Construction method of characteristic chromatogram of traditional Chinese medicine compound preparation and content determination method of seven index components of traditional Chinese medicine compound preparation

Abstract

The invention provides a construction method of a characteristic chromatogram of a traditional Chinese medicine compound preparation and a content determination method of seven index components of the traditional Chinese medicine compound preparation. The traditional Chinese medicine compound preparation is a mixture of extracts of radix puerariae, radix pseudostellariae and herba epimedii. The construction method of the specific chromatogram comprises the following steps: preparing a test solution from a traditional Chinese medicine compound preparation to be tested; and performing liquid chromatography detection on the test solution to obtain a characteristic chromatogram of the traditional Chinese medicine compound preparation, the conditions of liquid chromatography detection are as follows: a mobile phase comprises a mobile phase A and a mobile phase B, the mobile phase A is a phosphate solution, and the molar concentration of phosphate contained in the phosphate solution is 10mmol / L-50mmol / L; the mobile phase B is acetonitrile; gradient elution is adopted. The method can realize simultaneous detection of polar saccharide components and weak polar flavone / isoflavone components.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine detection, in particular to a construction method of a characteristic chromatogram of a traditional Chinese medicine compound preparation and a content determination method of seven index components thereof. BACKGROUND

[0002] Shenqi Bushen Capsule is a traditional Chinese medicine compound preparation composed of three medicinal materials, i.e., radix pseudostellariae, radix puerariae and herba epimedii, and has the effects of benefiting qi, nourishing yin and tonifying kidney. It is mainly used for the treatment of mild to moderate depression. The medicinal components of the medicinal materials in the compound are significantly different. Radix pseudostellariae is mainly composed of polysaccharides, which are high-molecular-weight carbohydrates formed by the dehydration condensation of multiple monosaccharide molecules, and the polysaccharides mainly contain glucose and galactose. Radix puerariae is rich in isoflavones such as puerarin, and herba epimedii is characterized by flavonol glycosides (the index components specified in the 2025 edition of the Pharmacopoeia of the People's Republic of China are tsushamin A, B, C and icariin). At present, there are many reports on liquid chromatography detection methods for single type of components (such as flavones or isoflavones), but due to the significant differences in polarity, solubility and chromatographic behavior between saccharide components (such as glucose and galactose, which are hydrolysis products of polysaccharides) and flavones / isoflavones, it is difficult to realize the simultaneous quantitative analysis of multiple types of components under the same chromatographic conditions by using traditional methods.

[0003] However, the quality control of traditional Chinese medicine compound preparations often needs to consider multiple index components, and the traditional method often needs to use different detection systems to determine saccharide components and weakly polar components respectively, resulting in low analysis efficiency, poor repeatability, and difficulty in comprehensively reflecting the overall quality of the compound. Therefore, it is of great significance to develop a liquid chromatography method for simultaneously detecting polar saccharide components (such as monosaccharides) and weakly polar flavone / isoflavone components (such as puerarin, tsushamin A-C and icariin) to improve the quality control level of traditional Chinese medicine compounds. SUMMARY

[0004] Therefore, it is necessary to provide a construction method of a characteristic chromatogram of a traditional Chinese medicine compound preparation and a content determination method of seven index components thereof, so as to realize the simultaneous detection of polar saccharide components and weakly polar flavone / isoflavone components.

[0005] The first aspect of the present application provides a construction method of a characteristic chromatogram of a traditional Chinese medicine compound preparation, wherein the traditional Chinese medicine compound preparation is a mixture comprising extracts of radix puerariae, radix pseudostellariae and herba epimedii; and the construction method of the characteristic chromatogram comprises the following steps:

[0006] preparing a test solution from the tested traditional Chinese medicine compound preparation; and

[0007] detecting the test solution by liquid chromatography to obtain the characteristic chromatogram of the traditional Chinese medicine compound preparation;

[0008] The liquid chromatography detection condition comprises:

[0009] The mobile phase comprises a mobile phase A and a mobile phase B, the mobile phase A is a phosphate solution, the phosphate contained in the phosphate solution has a molar concentration of 10 mmol / L-50 mmol / L; the mobile phase B is acetonitrile;

[0010] Gradient elution is adopted, and the program of the gradient elution comprises:

[0011] 0-2 min, maintaining the volume percentage of the mobile phase A as 85%, and the volume percentage of the mobile phase B as 15%; 2 min-8 min, reducing the volume percentage of the mobile phase A from 85% to 83%, and increasing the volume percentage of the mobile phase B from 15% to 17%; 8 min-20 min, reducing the volume percentage of the mobile phase A from 83% to 82%, and increasing the volume percentage of the mobile phase B from 17% to 18%; 20 min-36 min, reducing the volume percentage of the mobile phase A from 82% to 60%, and increasing the volume percentage of the mobile phase B from 18% to 40%; 36 min-40 min, maintaining the volume percentage of the mobile phase A as 60%, and the volume percentage of the mobile phase B as 40%; 40 min-42 min, increasing the volume percentage of the mobile phase A from 60% to 85%, and reducing the volume percentage of the mobile phase B from 40% to 15%.

[0012] In some embodiments, the phosphate contained in the phosphate solution comprises one or more of sodium phosphate and potassium phosphate;

[0013] Optionally, the phosphate contained in the phosphate solution comprises one or more of sodium dihydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate and dipotassium hydrogen phosphate.

[0014] In some embodiments, the liquid chromatography detection condition further comprises at least one of the following:

[0015] (1) the filler of the chromatographic column is octadecylsilane bonded silica gel;

[0016] (2) the column temperature is 25℃-40℃;

[0017] (3) the detection wavelength is 250 nm-260 nm;

[0018] (4) the flow rate is 0.5 mL / min-1.5 mL / min; and,

[0019] (5) the injection volume is 10 μL-20 μL;

[0020] Optionally, the length of the chromatographic column is 250 mm, the inner diameter is 4.6 mm, and the filler particle size is 5 μm.

[0021] In some embodiments, the step of preparing the test solution from the test traditional Chinese medicine compound preparation comprises:

[0022] mixing the test traditional Chinese medicine compound preparation with anhydrous ethanol, ultrasonically extracting, centrifuging, taking the supernatant after centrifugation, and preparing a first-step sample solution;

[0023] drying the residue after centrifugation to a constant weight, then mixing with an acid solution to perform a hydrolysis reaction, adjusting the pH of the solution after the hydrolysis reaction to neutral with an alkaline solution, diluting with water, and preparing a diluted reaction solution A;

[0024] mixing the diluted reaction solution A with a sodium hydroxide aqueous solution and a 1-phenyl-3-methyl-5-pyrazolone methanol solution, heating, mixing with an acidic aqueous solution after cooling, diluting with water, and preparing a diluted reaction solution B;

[0025] mixing the diluted reaction solution B with chloroform, shaking and extracting, taking the upper aqueous phase layer, and preparing a second-step sample solution; and

[0026] mixing the first-step sample solution and the second-step sample solution, diluting with an ethanol aqueous solution, and preparing a sample solution;

[0027] Optionally, the mass-volume ratio of the test traditional Chinese medicine compound preparation to anhydrous ethanol is (0.3-0.5) g:(25-40) mL;

[0028] Optionally, the acid solution comprises one or more of a trifluoroacetic acid aqueous solution, a hydrochloric acid aqueous solution, and a sulfuric acid aqueous solution;

[0029] Optionally, the acid solution is a trifluoroacetic acid aqueous solution, and the molar concentration of trifluoroacetic acid in the trifluoroacetic acid aqueous solution is 2 mol / L-4 mol / L;

[0030] Optionally, the temperature of the hydrolysis reaction is 90°C-120°C, and the time is 0.5 h-6 h;

[0031] Optionally, the alkaline solution comprises one or more of a sodium hydroxide aqueous solution and an ammonia aqueous solution, and the molar concentration of the alkaline substance in the alkaline solution is 2 mol / L-4 mol / L;

[0032] Optionally, the molar concentration of sodium hydroxide in the sodium hydroxide aqueous solution is 0.2 mol / L-0.5 mol / L, and the volume ratio of the diluted reaction solution A to the sodium hydroxide aqueous solution is 1:(1-3);

[0033] Optionally, the molar concentration of 1-phenyl-3-methyl-5-pyrazolone in the 1-phenyl-3-methyl-5-pyrazolone methanol solution is 0.5 mol / L-1 mol / L, and the volume ratio of the dilution reaction solution A to the 1-phenyl-3-methyl-5-pyrazolone methanol solution is 1:(1-2);

[0034] Optionally, the heating temperature is 60℃-90℃, and the time is 60 min-90 min;

[0035] Optionally, the acidic aqueous solution contains one or more of acetic acid and hydrochloric acid;

[0036] Optionally, the molar concentration of the acidic substance contained in the acidic aqueous solution is 0.2 mol / L-0.5 mol / L, and the volume ratio of the dilution reaction solution A to the acidic aqueous solution is 1:(1-2);

[0037] Optionally, the volume ratio of the dilution reaction solution B to the chloroform is (2-4):(4-6);

[0038] Optionally, the volume ratio of the first-step sample solution to the second-step sample solution is (2-5):(0.5-1).

