A method for detecting multiple active ingredients of Shuangshitonglin capsules

Abstract

The present application belongs to the technical field of detection, and relates to a detection method for multiple active ingredients of Shishi Tonglin capsules, comprising simultaneously determining the contents of salvianolic acid B, protodioscin, dioscin, hederagenin and tanshinone II A in Shishi Tonglin capsules based on UPLC-QAMS method, or / and simultaneously determining the contents of salvianolic acid B, verbascoside, berberine hydrochloride, pachymic acid A and tanshinone II A in Shishi Tonglin capsules based on HPLC-QAMS method. The present application utilizes the method of combining liquid chromatography with one measurement multiple evaluation, determines one stable, easily obtained and low-priced active ingredient, calculates the contents of other ingredients by using the relative correction factor method, realizes the simultaneous determination of multiple ingredients in Shishi Tonglin capsules, and has the advantages of simple determination method, good accuracy of detection results and reduced detection cost.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of detection, and relates to a detection method for multiple active ingredients of Shishitonglin capsules. BACKGROUND

[0002] The Shishitonglin capsule is derived from the Medical Heart Enlightenment by Cheng Guopeng in the Qing Dynasty, and is formulated by adding and subtracting the Bixie Fenshui Decoction of Cheng. The Shishitonglin capsule has the functions of clearing heat and removing dampness, separating clear and turbid, activating blood and removing stasis, and harmonizing the center and calming the mind. The Cortex Phellodendri in the prescription has the functions of cold and bitter, and can strengthen the body resistance, clear away heat, and remove dampness and toxicity. The Bixie Fenshui can clear heat and remove dampness, separate clear and turbid, and remove damp-heat and turbid from the lower Jiaochong, separate clear and turbid, and dredge the bladder. The two herbs can remove damp-heat and turbid from the lower Jiaochong, separate clear and turbid, and dredge the bladder, and are the monarch herbs. The Herba Bajajao, Indigofera tinctoria L. can clear heat and remove toxicity, and cool blood and remove stasis. The Natrii Carasii Semen and the Plantaginis can clear heat and remove dampness, dredge the urinary bladder, and stop pain. The four herbs can enhance the functions of the monarch herbs, cool blood and remove dampness, and dredge the urinary bladder, and are the minister herbs. The Rhizoma Pinelliae can dispel dampness with fragrance, the Poria cocos can invigorate the spleen and remove dampness, the Atractylodes lancea can transport the spleen and dry dampness, the Radix Salviae Miltiorhizae can cool blood and remove stasis, and the four herbs can invigorate the spleen and stomach, regulate water channels, remove stasis, and dredge stasis, and are the assistant herbs. The whole prescription can clear heat and remove dampness, and remove turbid and dredge the urinary bladder.

[0003] The existing detection method for the active ingredients of the Shishitonglin capsule can only detect one ingredient at a time. Since the Shishitonglin capsule has complex and various ingredients, the single ingredient detection cannot control the quality of the whole preparation. If the content of multiple ingredients is detected for quality control, the time cost and raw material cost are high. In addition, the multiple index control mode is also used to detect multiple ingredients at the same time, but the control products need to be selected, and there are defects such as not easy to obtain, large amount, complex content determination method, and high test cost. SUMMARY

[0004] In view of the technical problem of the existing Shishitonglin capsule detection method being complex, the application provides a detection method for multiple active ingredients of the Shishitonglin capsule. By detecting one stable, easily obtained, and low-cost active ingredient, and using the relative correction factor method to calculate the content of other ingredients, the multiple ingredients in the Shishitonglin capsule are simultaneously detected, the detection method is simple, the detection result is accurate, and the detection cost is reduced.

[0005] In order to achieve the above purpose, the technical scheme adopted by the application is as follows:

[0006] A detection method for multiple active ingredients of the Shishitonglin capsule, the detection method comprises simultaneously detecting the contents of salvianolic acid B, protodioscin, dioscin, hederacoside C, and tanshinone II A in the Shishitonglin capsule based on the UPLC-QAMS method, or / and

[0007] Simultaneously detecting the contents of salvianolic acid B, verbascoside, berberine hydrochloride, pachymic acid A, and tanshinone II A in the Shishitonglin capsule based on the HPLC-QAMS method.

[0008] Simultaneous determination of the contents of Danshensu, protodioscin, dioscin, hederacoside C and tanshinone II A in Shishitonglin Capsules based on UPLC-QAMS method, including the following steps:

[0009] 1) Solution preparation

[0010] 1.1) Respectively take Danshensu reference substance, protodioscin reference substance, dioscin reference substance, hederacoside C reference substance and tanshinone II A reference substance, and prepare a mixed reference substance solution by using methanol.

[0011] 1.2) Take Shishitonglin Capsules, remove the shell, and add methanol to the content to prepare a test sample solution by ultrasonic treatment.

[0012] 2) Obtain a liquid chromatogram

[0013] For the mixed reference substance solution and the test sample solution, respectively use UPLC chromatography to obtain the corresponding mixed reference substance chromatogram and test sample chromatogram.

[0014] 3) Determine the relative correction factor

[0015] From the mixed reference substance chromatogram of step 2), respectively obtain the peak areas of the five active ingredients Danshensu, protodioscin, dioscin, hederacoside C and tanshinone II A, and calculate the relative correction factors of the four active ingredients protodioscin, dioscin, hederacoside C and tanshinone II A respectively with Danshensu as the internal reference.

[0016] 4) QAMS method quantitative detection

[0017] From the test sample chromatogram of step 2), obtain the peak areas of each active ingredient to be tested in the test sample solution, and combine the relative correction factors obtained in step 3) to calculate the contents of Danshensu, protodioscin, dioscin, hederacoside C and tanshinone II A in the test sample solution.

[0018] Further limited, the specific preparation method of the mixed reference substance solution of step 1.1) is: respectively accurately weigh Danshensu reference substance, protodioscin reference substance, dioscin reference substance, hederacoside C reference substance and tanshinone II A reference substance, and prepare mixed reference substance solutions with concentrations of 0.2278 mg / mL, 0.2660 mg / mL, 0.2795 mg / mL, 0.1632 mg / mL and 0.2070 mg / mL respectively by using methanol.

[0019] Further limit, the specific preparation method of the test sample solution of step 1.2) is: 3g of Shishitonglin capsule content is weighed, 50mL of methanol is added, ultrasonic extraction is carried out for 50min, the difference is made up after filtration, and the filtrate is filtered through a 0.22μm microporous filter to obtain the test sample solution.

[0020] Further limit, the conditions of the UPLC chromatography of step 2) are:

[0021] The chromatographic column is an ACQUITY UPLC BEH C 18 column, an ACQUITY UPLC HSS T3 column or a Thermo Hypersil GOLD column; the detection wavelength is 202nm-205nm; the volume flow rate is 0.15mL / min-0.25mL / min; the column temperature is 20℃-30℃; the injection amount is 1μL-12μL, and the theoretical plate number is not less than 4000 calculated by salvianolic acid B; and the mobile phase is acetonitrile (A)-0.1% phosphoric acid water (B) for gradient elution, as shown in the following table:

[0022]

[0023]

[0024] Further limit, the conditions of the UPLC chromatography are:

[0025] The chromatographic column is an ACQUITY UPLC BEH C 18 column, with a specification of 2.1x100mm, 1.7μm; the detection wavelength is 203nm; the volume flow rate is 0.25mL / min; the column temperature is 30℃; and the injection amount is 5μL.

[0026] Further limit, in step 3), the relative correction factor of each component is obtained by taking salvianolic acid B as an internal reference:

[0027] Ingredient Relative correction factor f s / i ]]> Danshensuan B 1.0000 Protopanax Saponins 1.2102 Dioscorea Saponins 1.0681 Hederagenin 0.8754 Tanshinone ⅡA 1.6448

[0028] The application also provides another method for simultaneously determining the contents of salvianolic acid B, verbascoside, berberine hydrochloride, pachymic acid A and tanshinone IIA in Shishitonglin capsules based on HPLC-QAMS, comprising the following steps:

[0029] 1) Solution preparation

[0030] 1.1) The verbascoside control sample, salvianolic acid B control sample, berberine hydrochloride control sample, pachymic acid A control sample and tanshinone IIA control sample are weighed respectively, and a mixed control sample solution is prepared by using methanol;

[0031] 1.2) The Shishitonglin capsules are shelled, the content is added with methanol, and the test sample solution is prepared by ultrasonic treatment.

[0032] 2) Liquid chromatography detection

[0033] The mixed control solution and the test solution are respectively subjected to HPLC chromatography to obtain the mixed control chromatogram and the test chromatogram;

[0034] 3) Determining the relative correction factor

[0035] From the mixed control chromatogram of step 2), the peak areas of the five active ingredients of verbascoside, salvianolic acid B, berberine hydrochloride, pachymic acid A and tanshinone IIA are obtained, and the relative correction factors of the four active ingredients of verbascoside, salvianolic acid B, pachymic acid A and tanshinone IIA are calculated by using the multi-point correction method with berberine hydrochloride as the internal reference;

[0036] 4) QAMS method quantitative detection

[0037] From the test chromatogram of step 2), the peak areas of the active ingredients to be measured in the test solution are obtained, and the relative correction factors obtained in step 3) are combined; the contents of berberine hydrochloride, verbascoside, salvianolic acid B, pachymic acid A and tanshinone IIA in the test solution are calculated respectively.

