuerarin, puerarin-6-O xyloside, 3'-methoxypuerarin, puerarin apigenin, rutin, and salvianolic acid B to prepare reference stock solutions. The preparation results are shown in Table 5. Then, take appropriate amounts of the reference stock solutions and prepare mixed reference solutions. The preparation process is shown in Table 6.

[0109] Table 5 Preparation of reference solutions

[0110]

[0111] Table 6. Preparation of Mixed Standard Solutions

[0112]

[0113] 2.1.1.2 Operation

[0114] After system equilibration, inject blank solvent once, inject reference solution 1 five times consecutively, inject reference solution 2 twice, inject system reconfirmation solution (reference solution 1) once, and inject test solution twice. The injection volume is 10 μl, and the chromatogram is recorded.

[0115] 2.1.1.3 System Suitability Test Results

[0116] The blank solvent and negative control did not interfere with the determination, and the resolution of the chromatographic peak in the test solution was greater than 1.5, indicating that the system suitability met the requirements. The results are shown in Table 7.

[0117] Table 7 System Adaptability Test Results

[0118]

[0119]

[0120] 2.1.1.4 Precision determination of reference standard

[0121] Take 1 ml of the mixed reference solution and inject it into 6 syringes. Calculate the RSD (%) of the peak area. The required RSD is less than 3%. The results of this experiment show that the RSD is less than 3%, indicating that the instrument's precision meets the requirements. The results are shown in Table 8.

[0122] Table 8 Results of Precision Determination of Reference Standards

[0123]

[0124] 2.1.2 Stability

[0125] Take the reference solution and inject it at 0, 6, 10, 24, and 48 h, respectively. Measure the peak area and calculate the RSD (%) of the peak area. The results are shown in Tables 9 and 10. Take the test solution and inject it at 0, 10, 20, 24, 30, and 48 h, respectively. Measure the peak area and calculate the RSD (%) of the peak area.

[0126] Table 9 Results of stability test of reference standards

[0127]

[0128]

[0129] Table 10 Results of Sample Stability Tests

[0130]

[0131] 2.1.3 Precision determination

[0132] 2.1.3.1 Sample repeatability

[0133] (1) Preparation of the test solution

[0134] Experimenter 1 prepared 6 test solutions in parallel according to the preparation method of the test solution.

[0135] (2) Operation

[0136] Inject each sample solution once with an injection volume of 10 μl, record the peak area, and calculate the sample content. The results and calculations are shown in Table 11.

[0137] Table 11 Repeatability test results for Experimenter 1

[0138]

[0139]

[0140] 2.1.3.2 Intermediate Precision

[0141] (1) Preparation of the test solution

[0142] Experimenter 2 prepared 6 test solutions in parallel according to the preparation method of the test solution.

[0143] (2) Operation

[0144] One injection of each test solution was performed, with an injection volume of 10 μl. The peak area was recorded, and the sample content was calculated. The results and calculations are shown in Table 12. The intermediate precision determination results and calculations are shown in Table 13.

[0145] Table 12 Repeatability test results for Experimenter 2

[0146]

[0147]

[0148] Table 13 Results of intermediate precision determination

[0149]

[0150]

[0151]

[0152] 2.1.4 Linear

[0153] 2.1.4.1 Solution Preparation

[0154] (1) Preparation of reference stock solution: according to the preparation method of reference solution.

[0155] (2) Preparation of a series of solubility solutions: Take the reference standard stock solution and dilute it to different concentrations. The concentration configurations are shown in Table 14.

[0156] 2.1.4.2 Operation

[0157] One injection was performed for each linear sample, and the chromatogram was recorded.

[0158] 2.1.4.3 Linearity Test Results

[0159] The linear standard curve, linear range, and correlation coefficient are shown in Tables 14-15.

[0160] Table 14 Standard Curve Concentration Results

[0161]

[0162] Table 15 Results of linearity standard curve, linear range, and correlation coefficient

[0163]

[0164] 2.1.5 Accuracy Experiment

[0165] Accurately measure 0.25 g of the known content of the hypoglycemic and blood vessel-clearing capsule powder, prepare six parallel aliquots, add an appropriate amount of mixed reference solution, and prepare the test solution according to the preparation method of the test solution. Calculate the recovery rate and RSD. For content between 0.1% and 0.01%, the recovery rate should be 85%–110%, and the RSD (%) should be less than 4%; for content between 0.01% and 0.001%, the recovery rate should be 80%–115%, and the repeatability RSD (%) should be less than 6%. The test results show that the requirements are met. The results are shown in Table 16.

[0166] Table 16 Results of Spike Recovery Test

[0167]

[0168]

[0169] 2.1.6 Content Determination

[0170] Fifteen batches of Tongmai Jiangtang Capsules were collected, and test solutions were prepared according to the test solution preparation method. The solutions were injected, peak areas were measured, and the contents were calculated by substituting the results into the linear equation. The average contents of 3'-OH puerarin, puerarin, puerarin-6-O xyloside, 3'-methoxypuerarin, puerarin apigenin, rutin, and salvianolic acid B were 0.1333, 0.6009, 0.0419, 0.1234, 0.1546, 0.3038, and 0.4936%, respectively. The results are shown in Table 17.