[0039] The second aspect of the present application provides a method for constructing a characteristic map of a traditional Chinese medicine compound preparation, wherein the traditional Chinese medicine compound preparation is a mixture of extracts of Pueraria, Radix Pseudostellariae and Herba Epimedii; the method for constructing the characteristic map comprises the following steps:

[0040] Preparation of a test solution from the test traditional Chinese medicine compound preparation; and,

[0041] Liquid chromatography detection of the test solution to obtain a characteristic map of the traditional Chinese medicine compound preparation;

[0042] The conditions of the liquid chromatography detection include:

[0043] The mobile phase includes mobile phase A and mobile phase B, the mobile phase A is a phosphate solution, and the molar concentration of the phosphate in the phosphate solution is 10 mmol / L-50 mmol / L; the mobile phase B is acetonitrile;

[0044] Gradient elution is adopted, and the program of the gradient elution includes:

[0045] 0-8min, the volume percentage of the mobile phase A is maintained at 85%, and the volume percentage of the mobile phase B is 15%; 8min-15min, the volume percentage of the mobile phase A is decreased from 85% to 76%, and the volume percentage of the mobile phase B is increased from 15% to 24%; 15min-20min, the volume percentage of the mobile phase A is decreased from 76% to 74%, and the volume percentage of the mobile phase B is increased from 24% to 26%; 20min-36min, the volume percentage of the mobile phase A is decreased from 74% to 70%, and the volume percentage of the mobile phase B is increased from 26% to 30%; 36min-40min, the volume percentage of the mobile phase A is decreased from 70% to 60%, and the volume percentage of the mobile phase B is increased from 30% to 40%; 40min-42min, the volume percentage of the mobile phase A is increased from 60% to 85%, and the volume percentage of the mobile phase B is decreased from 40% to 15%.

[0046] In some embodiments, the phosphate salt contained in the phosphate salt solution comprises one or more of sodium phosphate salt and potassium phosphate salt;

[0047] Optionally, the phosphate salt contained in the phosphate salt solution comprises one or more of sodium dihydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate and dipotassium hydrogen phosphate.

[0048] In some embodiments, the conditions of the liquid chromatography detection further comprise at least one of the following:

[0049] (1) the packing material of the chromatographic column is octadecylsilane bonded silica gel;

[0050] (2) the column temperature is 25-40℃;

[0051] (3) the detection wavelength is 250-260nm;

[0052] (4) the flow rate is 0.5-1.5mL / min; and,

[0053] (5) the injection volume is 10-20μL;

[0054] Optionally, the length of the chromatographic column is 250mm, the inner diameter is 4.6mm, and the packing material particle size is 5μm.

[0055] In some embodiments, the step of preparing the test solution from the test traditional Chinese medicine compound preparation comprises:

[0056] mixing the test traditional Chinese medicine compound preparation with anhydrous ethanol, ultrasonically extracting, centrifuging, taking the supernatant after centrifugation, and preparing a first-step sample solution;

[0057] The centrifuged residue is dried to constant weight, then mixed with an acid solution for hydrolysis reaction, an alkaline solution is used to adjust the pH of the solution after hydrolysis reaction to neutral, water is added for dilution to prepare a diluted reaction solution A;

[0058] The diluted reaction solution A is mixed with a sodium hydroxide aqueous solution and a 1-phenyl-3-methyl-5-pyrazolone methanol solution, heated, mixed with an acidic aqueous solution after cooling, and diluted with water to prepare a diluted reaction B;

[0059] The diluted reaction B is mixed with chloroform, shaken and extracted, and the upper aqueous phase layer is absorbed to prepare a second step sample solution; and,

[0060] The first step sample solution and the second step sample solution are mixed, and an ethanol aqueous solution is added for dilution to prepare a sample solution;

[0061] Optionally, the mass-volume ratio of the test traditional Chinese medicine compound preparation to anhydrous ethanol is (0.3-0.5) g:(25-40) mL;

[0062] Optionally, the acid solution comprises one or more of a trifluoroacetic acid aqueous solution, a hydrochloric acid aqueous solution, and a sulfuric acid aqueous solution;

[0063] Optionally, the acid solution is a trifluoroacetic acid aqueous solution, and the molar concentration of trifluoroacetic acid in the trifluoroacetic acid aqueous solution is 2 mol / L-4 mol / L;

[0064] Optionally, the temperature of the hydrolysis reaction is 90℃-120℃, and the time is 0.5h-6h;

[0065] Optionally, the alkaline solution comprises one or more of a sodium hydroxide aqueous solution and an ammonia aqueous solution, and the molar concentration of the alkaline substance in the alkaline solution is 2 mol / L-4 mol / L;

[0066] Optionally, the molar concentration of sodium hydroxide in the sodium hydroxide aqueous solution is 0.2 mol / L-0.5 mol / L, and the volume ratio of the diluted reaction solution A to the sodium hydroxide aqueous solution is 1:(1-3);

[0067] Optionally, the molar concentration of 1-phenyl-3-methyl-5-pyrazolone in the 1-phenyl-3-methyl-5-pyrazolone methanol solution is 0.5 mol / L-1 mol / L, and the volume ratio of the diluted reaction solution A to the 1-phenyl-3-methyl-5-pyrazolone methanol solution is 1:(1-2);

[0068] Optionally, the heating temperature is 60℃-90℃, and the time is 60min-90min;

[0069] Optionally, the acidic aqueous solution comprises one or more of acetic acid and hydrochloric acid;

[0070] Optionally, the molar concentration of the acidic substance contained in the acidic aqueous solution is 0.2-0.5 mol / L, and the volume ratio of the dilution reaction solution A to the acidic aqueous solution is 1:(1-2);

[0071] Optionally, the volume ratio of the dilution reaction solution B to the chloroform is (2-4):(4-6);

[0072] Optionally, the volume ratio of the first-step sample solution to the second-step sample solution is (2-5):(0.5-1).