[0038] Further limitation, the specific preparation method of the mixed control solution of step 1.1) is: the verbascoside control, salvianolic acid B control, berberine hydrochloride control, pachymic acid A control and tanshinone IIA control are respectively accurately weighed, and methanol is used to prepare a mixed control solution with concentrations of 0.0474 mg / mL, 0.2278 mg / mL, 0.0921 mg / mL, 0.13328 mg / mL and 0.1035 mg / mL respectively.

[0039] Further limitation, the specific preparation method of the test solution of step 1.2) is: 2.5 g of Shuangshi Tonglin Capsule content is weighed, 30 mL of 70% methanol is added, the mass is weighed, and after ultrasonic extraction for 45 min, the difference is made up with 70% methanol, and then filtered. The filtrate is filtered through a 0.22 μm microporous filter membrane to obtain the test solution.

[0040] Further limitation, the HPLC chromatography condition of step 2) is:

[0041] The chromatographic column is Welch Ultimate XB-C 18 The column; the mobile phase is acetonitrile (A)-0.02 mol / L potassium dihydrogen phosphate (B); the detection wavelength is 254 nm; the volume flow rate is 1.0 mL / min; the column temperature is 30℃; the injection volume is 10 μL; and the gradient elution is shown in the following table:

[0042]

[0043]

[0044] Further limit, in the HPLC chromatography detection of step 2), the theoretical plate number is not less than 6000 calculated by berberine hydrochloride, and the separation degree of each component is greater than 1.5.

[0045] Further limit, in step 3), using berberine hydrochloride as an internal reference, the relative correction factor of each component obtained by using multi-point correction method is:

[0046] Ingredient Relative correction factor Berberine Hydrochloride 1.0000 Vaccinin 2.8231 Danshensuan B 1.5950 Tremella Acid A 1.5325 Tanshinone ⅡA 0.2935

[0047] Compared with the prior art, the beneficial effects of the present application are:

[0048] 1. The present application utilizes the intrinsic functional relationship and proportional relationship of active ingredients, and uses the common component with cheap and easy-to-obtain reference substance, peak area and relatively stable retention time as the internal reference to calculate the relative correction factor between other active ingredients to be measured, so that the synchronous determination of multiple active ingredients to be measured is realized, and the method has the advantages of low detection cost and high analysis efficiency.

[0049] 2. The present application uses Danshensuan B as an internal reference to simultaneously determine the contents of protodioscin, dioscin, hederacoside C and tanshinone II A in Shuishitonglin Capsules; and uses berberine hydrochloride as an internal reference to determine the contents of verbascoside, Danshensuan B, tenuiculinic acid A and tanshinone II A, thereby establishing a multi-index component quality control mode for Shuishitonglin Capsules and providing a basis for comprehensively evaluating the overall quality, and further ensuring the stability and reliability of clinical medication.

[0050] 3. The present application combines the one measurement multiple evaluation method and liquid chromatography to determine the contents of eight active ingredients in Shuishitonglin Capsules, i.e. Danshensuan B, protodioscin, dioscin, hederacoside C, tanshinone II A, verbascoside, tenuiculinic acid A and berberine hydrochloride, and the detection method is simple and can comprehensively evaluate the overall quality.

[0051] 4. In the present application, by comparing the fingerprints of multiple samples, the corresponding characteristic peaks of active ingredients such as Danshensuan B, protodioscin, dioscin, hederacoside C, tanshinone II A, verbascoside, tenuiculinic acid A and berberine hydrochloride are common to all samples, and the corresponding characteristic peak response values are high and stable, and have good characteristics, which can ensure that the established fingerprint of the present application can more specifically control the quality of the sample, the detection method has good repeatability, the detection result is accurate, stable and has high precision. BRIEF DESCRIPTION OF DRAWINGS

[0052] Figure 1 The chromatogram of the test solution of Shuishitonglin Capsules under the chromatographic conditions of Example 1;

[0053] Figure 2 Chromatogram of the mixed reference solution of Example 1;

[0054] Figure 3 Chromatogram of the test solution of Shishitonglin Capsules under the chromatographic conditions of Example 2;

[0055] Figure 4 Chromatogram of the mixed reference solution of Example 2;

[0056] Figure 5 Fingerprint of 10 batches of Shishitonglin Capsules of Example 1;

[0057] Figure 6 Standard control fingerprint of Example 1;

[0058] Figure 7 Fingerprint of 10 batches of Shishitonglin Capsules of Example 2;

[0059] Figure 8 Standard control fingerprint of Example 2;

[0060] Figure 9 is the fingerprint of the test solution of Example 1 under different ultrasonic time of methanol; Figure 9A ultrasonic extraction for 15 min of methanol; Figure 9B ultrasonic extraction for 30 min of methanol; Figure 9C ultrasonic extraction for 50 min of methanol;

[0061] Figure 10 is the fingerprint of the test solution of Example 1 under different mobile phases; Figure 10A methanol-water system mobile phase; Figure 10B water-acetonitrile system mobile phase, Figure 10C acetonitrile-0.1% phosphoric acid water mobile phase;

[0062] Figure 11 is the fingerprint of the test solution of Example 1 under different detection wavelengths Figure 11A 203 nm analysis wavelength, Figure 11B 254 nm analysis wavelength, Figure 11C 270 nm analysis wavelength;

[0063] Figure 12 is the fingerprint of the test solution of Example 2 under different mobile phases Figure 12C methanol-water, Figure 12D acetonitrile-water, Figure 12E acetonitrile-0.1% phosphoric acid aqueous solution; Figure 12F acetonitrile-0.02 mol / L potassium dihydrogen phosphate; Figure 12G acetonitrile-0.02 mol / L potassium dihydrogen phosphate;

[0064] Figure 13 is the corresponding fingerprint spectrum of the test sample of Example 2 at different wavelengths Figure 13M the corresponding fingerprint spectrum at a wavelength of 270 nm, Figure 13N the corresponding fingerprint spectrum at a wavelength of 254 nm, Figure 13O the corresponding fingerprint spectrum at a wavelength of 345 nm);

[0065] Figure 14 is the corresponding fingerprint spectrum of the test sample at 254 nm (A) and 345 nm (B) single wavelength conditions.

[0066] Figure 15 is the fingerprint spectrum of the peak position of hydroxyphellodendrine, berberine hydrochloride, salvianolic acid B, pachymic acid A, salvianolic acid, verbascoside, tanshinone II A, protodioscin, dioscin in the test sample of Example 2 Figure 15A the peak position fingerprint spectrum of hydroxyphellodendrine; Figure 15B the peak position fingerprint spectrum of berberine hydrochloride; Figure 15C the peak position fingerprint spectrum of salvianolic acid B; Figure 15D the peak position fingerprint spectrum of pachymic acid A; Figure 15E the peak position fingerprint spectrum of salvianolic acid; Figure 15F the peak position fingerprint spectrum of verbascoside; Figure 15G the peak position fingerprint spectrum of tanshinone II A; Figure 15H the peak position fingerprint spectrum of protodioscin; Figure 15I the peak position fingerprint spectrum of dioscin). DETAILED DESCRIPTION

[0067] In order to make the purpose, technical solutions and advantages of the present application clearer, the technical solutions in the embodiments of the present application are described clearly and completely below in combination with the drawings. Obviously, the described embodiments are only some of the embodiments of the present application, not all.

[0068] The present application will be described in detail below in combination with the drawings and embodiments, but this should not be taken as a limitation on the scope of protection of the present application.

[0069] Example 1

[0070] This example is a detection method for multiple active ingredients in Shishitonglin Capsules based on UPLC-QAMS method. Salvianolic acid B is used as an internal reference, and the content of active ingredient A (gymnemic saponin, protodioscin, diosgenin and tanshinone II A) in Shishitonglin Capsules is determined.

[0071] The raw reagents used in this example are as follows:

[0072] Table 1 is a list of raw reagents for Example 1

[0073]

[0074] The instrument used in this example is as follows.

[0075] Table 2 is a list of detection instruments of Example 1

[0076]

[0077]

[0078] The chromatographic column of the ultra-high performance liquid chromatograph is ACQUITY UPLC BEH C 18 column (2.1x100mm, 1.7μm), ACQUITY UPLC HSS T3 column (2.1x100mm, 1.8μm) or Thermo Hypersil GOLD column (2.1x100mm, 1.7μm).

[0079] The detection method of the active ingredients in Shishitonglin Capsules provided by the embodiment based on UPLC-QAMS method comprises the following steps.

[0080] 1) Solution preparation

[0081] Prepare the mixed reference solution of known concentration and the test sample solution of different batches of Shishitonglin Capsules respectively;

[0082] The mixed reference solution is prepared by mixing salvianolic acid B, protodioscin, dioscin, hederagenin, tanshinone II A and methanol.

