An ultra-high performance liquid chromatography detection method for the content of active ingredients and fingerprint of compound sophora flavescens injection

Abstract

The invention belongs to the field of medical technology, and specifically relates to an ultra-high performance liquid chromatography detection method for the active ingredient content and fingerprint of a compound sophora flavescens injection, wherein the method adopts ultra-high performance liquid chromatography for detection, and the ultra-high performance liquid chromatography conditions include a chromatographic column of ACQUITY UPLC CSH TM C 18 , with specifications of 1.7 μm and 2.1 mm×100 mm; the active ingredients are matrine, oxymatrine, methyl azoxymethanol primulin glycoside, sophocarpine, oxysophocarpine and sophoridine. The present invention can provide an improved detection method for compound sophora flavescens injection, which can simultaneously determine the six components in the compound sophora flavescens injection, and use the method to construct a chromatographic fingerprint, providing a fast and efficient technical method for quality control in the compound sophora flavescens injection, and reducing the inspection workload.

Description

Technical Field

[0001] The invention belongs to the technical field of medicines, and in particular relates to an improved UPLC detection method for compound sophora flavescens injection. Background Art

[0002] Compound Sophora flavescens injection is a Chinese medicine injection made of two Chinese medicines, Sophora flavescens and Poria cocos, refined by modern scientific methods. It is included in the national drug standards and has the effects of clearing away heat and dampness, cooling blood and detoxifying, dispersing knots and relieving pain. It is used for cancer pain and bleeding. Modern research shows that it has multiple pharmacological effects such as anti-tumor, anti-inflammatory, analgesic, and improving the body's immunity. It is widely used in clinical practice as an auxiliary treatment for serious diseases such as non-small cell lung cancer, primary liver cancer, digestive tract cancer, and malignant pleural effusion.

[0003] Alkaloids are the main medicinal ingredients of compound sophora flavescens injection. The existing national drug standard for compound sophora flavescens injection (WS3-B-2752-97-2014) only contains the HPLC content determination method and fingerprint detection of matrine, oxymatrine and methyl azoxymethanol primulin glycoside, lacking a variety of scientific quality evaluation methods. Ultra-high performance liquid chromatography has the characteristics of high separation efficiency, fast analysis speed and high detection sensitivity, and has been widely used in the detection of traditional Chinese medicine and Chinese patent medicine. By reviewing relevant literature, it was found that there were few studies on sophora flavescens in UPLC, while there were no studies on white radix turans.

[0004] Zhang Huangqin et al.'s "Analysis and evaluation of alkaloids and flavonoids in Sophora flavescens flowers from Shanxi" used UPLC-TQ-MS technology to analyze and determine 7 alkaloids and 7 flavonoids in Sophora flavescens flowers from Changzhi, Shanxi. This method uses acetonitrile-ammonium acetate as the mobile phase, which is similar to the fingerprint conditions in the standard. The chromatographic peak shape of the injection solution is poor under this condition.

[0005] In the study "Analysis of chemical components of alkaloids in seeds of Sophora flavescens produced in Shanxi and discussion on their resource value", UPLC-QTOF-MS and UPLC-TQ-MS were used to analyze the composition and content of alkaloid resource chemical components in mature and dried seeds of Sophora flavescens. This method also used acetonitrile-ammonium acetate as the mobile phase, which is not very suitable for compound Sophora flavescens injection.

[0006] Therefore, in order to more scientifically and comprehensively evaluate the intrinsic quality of the compound sophora flavescens injection product, it is necessary to provide a method that can simultaneously complete the content determination and fingerprint spectrum detection of multiple components of the compound sophora flavescens injection, provide a fast and efficient technical method for quality control in the compound sophora flavescens injection, and reduce the inspection workload. Summary of the invention

[0007] In view of the above technical status, the present invention provides an improved method for detecting the active ingredient content and fingerprint of compound sophora flavescens injection using UPLC. The specific invention is as follows:

[0008] The method for detecting the active ingredient content and fingerprint of the compound sophora flavescens injection of the present invention adopts ultra-high performance liquid chromatography for detection, and the ultra-high performance liquid chromatography conditions include a chromatographic column of ACQUITY UPLC CSH TM C 18 The active ingredients are matrine, oxymatrine, methyl azoxymethanol primrose glycoside, sophoracarpine, oxysophoracarpine and sophoridine.

[0009] In the method of the present invention, as one of the embodiments, the mobile phase in the ultra-high performance liquid chromatography conditions is 0.15%-0.25% potassium dihydrogen phosphate solution-methanol gradient elution, preferably 0.2% potassium dihydrogen phosphate solution-methanol gradient elution.

[0010] In the method of the present invention, as one of the embodiments, the elution gradient in the ultra-high performance liquid chromatography conditions is as follows:

[0011]

[0012] In the method of the present invention, as one of the embodiments, the ultra-high performance liquid chromatography conditions include a column temperature of 28°C-32°C, preferably 30°C.

[0013] In the method of the present invention, as one of the embodiments, the ultra-high performance liquid chromatography conditions include a flow rate of 0.3 ml / min.

[0014] In the method of the present invention, as one of the embodiments, the ultra-high performance liquid chromatography conditions include a detection wavelength of 209 nm-213 nm, preferably 211 nm;

[0015] In the method of the present invention, as one of the embodiments, the ultra-high performance liquid chromatography conditions include an injection volume of 0.8 μl-1.2 μl, preferably 1 μl.

[0016] In the method of the present invention, as one of the embodiments, the ultra-high performance liquid chromatography further comprises:

[0017] (1) Preparation of blank solution: adjust the pH value of potassium dihydrogen phosphate solution to 3.0 with phosphoric acid, and filter a mixed solution of 0.2% potassium dihydrogen phosphate solution and methanol in a ratio of 85:15.

[0018] In the method of the present invention, as one of the embodiments, the ultra-high performance liquid chromatography method further comprises:

[0019] (2) Preparation of reference solution:

[0020] Accurately weigh appropriate amounts of matrine reference substance, oxidized matrine reference substance, and oxidized sophocarpine reference substance, add blank solution to prepare a mixed reference solution I containing 0.33 mg of matrine, 0.85 mg of oxidized matrine, and 0.25 mg of oxidized sophocarpine per 1 ml, and shake well to obtain;

[0021] Accurately weigh appropriate amounts of sophoracarpine reference substance, sophoridine reference substance, and methyl azoxymethanol primulosyl glycoside reference substance, add blank solution to prepare a mixed reference substance solution II containing 0.09 mg sophoracarpine, 0.08 mg sophoridine, and 0.08 mg methyl azoxymethanol primulosyl glycoside per 1 ml, shake well, and obtain;

[0022] Accurately measure 2 ml of mixed reference solution I and II, place in a 10 ml volumetric flask, dilute to the mark with blank solution, and shake well; optionally, prepare 2 portions in the same way.

[0023] As one of the embodiments of the method of the present invention, the content of the reference substance in the reference substance solution can be within the following range:

[0024] The preferred range of matrine is 0.28-0.40 mg, and the optimal range is 0.33 mg; the preferred range of oxymatrine is 0.72-1.06 mg, and the optimal range is 0.85 mg; the preferred range of oxysophocarpine is 0.21-0.31 mg, and the optimal range is 0.25 mg; the preferred ranges of sophocarpine, sophoridine, and methyl azoxymethanol primulin glycoside are all 0.07-0.11 mg, and the optimal ranges are 0.09 mg, 0.08 mg, and 0.08 mg, respectively.

[0025] In the method of the present invention, as one of the embodiments, the ultra-high performance liquid chromatography further comprises:

[0026] (3) Preparation of test solution: Accurately measure 1 ml of compound Sophora flavescens injection and place it in a 50 ml volumetric flask. Add blank solution to the mark, shake well, filter, and take the filtrate as the test solution.

[0027] In the method of the present invention, as one of the embodiments, the method for detecting the content of active ingredients in the compound sophora flavescens injection of the present invention comprises:

[0028] The ultra-high performance liquid chromatography conditions were:

[0029]

[0030] (1) Preparation of blank solution: adjust the pH value of potassium dihydrogen phosphate solution to 3.0 with phosphoric acid, and filter a mixed solution of 0.2% potassium dihydrogen phosphate solution and methanol in a ratio of 85:15.