[0073] The third aspect of the present application provides a method for determining the content of seven index components in a traditional Chinese medicine compound preparation, wherein the traditional Chinese medicine compound preparation is a mixture comprising extracts of Pueraria lobata, Radix Pseudostellariae and Herba Epimedii; the seven index components are successively puerarin, glucose, galactose, desfenolid A, desfenolid B, desfenolid C and icariin; and the method comprises the following steps:

[0074] A control solution containing the seven index components is prepared, and the control solution is detected by liquid chromatography at different injection volumes to construct standard curves of the mass and peak area of puerarin, glucose, galactose, desfenolid A, desfenolid B, desfenolid C and icariin;

[0075] A sample solution of the traditional Chinese medicine compound preparation to be tested is prepared; and

[0076] The sample solution is detected by liquid chromatography to obtain the peak area of puerarin, glucose, galactose, desfenolid A, desfenolid B, desfenolid C and icariin in the sample solution, which is substituted into the corresponding standard curve for calculation;

[0077] The conditions for the liquid chromatography detection include:

[0078] The mobile phase comprises mobile phase A and mobile phase B, wherein the mobile phase A is a phosphate solution containing phosphate at a molar concentration of 10-50 mmol / L, and the mobile phase B is acetonitrile;

[0079] Gradient elution is adopted, and the program of the gradient elution comprises:

[0080] 0-2 min, maintaining the volume percentage of the mobile phase A at 85% and the volume percentage of the mobile phase B at 15%; 2 min-8 min, reducing the volume percentage of the mobile phase A from 85% to 83% and increasing the volume percentage of the mobile phase B from 15% to 17%; 8 min-20 min, reducing the volume percentage of the mobile phase A from 83% to 82% and increasing the volume percentage of the mobile phase B from 17% to 18%; 20 min-36 min, reducing the volume percentage of the mobile phase A from 82% to 60% and increasing the volume percentage of the mobile phase B from 18% to 40%; 36 min-40 min, maintaining the volume percentage of the mobile phase A at 60% and the volume percentage of the mobile phase B at 40%; 40 min-42 min, increasing the volume percentage of the mobile phase A from 60% to 85% and reducing the volume percentage of the mobile phase B from 40% to 15%.

[0081] In some embodiments, the phosphate salt contained in the phosphate salt solution comprises one or more of sodium phosphate salt and potassium phosphate salt;

[0082] Optionally, the phosphate salt contained in the phosphate salt solution comprises one or more of sodium dihydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate and dipotassium hydrogen phosphate.

[0083] In some embodiments, the conditions of the liquid chromatography detection further comprise at least one of:

[0084] (1) the packing material of the chromatographic column is octadecylsilane bonded silica gel;

[0085] (2) the column temperature is 25-40℃;

[0086] (3) the detection wavelength is 250-260 nm;

[0087] (4) the flow rate is 0.5-1.5 mL / min; and,

[0088] (5) the injection volume is 10-20 μL;

[0089] Optionally, the length of the chromatographic column is 250 mm, the inner diameter is 4.6 mm, and the packing material particle size is 5 μm.

[0090] In some embodiments, taking the traditional Chinese medicine compound preparation to be tested, the step of preparing the to-be-tested product solution comprises:

[0091] mixing the traditional Chinese medicine compound preparation to be tested with anhydrous ethanol, ultrasonically extracting, centrifuging, taking the supernatant after centrifugation, and preparing the first-step to-be-tested product solution;

[0092] The centrifuged residue is dried to constant weight, then mixed with acid solution for hydrolysis reaction, the pH of the solution after hydrolysis reaction is adjusted to neutral by alkaline solution, and then diluted with water to prepare dilution reaction liquid A;

[0093] The dilution reaction liquid A is mixed with sodium hydroxide aqueous solution and 1-phenyl-3-methyl-5-pyrazolone methanol solution, heated, mixed with acidic aqueous solution after cooling, and diluted with water to prepare dilution reaction B;

[0094] The dilution reaction liquid B is mixed with chloroform, shaken and extracted, and the upper aqueous layer is absorbed to prepare the second step test product solution; and,

[0095] The first step test product solution and the second step test product solution are mixed, and diluted with ethanol aqueous solution to prepare a test product solution;

[0096] Optionally, the mass-volume ratio of the test traditional Chinese medicine compound preparation to anhydrous ethanol is (0.3-0.5) g:(25-40) mL;

[0097] Optionally, the acid solution includes one or more of trifluoroacetic acid aqueous solution, hydrochloric acid aqueous solution and sulfuric acid aqueous solution;

[0098] Optionally, the acid solution is trifluoroacetic acid aqueous solution, and the molar concentration of trifluoroacetic acid in the trifluoroacetic acid aqueous solution is 2 mol / L-4 mol / L;

[0099] Optionally, the temperature of the hydrolysis reaction is 90℃-120℃, and the time is 0.5h-6h;

[0100] Optionally, the alkaline solution includes one or more of sodium hydroxide aqueous solution and ammonia aqueous solution, and the molar concentration of alkaline substances in the alkaline solution is 2 mol / L-4 mol / L;

[0101] Optionally, the molar concentration of sodium hydroxide in the sodium hydroxide aqueous solution is 0.2 mol / L-0.5 mol / L, and the volume ratio of the dilution reaction liquid A to the sodium hydroxide aqueous solution is 1:(1-3);

[0102] Optionally, the molar concentration of 1-phenyl-3-methyl-5-pyrazolone in the 1-phenyl-3-methyl-5-pyrazolone methanol solution is 0.5 mol / L-1 mol / L, and the volume ratio of the dilution reaction liquid A to the 1-phenyl-3-methyl-5-pyrazolone methanol solution is 1:(1-2);

[0103] Optionally, the heating temperature is 60℃-90℃, and the time is 60min-90min;

[0104] Optionally, the acidic aqueous solution comprises one or more of acetic acid and hydrochloric acid;

[0105] Optionally, the molar concentration of the acidic substance contained in the acidic aqueous solution is 0.2-0.5 mol / L, and the volume ratio of the dilution reaction solution A to the acidic aqueous solution is 1:(1-2);

[0106] Optionally, the volume ratio of the dilution reaction solution B to the chloroform is (2-4):(4-6);

[0107] Optionally, the volume ratio of the first-step test sample solution to the second-step test sample solution is (2-5):(0.5-1).

[0108] The fourth aspect of the present application provides a method for determining the content of seven index components in a traditional Chinese medicine compound preparation, the traditional Chinese medicine compound preparation being a mixture comprising extracts of Pueraria lobata, Radix Pseudostellariae and Herba Epimedii; the seven index components being successively puerarin, glucose, galactose, desmodidin A, desmodidin B, desmodidin C and icariin; and the method comprising the following steps:

[0109] A control sample solution containing the seven index components is prepared, and the control sample solution is subjected to liquid chromatography detection at different injection volumes to construct standard curves of the mass and peak area of puerarin, glucose, galactose, desmodidin A, desmodidin B, desmodidin C and icariin;

[0110] A test traditional Chinese medicine compound preparation is taken to prepare a test sample solution; and

[0111] The test sample solution is subjected to liquid chromatography detection to obtain the peak area of puerarin, glucose, galactose, desmodidin A, desmodidin B, desmodidin C and icariin in the test sample solution, which is substituted into the corresponding standard curve for calculation;

[0112] The conditions for the liquid chromatography detection include:

[0113] The mobile phase comprises mobile phase A and mobile phase B, the mobile phase A being a phosphate solution, the phosphate solution containing phosphate at a molar concentration of 10-50 mmol / L; and the mobile phase B being acetonitrile;

[0114] Gradient elution is adopted, and the program of the gradient elution comprises:

[0115] 0-8min, maintaining the volume percentage of the mobile phase A at 85%, the volume percentage of the mobile phase B at 15%; 8min-15min, reducing the volume percentage of the mobile phase A from 85% to 76%, increasing the volume percentage of the mobile phase B from 15% to 24%; 15min-20min, reducing the volume percentage of the mobile phase A from 76% to 74%, increasing the volume percentage of the mobile phase B from 24% to 26%; 20min-36min, reducing the volume percentage of the mobile phase A from 74% to 70%, increasing the volume percentage of the mobile phase B from 26% to 30%; 36min-40min, reducing the volume percentage of the mobile phase A from 70% to 60%, increasing the volume percentage of the mobile phase B from 30% to 40%; 40min-42min, increasing the volume percentage of the mobile phase A from 60% to 85%, reducing the volume percentage of the mobile phase B from 40% to 15%.

[0116] In some embodiments, the phosphate salt contained in the phosphate salt solution comprises one or more of sodium phosphate salt and potassium phosphate salt;

[0117] Optionally, the phosphate salt contained in the phosphate salt solution comprises one or more of sodium dihydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate and dipotassium hydrogen phosphate.