[0083] Specifically, accurately weigh salvianolic acid B reference substance, protodioscin reference substance, dioscin reference substance, hederagenin reference substance and tanshinone II A reference substance, and add methanol to prepare a mixed reference solution with concentrations of 0.2278mg / mL, 0.2660mg / mL, 0.2795mg / mL, 0.1632mg / mL and 0.2070mg / mL respectively.

[0084] The test sample solution is prepared by adding 50mL of methanol to 3g of Shishitonglin Capsule content, ultrasonic extraction for 50min, constant volume with methanol, and filtration.

[0085] Specifically, take 3.0g of Shishitonglin Capsule content, accurately weigh, place in a conical flask with a stopper, accurately add 50mL of methanol, weigh, ultrasonic extraction for 50min, then make up the difference with methanol, filter, take the filtrate, and pass through a 0.22μm microporous filter membrane to obtain the test sample solution.

[0086] 2) Obtain chromatogram

[0087] The mixed control solution and the test solution were subjected to UPLC chromatography to obtain the mixed control chromatogram and the test chromatogram, respectively.

[0088] Specifically, the test product (Shishitonglin Capsule) chromatogram is shown in Figure 1 , and the mixed control chromatogram is shown in Figure 2 .

[0089] The chromatographic column was an ACQUITY UPLC BEH C 18 column, an ACQUITY UPLC HSS T3 column, or a Thermo Hypersil GOLD column; the detection wavelength was 202-205 nm; the volume flow rate was 0.15-0.25 mL / min; the column temperature was 20-30 °C; the injection volume was 1-12 μL, and the theoretical plate number was not less than 4000 calculated based on Danshensuan B.

[0090] The mobile phase was acetonitrile (A)-0.1% phosphoric acid water (B), and the gradient elution is shown in Table 3.

[0091] Table 3 Gradient elution parameters

[0092]

[0093]

[0094] Further preferably, the chromatographic detection conditions are as follows: the chromatographic column is an ACQUITY UPLC BEH C 18 column (2.1 x 100 mm, 1.7 μm); the mobile phase is acetonitrile (A)-0.1% phosphoric acid water (B) for gradient elution, as shown in Table 3; the detection wavelength is 203 nm; the volume flow rate is 0.25 mL / min; the column temperature is 30 °C; and the injection volume is 5 μL. The mobile phase B is 0.1% phosphoric acid water by mass fraction.

[0095] 3) Calculate the relative correction factor

[0096] From the mixed control chromatogram of step 2), the peak areas of Danshensuan B, Yuanshujiusuan, Shujiansuan, Chongchunjiusuan, and Danshentong II A, five active ingredients, are obtained, respectively, and the relative correction factors f of Yuanshujiusuan, Shujiansuan, Chongchunjiusuan, and Danshentong II A, four active ingredients, are calculated based on Danshensuan B as the internal reference. s / i .

[0097] 3.1), determination of the relative correction factor

[0098] Based on Danshensuan B as the internal reference, the relative correction factor f is calculated according to the formula: s / i

[0099] ​

[0100] (A s C is the peak area of salvianolic acid B in the mixed reference substance chromatogram s A is the mass concentration of salvianolic acid B in the mixed reference substance solution i C is the peak area of other components to be tested in the mixed reference substance chromatogram i A is the mass concentration of other components to be tested in the mixed reference substance solution), the relative correction factors of the other four components are calculated, and the results are shown in Table 4.

[0101] Table 4 relative correction factors of salvianolic acid B as the internal reference (n = 3)

[0102]

[0103]

[0104] In this embodiment, a quantitative method of one test for multiple evaluations is established, and the selection of the internal reference is the key. The criteria for selecting the internal reference are easy to obtain, inexpensive, stable in properties, stable in peak area and retention time, and high content in the test sample. Salvianolic acid B has the above advantages in the test sample solution, and therefore salvianolic acid B is selected as the internal reference in this embodiment.

[0105] 4) Component content detection

[0106] The chromatogram is obtained according to the method of step 2), the content of salvianolic acid B in the test sample solution is determined by the external standard method, and the peak areas of other components to be tested in the test sample solution, i.e., protodioscin, dioscin, hederagenin and tanshinone IIA, are obtained, and the relative correction factor f s / i obtained in step 3) is combined; and the component contents of salvianolic acid B, protodioscin, dioscin, hederagenin and tanshinone IIA in the test sample solution are calculated to be 1.3429-1.6757 mg / g, 0.5497-0.5835 mg / g, 0.6808-0.7715 mg / g, 0.8559-0.9546 mg / g and 0.8759-0.9731 mg / g, respectively.

[0107] Example 2

[0108] In this embodiment, a method for detecting multiple active ingredients in Shishitonglin Capsules based on HPLC-QAMS is provided, in which berberine hydrochloride is used as an internal standard, and the contents of active ingredients B (mucronatin, salvianolic acid B, pachymic acid A and tanshinone IIA) in Shishitonglin Capsules are determined.

[0109] 1. Instruments and materials used

[0110] 1.1 Instruments

[0111] The instruments used in this embodiment are as follows.

[0112] Table 5 is a list of detection instruments used in Example 2

[0113] Name Model Manufacturer High-performance liquid chromatograph UltiMate 3000 type Thermo Fisher Corporation Electronic balance JY3002 Shanghai Puchun Measurement Instrument Co., Ltd. Ultrasonic cleaner PL-S40 FCC RoSH Corporation

[0114] 1.2 Reagents and raw materials

[0115] Table 6 is a list of reagents and raw materials used in Example 2

[0116]

[0117]

[0118] 2. Using the above instruments and materials, the HPLC-QAMS method provided in this embodiment for detecting the active ingredient B in Shishitonglin Capsules includes the following steps:

[0119] 1) Solution preparation

[0120] Prepare a mixed control solution of known concentration and a test sample solution of different batches of Shishitonglin Capsules, respectively;

[0121] The mixed control solution is prepared by dissolving the control samples of verbascoside, salvianolic acid B, berberine hydrochloride, pachymic acid A, and tanshinone II A in methanol solution, respectively.

[0122] Specifically, accurately weigh the control samples of verbascoside, salvianolic acid B, berberine hydrochloride, pachymic acid A, and tanshinone II A, and prepare a mixed control solution with concentrations of 0.0474 mg / mL, 0.2278 mg / mL, 0.0921 mg / mL, 0.13328 mg / mL, and 0.1035 mg / mL, respectively, using methanol.

[0123] The test sample solution is prepared by adding 30 mL of 70% methanol to 2.5 g of the contents of Shishitonglin Capsules, ultrasonically extracting for 45 min, and then diluting with methanol to obtain the filtrate.

[0124] Specifically, take 2.5 g of the contents of Shishitonglin Capsules, accurately weigh, and place in a conical flask with a stopper. Accurately add 30 mL of 70% methanol, weigh, ultrasonically extract for 45 min, and then dilute with 70% methanol to make up the difference in weight, filter, and then pass the filtrate through a 0.22 μm microporous filter membrane to obtain the test sample solution.

[0125] 2) Liquid chromatography detection

[0126] The mixed control solution and the test sample solution are subjected to HPLC chromatography to obtain the corresponding mixed control chromatogram and test sample chromatogram, respectively.

[0127] Specifically, see the chromatogram of the test solution Figure 3 , and see the chromatogram of the mixed reference solution Figure 4 .

[0128] The chromatographic column was Welch Ultimate XB-C 18 (4.5 x 250 mm, 5 μm);

[0129] The mobile phase was acetonitrile (A) - 0.02 mol / L potassium dihydrogen phosphate (phosphoric acid was used to adjust the pH value to about 4) (B), and the gradient elution is shown in Table 7.

[0130] The detection wavelength was 254 nm.

[0131] The volume flow rate was 1.0 mL / min.

[0132] The column temperature was 30°C, and the injection volume was 10 μL.

[0133] Under the chromatographic conditions, the theoretical plate number calculated based on berberine hydrochloride was not less than 6000, and the separation degree of each component was good (separation degree > 1.5).

[0134] Table 7 Gradient elution table

[0135] Time (min) Acetonitrile (A) % 0.02Mol / L of potassium dihydrogen phosphate (B) % 0 15 85 10 19 81 15 25 75 45 25 75 47 48 52 50 62 38 55 63 37 75 63 37 81 68 32 85 15 85

[0136] 3) Calculate the relative correction factor

[0137] From the mixed reference chromatogram of step 2), the peak area corresponding to each of the five active ingredients, berberine hydrochloride, verbascoside, salvianolic acid B, pachymic acid A and tanshinone IIA, was obtained. Taking berberine hydrochloride as the internal standard, the relative correction factors f s / i of verbascoside, salvianolic acid B, pachymic acid A and tanshinone IIA were calculated respectively.

[0138] Taking berberine hydrochloride as the internal standard, the relative correction factor f s / i was calculated according to the formula of Example 1.

[0139] The results are shown in Table 8.

[0140] Table 8 Relative correction factor taking berberine hydrochloride as the internal standard (n = 3)

[0141]

[0142]

[0143] In this example, berberine hydrochloride has the advantages of being easy to obtain, inexpensive, stable in nature, stable in peak area and retention time, and high content in the test solution. Therefore, berberine hydrochloride was selected as the internal standard in this example.