[0031] (2) Preparation of reference solution:

[0032] Accurately weigh appropriate amounts of matrine reference substance, oxidized matrine reference substance, and oxidized sophocarpine reference substance, add blank solution to prepare a mixed reference solution I containing 0.33 mg of matrine, 0.85 mg of oxidized matrine, and 0.25 mg of oxidized sophocarpine per 1 ml, and shake well to obtain;

[0033] Accurately weigh appropriate amounts of sophoracarpine reference substance, sophoridine reference substance, and methyl azoxymethanol primulosyl glycoside reference substance, add blank solution to prepare a mixed reference substance solution II containing 0.09 mg sophoracarpine, 0.08 mg sophoridine, and 0.08 mg methyl azoxymethanol primulosyl glycoside per 1 ml, shake well, and obtain;

[0034] Accurately measure 2 ml of mixed reference solution I and II, place in a 10 ml volumetric flask, dilute to scale with blank solution, and shake well; optionally, prepare 2 portions in the same way;

[0035] (3) Preparation of test solution: Accurately measure 1 ml of compound Sophora flavescens injection and place it in a 50 ml volumetric flask. Add blank solution to the mark, shake well, filter, and take the filtrate as the test solution.

[0036] (4) Inject the blank solution, reference substance, and test substance into the liquid chromatograph in the order of injection, record the chromatogram, and calculate the content of each active ingredient by the external standard method.

[0037] In the method of the present invention, as one of the embodiments, the method for detecting the fingerprint of the compound sophora flavescens injection of the present invention is characterized in that the method comprises constructing a fingerprint of the compound sophora flavescens injection containing matrine, oxymatrine, methyl azoxymethanol primulin, sophorocarpine, oxysophocarpine, sophoridine, guava acid, sophoramine, and trifoliate bean glycoside.

[0038] In the method of the present invention, as one of the embodiments, the method comprises: the ultra-high performance liquid chromatography conditions are:

[0039]

[0040] (1) Preparation of blank solution: adjust the pH value of potassium dihydrogen phosphate solution to 3.0 with phosphoric acid, and filter a mixed solution of 0.2% potassium dihydrogen phosphate solution and methanol in a ratio of 85:15.

[0041] (2) Preparation of reference solution:

[0042] Accurately weigh appropriate amounts of matrine reference substance, oxidized matrine reference substance, and oxidized sophocarpine reference substance, add blank solution to prepare a mixed reference solution I containing 0.33 mg of matrine, 0.85 mg of oxidized matrine, and 0.25 mg of oxidized sophocarpine per 1 ml, and shake well to obtain;

[0043] Accurately weigh appropriate amounts of sophoracarpine reference substance, sophoridine reference substance, and methyl azoxymethanol primulosyl glycoside reference substance, add blank solution to prepare a mixed reference substance solution II containing 0.09 mg sophoracarpine, 0.08 mg sophoridine, and 0.08 mg methyl azoxymethanol primulosyl glycoside per 1 ml, shake well, and obtain;

[0044] Accurately measure 2 ml of mixed reference solution I and II, place in a 10 ml volumetric flask, dilute to scale with blank solution, shake well, and prepare 2 portions in the same way;

[0045] (3) Preparation of test solution: Accurately measure 1 ml of compound Sophora flavescens injection and place it in a 50 ml volumetric flask. Add blank solution to the mark, shake well, filter, and take the filtrate as the test solution.

[0046] (4) Inject the blank solution, reference substance, and test substance in the order of injection to construct the fingerprint of the compound sophora flavescens injection containing matrine, oxymatrine, methyl azoxymethanol primulin, sophorocarpine, oxysophocarpine, sophoridine, guava acid, sophoramine, and trifoliate bean glycoside

[0047] (5) Inject the blank solution, reference substance, and test substance into the liquid chromatograph in the order of injection, record the chromatogram, and calculate the content of each active ingredient by the external standard method.

[0048] In the method of the present invention, as one of the embodiments, the method comprises:

[0049] Taking oxidized matrine as a reference, the relative retention times of the common characteristic peaks are: the relative retention time of the peak of sophoramine is 0.389±0.02; the relative retention time of the peak of methyl azoxy primulin is 0.469±0.04; the relative retention time of the peak of matrine is 0.637±0.04; the relative retention time of the peak of sophoracarpine is 0.767±0.03; the relative retention time of the peak of sophoridine is 0.819±0.02; the relative retention time of the peak of oxidized sophoracarpine is 0.925±0.02; the relative retention time of the peak of oxidized matrine is 1.0; the relative retention time of the peak of guava acid is 1.055±0.03; and the relative retention time of the peak of trifoliate bean pterostilbene is 2.097±0.05.

[0050] In the method of the present invention, as one of the implementation schemes, the method further includes that the RSD of the peak areas of oxymatrine, matrine, and oxysophocarpine shall not be greater than 2.0%, the RSD of the retention time shall not be greater than 2.0%, the RSD of the peak areas of sophoracarpine, sophoridine, and methyl azoxymethanol primulosidoside shall not be greater than 3.0%, the RSD of the retention time shall not be greater than 3.0%, the theoretical plate number of the content detection index shall not be less than 3000, and the tailing factor shall not be greater than 2.0.

[0051] In the present invention, as one of the implementation schemes, with peak No. 7 as a reference, the relative peak areas of the common characteristic peaks are: the relative peak area of ​​peak No. 1 is 0.032; the relative peak area of ​​peak No. 2 is 0.073; the relative peak area of ​​peak No. 3 is 0.466; the relative peak area of ​​peak No. 4 is 0.183; the relative peak area of ​​peak No. 5 is 0.107; the relative peak area of ​​peak No. 6 is 0.422; the relative peak area of ​​peak No. 7 is 1.0; the relative peak area of ​​peak No. 8 is 0.222; the relative peak area of ​​peak No. 9 is 0.056; wherein peak No. 1 is sophoramine, peak No. 2 is methyl azoxy primulin glycoside, peak No. 3 is matrine, peak No. 4 is sophorocarpine, peak No. 5 is sophoridine, peak No. 6 is oxysophocarpine, peak No. 7 is oxymatrine, peak No. 8 is guava acid, and peak No. 9 is trifoliate bean pterostilbene.

[0052] Compared with the existing detection method of compound sophora flavescens injection, the present invention adopts ultra-high performance liquid chromatography, which can simultaneously determine the six components in the compound sophora flavescens injection, and uses this method to construct a chromatographic fingerprint, providing a fast and efficient technical method for quality control in the compound sophora flavescens injection and reducing the inspection workload. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 The blank and negative sample results of Example 1 are shown in FIG.

[0054] Figure 2 This is a linear graph of the six indicator components of Example 1.

[0055] Figure 3-1 This is the standard reference fingerprint of Example 2.

[0056] Figure 3-2 This is the fingerprint of the test sample in Example 2.

[0057] Figure 4 This is the repetitive fingerprint of Example 2.

[0058] Figure 5 This is the precision fingerprint of Example 2.

[0059] Figure 6 This is the stability fingerprint of Example 2.

[0060] Figure 7-1 These are the fingerprints of different chromatographic columns in Example 2.

[0061] Figure 7-2 These are the fingerprints of different instruments in Example 2.

[0062] Figure 7-3 The overlay graphs are for 10 batches of samples in the chromatographic column 2 of the instrument 1 of Example 2.

[0063] Figure 7-4This is the reference fingerprint of instrument 1 chromatographic column 2 in Example 2.

[0064] Figure 7-5 The overlay graphs are for 10 batches of samples in the chromatographic column 3 of the instrument 1 of Example 2.

[0065] Figure 7-6 This is the reference fingerprint of the instrument 1 chromatographic column 3 of Example 2.

[0066] Figure 7-7 The overlay graphs are for 10 batches of samples in the chromatographic column 1 of the instrument 2 of Example 2.

[0067] Figure 7-8 This is the reference fingerprint of the chromatographic column 1 of instrument 2 in Example 2.

[0068] Figure 7-9 The overlay graphs are for 10 batches of samples in the chromatographic column 1 of the instrument 3 of Example 2.

[0069] Figure 7-10 This is the reference fingerprint of the instrument 3 chromatographic column 1 in Example 2.

[0070] Figure 7-11 This is an overlay of the control fingerprints under different conditions in Example 2.