[0118] In some embodiments, the conditions of the liquid chromatography detection further comprise at least one of:

[0119] (1) the packing material of the chromatographic column is octadecylsilane bonded silica gel;

[0120] (2) the column temperature is 25-40℃;

[0121] (3) the detection wavelength is 250-260nm;

[0122] (4) the flow rate is 0.5-1.5mL / min; and,

[0123] (5) the injection volume is 10-20μL;

[0124] Optionally, the length of the chromatographic column is 250mm, the inner diameter is 4.6mm, and the packing material particle size is 5μm.

[0125] In some embodiments, taking the traditional Chinese medicine compound preparation to be tested, the step of preparing the to-be-tested product solution comprises:

[0126] mixing the traditional Chinese medicine compound preparation to be tested with anhydrous ethanol, ultrasonically extracting, centrifuging, taking the supernatant after centrifugation, and preparing the first-step to-be-tested product solution;

[0127] The centrifuged residue is dried to constant weight, then mixed with acid solution for hydrolysis reaction, the pH of the solution after hydrolysis reaction is adjusted to neutral by alkaline solution, and then diluted with water to prepare dilution reaction liquid C;

[0128] The dilution reaction liquid C is mixed with sodium hydroxide aqueous solution and 1-phenyl-3-methyl-5-pyrazolone methanol solution, heated, mixed with acidic aqueous solution after cooling, and diluted with water to prepare dilution reaction D;

[0129] The dilution reaction liquid D is mixed with chloroform, shaken and extracted, and the upper aqueous phase layer is absorbed to prepare a second-step test sample solution; and,

[0130] The first-step test sample solution and the second-step test sample solution are mixed, and diluted with ethanol aqueous solution to prepare a test sample solution;

[0131] Optionally, the mass-volume ratio of the test traditional Chinese medicine compound preparation to anhydrous ethanol is (0.3-0.5) g:(25-40) mL;

[0132] Optionally, the acid solution comprises one or more of trifluoroacetic acid aqueous solution, hydrochloric acid aqueous solution and sulfuric acid aqueous solution;

[0133] Optionally, the acid solution is trifluoroacetic acid aqueous solution, and the molar concentration of trifluoroacetic acid in the trifluoroacetic acid aqueous solution is 2 mol / L-4 mol / L;

[0134] Optionally, the temperature of the hydrolysis reaction is 90℃-120℃, and the time is 0.5h-6h;

[0135] Optionally, the alkaline solution comprises one or more of sodium hydroxide aqueous solution and ammonia aqueous solution, and the molar concentration of alkaline substances in the alkaline solution is 2 mol / L-4 mol / L;

[0136] Optionally, the molar concentration of sodium hydroxide in the sodium hydroxide aqueous solution is 0.2 mol / L-0.5 mol / L, and the volume ratio of the dilution reaction liquid C to the sodium hydroxide aqueous solution is 1:(1-3);

[0137] Optionally, the molar concentration of 1-phenyl-3-methyl-5-pyrazolone in the 1-phenyl-3-methyl-5-pyrazolone methanol solution is 0.5 mol / L-1 mol / L, and the volume ratio of the dilution reaction liquid C to the 1-phenyl-3-methyl-5-pyrazolone methanol solution is 1:(1-2);

[0138] Optionally, the heating temperature is 60℃-90℃, and the time is 60min-90min;

[0139] Optionally, the acidic aqueous solution comprises one or more of acetic acid and hydrochloric acid.

[0140] Optionally, the molar concentration of the acidic substance contained in the acidic aqueous solution is 0.2-0.5 mol / L, and the volume ratio of the dilution reaction solution C to the acidic aqueous solution is 1:(1-2).

[0141] Optionally, the volume ratio of the dilution reaction solution D to the chloroform is (2-4):(4-6).

[0142] Optionally, the volume ratio of the first-step test sample solution to the second-step test sample solution is (2-5):(0.5-1).

[0143] The above technical solution realizes the characterization of 7 index components (puerarin, glucose, galactose, damianin A, damianin B, damianin C and icariin) in the traditional Chinese medicine compound preparation based on a suitable liquid chromatography analysis method, the chromatographic peak separation degrees of the index components are good, and the index components are not interfered with each other, the content determination can be realized in the same set of chromatographic detection system, thereby providing a reference for the quality control of the traditional Chinese medicine compound preparation.

[0144] The above technical solution realizes the synchronous determination of the contents of the 7 index components in the traditional Chinese medicine compound preparation, reduces the complex operation of the existing standard for separately detecting the 7 index components, improves the inspection efficiency, and reduces the amount of organic reagent. BRIEF DESCRIPTION OF DRAWINGS

[0145] In order to more clearly illustrate the technical solutions in the embodiments of the present application, more completely understand the present application and its beneficial effects, the following will briefly introduce the drawings needed to be used in the embodiment description. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0146] Figure 1 The chromatograms of the test sample solution, the control sample solution and the blank solution of Example 1. DETAILED DESCRIPTION

[0147] In order to facilitate the understanding of the present application, the present application will be described more fully below with reference to the related embodiments. The following gives the preferred embodiments of the present application. However, the present application can be realized in many different forms, and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive.

[0148] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the application.

[0149] The selection scope of the terms "and / or", "or / and", "and / or" used herein includes any one of two or more related listed items, and also includes any and all combinations of the related listed items, including any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", "and / or", it should be understood that in this application, the technical solution undoubtedly includes the technical solution connected by "logical and", and also undoubtedly includes the technical solution connected by "logical or".

[0150] In this application, in the technical features described in an open manner, both the closed technical solution consisting of the listed features and the open technical solution containing the listed features are included.

[0151] In this application, when referring to a numerical interval, unless otherwise specified, the numerical interval is considered to be continuous and includes the minimum value and the maximum value of the range, as well as every value between the minimum value and the maximum value. Further, when the range refers to an integer, every integer between the minimum value and the maximum value of the range is included. In addition, when multiple ranges are provided to describe a feature or a characteristic, the ranges can be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all sub-ranges encompassed therein.

[0152] Only some numerical ranges are specifically disclosed herein. However, any lower limit can be combined with any upper limit to form a range not explicitly recited; and any lower limit can be combined with other lower limits to form a range not explicitly recited, and likewise any upper limit can be combined with any other upper limit to form a range not explicitly recited. In addition, each individual disclosed point or single numerical value can itself be combined as a lower limit or an upper limit with any other point or single numerical value or with other lower limits or upper limits to form a range not explicitly recited.

[0153] In this application, the temperature parameters, unless otherwise specified, allow for constant temperature processing, and also allow for processing within a certain temperature range. The constant temperature processing allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within a range such as ±5°C, ±4°C, ±3°C, ±2°C, ±1°C are allowed.

[0154] In the present application, the term "suitable" in "suitable combination", "suitable manner", "any suitable manner" and the like means that the technical solutions of the present application can be implemented, the technical problems of the present application can be solved, and the expected technical effects of the present application can be achieved.

[0155] In the present application, the terms "further", "still further", "in particular" and the like are used for the purpose of description and represent differences in content, but should not be understood as limiting the scope of protection of the present application.

[0156] In the present application, the terms "optionally", "optional" and "optional" mean that something can or can not be present, i.e. either of the two parallel schemes "with" or "without" is selected. If there are multiple "options" in a technical solution, and there is no special description, and there is no contradictory relationship or mutual restriction, each "option" is independent.

[0157] In the description of the application, the meaning of "multiple" is at least two, for example, two, three, etc., unless otherwise specifically limited.

[0158] If not specifically stated, all embodiments and optional embodiments of the present application can be combined to form new technical solutions. If not specifically stated, all technical features and optional technical features of the present application can be combined to form new technical solutions.

[0159] If not specifically stated, all steps of the present application can be performed in sequence or randomly, preferably in sequence.

[0160] At present, there are many reports on liquid chromatography detection methods for single type components (such as flavonoids or isoflavones), but due to the significant differences in polarity, solubility and chromatographic behavior between saccharide components (such as polysaccharide hydrolysate glucose, galactose) and flavonoids, isoflavones, it is difficult to realize the simultaneous quantitative analysis of multiple components under the same chromatographic conditions by traditional methods.