[0171] Table 17 Results of content determination in Tongmai Jiangtang Capsules samples

[0172]

[0173]

[0174] 3. Conclusion

[0175] 3.1 The method for determining the content of seven components in Tongmai Jiangtang Capsules was optimized.

[0176] 3.2 Methodological validation was performed for the determination of the content of seven components in Tongmai Jiangtang Capsules. This method can be used for the determination of the content of seven components in Tongmai Jiangtang Capsules.

[0177] 3.3 Content determination was conducted on 15 batches. The content of 7 components varied significantly among the 15 batches. Based on the accuracy of quality control, it is necessary to determine the active ingredients of Tongmai Jiangtang Capsules. Using the active ingredients as indicators, the best quality control method can be selected through big data analysis.

Claims

1. A method for detecting the content of multiple components in a traditional Chinese medicine for promoting blood circulation and lowering blood sugar, characterized in that, The detection method includes the following steps: (1) Preparation of the test solution: Take the Chinese herbal medicine of the present invention, weigh it, add methanol, soak it, sonicate it, cool it, weigh it again, make up the weight loss with methanol, shake it well, filter it, and take the filtrate to obtain the test solution. (2) Preparation of reference solution: Weigh 3'-hydroxypuerarin, puerarin, puerarin-6-O xyloside, 3'-methoxypuerarin, puerarin apigenin, rutin, and salvianolic acid B, and prepare a reference solution with methanol. (3) Chromatographic conditions and system suitability test: The chromatographic column was an Agilent XDB-C18, 4.6 × 250 mm, 5 μm. Acetonitrile was used as mobile phase A, and 0.02–0.04% phosphoric acid solution was used as mobile phase B. The gradient elution ratios were as follows: 0–10 min, mobile phase A 3% → 10%, mobile phase B 97% → 90%; 10–14 min, mobile phase A 10% → 12%, mobile phase B 90% → 88%; 14–34 min, mobile phase A 12% → 12.5%, mobile phase B 88% → 87.5%; 34–44 min, mobile phase A 12.5% ​​→ 20.9%, mobile phase B 87.5% → 79.1%; 44–60 min, mobile phase A 20.9% → 23.6%, mobile phase B 79.1% → 76.4%; 60–74 min, mobile phase B 97% → 8 ... The mobile phase composition was as follows: 74–75 min, mobile phase A 23.6% → 60%, mobile phase B 76.4% → 40%; 74–75 min, mobile phase A 60% → 3%, mobile phase B 40% → 97%; 75–80 min, mobile phase A 3%, mobile phase B 97%; detection wavelengths were 210 nm and 250 nm. (4) Content determination: inject the reference solution and the test solution into the liquid chromatograph, measure the content, and perform the corresponding conversion to obtain the content.

2. The content detection method as described in claim 1, characterized in that, The concentration of methanol in steps (1) and (2) of the detection method is 40% to 60%.

3. The content detection method as described in claim 2, characterized in that, The concentration of methanol in steps (1) and (2) of the detection method is 50%.

4. The content detection method as described in claim 1, characterized in that, The ultrasonic treatment in step (1) of the detection method Power 400-600W, ultrasonic frequency 30-50kHz, ultrasonic time 40-50 minutes.

5. The content detection method as described in claim 4, characterized in that, The ultrasonic treatment in step (1) of the detection method Power 500W, ultrasonic frequency 40kHz, ultrasonic time 45 minutes.

6. The content detection method as described in claim 1, characterized in that, In step (2) of the detection method, the concentrations of 3'-hydroxypuerarin in the reference solution are 0.0039~0.0774 mg / ml, puerarin concentration is 0.0242~0.4834 mg / ml, puerarin-6-O xyloside concentration is 0.0015~0.0306 mg / ml, 3'-methoxypuerarin concentration is 0.0044~0.0873 mg / ml, puerarin apigenin concentration is 0.0065~0.1307 mg / ml, rutin concentration is 0.0041~0.0819 mg / ml, and salvianolic acid B concentration is 0.0188~0.3764 mg / ml.

7. The content detection method as described in claim 6, characterized in that, In step (2) of the detection method, the concentrations of 3'-hydroxypuerarin, puerarin, puerarin-6-O xyloside, 3'-methoxypuerarin, puerarin apigenin, rutin, and salvianolic acid B in the reference solution are 0.03 mg / ml, 0.10 mg / ml, 0.10 mg / ml, 0.05 mg / ml, 0.07 mg / ml, 0.04 mg / ml, and 0.2 mg / ml.

8. The content detection method as described in claim 1, characterized in that, In step (3) of the detection method, the concentration of the mobile phase B phosphoric acid solution is 0.03%.

9. The content detection method as described in claim 1, characterized in that, Following the preparation of the test solution in step (1), the preparation of the reference solution in step (2), and the chromatographic conditions in step (3), the content of the traditional Chinese medicine for promoting blood circulation and lowering blood sugar was determined. The contents of the following were: 3'-OH puerarin (0.0923%~0.2317%), puerarin (0.5320%~0.6843%), puerarin-6-O xyloside (0.0320%~0.0412%), 3'-methoxypuerarin (0.0972%~0.1615%), puerarin apigenin (0.1374%~0.1712%), rutin (0.1490%~0.3698%), and salvianolic acid B (0.3849%~0.8152%).

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