[0071] Figure 8 This is the fingerprint of the key points of the production process of Example 2.

[0072] Fig. 9 This is the result diagram of Example 3-1, and the curves from bottom to top are: Test Sample 1, Test Sample 2.

[0073] Figure 10-1 The result diagram of the chromatographic condition 1 in Example 3-2, the curves from bottom to top are: sample, sophocarpine, oxidized sophocarpine, matrine and oxidized matrine;

[0074] Figure 10-2 This is the result diagram of chromatographic condition 2 in Example 3-2;

[0075] Figure 10-3 This is the result diagram of chromatographic condition 3 in Example 3-2.

[0076] Figure 10-4 This is the result diagram of chromatographic condition 4 in Example 3-2;

[0077] Figure 10-5 They are respectively the result graphs of chromatographic condition 5 in Example 3-2;

[0078] Figure 10-6This is an overlay of the reference substances in Example 3-2 after injection under the above conditions and the test substances, wherein the chromatographic peak positions (curve from bottom to top) are: sample, matrine, oxymatrine, sophorocarpine, oxysophocarpine, sophoridine, methylazoxymethanol primulin, guava acid, trifoliate bean glycoside, sophoramine, and N-methylcytisine.

[0079] Figure 11-1 This is the result diagram of method 1 in Example 3-3.

[0080] Figure 11-2 This is the result diagram of method 2 in Example 3-3;

[0081] Figure 11-3 This is the result diagram of method 3 in Example 3-3.

[0082] Figure 11-4 This is a result diagram of method 4 of Example 3-3.

[0083] Figure 11-5 This is the result diagram of method 5 in Example 3-3.

[0084] Figure 11-6 This is the result diagram of method 6 in Example 3-3;

[0085] Figure 12-1 This is the result diagram of investigating condition 1 in Example 3-4;

[0086] Figure 12-2 This is the result diagram of the investigation condition 2 in Example 3-4;

[0087] Figure 12-3 This is a result diagram of the investigation condition 3 in Example 3-4;

[0088] Figure 12-4 This is a result diagram of the investigation condition 4 in Example 3-4;

[0089] Figure 12-5 This is a result diagram of the investigation condition 5 in Example 3-4;

[0090] Figure 12-6 This is a result diagram of the investigation condition 6 in Example 3-4;

[0091] Figure 12-7 This is a graph showing the results of investigating condition 7 in Example 3-4. DETAILED DESCRIPTION

[0092] The following examples and test examples are used to further illustrate the present invention, but are not intended to limit the effective scope of the present invention in any way.

[0093] instrument

[0094]

[0095] Reference

[0096]

[0097]

[0098] Test sample of Example 1

[0099] name batch number source Compound Sophora flavescens injection 20181138 Shanxi Zhendong Pharmaceutical Co., Ltd. Compound Sophora flavescens injection 20181034 Shanxi Zhendong Pharmaceutical Co., Ltd. Compound Sophora flavescens injection 20181139 Shanxi Zhendong Pharmaceutical Co., Ltd. Compound Sophora flavescens injection 20181203 Shanxi Zhendong Pharmaceutical Co., Ltd. Compound Sophora flavescens injection 20181204 Shanxi Zhendong Pharmaceutical Co., Ltd. Compound Sophora flavescens injection 20181209 Shanxi Zhendong Pharmaceutical Co., Ltd. Compound Sophora flavescens injection 20181212 Shanxi Zhendong Pharmaceutical Co., Ltd. Compound Sophora flavescens injection 20181213 Shanxi Zhendong Pharmaceutical Co., Ltd. Compound Sophora flavescens injection 20181214 Shanxi Zhendong Pharmaceutical Co., Ltd. Compound Sophora flavescens injection 20181215 Shanxi Zhendong Pharmaceutical Co., Ltd.

[0100] Test sample of Example 2

[0101]

[0102]

[0103] Reagents

[0104] name batch number source level Potassium dihydrogen phosphate 018823 MREAD HPLC Phosphoric acid 0160318 Beijing Chemical Plant Analytical level Methanol 10985407 902 merck keck gmbh & co. kg HPLC

[0105] Example 1 Detection method of active ingredients of compound sophora flavescens injection

[0106] 1. Chromatographic conditions, sample preparation, system suitability requirements, calculation formulas, and limit requirements

[0107]

[0108] 2. Verify the specific content

[0109] 2.1 System Applicability

[0110] (1) Experimental procedures

[0111] Preparation of blank solution: 0.2% potassium dihydrogen phosphate solution-methanol = 85:15 mixed solution, filter and obtain.

[0112] Preparation of reference solution: accurately weigh appropriate amount of matrine reference substance, oxidized matrine reference substance and oxidized sophocarpine reference substance, add blank solution to prepare mixed reference solution I containing 0.33 mg of matrine, 0.85 mg of oxidized matrine and 0.25 mg of oxidized sophocarpine per 1 ml, shake well to obtain;

[0113] Accurately weigh appropriate amounts of sophoracarpine reference substance, sophoridine reference substance, and methylazooxymethanol primulosyl glycoside reference substance, add blank solution to prepare a mixed reference solution II containing 0.09 mg sophoracarpine, 0.08 mg sophoridine, and 0.08 mg methylazooxymethanol primulosyl glycoside per 1 ml, shake well, and obtain.

[0114] Accurately measure 2 ml of mixed reference solution I and II, place in a 10 ml volumetric flask, dilute to the mark with blank solution, and shake well.

[0115] Preparation of test solution: Accurately measure 1 ml of compound sophora flavescens injection (batch number 20181138), place in a 50 ml volumetric flask, add blank solution to the scale, shake well, filter, and take the filtrate as the test solution.

[0116] Injection procedure requirements

[0117] Injection order

[0118] order sample Number of needles 1 blank solution 1 needle 2 Reference solution 5 needles (continuous needle insertion) 3 Test solution 1 needle

[0119] (2) Result report

[0120] The RSD values ​​of the peak area and retention time of the reference solution after 5 consecutive injections.

[0121] Table 2.1-1 Peak area and retention time results of reference solution

[0122]

[0123] Table 2.1-2 System suitability results

[0124]

[0125]

[0126] (3) Conclusion

[0127] From the results, the reference solution was injected 5 times continuously, and the RSDs of the peak areas of oxymatrine, matrine and oxysophocarpine were all less than 2.0%, and the RSDs of the retention times were all less than 2.0%. The RSDs of the peak areas of sophoracarpine, sophoridine and methyl azoxymethanol primulosidoside were all less than 3.0%, and the RSDs of the retention times were all less than 3.0%. The theoretical plate numbers of the six indicator components were greater than 3000, and the tailing factors were all less than 2.0, which met the requirements.

[0128] 2.2 Exclusivity

[0129] (1) Experimental procedures

[0130] Preparation of blank solution: 0.2% potassium dihydrogen phosphate solution-methanol = 85:15 mixed solution, filter and obtain.

[0131] Preparation of negative sample solution: Accurately measure 1 ml of Sophora flavescens injection (wild and cultivated) and 1 ml of Sophora flavescens injection, place in a 50 ml volumetric flask, add blank solution to the scale, shake well, filter, and take the filtrate as the negative sample solution.

[0132] Preparation of 0.25% Tween solution: weigh 0.25g Tween 80, add water to dissolve to 100ml, shake well, filter, and take the filtrate as 0.25% Tween solution.

[0133] Preparation of reference solution: refer to the preparation method of reference solution under 2.1.

[0134] Preparation of test solution: Accurately measure 1 ml of compound sophora flavescens injection, place in a 50 ml volumetric flask, add blank solution to the scale, shake well, and set aside.

[0135] Preparation of filter interference samples: Take the test solution (batch number 20181138), centrifuge one portion, filter one portion and discard different volumes (1ml, 3ml, 5ml, 7ml, 9ml).

[0136] Injection procedure requirements

[0137] Injection order

[0138]

[0139]

[0140] (2) Results report Figure 1 And the table below.

[0141] Table 2.2-1 Results of filter interference experiment (discarded different volumes relative to the area percentage of centrifuged samples)

[0142]

[0143] (3) Conclusion

[0144] From the results, the blank solution, blank mobile phase and 0.25% Tween 80 solution had no interference on the samples, the single Sophora flavescens negative sample solution (wild Sophora flavescens and cultivated Sophora flavescens) had no interference on methyl azoxymethanol primulin glycoside, and the single white turmeric negative sample solution had no interference on alkaloids.