[0161] Under the above problems, one or more embodiments of the present application provide a method for constructing a characteristic map of a traditional Chinese medicine compound preparation, the traditional Chinese medicine compound preparation being a mixture of extracts of Puerariae radix, Radix pseudoginseng and Herba epimedii; the method for constructing the characteristic map comprising the following steps: preparing a test solution from the test traditional Chinese medicine compound preparation; and performing liquid chromatography detection on the test solution to obtain the characteristic map of the traditional Chinese medicine compound preparation; the conditions for the liquid chromatography detection comprising: the mobile phase comprising mobile phase A and mobile phase B, the mobile phase A being a phosphate solution, the phosphate solution containing phosphate at a molar concentration of 10-50 mmol / L; the mobile phase B being acetonitrile; gradient elution is adopted, the program of the gradient elution comprising: 0-2 min, maintaining the volume percentage of the mobile phase A at 85% and the volume percentage of the mobile phase B at 15%; 2-8 min, reducing the volume percentage of the mobile phase A from 85% to 83% and increasing the volume percentage of the mobile phase B from 15% to 17%; 8-20 min, reducing the volume percentage of the mobile phase A from 83% to 82% and increasing the volume percentage of the mobile phase B from 17% to 18%; 20-36 min, reducing the volume percentage of the mobile phase A from 82% to 60% and increasing the volume percentage of the mobile phase B from 18% to 40%; 36-40 min, maintaining the volume percentage of the mobile phase A at 60% and the volume percentage of the mobile phase B at 40%; 40-42 min, increasing the volume percentage of the mobile phase A from 60% to 85% and reducing the volume percentage of the mobile phase B from 40% to 15%.

[0162] Understandably, the technical solution of the present application realizes the characterization of 7 index components (puerarin, glucose, galactose, epimedin A, epimedin B, epimedin C and icariin) of the traditional Chinese medicine compound preparation based on the characteristic map of the traditional Chinese medicine compound preparation established by the suitable liquid chromatography analysis method, the chromatographic peak separation degrees of the index components are good, and the index components do not interfere with each other, the content determination can be realized in the same set of chromatography detection system, thereby providing a reference for the quality control of the traditional Chinese medicine compound preparation.

[0163] In the present application, the traditional Chinese medicine compound preparation is a mixture of extracts of Puerariae radix, Radix pseudoginseng and Herba epimedii.

[0164] In the present application, the 7 index components of the traditional Chinese medicine compound preparation are puerarin, glucose, galactose, epimedin A, epimedin B, epimedin C and icariin in turn.

[0165] It should be noted that the glucose and galactose mentioned in the context are D(+)-glucose and D(-)-galactose respectively, which are formed by hydrolysis of Radix pseudoginseng polysaccharide under acid conditions.

[0166] As a non-limiting example, the molar concentration of the phosphate salt contained in the phosphate salt solution can be, but is not limited to, 10 mmol / L, 15 mmol / L, 20 mmol / L, 25 mmol / L, 30 mmol / L, 35 mmol / L, 40 mmol / L, 45 mmol / L, 50 mmol / L, or a range between any two of the aforementioned values, etc.

[0167] In some embodiments, the phosphate salt contained in the phosphate salt solution comprises one or more of sodium phosphate salts and potassium phosphate salts.

[0168] As a possible implementation, the phosphate salt contained in the phosphate salt solution comprises one or more of sodium dihydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, and dipotassium hydrogen phosphate.

[0169] In some alternative implementations, the packing of the chromatographic column is octadecylsilane-bonded silica gel.

[0170] As a possible implementation, the length of the chromatographic column is 250 mm, the inner diameter is 4.6 mm, and the packing particle size is 5 μm.

[0171] In some exemplary implementations, the column temperature is 25-40 °C; for example, it can be, but is not limited to, 25 °C, 30 °C, 35 °C, 40 °C, or a range between any two of the aforementioned temperatures, etc.

[0172] In some of the implementations, the detection wavelength is 250-260 nm; for example, it can be, but is not limited to, 250 nm, 255 nm, 260 nm, or a range between any two of the aforementioned wavelengths, etc.

[0173] As a possible implementation, the flow rate is 0.5-1.5 mL / min; for example, it can be, but is not limited to, 0.5 mL / min, 0.6 mL / min, 0.7 mL / min, 0.8 mL / min, 0.9 mL / min, 1 mL / min, 1.1 mL / min, 1.2 mL / min, 1.3 mL / min, 1.4 mL / min, 1.5 mL / min, or a range between any two of the aforementioned flow rates, etc.

[0174] In some alternative implementations, the injection volume is 10-20 μL; for example, it can be, but is not limited to, 10 μL, 12 μL, 14 μL, 16 μL, 18 μL, 20 μL, or a range between any two of the aforementioned values, etc.

[0175] In some embodiments, the step of preparing the test solution from the test traditional Chinese medicine compound preparation comprises:

[0176] The test traditional Chinese medicine compound preparation is mixed with anhydrous ethanol, ultrasonically extracted, centrifuged, and the supernatant after centrifugation is taken to prepare a first-step sample solution; the residue after centrifugation is dried to a constant weight, then mixed with an acid solution to perform a hydrolysis reaction, a basic solution is used to adjust the pH of the solution after the hydrolysis reaction to neutral, water is added for dilution to prepare a diluted reaction solution A; the diluted reaction solution A is mixed with a sodium hydroxide aqueous solution and a 1-phenyl-3-methyl-5-pyrazolone methanol solution, heated, then mixed with an acidic aqueous solution after cooling, and diluted with water to prepare a diluted reaction solution B; the diluted reaction solution B is mixed with chloroform, shaken, extracted, and the upper aqueous phase layer is taken to prepare a second-step sample solution; and the first-step sample solution and the second-step sample solution are mixed, diluted with an ethanol aqueous solution to prepare a sample solution.

[0177] It should be noted that glucose and galactose do not contain chromophores and cannot be detected by liquid chromatography alone, and must be derivatized with 1-phenyl-3-methyl-5-pyrazolone (PMP) under alkaline conditions to generate monosaccharide-PMP derivatives, which can be detected under liquid chromatography conditions at 254 nm.

[0178] In some optional embodiments, the mass-volume ratio of the test traditional Chinese medicine compound preparation to anhydrous ethanol is (0.3-0.5) g:(25-40) mL; for example, it can be but is not limited to 0.3 g:40 mL, 0.4 g:40 mL, 0.5 g:40 mL, 0.4 g:30 mL, 0.5 g:30 mL, 0.3 g:25 mL, 0.4 g:25 mL, 0.5 g:25 mL, or a range between any two of the above mass-volume ratios.

[0179] As a possible embodiment, the acid solution includes one or more of a trifluoroacetic acid aqueous solution, a hydrochloric acid aqueous solution, and a sulfuric acid aqueous solution.

[0180] In some optional embodiments, the acid solution is a trifluoroacetic acid aqueous solution, and the molar concentration of trifluoroacetic acid in the trifluoroacetic acid aqueous solution is 2 mol / L-4 mol / L; for example, it can be but is not limited to 2 mol / L, 2.5 mol / L, 3 mol / L, 3.5 mol / L, 4 mol / L, or a range between any two of the above molar concentrations.

[0181] As a possible embodiment, the temperature of the hydrolysis reaction is 90℃-120℃; for example, it can be but is not limited to 90℃, 100℃, 110℃, 120℃, or a range between any two of the above temperatures.

[0182] In some optional embodiments, the time of the hydrolysis reaction is 0.5h-6h; for example, it can be but is not limited to 0.5h, 1h, 2h, 3h, 4h, 5h, 6h, or a range between any two of the above times.

[0183] As a possible implementation, the alkaline solution comprises one or more of a sodium hydroxide aqueous solution and an aqueous ammonia solution, and the molar concentration of the alkaline substance in the alkaline solution is 2-4 mol / L; for example, it can be but is not limited to 2 mol / L, 3 mol / L, 4 mol / L, or a range between any two of the above values, etc.