[0144] 4) QAMS method for quantitative detection

[0145] From the chromatogram of the test sample solution of step 2), the peak area of each component to be detected in the test sample solution is obtained, and the relative correction factor f obtained in step 3) is combined s / i ; the hydrochloric acid berberine in the test sample solution is calculated as 1.8546-1.9963 mg / g, the verbascoside is calculated as 0.4009-0.4386 mg / g, the salvianolic acid B is calculated as 4.6373-4.8395 mg / g, the pachymic acid A is calculated as 1.1070-1.1975 mg / g, and the tanshinone II A is calculated as 0.4569-0.4692 mg / g.

[0146] Example 3

[0147] In this embodiment, the salvianolic acid B is used as an internal reference, and the contents of the active components of dianshi tonglin capsules, including diosgenin, dioscin, hederagenin, and tanshinone II A, are determined simultaneously by the method of Example 1.

[0148] Then, the hydrochloric acid berberine is used as an internal reference, and the contents of the active components of dianshi tonglin capsules, including verbascoside, salvianolic acid B, pachymic acid A, and tanshinone II A, are determined by the method of Example 2.

[0149] This embodiment can determine the contents of eight active components in dianshi tonglin capsules, including salvianolic acid B, diosgenin, dioscin, hederagenin, tanshinone II A, verbascoside, pachymic acid A, and hydrochloric acid berberine.

[0150] In order to verify the technical effect of the present application, the applicant has conducted a large number of experiments for verification, which are specifically described as follows:

[0151] 1. Fingerprint spectrum establishment

[0152] 1.1 UPLC fingerprint spectrum establishment of active components A: salvianolic acid B, diosgenin, dioscin, hederagenin, and tanshinone II A

[0153] Take 10 batches of dianshi tonglin capsule contents, labeled as S1-S10, and prepare the test sample solution according to the method of step 1) in Example 1, and continuously sample under the chromatographic conditions (preferred conditions) of step 2) in Example 1, and record the chromatogram Figure 5 . The chromatograms of the 10 batches of samples are imported into the 2012 version of traditional Chinese medicine fingerprint spectrum software for data processing and analysis.

[0154] Specifically, the S1 sample spectrum is used as the reference spectrum, a multi-point correction method (time window width of 0.1 min) is used, and the average method is used to generate the test sample fingerprint spectrum (see Figure 5 ) and the standard control fingerprint spectrum ( Figure 6).

[0155] From Figure 5 the fingerprint analysis of 10 batches of samples, it was found that 66 peaks were common peaks in the chromatograms of all test solutions, therefore, the 66 peaks were determined as common peaks of the contents of Shishi Tonglin capsules. Among them, the separation degree and symmetry of No. 39 component peak (Danshensan B) were good, so it was used as a reference peak (S), and four other component peaks were determined, which were No. 42 component peak (prosaoside), No. 58 component peak (saoside), No. 59 component peak (hederacoside), and No. 63 component peak (Danshenshan II A).

[0156] The similarity of the fingerprint chromatograms of the contents of 10 batches of Shishi Tonglin capsules was calculated, and the results of the similarity of S1-S10 samples are shown in Table 9.

[0157] Table 9 Similarity analysis results of the contents of Shishi Tonglin capsules

[0158]

[0159] The results showed that the similarity of 10 batches of Shishi Tonglin capsules was greater than 0.9, indicating that the quality of the contents of 10 batches of Shishi Tonglin capsules was very stable.

[0160] Further, 10 batches of test samples ( Figure 5 ) were used to identify the common peaks, and the relative retention time was compared with the standard control fingerprint ( Figure 6 ), and five common peaks were determined, which were No. 1 peak (Danshensan B), No. 2 peak (prosaoside), No. 3 peak (saoside), No. 4 peak (hederacoside), and No. 5 peak (Danshenshan II A). The results showed that there was a good correlation between Shishi Tonglin capsules and its component medicinal materials.

[0161] 1.2 Active ingredient B: HPLC fingerprint chromatogram of verbascoside, danshensan B, berberine hydrochloride, pachymic acid A, and danshenshan II A

[0162] Ten batches of contents of Shishi Tonglin capsules were taken, labeled as S1-S10, and the chromatograms were obtained according to the method of Example 2. The chromatograms of 10 batches of samples were imported into the "Traditional Chinese Medicine Fingerprint Software" (2012 edition) software for data processing and analysis, and the chromatogram of S1 sample was set as the reference chromatogram, and the multi-point correction method (time window width of 0.1 min) was used to generate the fingerprint chromatogram ( Figure 7 ) and the control fingerprint chromatogram ( Figure 8 ).

[0163] Through the fingerprint analysis of 10 batches of samples, it was found that there were 158 peaks in the figure which were common chromatographic peaks in the chromatograms of all sample solutions, therefore, the 158 chromatographic peaks were determined as the common peaks of Shishitonglin Capsule contents. Among them, 76 (berberine hydrochloride) had good separation degree and symmetry, so it was used as a reference peak (S), and other 4 component peaks were determined, which were peak 45 (mussaenoside), peak 60 (salvianolic acid B), peak 149 (pachymic acid A) and peak 156 (tanshinone IIA).

[0164] The similarity of the fingerprint of 10 batches of Shishitonglin Capsule contents was calculated, and the results of S1-S10 samples were shown in Table 10.

[0165] Table 10 is the similarity analysis results of Shishitonglin Capsule contents

[0166]

[0167] As shown in Table 10, the similarity of 10 batches of Shishitonglin Capsule was greater than 0.9, indicating that the quality of the contents of 10 batches of Shishitonglin Capsule was very stable.

[0168] Further, 10 batches of test samples ( Figure 7 ) were used to identify the common peaks, and the relative retention time was compared with the standard control fingerprint ( Figure 8 ), and 5 common peaks were determined, which were peak 1 (mussaenoside), peak 2 (salvianolic acid B), peak 3 (berberine hydrochloride), peak 4 (pachymic acid A) and peak 5 (tanshinone IIA), and the results showed that Shishitonglin Capsule had good correlation with its component medicinal materials.

[0169] 2, the extraction method of test sample solution in step 1) and the chromatographic condition screening in step 2)

[0170] 2.1 Detection of active ingredients A: salvianolic acid B, protodioscin, dioscin, hederagenin and tanshinone IIA

[0171] 2.1.1 Extraction method screening

[0172] The screening of different extraction methods of test sample solution was investigated.

[0173] Different extraction methods (ultrasonic extraction, reflux extraction), different extraction solvents (analytical pure methanol, analytical pure ethanol, 70% methanol, 70% ethanol), different extraction time (15 min, 30 min, 50 min).

[0174] Referring to Figure 9, the fingerprint of the test sample solution obtained by ultrasonic extraction with analytical pure methanol for 15 min, 30 min and 50 min was obtained. Figure 9Aultrasonic extraction for 15 min with methanol; Figure 9B ultrasonic extraction for 30 min with methanol; Figure 9C ultrasonic extraction for 50 min with methanol; in Fig. 9, peak 1 (salvianolic acid B), peak 2 (prosaikosaponin), peak 3 (saikosaponin), peak 4 (hederacoside), and peak 5 (tanshinone II A)

[0175] The results show that the ultrasonic extraction is easier to filter than the reflux extraction; when methanol is used as the extraction solvent, the target component peaks are less interfered, the peak shape of each target component in the obtained fingerprint spectrum is better, and the separation degree is good. Referring to Fig. 9, the peak area of the obtained fingerprint spectrum of the sample prepared by the extraction for 15 min and the sample prepared by the extraction for 30 min is smaller, indicating that the extraction rate is lower, and the peak area of the fingerprint spectrum of the sample prepared by the extraction for 50 min is larger, indicating that the extraction rate is higher, so the preparation method of the test sample solution is selected as ultrasonic extraction for 50 min with methanol.

[0176] 2.1.2 Mobile phase screening

[0177] The present application investigates different systems of mobile phases, such as methanol-water solution, acetonitrile-water solution, methanol-0.1% formic acid aqueous solution, acetonitrile-0.1% formic acid aqueous solution, methanol-0.1% phosphoric acid aqueous solution, and acetonitrile-0.1% phosphoric acid aqueous solution. Other conditions adopt the chromatographic optimization conditions in Example 1. Taking the separation effects of the measured components salvianolic acid B, prosaikosaponin, saikosaponin, hederacoside, and tanshinone II A in Shuishitonglin capsules as indexes, the chromatogram results corresponding to part of the mobile phases are given. Referring to Fig. 10, wherein Figure 10A is the methanol-water system mobile phase; Figure 10B is the water-acetonitrile system mobile phase, Figure 10C is the acetonitrile-0.1% phosphoric acid water mobile phase. In Fig. 10, peak 1 (salvianolic acid B), peak 2 (prosaikosaponin), peak 3 (saikosaponin), peak 4 (hederacoside), and peak 5 (tanshinone II A).