[0145] The relative content of the area of ​​the index component of the test solution after discarding different volumes and the area of ​​the index component of the centrifuged test solution is between 95.0% and 102.0%, and the adsorption is negligible.

[0146] 2.3 Linearity and range

[0147] (1) Experimental procedures

[0148] Preparation of blank solution: 0.2% potassium dihydrogen phosphate solution-methanol = 85:15 mixed solution, filter and obtain.

[0149] Preparation of linear stock solution: accurately weigh appropriate amount of matrine reference substance, oxidized matrine reference substance, and oxidized sophoracarpine reference substance, add blank solution to prepare mixed reference solution I containing 0.33 mg of matrine, 0.85 mg of oxidized matrine, and 0.25 mg of oxidized sophoracarpine per 1 ml, shake well to obtain; accurately weigh appropriate amount of sophoracarpine reference substance, sophoridine reference substance, and methyl azoxymethanol primulosin reference substance, add blank solution to prepare mixed reference solution II containing 0.09 mg of sophoracarpine, 0.08 mg of sophoridine, and 0.08 mg of methyl azoxymethanol primulosin per 1 ml, shake well to obtain.

[0150] Reference solution 1: Accurately measure 0.5 ml of each of the mixed reference solutions I and II, place them in a 10 ml volumetric flask, dilute to the mark with the blank solution, and shake well.

[0151] Reference solution 2: Accurately measure 1 ml each of the mixed reference solutions I and II, place in a 10 ml volumetric flask, dilute to the mark with the blank solution, and shake well.

[0152] Reference solution 3: Accurately measure 2 ml of mixed reference solution I and II, place in a 10 ml volumetric flask, dilute to the mark with blank solution, and shake well.

[0153] Reference solution 4: Accurately measure 3 ml of mixed reference solution I and II, place in a 10 ml volumetric flask, dilute to the mark with blank solution, and shake well.

[0154] Reference solution 5: Accurately measure 4 ml of mixed reference solution I and II, place in a 10 ml volumetric flask, dilute to the mark with blank solution, and shake well.

[0155] Injection procedure requirements

[0156] Injection order

[0157]

[0158]

[0159] ( 2) Result Report

[0160] The regression equations, correlation coefficients and linear graph results of each indicator component can be found in Figure 2 .

[0161] (3) Conclusion

[0162] The linear correlation coefficient was ≥0.999, which met the standard.

[0163] 2.4 Accuracy

[0164] (1) Experimental procedures

[0165] Preparation of blank solution: 0.2% potassium dihydrogen phosphate solution-methanol = 85:15 mixed solution, filter and obtain.

[0166] Preparation of reference solution: Refer to the preparation method of reference solution under 2.1 and prepare two portions in the same way.

[0167] Preparation of 50% recovery solution: Accurately measure 0.5 ml of compound sophora flavescens injection and place it in a 50 ml volumetric flask. Add 2.5 ml of mixed reference solution I and II respectively, then add blank solution to the scale, shake well, filter, and use this as the 50% recovery solution (prepare 3 portions in the same way).

[0168] Preparation of 100% recovery solution: Accurately measure 0.5 ml of compound sophora flavescens injection and place it in a 50 ml volumetric flask. Add 5 ml of mixed reference solution I and II respectively, then add blank solution to the scale, shake well, filter, and use this as the 100% recovery solution (prepare 3 portions in the same way).

[0169] Preparation of 150% recovery solution: Accurately measure 0.5 ml of compound Sophora flavescens injection and place it in a 50 ml volumetric flask. Add 7.5 ml of mixed reference solution I and II respectively, then add blank solution to the scale, shake well, filter, and use this as 150% recovery solution (prepare 3 portions in the same way).

[0170] Injection order

[0171]

[0172]

[0173] (2) Result report

[0174] Table 2.4-1 Test results of recovery rate of methyl azoxymethanol primrose glycoside content

[0175]

[0176] Table 2.4-2 Matrine content recovery test results

[0177]

[0178] Table 2.4-3 Sophoracarpine content recovery test results

[0179]

[0180] Table 2.4-4 Sophoridine content recovery test results

[0181]

[0182]

[0183] Table 2.4-5 Test results of recovery rate of oxidized sophoracarpine content

[0184]

[0185] Table 2.4-6 Test results of recovery rate of oxymatrine content

[0186] (3) Conclusion

[0187] The recoveries of matrine, oxymatrine, oxysophocarpine, sophoracarpine, sophoridine, and methylazoxymethanol primulosidoside in the test samples were all between 95% and 105%, and the RSDs of the 9 recoveries were all less than 3%, which met the requirements.

[0188] 2.5 Repeatability

[0189] (1) Experimental procedures

[0190] Preparation of blank solution: 0.2% potassium dihydrogen phosphate solution-methanol = 85:15 mixed solution, filter and obtain.

[0191] Preparation of reference solution: Refer to the preparation method of reference solution under 2.1 and prepare two portions in the same way.

[0192] Preparation of test solution: accurately measure 1 ml of compound sophora flavescens injection (batch number 2081138), place in a 50 ml volumetric flask, add blank solution to the mark, shake well, filter, and take the filtrate as the test solution. Repeat 6 times in parallel.

[0193] Injection procedure requirements

[0194] Injection order

[0195] order sample Number of needles 1 blank solution 1 needle 2 Reference substance 1 solution Pin 5 (Continuity Test) 3 Reference substance 2 solution 2-pin 4 Test sample-1 solution 1 needle 5 Test sample-2 solution 1 needle 6 Test sample-3 solution 1 needle 7 Test sample-4 solution 1 needle 8 Test sample-5 solution 1 needle 9 Test sample-6 solution 1 needle 10 Reference substance 1 solution 1 needle

[0196] (2) Result report

[0197] Table 2.5 Repeatability test results

[0198]

[0199] (3) Conclusion

[0200] From the results, the RSDs of matrine, oxymatrine and oxysophocarpine in the six samples were all less than 3.0%, and the RSDs of sophocarpine, sophoridine and methylazoxymethanol primulosidoside were all less than 4.0%, indicating that the samples had good repeatability.

[0201] 2.6 Precision

[0202] (1) Experimental procedures

[0203] Preparation of blank solution: 0.2% potassium dihydrogen phosphate solution-methanol = 85:15 mixed solution, filter and obtain.

[0204] Preparation of reference solution: Refer to the preparation method of reference solution under 2.1 and prepare two portions in the same way.

[0205] Preparation of test solution: Accurately measure 1 ml of compound sophora flavescens injection (batch number 20181138), place in a 50 ml volumetric flask, add blank solution to the scale, shake well, filter, and take the filtrate as the test solution.

[0206] Injection procedure requirements

[0207] Injection order

[0208]

[0209]

[0210] (2) Result report

[0211] Table 2.6 Precision test results

[0212]

[0213] (3) Conclusion

[0214] From the results, it can be seen that after 6 consecutive injections, the RSDs of the contents of matrine, oxymatrine and oxysophocarpine in the test sample were all less than 3.0%, and the RSDs of the contents of sophoracarpine, sophoridine and methylazoxymethanol primulosidoside were all less than 4.0%, indicating that the test sample had good precision.

[0215] 2.7 Solution stability

[0216] (1) Experimental procedures

[0217] Preparation of blank solvent: 0.2% potassium dihydrogen phosphate solution-methanol = 85:15 mixed solution, filter and obtain.

[0218] Preparation of reference solution: Refer to the preparation method of reference solution under 2.1 and prepare two portions in the same way.

[0219] Preparation of test solution: Accurately measure 1 ml of compound sophora flavescens injection (batch number 20181138), place in a 50 ml volumetric flask, add blank solution to the scale, shake well, filter, and take the filtrate as the test solution.