[0184] In some optional implementations, the molar concentration of sodium hydroxide in the sodium hydroxide aqueous solution is 0.2-0.5 mol / L; for example, it can be but is not limited to 0.2 mol / L, 0.3 mol / L, 0.4 mol / L, 0.5 mol / L, or a range between any two of the above values, etc. The volume ratio of the diluted reaction solution A to the sodium hydroxide aqueous solution is 1:(1-3); for example, it can be but is not limited to 1:1, 1:2, 1:3, or a range between any two of the above values, etc.

[0185] In some of the implementations, the molar concentration of 1-phenyl-3-methyl-5-pyrazolone in the 1-phenyl-3-methyl-5-pyrazolone methanol solution is 0.5-1 mol / L; for example, it can be but is not limited to 0.5 mol / L, 0.6 mol / L, 0.7 mol / L, 0.8 mol / L, 0.9 mol / L, 1 mol / L, or a range between any two of the above values, etc. The volume ratio of the diluted reaction solution A to the 1-phenyl-3-methyl-5-pyrazolone methanol solution is 1:(1-2); for example, it can be but is not limited to 1:1, 1:1.5, 1:2, or a range between any two of the above values, etc.

[0186] As a possible implementation, the heating temperature is 60-90℃; for example, it can be but is not limited to 60℃, 70℃, 80℃, 90℃, or a range between any two of the above values, etc. The heating time is 60-90 min; for example, it can be but is not limited to 60 min, 70 min, 80 min, 90 min, or a range between any two of the above values, etc.

[0187] In some optional implementations, the acidic aqueous solution comprises one or more of acetic acid and hydrochloric acid.

[0188] In some embodiments, the molar concentration of the acidic substance contained in the acidic aqueous solution is 0.2-0.5 mol / L; for example, it can be, but is not limited to, 0.2 mol / L, 0.3 mol / L, 0.4 mol / L, 0.5 mol / L, or a range between any two of the above molar concentrations, etc. The volume ratio of the diluted reaction solution A to the acidic aqueous solution is 1:(1-2); for example, it can be, but is not limited to, 1:1, 1:1.5, 1:2, or a range between any two of the above values, etc.

[0189] In some embodiments, the volume ratio of the diluted reaction solution B to chloroform is (2-4):(4-6); for example, it can be, but is not limited to, 2:6, 3:6, 4:6, 4:5, 4:4, or a range between any two of the above ratios, etc.

[0190] As a possible embodiment, the volume ratio of the first-step sample solution to the second-step sample solution is (2-5):(0.5-1); for example, it can be, but is not limited to, 2:1, 2:0.5, 3:1, 5:1, 5:0.9, 5:0.8, 5:0.7, 5:0.6, 5:0.5, 5:0.9, 5:0.9, or a range between any two of the above ratios, etc.

[0191] In some embodiments, the characteristic spectrum of the traditional Chinese medicine compound preparation includes 7 characteristic peaks, which are peak 1, peak 2, peak 3, peak 4, peak 5, peak 6 and peak 7; wherein peak 1 is puerarin, peak 2 is glucose, peak 3 is galactose, peak 4 is damianin A, peak 5 is damianin B, peak 6 is damianin C and peak 7 is icariin.

[0192] One or more embodiments of the present application provide a method for constructing a characteristic map of a traditional Chinese medicine compound preparation, the traditional Chinese medicine compound preparation being a mixture of extracts including Puerariae radix, Radix pseudoginseng and Herba epimedii; the method for constructing the characteristic map comprising the following steps: preparing a test solution from the test traditional Chinese medicine compound preparation; and performing liquid chromatography detection on the test solution to obtain the characteristic map of the traditional Chinese medicine compound preparation; the conditions for the liquid chromatography detection comprising: the mobile phase comprising mobile phase A and mobile phase B, the mobile phase A being a phosphate solution, the phosphate solution containing phosphate at a molar concentration of 10 mmol / L-50 mmol / L; the mobile phase B being acetonitrile; gradient elution is used, the program for the gradient elution comprising: 0-8 min, maintaining the volume percentage of the mobile phase A at 85% and the volume percentage of the mobile phase B at 15%; 8 min-15 min, reducing the volume percentage of the mobile phase A from 85% to 76% and increasing the volume percentage of the mobile phase B from 15% to 24%; 15 min-20 min, reducing the volume percentage of the mobile phase A from 76% to 74% and increasing the volume percentage of the mobile phase B from 24% to 26%; 20 min-36 min, reducing the volume percentage of the mobile phase A from 74% to 70% and increasing the volume percentage of the mobile phase B from 26% to 30%; 36 min-40 min, reducing the volume percentage of the mobile phase A from 70% to 60% and increasing the volume percentage of the mobile phase B from 30% to 40%; 40 min-42 min, increasing the volume percentage of the mobile phase A from 60% to 85% and reducing the volume percentage of the mobile phase B from 40% to 15%.

[0193] Understandably, the technical solution of the present application realizes the characterization of 7 index components (puerarin, glucose, galactose, epimedin A, epimedin B, epimedin C and icariin) of the traditional Chinese medicine compound preparation based on the characteristic map of the traditional Chinese medicine compound preparation established by the suitable liquid chromatography analysis method, the chromatographic peak separation degrees of the index components are good and the index components do not interfere with each other, the content determination can be realized in the same set of chromatography detection system, thereby providing a reference for the quality control of the traditional Chinese medicine compound preparation.

[0194] As non-limiting examples, the molar concentration of the phosphate contained in the phosphate solution can be, but is not limited to, 10 mmol / L, 15 mmol / L, 20 mmol / L, 25 mmol / L, 30 mmol / L, 35 mmol / L, 40 mmol / L, 45 mmol / L, 50 mmol / L or a range between any two of the above values, etc.

[0195] In some embodiments, the phosphate contained in the phosphate solution includes one or more of sodium phosphate and potassium phosphate.

[0196] As a possible implementation, the phosphate salt contained in the phosphate salt solution includes one or more of sodium dihydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, and dipotassium hydrogen phosphate.

[0197] In some alternative embodiments, the packing of the chromatographic column is octadecylsilane bonded silica gel.

[0198] As a possible implementation, the length of the chromatographic column is 250 mm, the inner diameter is 4.6 mm, and the packing particle size is 5 μm.

[0199] In some exemplary embodiments, the column temperature is 25-40°C; for example, it can be but is not limited to 25°C, 30°C, 35°C, 40°C, or a range between any two of the above temperatures, etc.

[0200] In some of the embodiments, the detection wavelength is 250-260 nm; for example, it can be but is not limited to 250 nm, 255 nm, 260 nm, or a range between any two of the above wavelengths, etc.

[0201] As a possible implementation, the flow rate is 0.5-1.5 mL / min; for example, it can be but is not limited to 0.5 mL / min, 0.6 mL / min, 0.7 mL / min, 0.8 mL / min, 0.9 mL / min, 1 mL / min, 1.1 mL / min, 1.2 mL / min, 1.3 mL / min, 1.4 mL / min, 1.5 mL / min, or a range between any two of the above flow rates, etc.

[0202] In some alternative embodiments, the sample injection volume is 10-20 μL; for example, it can be but is not limited to 10 μL, 12 μL, 14 μL, 16 μL, 18 μL, 20 μL, or a range between any two of the above values, etc.

[0203] In some embodiments, the step of preparing the test solution from the test traditional Chinese medicine compound preparation includes:

[0204] The test traditional Chinese medicine compound preparation is mixed with anhydrous ethanol, ultrasonically extracted, centrifuged, and the supernatant after centrifugation is taken to prepare a first-step sample solution; the residue after centrifugation is dried to a constant weight, then mixed with an acid solution to perform a hydrolysis reaction, a basic solution is used to adjust the pH of the solution after the hydrolysis reaction to neutral, water is added for dilution to prepare a diluted reaction solution A; the diluted reaction solution A is mixed with a sodium hydroxide aqueous solution and a 1-phenyl-3-methyl-5-pyrazolone methanol solution, heated, then mixed with an acidic aqueous solution after cooling, and diluted with water to prepare a diluted reaction solution B; the diluted reaction solution B is mixed with chloroform, shaken, extracted, and the upper aqueous phase layer is taken to prepare a second-step sample solution; and the first-step sample solution and the second-step sample solution are mixed, diluted with an ethanol aqueous solution to prepare a sample solution.