[0178] Referring to Fig. 10, the results show that when the methanol-water system ( Figure 10A ) is selected, the peak time of the five target components is longer, and the detection efficiency is lower; when the water-acetonitrile system ( Figure 10B ) is selected, the chromatographic peak of prosaikosaponin is bifurcated, which may be because there are two molecular states of prosaikosaponin in pure water, and the two molecular states are weakly separated under the chromatographic conditions, resulting in inaccurate quantification; when the acetonitrile-0.1% formic acid aqueous solution is selected ( Figure 10CUnder these chromatographic conditions, hedyotis diffusin did not produce a peak, affecting the experimental results. When acetonitrile-0.1% phosphoric acid aqueous solution was selected as the mobile phase, the analysis time for the five target components was shorter, the chromatographic peak shapes were better, the separation between peaks was good, and the baseline was more stable. Therefore, acetonitrile-0.1% phosphoric acid aqueous solution was selected as the mobile phase.

[0179] 2.1.3 Selection of detection wavelength

[0180] This invention measures a wide variety of chemical components; therefore, sample spectra at wavelengths of 203, 254, and 270 nm were examined. See Figure 11. Figure 11A The analysis wavelength is 203nm. Figure 11B The analysis wavelength is 254nm. Figure 11C The analysis wavelength is 270 nm. In Figure 11, peak 1 (tanshinone B), peak 2 (prodiosgenin), peak 3 (diosgenin), peak 4 (hederonogenin), and peak 5 (tanshinone IIA).

[0181] Through comparative analysis, at a wavelength of 254nm ( Figure 11B Under these conditions, the peak shape is poor, the baseline is unstable, and the peak areas of the target components, salvianolic acid B, protodiosgenin, and tanshinone IIA, are small. Diosgenin and hedyotis diffusive substances do not produce peaks and there are many impurity peaks. At a wavelength of 270 nm... Figure 11C Under these conditions, the number of peaks is relatively small, and the peak areas of the target components, salvianolic acid B and protodiosgenin, are small. Diosgenin, hederaponin, and tanshinone IIA do not show any peaks. (203nm wavelength) Figure 11A Compared with the 254nm and 270nm wavelengths, the 203nm wavelength has more ideal peaks, peak shape, resolution, symmetry factor and theoretical plate number. The results show that more peaks are detected in the spectrum at the 203nm wavelength, the peak areas of the five target components are large, the baseline is stable, the peak shape is good and there is less interference. Therefore, 203nm was selected as the wavelength for content analysis.

[0182] 2.2 Detection of active ingredient B: verbascoside, salvianolic acid B, berberine hydrochloride, pachymetic acid A, tanshinone IIA

[0183] 2.2.1 Mobile phase screening

[0184] This experiment used high separation degree, significant separation, and a theoretical plate number meeting the standard as the basic objectives for separating the target components. Methanol-water solution, acetonitrile-water solution, acetonitrile-0.1% phosphoric acid water solution, and acetonitrile-0.02 mol / L potassium dihydrogen phosphate solutions were investigated as mobile phases. Referring to Figure 12, the investigation revealed that:

[0185] Methanol-water: The test sample showed virtually no obvious peak shape, and no obvious component peaks were observed. Figure 12C )

[0186] Acetonitrile-water: The test sample peaks were prominent, but the separation between component peaks was not high, and time stacking occurred, so that the characteristic peaks were not accurately identified using the external standard method. Figure 12D

[0187] Therefore, methanol was excluded as the organic phase, and acetonitrile was used with other aqueous solutions to form the mobile phase.

[0188] Acetonitrile-0.1% phosphoric acid aqueous solution: The test sample peaks were prominent, but the peak shape was crossed, the separation was low, the number was relatively small, and the peaks were not completely separated. Figure 12E

[0189] Acetonitrile-0.02 mol / L potassium dihydrogen phosphate: The test sample solution peaks were relatively good in shape and high in separation between component peaks, but some component peaks were tailing. Figure 12F

[0190] Acetonitrile-0.02 mol / L potassium dihydrogen phosphate (phosphoric acid adjusted to a pH of about 4): The test sample had more peaks, good peak shape, high separation, no tailing, and more components were separated. Figure 12G

[0191] From the above Figure 12C-Figure 12G it can be seen that when acetonitrile-0.02 mol / L potassium dihydrogen phosphate (phosphoric acid adjusted to a pH of about 4) is used as the mobile phase, the component peaks are well separated, the peak shape is good, and the baseline is flat, achieving the purpose of complete separation of components.

[0192] 2.2.2 Optimization of elution program screening

[0193] In this experiment, the high separation of target components, the significant separation, and the theoretical plate number meeting the standard were used as the basic target for separating components, and the components were separated as comprehensively as possible. Therefore, the component exploration experiment was carried out from small polarity to large polarity in the early stage.

[0194] Here are some representative examples of optimized gradient programs:

[0195] Table 11: Gradient 12

[0196]

[0197]

[0198] Table 12: Gradient 18

[0199] Time Acetonitrile 0.02mol / L of potassium dihydrogen phosphate (phosphoric acid adjusts pH value to about 4) 0 10 90 5 15 85 8 25 75 23 25 75 25 48 52 27 63 37 67 68 32 74 70 30 80 72 28 90 10 90

[0200] Table 13: Gradient 21

[0201] Time Acetonitrile 0.02mol / L of potassium dihydrogen phosphate (phosphoric acid adjusts pH value to about 4) 0 10 90 10 18 85 15 25 75 40 25 75 45 48 52 50 48 52 55 52 48 60 57 43 63 63 37 70 10 90

[0202] ​​​​Table 14: Gradient 24-14

[0203]

[0204]

[0205] Table 15: Gradient 26

[0206] Time Acetonitrile 0.02mol / L of potassium dihydrogen phosphate (phosphoric acid adjusts pH value to about 4) 0 15 85 10 19 81 15 25 75 45 25 75 47 48 52 50 62 38 55 63 37 75 63 37 81 68 32 85 15 85

[0207] According to the above table, under gradient 26, the separation degree of each component is high, the separation effect is good, and the peak shape is better, so gradient method 26 is selected for determination.

[0208] 2.2.3 Selection of detection wavelength

[0209] The liquid chromatograph used in the application is equipped with a VWD detector, which is a variable wavelength detector. In the active ingredient B, the maximum absorption wavelength of salvianolic acid B, tanshinone II A and pachymic acid A is about 270 nm; berberine hydrochloride is about 345 nm; and verbascoside is about 254 nm. Therefore, the variable wavelength method is used for determination at the beginning of the test, such as Figure 13M-Figure 13O ), Fig. 13 is the corresponding fingerprint spectrum of the test sample in example 2 under different wavelengths, Figure 13M the corresponding fingerprint spectrum under the condition of wavelength 270 nm, Figure 13N the corresponding fingerprint spectrum under the condition of wavelength 254 nm, Figure 13O the corresponding fingerprint spectrum under the condition of wavelength 345 nm.

[0210] As shown in Fig. 13, after using multi-band determination, baseline shift occurs because the two wavelength spans switched are large; at the same time, components are being out of peak at the time of switching wavelength, which will affect the absorption of the component and thus affect the accuracy of determination. Therefore, single wavelength determination is carried out at 254 nm and 345 nm (see Figs. 14 and 15, respectively). Figure 14A 、 Figure 14B )。

[0211] Considering that 345 nm is the best absorption wavelength of berberine hydrochloride, but there is still absorption under 254 nm; the remaining to-be-tested components have the maximum absorption at about 254 nm, therefore, 254 nm is used as the detection wavelength for determination.

[0212] 2.2.4 Selection of internal reference

[0213] The experiment was analyzed by determining 10 batches of Shuangshi Tonglin Capsules. Berberine hydrochloride, verbascoside, salvianolic acid B, pachymic acid A, and tanshinone II A were common to most samples. The response values of the above five characteristic peaks were high, stable, and had good characteristics. Therefore, the five peaks were determined as common peaks of the fingerprint to establish a method for the determination of fingerprint by multi-attribute determination. See Figure 15A-15I , Figure 15A The peak position fingerprint of phellodendrine hydrochloride is shown in the following table: Figure 15B The peak position fingerprint of berberine hydrochloride is shown in the following table: Figure 15C The peak position fingerprint of salvianolic acid B is shown in the following table: Figure 15D The peak position fingerprint of pachymic acid A is shown in the following table: Figure 15E The peak position fingerprint of paeonol is shown in the following table: Figure 15F The peak position fingerprint of verbascoside is shown in the following table: Figure 15G The peak position fingerprint of tanshinone II A is shown in the following table: Figure 15H The peak position fingerprint of protodioscin is shown in the following table: Figure 15I The peak position fingerprint of dioscin is shown in the following table: Figure 15A-15I As can be seen, although phellodendrine hydrochloride peaks in the test sample, the peak shape is not good; in the test sample chromatography, there is a large amount of component accumulation at the relative position of paeonol, which is not completely separated; protodioscin and dioscin do not peak, so the above components are discarded. Berberine hydrochloride, verbascoside, salvianolic acid B, pachymic acid A, and tanshinone II A with high separation degree and the same retention time in the test sample position are selected as index components. Berberine hydrochloride has high separation degree, good peak shape, and less interference components at the middle, left and right of the gradient elution time, and the peak shape is relatively independent, so berberine hydrochloride is selected as the internal reference.