[0220] Injection procedure requirements

[0221] Injection order

[0222] order sample Number of needles 1 blank solution 1 needle 2 Reference substance 1 solution Pin 5 (Continuity Test) 3 Reference substance 2 solution 2-pin 4 Test product - 0h 1 needle 5 Test product - 1h 1 needle 6 Test product - 2h 1 needle 7 Test product - 4h 1 needle 8 Test product - 8h 1 needle 9 Test product - 16h 1 needle 10 Test product - 24h 1 needle 11 Reference substance 1 solution 1 needle

[0223] (2) Result report

[0224] Table 2.7 Stability test results

[0225]

[0226]

[0227] (3) Conclusion

[0228] From the results, it can be seen that after the reference solution and the test solution were placed for 24 hours, the RSD of the contents of matrine, oxymatrine and oxysophocarpine in the test sample was less than 3.0%, and the RSD of the contents of sophocarpine, sophoridine and methyl azoxymethanol primulosidoside was less than 4.0%, indicating that the test sample was stable within 24 hours.

[0229] Calculate the percentage of the index component area at each time point to the index component area at 0 hours. The results of the reference and test solutions at each time point are compared with the initial results.

[0230] Between 98.0% and 102.0%, the solution stability is good.

[0231] 2.8 Durability

[0232] (1) Experimental procedures

[0233] The changed chromatographic conditions are shown in the table below.

[0234]

[0235] Preparation of blank solution: 0.2% potassium dihydrogen phosphate solution-methanol = 85:15 mixed solution, filter and obtain.

[0236] Preparation of reference solution: refer to the preparation method of reference solution under 2.1.

[0237] Preparation of test solution: Accurately measure 1 ml of compound sophora flavescens injection from each batch, place in a 50 ml volumetric flask, add blank solution to the scale, shake well, filter, and take the filtrate as the test solution.

[0238] Injection procedure requirements.

[0239] (Different inspection conditions are sampled in this order)

[0240] Injection order

[0241] order sample Number of needles 1 blank solution 1 needle 2 Reference substance 1 solution 5-pin 3 Reference substance 2 solution 2-pin 4 Test sample-1 1 needle 5 Test sample-2 1 needle 6 Sample-3~Sample-9 1 injection per test sample 7 Test sample-10 1 needle 8 Reference substance 1 solution 1 needle

[0242] Note: If the reference substance and test solution are stable within the detection time range, there is no need to re-prepare. If the solution is unstable, when the chromatographic conditions are changed, the reference substance and test solution need to be re-prepared before use.

[0243] (2) Result report

[0244] Table 2.9 Content durability test results

[0245]

[0246]

[0247]

[0248] (3) Conclusion

[0249] The test solution, under different conditions, the contents of matrine, sophorocarpine, oxysophorocarpine, and oxymatrine were basically the same, and the RSD values ​​of each batch of samples were less than 3%, and the RSD value of methyl azoxymethanol primulin glycoside was less than 5%, with good durability. Under the conditions of instrument 2 chromatographic column 1, the content of each batch of sophoridine samples was low, so the RSD value was less than 10%, and the durability was within an acceptable range.

[0250] Example 2 Verification of UPLC fingerprint of compound sophora flavescens injection

[0251] 1. Chromatographic conditions, elution conditions, sample preparation, etc.

[0252]

[0253] 2 Verify content

[0254] 2.1 System suitability

[0255] (1) Experimental procedures

[0256] Preparation of blank solution: methanol-0.2% potassium dihydrogen phosphate = 15:85, filter and obtain.

[0257] Preparation of reference solution: accurately weigh appropriate amount of matrine reference substance, oxidized matrine reference substance, and oxidized sophoracarpine reference substance, add blank solution to prepare mixed reference solution I containing 0.33 mg of matrine, 0.85 mg of oxidized matrine, and 0.25 mg of oxidized sophoracarpine per 1 ml, shake well to obtain; accurately weigh appropriate amount of sophoracarpine reference substance, sophoridine reference substance, and methyl azoxymethanol primulosin reference substance, add blank solution to prepare mixed reference solution II containing 0.09 mg of sophoracarpine, 0.08 mg of sophoridine, and 0.08 mg of methyl azoxymethanol primulosin per 1 ml, shake well to obtain; accurately weigh appropriate amount of trifoliate bean pterostilbene glycoside reference substance, sophoramine reference substance, and guava acid reference substance, add blank solution to prepare mixed reference solution III containing 0.08 mg of trifoliate bean pterostilbene glycoside, 0.08 mg of sophoramine, and guava acid per 1 ml, shake well to obtain. Accurately measure 2 ml each of mixed reference solution I, II, and III, place in a 10 ml volumetric flask, dilute to the mark with blank solution, shake well, and the mixture is ready.

[0258] Test solution: Accurately measure 1 ml of compound sophora flavescens injection, place in a 50 ml volumetric flask, add blank solution to the scale, shake well, filter, and take the filtrate as the test solution.

[0259] The injection sequence and requirements are shown in the table below.

[0260] Injection order

[0261] order sample Number of needles 1 blank solution 1 needle 2 Reference solution 5 needles (continuous needle insertion) 3 Test solution 1 needle

[0262] (2) Result report

[0263] The RSD values ​​of the peak area and retention time of the reference solution after 5 consecutive injections.

[0264] Table 2.1-1 Peak area and retention time results of reference solution

[0265]

[0266] Table 2.1-2 System suitability results

[0267]

[0268] (3) Conclusion

[0269] From the results, the reference solution was injected 5 times continuously, and the RSDs of the peak areas of oxymatrine, matrine and oxysophocarpine were all less than 2.0%, and the RSDs of the retention times were all less than 2.0%. The RSDs of the peak areas of sophoracarpine, sophoridine and methyl azoxymethanol primulosidoside were all less than 3.0%, and the RSDs of the retention times were all less than 3.0%. The theoretical plate numbers of the six indicator components were greater than 3000, and the tailing factors were less than 2.0, which met the requirements.

[0270] 2.2 Fingerprint establishment

[0271] (1) Experimental procedures

[0272] Blank solution: methanol-0.2% potassium dihydrogen phosphate = 15:85 mixed solution, filtered to obtain.

[0273] Preparation of reference solution: refer to the preparation method of reference solution under 2.1.

[0274] Each batch of test solution: accurately measure 1 ml of compound sophora flavescens injection from each batch, place it in a 50 ml volumetric flask, add blank solution to the scale, shake well, filter, and take the filtrate as the test solution.

[0275] The injection sequence and requirements are shown in the table below.

[0276] Injection order

[0277]

[0278]

[0279] (2) Result report

[0280] Based on the chromatographic fingerprints of 10 batches of compound Kushen injection, the 2012 edition of the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" recommended by the Pharmacopoeia Committee was used for data processing. The chromatographic peak of test sample 1 (S1) was used as the reference spectrum. The median method was used with a time window of 0.1. After multi-point correction, full peak matching was performed to generate standard reference fingerprints and common patterns. Figure 3-1 to Figure 3-2 And the table below.

[0281] Table 2.2-1 Similarity results between fingerprints of each batch and control fingerprints

[0282]

[0283]

[0284] Table 2.2-2 Non-common peak results of each batch

[0285] Test product Non-common peak area Total peak area Non-common peak area % 20181138 74.01 2242.64 3.30 20181139 66.45 2476.85 2.68 20181203 46.58 2329.11 2.00 20181204 56.14 2279.62 2.46 20181209 57.61 2291.59 2.51 20181212 53.14 2381.77 2.23 20181213 52.58 2473.87 2.13 20181214 38.36 2328.47 1.65 20181215 47.41 2395.86 1.98 20181034 39.67 2413.80 1.64

[0286] (3) Conclusion

[0287] After comparison with the reference substance, it can be concluded that peak 1 is sophoramine, peak 2 is methyl azoxyprimulosidoside, peak 3 is matrine, peak 4 is sophoracarpine, peak 5 is sophoridine, peak 6 is oxysophocarpine, peak 7 is oxymatrine, peak 8 is guava acid, and peak 9 is pterostilbene glycoside.

[0288] From the results, the similarity between the 10 batches of samples and the control fingerprints is greater than 0.9, and the non-common peak area accounts for less than 5%.

[0289] 2.3 Repeatability

[0290] (1) Experimental procedures

[0291] Preparation of blank solution: methanol-0.2% potassium dihydrogen phosphate = 15:85, filter and obtain.

[0292] Preparation of reference solution: refer to the preparation method of reference solution under 2.1.

[0293] Preparation of test solution: Accurately measure 1 ml of 6 portions of compound sophora flavescens injection of the same batch number, place in a 50 ml volumetric flask, add blank solution to the scale, shake well, filter, and take the filtrate as the test solution.