[0205] In some optional embodiments, the mass-volume ratio of the test traditional Chinese medicine compound preparation to anhydrous ethanol is (0.3-0.5) g:(25-40) mL; for example, it can be but is not limited to 0.3 g:40 mL, 0.4 g:40 mL, 0.5 g:40 mL, 0.4 g:30 mL, 0.5 g:30 mL, 0.3 g:25 mL, 0.4 g:25 mL, 0.5 g:25 mL, or a range between any two of the above mass-volume ratios.

[0206] As a possible embodiment, the acid solution comprises one or more of a trifluoroacetic acid aqueous solution, a hydrochloric acid aqueous solution, and a sulfuric acid aqueous solution.

[0207] In some optional embodiments, the acid solution is a trifluoroacetic acid aqueous solution, and the molar concentration of trifluoroacetic acid in the trifluoroacetic acid aqueous solution is 2 mol / L-4 mol / L; for example, it can be but is not limited to 2 mol / L, 2.5 mol / L, 3 mol / L, 3.5 mol / L, 4 mol / L, or a range between any two of the above molar concentrations.

[0208] As a possible embodiment, the temperature of the hydrolysis reaction is 90℃-120℃; for example, it can be but is not limited to 90℃, 100℃, 110℃, 120℃, or a range between any two of the above temperatures.

[0209] In some optional embodiments, the time of the hydrolysis reaction is 0.5 h-6 h; for example, it can be but is not limited to 0.5 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, or a range between any two of the above times.

[0210] As a possible embodiment, the basic solution comprises one or more of a sodium hydroxide aqueous solution and an ammonia aqueous solution, and the molar concentration of the basic substance in the basic solution is 2 mol / L-4 mol / L; for example, it can be but is not limited to 2 mol / L, 3 mol / L, 4 mol / L, or a range between any two of the above values.

[0211] In some alternative embodiments, the molar concentration of sodium hydroxide in the aqueous sodium hydroxide solution is 0.2-0.5 mol / L; for example, it can be but is not limited to 0.2 mol / L, 0.3 mol / L, 0.4 mol / L, 0.5 mol / L, or a range between any two of the foregoing values, etc. The volume ratio of diluting reaction solution A to the aqueous sodium hydroxide solution is 1:(1-3); for example, it can be but is not limited to 1:1, 1:2, 1:3, or a range between any two of the foregoing values, etc.

[0212] In some embodiments, the molar concentration of 1-phenyl-3-methyl-5-pyrazolone in the methanolic 1-phenyl-3-methyl-5-pyrazolone solution is 0.5-1 mol / L; for example, it can be but is not limited to 0.5 mol / L, 0.6 mol / L, 0.7 mol / L, 0.8 mol / L, 0.9 mol / L, 1 mol / L, or a range between any two of the foregoing values, etc. The volume ratio of diluting reaction solution A to the methanolic 1-phenyl-3-methyl-5-pyrazolone solution is 1:(1-2); for example, it can be but is not limited to 1:1, 1:1.5, 1:2, or a range between any two of the foregoing values, etc.

[0213] As one possible embodiment, the heating temperature is 60-90 °C; for example, it can be but is not limited to 60 °C, 70 °C, 80 °C, 90 °C, or a range between any two of the foregoing values, etc. The heating time is 60-90 min; for example, it can be but is not limited to 60 min, 70 min, 80 min, 90 min, or a range between any two of the foregoing values, etc.

[0214] In some alternative embodiments, the aqueous acidic solution comprises one or more of acetic acid and hydrochloric acid.

[0215] In some embodiments, the molar concentration of the acidic substance in the aqueous acidic solution is 0.2-0.5 mol / L; for example, it can be but is not limited to 0.2 mol / L, 0.3 mol / L, 0.4 mol / L, 0.5 mol / L, or a range between any two of the foregoing values, etc. The volume ratio of diluting reaction solution A to the aqueous acidic solution is 1:(1-2); for example, it can be but is not limited to 1:1, 1:1.5, 1:2, or a range between any two of the foregoing values, etc.

[0216] In some embodiments, the volume ratio of diluting reaction solution B to chloroform is (2-4):(4-6); for example, it can be but is not limited to 2:6, 3:6, 4:6, 4:5, 4:4, or a range between any two of the foregoing values, etc.

[0217] As a possible implementation, the volume ratio of the first step sample solution and the second step sample solution is (2-5):(0.5-1); for example, it can be, but is not limited to, 2:1, 2:0.5, 3:1, 5:1, 5:0.9, 5:0.8, 5:0.7, 5:0.6, 5:0.5, 5:0.9, 5:0.9, or a range between any two of the above ratios.

[0218] One or more embodiments of the present application provide a content determination method of seven index components in a traditional Chinese medicine compound preparation, the traditional Chinese medicine compound preparation being a mixture of extracts including Puerariae radix, Radix pseudoginseng and Herba epimedii; the seven index components are successively puerarin, glucose, galactose, epimedin A, epimedin B, epimedin C and icariin; the content determination method comprises the following steps: preparing a reference solution containing the seven index components, detecting the reference solution by liquid chromatography at different injection volumes and constructing standard curves of the mass and peak area of puerarin, glucose, galactose, epimedin A, epimedin B, epimedin C and icariin; preparing a sample solution from the traditional Chinese medicine compound preparation to be tested; and detecting the sample solution by liquid chromatography to obtain the peak area of puerarin, glucose, galactose, epimedin A, epimedin B, epimedin C and icariin in the sample solution, which are respectively substituted into the corresponding standard curves for calculation.

[0219] The conditions of the liquid chromatography detection include: the mobile phase comprises mobile phase A and mobile phase B, the mobile phase A is a phosphate solution, the molar concentration of the phosphate contained in the phosphate solution is 10mmol / L-50mmol / L; the mobile phase B is acetonitrile; gradient elution is adopted, the program of the gradient elution comprises: 0-2min, maintaining the volume percentage of the mobile phase A at 85% and the volume percentage of the mobile phase B at 15%; 2min-8min, reducing the volume percentage of the mobile phase A from 85% to 83% and increasing the volume percentage of the mobile phase B from 15% to 17%; 8min-20min, reducing the volume percentage of the mobile phase A from 83% to 82% and increasing the volume percentage of the mobile phase B from 17% to 18%; 20min-36min, reducing the volume percentage of the mobile phase A from 82% to 60% and increasing the volume percentage of the mobile phase B from 18% to 40%; 36min-40min, maintaining the volume percentage of the mobile phase A at 60% and the volume percentage of the mobile phase B at 40%; 40min-42min, increasing the volume percentage of the mobile phase A from 60% to 85% and reducing the volume percentage of the mobile phase B from 40% to 15%.

[0220] The technical scheme of the present application realizes the synchronous determination of the contents of the seven index components in the traditional Chinese medicine compound preparation, reduces the complex operation of the existing standard for detecting the same by using separate liquid phase conditions, improves the inspection efficiency, and reduces the amount of organic reagents.

[0221] As a non-limiting example, the molar concentration of the phosphate contained in the phosphate solution can be, but is not limited to, 10 mmol / L, 15 mmol / L, 20 mmol / L, 25 mmol / L, 30 mmol / L, 35 mmol / L, 40 mmol / L, 45 mmol / L, 50 mmol / L, or a range between any two of the above values, etc.

[0222] In some embodiments, the phosphate contained in the phosphate solution includes one or more of sodium phosphate salts and potassium phosphate salts.

[0223] As a possible implementation, the phosphate contained in the phosphate solution includes one or more of sodium dihydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, and dipotassium hydrogen phosphate.

[0224] In some optional embodiments, the packing of the chromatographic column is octadecylsilane-bonded silica gel.