[0214] 3. Methodology verification

[0215] 3.1 Detection of active ingredients A: salvianolic acid B, protodioscin, dioscin, hederacoside, and tanshinone II A

[0216] 3.1.1 Precision

[0217] Precisely take the content sample of Shuangshi Tonglin Capsules (batch number: 220801) of the same batch, prepare the test sample solution according to the method of step 1) in Example 1, and continuously sample 6 times according to the chromatographic conditions of step 2), record the chromatogram, and calculate the relative retention time and relative peak area of the common peaks, as shown in Table 16.

[0218] Table 16 Precision results of Shuangshi Tonglin Capsules

[0219]

[0220]

[0221] The results show that the relative retention time RSD value and the relative peak area RSD value of each common peak indicate that the method has good precision.

[0222] 3.1.2 repeatability

[0223] The contents of the same batch of Shishi Tonglin Capsules (batch number: 220801) were precisely taken, and 6 sample solutions were prepared in parallel according to the method of step 1) in the example, and the chromatograms were recorded, and the relative retention time and relative peak area of the common peaks were calculated, as shown in Table 17.

[0224] Table 17 Shishi Tonglin Capsule repeatability results

[0225]

[0226]

[0227] The results show that the relative retention time RSD value and the relative peak area RSD value of each common peak indicate that the method has good repeatability.

[0228] 3.1.3 stability

[0229] The same sample solution (batch number: 220801) was precisely taken and placed at room temperature for 0, 2, 4, 6, 8, 10, 12, and 24 h, and the chromatograms were recorded, and the relative retention time and relative peak area of the common peaks were calculated, as shown in Table 18.

[0230] Table 18 Shishi Tonglin Capsule stability results

[0231]

[0232]

[0233] The results show that the relative retention time RSD value and the relative peak area RSD value of each common peak indicate that the sample solution is stable within 24 h after preparation.

[0234] 3.1.4 sample recovery

[0235] About 3 g of the same batch of sample (batch number: 220801) containing a known amount was taken, and 6 portions were precisely weighed, and the control solution containing the same amount of each component as the contents of Shishi Tonglin Capsules was added, respectively. The sample solution was prepared according to the method of step 1) in Example 1, and the chromatograms were recorded, and the sample recovery rate was calculated, as shown in Table 19.

[0236] Table 19 Shishi Tonglin Capsule sample recovery results

[0237]

[0238]

[0239]

[0240] The results show that the sample addition recoveries of salvianolic acid B, protodioscin, dioscin, hederagenin and tanshinone II A are 99%, 99%, 101%, 102% and 98% respectively, and the RSDs are 3.76%, 3.21%, 4.51%, 3.70% and 2.97% respectively, indicating that the accuracy of the method is good.

[0241] In summary, the UPLC detection conditions selected in Example 1 of the present application have good precision, repeatability, stability and accuracy of the obtained chromatogram; combined with the multi-estimation method, the relative correction factors of other active ingredients are calculated by taking the active ingredient salvianolic acid B in the mixed reference solution as the internal reference, and the content of the detected component in Shuishitonglin Capsules is further obtained, the content detection error is small, the reference substance is easy to obtain, the multi-estimation is realized, and the detection process is simple.

[0242] 3.2 Detection of active ingredient B: verbascoside, salvianolic acid B, berberine hydrochloride, pachymic acid A, tanshinone II A

[0243] 3.2.1 Linear relationship

[0244] 2, 4, 5, 8, 10, 15 and 20 μL of the mixed reference solution were precisely taken respectively, and the standard curve was drawn by taking the sample amount (μL) of the reference solution as the abscissa (X) and the peak area integral value as the ordinate (Y), the regression equation was obtained, and the results are shown in Table 20. Each component has a good linear relationship within its own range.

[0245] Table 20: Linear relationship investigation results of each component (n = 3)

[0246] Ingredient Linear equation R Vaccinin Y=9.6157X+0.1037 0.9999 Danshensuan B Y=17.47X+0.2544 1 Berberine Hydrochloride Y=33.449X-0.4441 1 Tremella Acid A Y=18.27X-0.0233 1 Tanshinone ⅡA Y=94.636X+0.2566 1

[0247] 3.2.2 Precision

[0248] The same mixed reference solution was precisely taken, the sample solution was prepared according to the method of Example 2, and was continuously injected 6 times according to the chromatographic conditions, the chromatogram was recorded, and the relative retention time and relative peak area of the target component peak were calculated, as shown in Table 21. The results show that the relative retention time RSD value of each component peak is less than 0.1%, and the relative peak area RSD value is less than 0.4%, indicating that the precision of the method is good.

[0249] Table 21: Precision investigation results

[0250]

[0251]

[0252] 3.2.3 repeatability

[0253] The contents of the same batch of Shishitonglin Capsules (batch number: 220801) were precisely taken, and 6 test sample solutions were prepared in parallel according to the method of Example 2, and were continuously injected according to the chromatographic conditions, and the chromatograms were recorded, and the relative retention time and relative peak area of the target component peaks were calculated, as shown in Table 22. The results showed that the relative retention time RSD values of each component peak were less than 0.1%, and the relative peak area RSD values were less than 1.6%, indicating that the method had good repeatability.

[0254] Table 22 Repeatability test results

[0255]

[0256]

[0257] 3.2.4 stability

[0258] The same test sample solution (batch number: 220801) was precisely taken and placed at room temperature, and was continuously injected according to the chromatographic conditions of Example 2 at 0, 2, 4, 8, 10, 12, 24 h, and the chromatograms were recorded, and the relative retention time and relative peak area of the common peaks were calculated, as shown in Table 23. The results showed that the relative retention time RSD values of each common peak were less than 1%, and the relative peak area RSD values were less than 5%, indicating that the test sample solution had good stability within 24 h after preparation.

[0259] Table 23 Stability test results

[0260]

[0261]

[0262] 3.2.5 sample addition recovery

[0263] Nine samples of Shishitonglin Capsules (batch number: 220801) with known content were precisely measured and divided into low, medium and high concentration groups, and 3 test sample solutions were prepared for each according to the method of Example 2, and approximately 50%, 100%, 150% of the concentration of each component in the sample was precisely added in parallel to each group, and was injected according to the chromatographic conditions of Example 2, and the chromatograms were recorded, and the sample addition recovery rate was calculated, as shown in Table 24.

[0264] Table 24 Sample addition recovery rate determination results

[0265]

[0266]

[0267]

[0268] The results show that the sample addition recovery rates of berberine hydrochloride, verbascoside, salvianolic acid B, pachymic acid A and tanshinone IIA are between 95% and 105%, and the RSD values are all less than 2.1%, indicating that the accuracy of the method is good.

[0269] 4. Investigation on the relative correction factor of QAMS

[0270] 4.1. Verification of the f of the active ingredient A with salvianolic acid B as the internal reference substance to calculate the f of the protodioscin, dioscin, hederacoside C saponin, tanshinone IIA s / i

[0271] 4.1.1. Investigation on the relative correction factor f s / i of the active ingredient A with salvianolic acid B as the internal reference substance to calculate the f of the protodioscin, dioscin, hederacoside C saponin, tanshinone IIA

[0272] In actual situations, due to system, operation and other errors, the calculated relative correction factor has a certain deviation from the true value. In order to eliminate the system error, reduce the accidental error, make the relative correction factor more accurate, verify the adaptability of the relative correction factor, ensure the accuracy, scientificity and rigor of the experiment, the influences of different injection volumes, chromatographic column types, column temperatures, volume flow rates and detection wavelengths on the f of the relative correction factor are investigated, and it is indicated that different factors have no significant influence on the relative correction factors of the components, and the f of the present application has good durability. s / i s / i

[0273] 4.1.2. Influence of different injection volumes on the f of the relative correction factor s / i

[0274] According to the chromatographic conditions preferred in step 2) of Example 1, the mixed reference solution of step 1) is injected in 1, 3, 5, 7, 9 and 11 μL respectively, the peak areas of the components are recorded, salvianolic acid B is used as the internal reference substance, and the f of the protodioscin, dioscin, hederacoside C saponin and tanshinone IIA is calculated according to the formula by using the multi-point correction method, and the results show that the RSD values of the f of the four to-be-tested components and the internal reference substance are all less than 1.90%, and are shown in Table 25. s / i s / i

[0275] Table 25. f of the components at different injection volumes s / i (n = 3)

[0276]

[0277] ​​​​​

[0278] 4.1.3 Influence of different chromatographic columns on relative correction factor f s / i

[0279] Influence of ACQUITY UPLC BEH C 18 18 column, ACQUITY UPLC HSS T3 column and Thermo Hypersil GOLD column on relative correction factor f s / i was investigated, other chromatographic conditions were preferred chromatographic conditions in step 2), and RSD was calculated, all less than 1.50%, indicating that different chromatographic columns had good applicability and no significant influence on relative correction factor f, and the results were shown in Table 26.