[0294] The injection sequence and requirements are shown in the table below.

[0295] Injection order

[0296] order sample Number of needles 1 blank solution 1 needle 2 Reference solution Pin 5 (Continuity Test) 3 Test sample-1 solution 1 needle 4 Test sample-2 solution 1 needle 5 Test sample-3 solution 1 needle 6 Test sample-4 solution 1 needle 7 Test sample-5 solution 1 needle 8 Test sample-6 solution 1 needle 9 Reference solution 1 needle

[0297] (2) Results report Figure 4

[0298] Table 2.3-1 Similarity results of repetitive common peak patterns

[0299]

[0300]

[0301] Table 2.3-2 Relative retention time results of repetitive common peaks

[0302]

[0303] Table 2.3-3 Repeatability common peak area results

[0304]

[0305] (3) Conclusion

[0306] From the results, we can see that the similarity among the 6 samples is greater than 0.99, and the RSD values ​​of the relative retention time and peak area of ​​each common peak are less than 3%, so the repeatability is good.

[0307] 2.6 Precision

[0308] (1) Experimental procedures

[0309] Preparation of blank solution: methanol-0.2% potassium dihydrogen phosphate = 15:85, filter and obtain.

[0310] Preparation of reference solution: refer to the preparation method of reference solution under 2.1.

[0311] Preparation of test solution: Accurately measure 1 ml of compound sophora flavescens injection, place in a 50 ml volumetric flask, add blank solution to the scale, shake well, filter, and take the filtrate as the test solution.

[0312] The injection sequence and requirements are shown in the table below.

[0313] Injection order

[0314] order sample Number of needles 1 blank solution 1 needle 2 Reference solution Pin 5 (Continuity Test) 3 Test solution 6-pin 4 Reference solution 1 needle

[0315] (2) Results report Figure 5 And the following table

[0316] Table 2.4-1 Precision common peak pattern similarity results

[0317]

[0318]

[0319] Table 2.4-2 Precision common peak relative retention time results

[0320]

[0321] Table 2.4-3 Precision common peak area results

[0322]

[0323]

[0324] ( 3) Conclusion

[0325] From the results, it can be seen that the similarity of the test sample after 6 consecutive injections was greater than 0.99, and the RSD values ​​of the relative retention time and peak area of ​​each common peak were less than 3%, which met the requirements.

[0326] 2.7 Solution stability

[0327] (1) Experimental procedures

[0328] Preparation of blank solution: methanol-0.2% potassium dihydrogen phosphate = 15:85, filter and obtain.

[0329] Preparation of reference solution: refer to the preparation method of reference solution under 2.1.

[0330] Preparation of test solution: Accurately measure 1 ml of compound sophora flavescens injection, place in a 50 ml volumetric flask, add blank solution to the scale, shake well, filter, and take the filtrate as the test solution.

[0331] The injection sequence and requirements are shown in the following table.

[0332] Injection order

[0333] order sample Number of needles 1 blank solution 1 needle 2 Reference solution Pin 5 (Continuity Test) 3 Test product - 0h 1 needle 4 Test product - 1h 1 needle 5 Test product - 2h 1 needle 6 Test product - 4h 1 needle 7 Test product - 8h 1 needle 8 Test product - 16h 1 needle 9 Test product - 24h 1 needle 10 Reference solution 1 needle

[0334] (2) Results report Figure 6 And the following table

[0335] Table 2.5-1 Stability common peak pattern similarity results

[0336]

[0337] Table 2.5-2 Relative retention time results of common stability peaks

[0338]

[0339] Table 2.5-3 Stability common peak area results

[0340]

[0341] ( 3) Conclusion

[0342] From the results, it can be seen that the similarity of the test samples within 24 hours is greater than 0.99, and the relative retention time and peak area RSD values ​​of each common peak are less than 3%, so the test samples are stable within 24 hours.

[0343] 2.8 Durability

[0344] (1) Experimental procedures

[0345] Preparation of blank solution: methanol-0.2% potassium dihydrogen phosphate = 15:85, filter and obtain.

[0346] Preparation of reference solution: refer to the preparation method of reference solution under 2.1.

[0347] Preparation of test solution: Accurately measure 1 ml of compound sophora flavescens injection and place it in a 50 ml volumetric flask, add blank solution to the scale, shake well, filter, and take the filtrate as the test solution.

[0348] Take 1 μl of the above solution and inject it into the HPLC under different conditions. The injection sequence and requirements are shown in the table below (different inspection conditions are injected according to this injection sequence).

[0349] Injection order

[0350]

[0351]

[0352] (3) Result report

[0353] Table 2.8-1 Parameters of Chromatographic Condition Changes

[0354]

[0355] (2) Result report

[0356] ① Using batch number 20181138 as the test sample, the durability of the test sample on the chromatographic column and instrument was investigated. The results are shown in Figure 7-1 And the table below.

[0357] Table 2.8-2 Similarity results of common peak patterns of different chromatographic columns

[0358]

[0359]

[0360] Table 2.8-3 Relative retention time results of common peaks in different chromatographic columns

[0361]

[0362] Table 2.8-4 Common peak area results of different chromatographic columns

[0363]

[0364] The durability results of different instruments can be found in Figure 7-2 And the following table

[0365] Table 2.8-5 Common peak pattern similarity results of different instruments

[0366]

[0367] Table 2.8-6 Relative retention time results of common peaks in different instruments

[0368]

[0369] Table 2.8-7 Common peak area results for different instruments

[0370]

[0371] ② Based on the chromatographic fingerprints of 10 batches of compound Sophora flavescens injection, fingerprints of different chromatographic columns and instruments were established and reference fingerprints were generated to examine the similarity of the generated reference fingerprints. Figure 7-3 to Figure 7-11 And the table below.

[0372] Table 2.8-8 Similarity results (Instrument 1 Column 2)

[0373]

[0374]

[0375] Table 2.8-9 Similarity results (Instrument 1 Column 3)

[0376]

[0377] Table 2.8-10 Similarity results (Instrument 2 Column 1)

[0378]

[0379]

[0380] Table 2.8-11 Similarity results (Instrument 3 Column 1)

[0381]

[0382] Table 2.8-12 Similarity results (different control fingerprints)

[0383]

[0384] (3) Conclusion

[0385] From the results, we can see that in the results of different chromatographic columns, the similarity of each common peak is greater than 0.99, the relative retention time RSD is less than 3%, and the peak area is less than 5%; in the results of different instruments, the similarity of each common peak is greater than 0.99, and the relative retention time and peak area RSD are less than 10%, indicating that the instrument has an impact on the method, but it is within an acceptable range.

[0386] The similarities of the 10 batches of samples under different inspection conditions were greater than 0.99, and the similarities between the generated control fingerprint maps were greater than 0.99. Therefore, judging from the similarity results, this method has good durability.

[0387] 2.9 Inspection of key points of production process

[0388] (1) Experimental procedures

[0389] Preparation of blank solution: methanol-0.2% potassium dihydrogen phosphate = 15:85, filter and obtain.

[0390] Preparation of reference solution: refer to the preparation method of reference solution under 2.1.

[0391] Preparation of test solution: Calculate the sampling volume according to the volume of each key point, where key point 1 is to take 1ml into a 25ml volumetric flask, key points 2 and 6 are to take 5ml into a 50ml volumetric flask, key point 4 is to take 2ml into a 25ml volumetric flask, key point 5 is to take 1ml into a 100ml volumetric flask, and the remaining key points are to take 1ml into a 50ml volumetric flask. Add blank solution to all samples to the scale, shake well, filter, and take the filtrate as the test solution.

[0392] The injection sequence and requirements are shown in the following table.

[0393] Injection order

[0394]

[0395]

[0396] (2) Results report Figure 8

[0397] Table 2.9-1 Similarity results of common peak patterns in production processes

[0398]

[0399]

[0400] (3) Conclusion

[0401] From the results, we can see that the similarity of the key points in the production process is between 0.451 and 0.754, and the similarity between the key points 8-22 (water-sinking solution-sterilized samples) in the production process is relatively high, greater than 0.9.