[0280] Table 26 Influence of different chromatographic columns on f s / i (n = 3)

[0281] Chromatographic column f 丹酚酸B / 原薯蓣皂苷 ]]> f 丹酚酸B / 薯蓣皂苷 ]]> f 丹酚酸B / 常春藤皂苷元 ]]> f 丹酚酸B / 丹参酮ⅡA ]]> ACQUITY UPLC BEH C 18 ]]> 1.2263 1.0762 0.8768 1.6368 ACQUITY UPLC HSS T3 1.2108 1.0886 0.8645 1.6563 Thermo Hypersil GOLD 1.1958 1.0671 0.8531 1.6482 Average value 1.2110 1.0773 0.8648 1.6471 RSD 1.26 1.00 1.37 0.60

[0282] 4.1.4 Influence of different column temperatures on relative correction factor f s / i

[0283] Using Waters I-CLASS ultra-high performance liquid chromatograph and ACQUITY UPLC BEH C 18 18 column (2.1 x 100 mm, 1.7 μm), the influence of column temperature at 20℃, 25℃ and 30℃ on f s / i was investigated, and RSD was calculated, all less than 2.60%, indicating that different column temperatures had no significant influence on the relative correction factor of each component, and the results were shown in Table 27.

[0284] Table 27 Influence of different column temperatures on f s / i (n = 3)

[0285]

[0286]

[0287] 4.1.5 Influence of different volumetric flow rates on relative correction factor f s / i

[0288] Using Waters I-CLASS ultra-high performance liquid chromatograph and ACQUITY UPLC BEH C 18 18 column (2.1 x 100 mm, 1.7 μm), the influence of volumetric flow rate at 0.15 mL / min, 0.20 mL / min and 0.25 mL / min on f s / i ​​​The influence of different volume flow rates on f was investigated, and the RSD values were calculated. The results were all less than 3.30%, indicating that the change in volume flow rate had no significant influence on the relative correction factors of the components. The results are shown in Table 28.

[0289] Table 28 Influence of different volume flow rates on f s / i (n = 3)

[0290]

[0291] 4.1.6 Influence of different detection wavelengths on f s / i

[0292] A Waters I-CLASS ultra-high performance liquid chromatograph and an ACQUITY UPLC BEH C 18 (2.1 x 100 mm, 1.7 μm) chromatographic column were used to investigate the influence of detection wavelengths of 202, 203 and 205 nm on f s / i , and the RSD values were calculated. The results were all less than 3%, indicating that the change in detection wavelength had no significant influence on f s / i . The results are shown in Table 29.

[0293] Table 29 Influence of different detection wavelengths on f s / i (n = 3)

[0294]

[0295] 4.1.7 Positioning of the chromatographic peaks of the components to be tested

[0296] The relative retention value method was used to position the chromatographic peaks. The mixed reference solution was taken, three different brands of chromatographic columns were selected, and the relative retention times (t s / i ) of the other four components to be tested were calculated with salvianolic acid B as the internal standard, the components to be tested were positioned, and the RSDs were calculated. The results showed that the RSDs of the relative retention values of the components to be tested were all less than 4.50%, indicating that the relative retention value method used to position the components to be tested was reasonable. The results are shown in Table 30.

[0297] Table 30 t s / i values measured by different chromatographic columns (n = 3)

[0298]

[0299] 4.2 Verification of f s / i of marrubiin, salvianolic acid B, pachymic acid A and tanshinone IIA in the active ingredient B with berberine hydrochloride as the internal standard

[0300] 4.2.1 Determination of f s / i at different injection volumes

[0301] According to the chromatographic conditions of Example 2 and the mixed reference solution, 2, 4, 5, 8, 10, and 15 μL were injected respectively, and the peak areas of each component were recorded. Berberine hydrochloride was used as an internal reference, and the f values ​​of verbascoside, salvianolic acid B, pamoatenic acid A, and tanshinone IIA were calculated using the multi-point calibration method according to the following formula. s / i The results showed that the f values ​​of the four analytes and the internal reference were... s / i The RSD values ​​are all less than 3.1%, as shown in Table 31.

[0302] Table 31 f for different injection volumes of each component s / i (n=3)

[0303]

[0304]

[0305] 4.2.2 Investigation of f using different chromatographic columns s / i Measurement

[0306] Welch Ultimate XB-C was examined 18 Column, Inertsil ODS-3 column, ThermoFisher Acclaim TM C 18 Three types of chromatographic columns for f s / i The influence of the column was investigated, and the RSD was calculated. The results were all less than 4.0%, indicating that the different chromatographic columns were well applicable. The results are shown in Table 32.

[0307] Table 32 Different chromatographic columns for f s / i The impact (n=3)

[0308]

[0309] 4.2.3 Effect of different column temperatures on f s / i Impact

[0310] ThermoFisher UltiMate 3000 high-performance liquid chromatograph and Welch Ultimate XB-C 18 The effect of column temperatures of 20, 25, and 30 °C on f was investigated using a column (4.5 × 250 mm, 5 μm). s / i The effects of different column temperatures on each component were investigated, and the RSD was calculated. The results were all less than 3.2%, indicating that different column temperatures had no significant effect on each component. The results are shown in Table 33.

[0311] Table 33 Effects of different column temperatures on f s / i The impact (n=3)

[0312] Column temperature / ℃ f 盐酸小檗碱 / 毛蕊花糖苷 ]]> f 盐酸小檗碱 / 丹酚酸B ]]> f 盐酸小檗碱 / 茯苓新酸A ]]> f 盐酸小檗碱 / 丹参酮ⅡA ]]> 25 3.1242 1.7498 1.7392 0.3898 30 3.2519 1.8314 1.8300 0.3660 35 3.2907 1.8301 1.8374 0.3814 Average value 3.2222 1.8038 1.8022 0.3791 RSD (%) 2.70 2.59 3.04 3.18

[0313] 4.2.4 Influence of different flow rates on f s / i

[0314] The experiment used ThermoFisher UltiMate 3000 high performance liquid chromatograph and Welch Ultimate XB-C 18 column (4.5 x 250 mm, 5 μm) to investigate the influence of different flow rates of 0.5 mL / min, 0.8 mL / min, 1.0 mL / min and 1.2 mL / min on f s / i , and the RSD values were calculated, all less than 3.0%, indicating that the change of different flow rates had no significant influence on f s / i of each component. The results are shown in Table 34.

[0315] Table 34 Influence of different flow rates on f s / i (n = 3)

[0316]

[0317] 4.2.5 Influence of different detection wavelengths on f s / i

[0318] The experiment used ThermoFisher UltiMate 3000 high performance liquid chromatograph and Welch Ultimate XB-C 18 column (4.5 x 250 mm, 5 μm) to investigate the influence of detection wavelengths of 230, 254, 270 and 345 nm on f s / i , and the RSD values were calculated, all less than 4.5%, indicating that the change of different detection wavelengths had no significant influence on f s / i . The results are shown in Table 35.

[0319] Based on the above several factors, it is shown that the method has good durability for f s / i

[0320] Table 35 Influence of different detection wavelengths on f s / i (n = 3)

[0321]

[0322] 4.2.6 Peak positioning of the components to be tested

[0323] The mixed reference solution was taken, 3 different brands of chromatographic columns were selected, and the determination was performed according to the chromatographic conditions of Example 2, with berberine hydrochloride as the internal standard, and the relative retention times (t s / i ​​​), and the RSD was calculated. The results showed that the RSD of the relative retention value of each component was less than 4.8%, indicating that the relative retention value method for positioning the components was reasonable. The results are shown in Table 36.

[0324] Table 36 t values of different chromatographic columns s / i (n = 3)

[0325]

[0326] 5. Reliability of the detection results of HPLC-QAMS

[0327] 5.1 Comparison of the detection results of Example 1 with the results of the external standard method

[0328] Ten batches of samples were prepared according to the method of step 1) of Example 1 to prepare the sample solution; and the contents of Danshensuan B, Yuanshujiusuan, Shujiansuan, Chongchunjiuansuan, and Danshensuan II A were determined by the external standard method under the preferred chromatographic conditions of step 2).

[0329] The relative error (RE) was used to represent the difference between the results measured by the two methods in steps 4) and 5). The calculation formula of the relative error is as follows:

[0330] RE = (W 本发明 -W 外标法 ) / W 外标法

[0331] Wherein: W 本发明 is the content measured by the method of the application, and W 外标法 is the content measured by the external standard method. The results are shown in Table 37.

[0332] Table 37 Mass concentrations of five components in ten batches of Shuangshitonglin capsules measured by Example 1 and the external standard method (n = 3)

[0333]

[0334]

[0335] The results show that the mass concentrations of the five components in the Shishitonglin capsules calculated in step 4) of Example 1 are basically consistent with the component contents detected by the external standard method, and there is no significant difference between the measured content values by the external standard method and the component content values measured by the method of the present application. The comprehensive quality scores of hydrochloric acid berberine, verbascoside, salvianolic acid B, pachymic acid A and tanshinone II A in the 10 batches of samples are 1.3429-1.6757, 0.5497-0.5835, 0.6808-0.7715, 0.8559-0.9546 and 0.8759-0.9731 mg / g respectively, and the contents of salvianolic acid B and tanshinone II A are higher.

[0336] 5.2 Comparison of the detection results of Example 2 with the results of the external standard method

[0337] Ten batches of samples were prepared into sample solutions according to the conditions of Example 2, and were detected under the chromatographic conditions of Example 2. The peak areas were recorded, and the f s / i The contents of each component in the 10 batches of Shishitonglin capsule samples were determined, and the contents of verbascoside, salvianolic acid B, pachymic acid A and tanshinone II A were determined by the external standard method. The relative error (RE) was used to represent the difference between the results of the two methods, RE = (W QAMS -W 外标法 ) / W 外标法 (W QAMS is the content measured by QAMS, and W 外标法 is the content measured by the external standard method). The results are shown in Table 38.

[0338] Table 38 Mass concentrations of five components in 10 batches of Shishitonglin capsules measured by QAMS and external standard method (n = 3)

[0339]

[0340] The results show that the component contents calculated by the external standard method and the QAMS method of the present application are basically consistent, and there is no significant difference between the measured content values by the external standard method and the component content values measured by the QAMS method. The comprehensive quality scores of hydrochloric acid berberine, verbascoside, salvianolic acid B, pachymic acid A and tanshinone II A in the 10 batches of samples are 1.8546-1.9963, 0.4009-0.4386, 4.6373-4..8395, 1.1070-1.1975 and 0.4569-0.4692 mg / g respectively, and the contents of salvianolic acid B, hydrochloric acid berberine and pachymic acid A are higher.

[0341] The HPLC-QAMS method of the application can realize simultaneous determination of multiple components and contents of the active components of Shuishitonglin Capsules, establish a multi-index compound quality control mode of Shuishitonglin Capsules, simplify the analysis process as a whole, greatly reduce the analysis cost, and provide support for comprehensively evaluating the overall quality of Shuishitonglin Capsules and ensuring stable and reliable clinical medication.

Claims

1. A method for detecting the multiple active ingredients of Shuangshitonglin Capsules, characterized in that, The detection method comprises simultaneously determining the contents of salvianolic acid B, protodioscin, dioscin, hederagenin and tanshinone II A in Shishitonglin Capsules based on UPLC-QAMS method, or / and The detection method comprises simultaneously determining the contents of salvianolic acid B, verbascoside, berberine hydrochloride, pachymic acid A and tanshinone II A in Shishitonglin Capsules based on HPLC-QAMS method. The detection method comprises simultaneously determining the contents of salvianolic acid B, protodioscin, dioscin, hederagenin and tanshinone II A in Shishitonglin Capsules based on UPLC-QAMS method, or / and A1) solution preparation A1.1) salvianolic acid B reference substance, protodioscin reference substance, dioscin reference substance, hederagenin reference substance and tanshinone II A reference substance are respectively weighed, and a mixed reference substance solution is prepared by using methanol; A1.2) Shishitonglin Capsules are taken, the shell is removed, the content is added with methanol, and a test sample solution is prepared by ultrasonic treatment; A2) obtaining a liquid chromatogram The mixed reference substance solution and the test sample solution are respectively subjected to UPLC chromatography to obtain corresponding mixed reference substance chromatograms and test sample chromatograms; A3) determining a relative correction factor From the mixed reference substance chromatogram of step A2), peak areas of salvianolic acid B, protodioscin, dioscin, hederagenin and tanshinone II A are obtained, and the relative correction factors of protodioscin, dioscin, hederagenin and tanshinone II A are calculated by taking salvianolic acid B as an internal reference substance; A4) QAMS method quantitative detection From the test sample chromatogram of step A2), the peak areas of each component to be detected in the test sample solution are obtained, and the relative correction factors obtained in step A3) are combined to calculate the component contents of salvianolic acid B, protodioscin, dioscin, hederagenin and tanshinone II A in the test sample solution; The specific preparation method of the test sample solution in step A1.2) is as follows: 3g of the content of Shishitonglin Capsules is weighed, 50mL of methanol is added, ultrasonic extraction is performed for 50min, the weight difference is supplemented with methanol, and then filtration is performed, the filtrate is filtered through a 0.22μm microporous filter, and the test sample solution is obtained; The conditions of the UPLC chromatography in step A2) are as follows: The chromatographic column was ACQUITY UPLC BEH C 18 The chromatographic column was ACQUITY UPLC BEH C 18 The chromatographic column was ACQUITY UPLC BEH C 18 The chromatographic column was ACQUITY UPLC BEH C 18 The chromatographic column was ACQUITY UPLC BEH C 18 The chromatographic column was ACQUITY UPLC BEH C 18 The chromatographic column was ACQUITY UPLC BEH C 18 The chromatographic column was ACQUITY UPLC BEH C 18 The chromatographic column was ACQUITY UPLC BEH C 18 The chromatographic column was ACQUITY UPLC BEH C 18 The chromatographic column was ACQUITY UPLC BEH C 18 The chromatographic column was ACQUITY UPLC BEH C 18 The chromatographic column was AC The detection method comprises simultaneously determining the contents of salvianolic acid B, verbascoside, berberine hydrochloride, pachymic acid A and tanshinone II A in Shishitonglin Capsules based on HPLC-QAMS method. B1) solution preparation B1.1) verbascoside reference substance, salvianolic acid B reference substance, berberine hydrochloride reference substance, pachymic acid A reference substance and tanshinone II A reference substance are respectively weighed, and a mixed reference substance solution is prepared by using methanol; B1.2) Shishitonglin Capsules are taken, the shell is removed, the content is added with 70% methanol, and a test sample solution is prepared by ultrasonic treatment; B2) liquid chromatography detection The mixed reference substance solution and the test sample solution are respectively subjected to HPLC chromatography to obtain corresponding mixed reference substance chromatograms and test sample chromatograms; B3) determining a relative correction factor From the mixed reference substance chromatogram of step B2), the peak areas of verbascoside, salvianolic acid B, berberine hydrochloride, pachymic acid A, and tanshinone IIA were obtained, respectively, and the relative correction factors of verbascoside, salvianolic acid B, pachymic acid A, and tanshinone IIA were calculated with berberine hydrochloride as the internal reference substance; B4) QAMS method for quantitative detection From the test substance chromatogram of step B2), the peak areas of the test substance solution were obtained, and the relative correction factors obtained in step B3) were combined to calculate the contents of berberine hydrochloride, verbascoside, salvianolic acid B, pachymic acid A, and tanshinone IIA in the test substance solution, respectively. The conditions of the HPLC chromatography of step B2) are as follows: The chromatographic column was Welch Ultimate XB-C 18 The column was Agilent ZORBAX SB-C18; the mobile phase was acetonitrile A-0.02 mol / L potassium dihydrogen phosphate B; the detection wavelength was 254 nm; the volume flow rate was 1.0 mL / min; the column temperature was 30°C; the injection volume was 10 μL; the gradient elution was as shown in the following table:

2. The method for detecting the multiple active ingredients of Shangshitonglin Capsules according to claim 1, characterized in that, The specific preparation method of the mixed reference substance solution of step A1.1) is as follows: accurately weigh the salvianolic acid B reference substance, the protodioscin reference substance, the dioscin reference substance, the hederagenin reference substance, and the tanshinone IIA reference substance, respectively, and prepare the mixed reference substance solution with a concentration of 0.2278 mg / mL, 0.2660 mg / mL, 0.2795 mg / mL, 0.1632 mg / mL, and 0.2070 mg / mL, respectively, using methanol.

3. The method for detecting multiple active ingredients in Shuangshitonglin capsules according to claim 1, characterized in that, The conditions of the UPLC chromatography are as follows: The chromatographic column was ACQUITY UPLC BEH C 18 column, 2.1 x 100 mm, 1.7 μm; detection wavelength 203 nm; volume flow rate 0.25 mL / min; column temperature 30 °C; injection volume 5 μL.

4. The method for detecting multiple active ingredients in Shuangshitonglin capsules according to claim 1, characterized in that, In step A3), the relative correction factors of the components are obtained with salvianolic acid B as the internal reference substance:

5. The method for detecting multiple active ingredients of Shangshitonglin Capsules according to claim 1, characterized in that, The specific preparation method of the mixed reference substance solution of step B1.1) is as follows: accurately weigh the verbascoside reference substance, the salvianolic acid B reference substance, the berberine hydrochloride reference substance, the pachymic acid A reference substance, and the tanshinone IIA reference substance, respectively, and prepare the mixed reference substance solution with a concentration of 0.0474 mg / mL, 0.2278 mg / mL, 0.0921 mg / mL, 0.13328 mg / mL, and 0.1035 mg / mL, respectively, using methanol.

6. The method for detecting multiple active ingredients of Shangshitonglin Capsules according to claim 1, characterized in that, The specific preparation method of the test substance solution of step B1.2) is as follows: weigh 2.5 g of the contents of Shuangshi Tonglin Capsules, add 30 mL of 70% methanol, weigh the mass, ultrasonically extract for 45 min, make up the difference with 70% methanol, filter, take the filtrate through a 0.22 μm microporous filter membrane, and obtain the test substance solution.

7. The method for detecting multiple active ingredients in Shuangshitonglin capsules according to claim 1, characterized in that, In the HPLC chromatography detection of step B2), the theoretical plate number calculated by berberine hydrochloride is not less than 6000, and the separation degree of each component is >1.

5.

8. The method for detecting multiple active ingredients in Shuangshitonglin capsules according to claim 1, characterized in that, In step B3), the relative correction factors of the active ingredients are obtained with berberine hydrochloride as the internal reference substance using the multiple-point correction method: In step B3), the relative correction factors of the active ingredients are obtained with berberine hydrochloride as the internal reference substance using the multiple-point correction method:

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