[0402] Examples 3-1 to 3-4 UPLC Conditions Exploration

[0403] Example 3-1 (Test sample preparation and investigation)

[0404] Experimental steps:

[0405] 3-1.1 Preparation of blank solution: adjust the pH value of potassium dihydrogen phosphate solution to 3.0 with phosphoric acid, and filter the mixed solution of 0.34% potassium dihydrogen phosphate solution-methanol = 85:15.

[0406] 3-1.2 Preparation of reference solution: Accurately measure appropriate amount of matrine, oxymatrine, oxysophocarpine, sophoracarpine, sophoridine, and methylazoxymethanol primulosidoside reference substances, and add blank solution to make reference solutions of 0.2 mg / ml, 0.4 mg / ml, 0.2 mg / ml, 0.05 mg / ml, 0.05 mg / ml, and 0.05 mg / ml, respectively.

[0407] 3-1.3 Preparation of test solution: Take the compound sophora flavescens injection sample, accurately measure 0.5 ml, place it in a 25 ml volumetric flask, dilute to the mark with blank solution, shake well, filter, and use as test solution 1. Take the compound sophora flavescens injection sample, accurately measure 1 ml, place it in a 50 ml volumetric flask, dilute to the mark with blank solution, shake well, filter, and use as test solution 2.

[0408] 3-1.4 Chromatographic conditions

[0409] Column: ACQUITY UPLC CSH TM C 18 1.7μm, 2.1mm×100mm; detection wavelength: 211nm, column temperature: 30℃, flow rate: 0.3ml / min; mobile phase A: methanol, mobile phase B: 0.2% potassium dihydrogen phosphate (pH adjusted to 3.0 with phosphoric acid) gradient elution.

[0410] 3-1.5 According to the above chromatographic conditions, inject 0.5 μl of sample 1 and 1 μl of sample 2 respectively.

[0411] 3-1.6 Experimental results: See Fig. 9 .

[0412] In summary, the test sample was prepared using the test solution 2 preparation method (1 ml of sample was diluted to 50 ml with blank solution).

[0413] Example 3-2 (mobile phase investigation)

[0414] Experimental steps:

[0415] 3-2.1 Blank solution preparation: See Example 3-1 for blank solution preparation.

[0416] 3-2.2 Preparation of reference solution: Accurately measure appropriate amounts of sophoramine, guava acid, N-methylcytisine, and pterostilbene glycoside reference substances, add blank solution to prepare 0.05 mg / ml, 0.1 mg / ml, 0.05 mg / ml, and 0.1 mg / ml reference solutions, respectively. The rest is the same as the reference solution preparation in Example 3-1.

[0417] 3-2.3 Preparation of test solution: Take a sample of compound Sophora flavescens injection, accurately measure 1 ml, place it in a 50 ml volumetric flask, add blank solution to dilute to the scale, shake well, filter, and use it as the test solution.

[0418] 3-2.4 Investigation of chromatographic conditions:

[0419] Chromatographic conditions 1:

[0420] Column: ACQUITY UPLC CSH TM C18 1.7μm, 2.1mm×100mm

[0421] Detection wavelength: 211nm, column temperature: 30℃, flow rate: 0.4ml / min, injection volume: 1μl

[0422] Mobile phase A: 0.01M ammonium formate aqueous solution B: acetonitrile: 0.01M ammonium formate aqueous solution (9:1) Elution conditions:

[0423]

[0424] Experimental results: see Figure 10-1 .

[0425] Chromatographic conditions 2:

[0426] Column: ACQUITY UPLC CSH TM C 18 1.7μm, 2.1mm×100mm

[0427] Detection wavelength: 211nm, column temperature: 30℃, flow rate: 0.3ml / min, injection volume: 1μl

[0428] Mobile phase A: acetonitrile B: 0.34% potassium dihydrogen phosphate (pH adjusted to 3.0 with phosphoric acid)

[0429]

[0430] Experimental results: see Figure 10-2 .

[0431] Chromatographic conditions 3:

[0432] The mobile phase was adjusted as follows, and the rest were the same as chromatographic conditions 2.

[0433] Mobile phase A: methanol B: 10mmol ammonium bicarbonate (pH 9.0)

[0434]

[0435] Experimental results: see Figure 10-3

[0436] Chromatographic conditions 4:

[0437] The mobile phase was adjusted as follows, and the rest was the same as condition 2.

[0438] Mobile phase A: Methanol B: 0.34% potassium dihydrogen phosphate (pH adjusted to 3.0 with phosphoric acid)

[0439]

[0440]

[0441] Experimental results: see Figure 10-4

[0442] Chromatographic conditions 5:

[0443] The mobile phase was adjusted as follows, and the rest was the same as condition 2.

[0444] Mobile phase A: Methanol B: 0.2% potassium dihydrogen phosphate (pH adjusted to 3.0 with phosphoric acid)

[0445]

[0446] Experimental results: see Figure 10-5 See 10-6 for the overlay diagram of each reference sample after injection under the above conditions and the test sample.

[0447] In summary, methanol-0.2% potassium dihydrogen phosphate is the best mobile phase.

[0448] Example 3-3 (mobile phase gradient investigation):

[0449] Experimental steps:

[0450] 3-3.1 Blank solution preparation: See Example 3-1 for blank solution preparation.

[0451] 3-3.2 Preparation of test solution: See Example 3-2 for preparation of test solution.

[0452] 3-3.3 Gradient condition investigation:

[0453] Column: ACQUITY UPLC CSH TM C 18 1.7μm, 2.1mm×100mm

[0454] Flow rate: 0.3 ml / min, column temperature: 30 °C, detection wavelength: 211 nm, injection volume: 1 μl

[0455] Mobile phase A: methanol B: 0.2% potassium dihydrogen phosphate (pH adjusted to 3.0 with phosphoric acid) Gradient elution procedures are: Method 1:

[0456]

[0457] Experimental results: see Figure 11-1 Method 2:

[0458]

[0459] Experimental results: see Figure 11-2 Method 3:

[0460]

[0461] Experimental results: see Figure 11-3 Method 4:

[0462]

[0463] Experimental results: see Figure 11-4 Method 5:

[0464]

[0465] Experimental results: see Figure 11-5 Method 6:

[0466]

[0467] Experimental results: see Figure 11-6 In summary, the elution program conditions of method 5 are optimal.

[0468] Example 3-4 (Investigation of Chromatographic Column, Column Temperature and Flow Rate)

[0469] Experimental steps:

[0470] 3-4.1 Blank solution preparation: See Example 3-1 for blank solution preparation.

[0471] 3-4.2 Preparation of test solution: See Example 3-1 for preparation of test solution 1.

[0472] 3-4.3 Conditions inspection:

[0473] Inspection Condition 1:

[0474] Column: Agilent Poroshell 120EC-C 18 4μm, 4.6mm×100mm

[0475] Flow rate: 0.3ml / min Column temperature: 30℃ Detection wavelength: 211nm Injection volume: 1μl

[0476] Mobile phase A: Methanol B: 0.34% potassium dihydrogen phosphate (pH adjusted to 3.0 with phosphoric acid)

[0477]

[0478] Experimental results: see Figure 12-1

[0479] Inspection Condition 2:

[0480] Column: ACQUITY UPLC CSH TM C 181.7μm, 2.1mm×100mm; detection wavelength: 211nm, injection volume: 1μl, flow rate: 0.3ml / min, column temperature: 35℃

[0481] The elution procedure is as in Example 3-3, and the gradient conditions are the same as those in Method 5 in Example 3-3.

[0482] Experimental results: see Figure 12-2

[0483] Inspection Condition 3:

[0484] Injection volume: 0.5ul, the rest is the same as investigation condition 2, and the gradient elution program is adjusted as follows:

[0485]

[0486] Experimental results: see Figure 12-3

[0487] Inspection Condition 4:

[0488] Injection volume: 0.5μl, flow rate: 0.3ml / min, 0.28ml / min, the rest is the same as investigation condition 2, and the elution program is adjusted as follows:

[0489]

[0490] Experimental results: see Figure 12-4

[0491] Inspection Condition 5:

[0492] Injection volume: 0.5μl, flow rate 0.28ml / min, the rest is the same as investigation condition 2, and the elution program is adjusted as follows:

[0493]

[0494]

[0495] Experimental results: see Figure 12-5

[0496] Inspection Condition 6:

[0497] Column temperature: 30℃, injection volume: 0.5μl, flow rate: 0.28ml / min, the rest is the same as investigation condition 2, and the elution program is adjusted as follows:

[0498]

[0499] Experimental results: see Figure 12-6

[0500] Inspection Condition 7:

[0501] Column temperature: 30℃, injection volume: 0.5μl, flow rate: 0.25ml / min, the rest is the same as investigation condition 2, and the elution program is adjusted as follows:

[0502]

[0503] Experimental results: see Figure 12-7

[0504] In summary, the best column is ACQUITY UPLC CSH TM C 18 1.7μm, 2.1mm×100mm, the optimal flow rate is 0.3ml / min, and the optimal column temperature is 30℃.

Claims

1. An ultra-high performance liquid chromatography detection method for the active ingredient content and fingerprint of compound sophora flavescens injection, characterized in that: The active ingredients are matrine, oxymatrine, methylazoxymethanol primulin glycoside, sophoracarpine, oxysophocarpine and sophoridine; the method comprises constructing a fingerprint of the compound sophora flavescens injection containing matrine, oxymatrine, methylazoxymethanol primulin glycoside, sophoracarpine, oxysophocarpine, sophoridine, guava acid, sophoramine and trifoliate glycoside, The ultra-high performance liquid chromatography conditions include a chromatographic column of ACQUITY UPLC CSH TM C 18 ; The mobile phase was 0.15%-0.25% potassium dihydrogen phosphate solution-methanol gradient elution; Column temperature is 28°C-32°C; Flow rate: 0.1ml / min-0.5ml / min; The detection wavelength is 209nm-213nm; The injection volume is 0.8μl-1.2μl; The elution gradient in the ultra-high performance liquid chromatography conditions is as follows: Ultra-high performance liquid chromatography further comprises: Preparation of blank solution: adjust the pH value of potassium dihydrogen phosphate solution to 3.0 with phosphoric acid, and filter a mixed solution of 0.2% potassium dihydrogen phosphate solution and methanol in a ratio of 85:15; Preparation of test solution: Accurately measure 1 ml of compound sophora flavescens injection, place in a 50 ml volumetric flask, add blank solution to the scale, shake well, filter, and take the filtrate as the test solution.

2. The method according to claim 1, characterized in that: The mobile phase in the ultra-high performance liquid chromatography conditions is 0.2% potassium dihydrogen phosphate solution-methanol gradient elution.

3. The method according to claim 1, characterized in that The ultra-high performance liquid chromatography conditions include a column temperature of 30°C.

4. The method according to claim 1, characterized in that: The ultra-high performance liquid chromatography conditions include a flow rate of 0.3 ml / min.

5. The method according to claim 1, characterized in that The ultra-high performance liquid chromatography conditions include a detection wavelength of 211 nm.

6. The method according to claim 1, characterized in that The ultra-high performance liquid chromatography conditions include an injection volume of 1 μl.

7. The method according to claim 1, characterized in that The ultra high performance liquid chromatography further comprises: Preparation of reference solution: Accurately weigh appropriate amounts of matrine reference substance, oxidized matrine reference substance, and oxidized sophocarpine reference substance, add blank solution to prepare a mixed reference solution I containing 0.33 mg of matrine, 0.85 mg of oxidized matrine, and 0.25 mg of oxidized sophocarpine per 1 ml, and shake well to obtain; Accurately weigh appropriate amounts of sophoracarpine reference substance, sophoridine reference substance, and methyl azoxymethanol primulosyl glycoside reference substance, add blank solution to prepare a mixed reference substance solution II containing 0.09 mg sophoracarpine, 0.08 mg sophoridine, and 0.08 mg methyl azoxymethanol primulosyl glycoside per 1 ml, shake well, and obtain; Accurately measure 2 ml of mixed reference solution I and II, place in a 10 ml volumetric flask, dilute to the mark with blank solution, and shake well.

8. The method according to any one of claims 1 to 7, characterized in that: The method comprises: The ultra-high performance liquid chromatography conditions were: (1) Preparation of blank solution: adjust the pH value of potassium dihydrogen phosphate solution to 3.0 with phosphoric acid, and filter a mixed solution of 0.2% potassium dihydrogen phosphate solution and methanol in a ratio of 85:15; (2) Preparation of reference solution: Accurately weigh appropriate amounts of matrine reference substance, oxidized matrine reference substance, and oxidized sophocarpine reference substance, add blank solution to prepare a mixed reference solution I containing 0.33 mg of matrine, 0.85 mg of oxidized matrine, and 0.25 mg of oxidized sophocarpine per 1 ml, and shake well to obtain; Accurately weigh appropriate amounts of sophoracarpine reference substance, sophoridine reference substance, and methyl azoxymethanol primulosyl glycoside reference substance, add blank solution to prepare a mixed reference substance solution II containing 0.09 mg sophoracarpine, 0.08 mg sophoridine, and 0.08 mg methyl azoxymethanol primulosyl glycoside per 1 ml, shake well, and obtain; Accurately measure 2 ml of mixed reference solution I and II, place in a 10 ml volumetric flask, dilute to scale with blank solution, and shake well; (3) Preparation of test solution: Accurately measure 1 ml of compound Sophora flavescens injection and place it in a 50 ml volumetric flask. Add blank solution to the mark, shake well, filter, and take the filtrate as the test solution. (4) Inject the blank solution, reference solution, and test solution into the liquid chromatograph in this order, record the chromatogram, and calculate the content of each active ingredient using the external standard method.

9. The method according to any one of claims 1 to 7, characterized in that: The method comprises: ultra-high performance liquid chromatography conditions are: (1) Preparation of blank solution: adjust the pH value of potassium dihydrogen phosphate solution to 3.0 with phosphoric acid, and filter a mixed solution of 0.2% potassium dihydrogen phosphate solution and methanol in a ratio of 85:15; (2) Preparation of reference solution: Accurately weigh appropriate amounts of matrine reference substance, oxidized matrine reference substance, and oxidized sophocarpine reference substance, add blank solution to prepare a mixed reference solution I containing 0.33 mg of matrine, 0.85 mg of oxidized matrine, and 0.25 mg of oxidized sophocarpine per 1 ml, and shake well to obtain; Accurately weigh appropriate amounts of sophoracarpine reference substance, sophoridine reference substance, and methyl azoxymethanol primulosyl glycoside reference substance, add blank solution to prepare a mixed reference substance solution II containing 0.09 mg sophoracarpine, 0.08 mg sophoridine, and 0.08 mg methyl azoxymethanol primulosyl glycoside per 1 ml, shake well, and obtain; Accurately weigh appropriate amounts of Pterostilbene glycoside reference substance, Sophoramine reference substance, and Guava acid reference substance, add blank solution to prepare mixed reference solution III, shake well, and obtain; Accurately measure 2 ml of each of the mixed reference solution Ⅰ, Ⅱ, and III, place them in a 10 ml volumetric flask, dilute to the mark with the blank solution, and shake well; (3) Preparation of test solution: Accurately measure 1 ml of compound Sophora flavescens injection and place it in a 50 ml volumetric flask. Add blank solution to the mark, shake well, filter, and take the filtrate as the test solution. (4) Inject the blank solution, reference solution, and test solution in the order of injection to construct the fingerprint of the compound sophora flavescens injection containing matrine, oxymatrine, methyl azoxymethanol primulin, sophorocarpine, oxysophocarpine, sophoridine, guava acid, sophoramine, and trifoliate glycoside; (5) Inject the blank solution, reference solution, and test solution into the liquid chromatograph in this order, record the chromatogram, and calculate the content of each active ingredient using the external standard method.

10. The method according to claim 9, characterized in that The method comprises: Taking oxymatrine as a reference, the relative retention times of the common characteristic peaks are: the relative retention time of sophoramine is 0.389±0.02; the relative retention time of methyl azoxyprimulosin is 0.469±0.04; the relative retention time of matrine is 0.637±0.04; the relative retention time of sophoracarpine is 0.767±0.03; the relative retention time of sophoridine is 0.819±0.02; the relative retention time of oxymatrine is 0.925±0.02; the relative retention time of oxymatrine is 1.0; the relative retention time of guava acid is 1.055±0.03; and the relative retention time of pterostilbene is 2.097±0.05.

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