[0225] As a possible implementation, the length of the chromatographic column is 250 mm, the inner diameter is 4.6 mm, and the packing particle size is 5 μm.

[0226] In some exemplary embodiments, the column temperature is 25-40℃; for example, it can be, but is not limited to, 25℃, 30℃, 35℃, 40℃, or a range between any two of the above temperatures, etc.

[0227] In some of the embodiments, the detection wavelength is 250-260 nm; for example, it can be, but is not limited to, 250 nm, 255 nm, 260 nm, or a range between any two of the above wavelengths, etc.

[0228] As a possible implementation, the flow rate is 0.5-1.5 mL / min; for example, it can be, but is not limited to, 0.5 mL / min, 0.6 mL / min, 0.7 mL / min, 0.8 mL / min, 0.9 mL / min, 1 mL / min, 1.1 mL / min, 1.2 mL / min, 1.3 mL / min, 1.4 mL / min, 1.5 mL / min, or a range between any two of the above flow rates, etc.

[0229] In some alternative embodiments, the sample injection volume is 10 μL to 20 μL; for example, it can be, but is not limited to, 10 μL, 12 μL, 14 μL, 16 μL, 18 μL, 20 μL, or a range between any two of the above values, etc.

[0230] In some embodiments, the step of preparing the solution of the test product from the test traditional Chinese medicine compound preparation includes:

[0231] The test traditional Chinese medicine compound preparation is mixed with anhydrous ethanol, ultrasonically extracted, centrifuged, and the supernatant after centrifugation is taken to prepare the solution of the test product in the first step; the residue after centrifugation is dried to a constant weight, then mixed with an acid solution containing trifluoroacetic acid to perform a hydrolysis reaction, an alkaline solution is used to adjust the pH of the solution after the hydrolysis reaction to neutral, water is added for dilution to prepare a diluted reaction solution C; the diluted reaction solution C is mixed with a sodium hydroxide aqueous solution and a 1-phenyl-3-methyl-5-pyrazolone methanol solution, heated, mixed with an acidic aqueous solution after cooling, and diluted with water to prepare a diluted reaction solution D; the diluted reaction solution D is mixed with chloroform, shaken, extracted, and the upper aqueous phase layer is taken to prepare the solution of the test product in the second step; and the solution of the test product in the first step and the solution of the test product in the second step are mixed, diluted with an ethanol aqueous solution to prepare the sample solution.

[0232] In some alternative embodiments, the mass-to-volume ratio of the test traditional Chinese medicine compound preparation to anhydrous ethanol is (0.3-0.5) g:(25-40) mL; for example, it can be, but is not limited to, 0.3 g:40 mL, 0.4 g:40 mL, 0.5 g:40 mL, 0.4 g:30 mL, 0.5 g:30 mL, 0.3 g:25 mL, 0.4 g:25 mL, 0.5 g:25 mL, or a range between any two of the above mass-to-volume ratios, etc.

[0233] As a possible embodiment, the acid solution includes one or more of a trifluoroacetic acid aqueous solution, a hydrochloric acid aqueous solution, and a sulfuric acid aqueous solution.

[0234] In some alternative embodiments, the acid solution is a trifluoroacetic acid aqueous solution, and the molar concentration of trifluoroacetic acid in the trifluoroacetic acid aqueous solution is 2 mol / L to 4 mol / L; for example, it can be, but is not limited to, 2 mol / L, 2.5 mol / L, 3 mol / L, 3.5 mol / L, 4 mol / L, or a range between any two of the above molar concentrations, etc.

[0235] As a possible embodiment, the temperature of the hydrolysis reaction is 90°C to 120°C; for example, it can be, but is not limited to, 90°C, 100°C, 110°C, 120°C, or a range between any two of the above temperatures, etc.

[0236] In some alternative embodiments, the hydrolysis reaction is performed for a time period of 0.5 h to 6 h; for example, but not limited to, 0.5 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, or a range between any two of the foregoing.

[0237] As one possible embodiment, the basic solution comprises one or more of a sodium hydroxide aqueous solution and an ammonia aqueous solution, and the molar concentration of the basic substance in the basic solution is 2 mol / L to 4 mol / L; for example, but not limited to, 2 mol / L, 3 mol / L, 4 mol / L, or a range between any two of the foregoing.

[0238] In some alternative embodiments, the molar concentration of sodium hydroxide in the sodium hydroxide aqueous solution is 0.2 mol / L to 0.5 mol / L; for example, but not limited to, 0.2 mol / L, 0.3 mol / L, 0.4 mol / L, 0.5 mol / L, or a range between any two of the foregoing. The volume ratio of the diluted reaction solution C to the sodium hydroxide aqueous solution is 1:(1-3); for example, but not limited to, 1:1, 1:2, 1:3, or a range between any two of the foregoing.

[0239] In some embodiments, the molar concentration of 1-phenyl-3-methyl-5-pyrazolone in the 1-phenyl-3-methyl-5-pyrazolone methanol solution is 0.5 mol / L to 1 mol / L; for example, but not limited to, 0.5 mol / L, 0.6 mol / L, 0.7 mol / L, 0.8 mol / L, 0.9 mol / L, 1 mol / L, or a range between any two of the foregoing. The volume ratio of the diluted reaction solution C to the 1-phenyl-3-methyl-5-pyrazolone methanol solution is 1:(1-2); for example, but not limited to, 1:1, 1:1.5, 1:2, or a range between any two of the foregoing.

[0240] As one possible embodiment, the heating temperature is 60°C to 90°C; for example, but not limited to, 60°C, 70°C, 80°C, 90°C, or a range between any two of the foregoing. The heating time is 60 min to 90 min; for example, but not limited to, 60 min, 70 min, 80 min, 90 min, or a range between any two of the foregoing.

[0241] In some alternative embodiments, the acidic aqueous solution comprises one or more of acetic acid and hydrochloric acid.

[0242] In some embodiments, the molar concentration of the acidic substance contained in the acidic aqueous solution is 0.2-0.5 mol / L; for example, it can be, but is not limited to, 0.2 mol / L, 0.3 mol / L, 0.4 mol / L, 0.5 mol / L, or a range between any two of the above molar concentrations, etc. The volume ratio of the diluted reaction solution C to the acidic aqueous solution is 1:(1-2); for example, it can be, but is not limited to, 1:1, 1:1.5, 1:2, or a range between any two of the above values, etc.

[0243] In some embodiments thereof, the volume ratio of the diluted reaction solution D to chloroform is (2-4):(4-6); for example, it can be, but is not limited to, 2:6, 3:6, 4:6, 4:5, 4:4, or a range between any two of the above ratios, etc.

[0244] As one possible embodiment, the volume ratio of the first-step test substance solution to the second-step test substance solution is (2-5):(0.5-1); for example, it can be, but is not limited to, 2:1, 2:0.5, 3:1, 5:1, 5:0.9, 5:0.8, 5:0.7, 5:0.6, 5:0.5, 5:0.9, 5:0.9, or a range between any two of the above ratios, etc.

[0245] One or more embodiments of the present application provide a method for determining the content of seven index components in a traditional Chinese medicine compound preparation, the traditional Chinese medicine compound preparation being a mixture comprising extracts of Pueraria lobata, Radix Pseudostellariae, and Herba Epimedii; the seven index components being successively puerarin, glucose, galactose, damianin A, damianin B, damianin C, and icariin; the method comprising the following steps: preparing a reference solution containing the seven index components, detecting the reference solution by liquid chromatography at different injection volumes, and constructing standard curves of the mass and peak area of puerarin, glucose, galactose, damianin A, damianin B, damianin C, and icariin; preparing a test substance solution from the traditional Chinese medicine compound preparation to be tested; and detecting the test substance solution by liquid chromatography to obtain the peak area of puerarin, glucose, galactose, damianin A, damianin B, damianin C, and icariin in the test substance solution, and substituting the peak area into the corresponding standard curve for calculation;

[0246] The conditions for liquid chromatography detection include: the mobile phase includes mobile phase A and mobile phase B, the mobile phase A is a phosphate solution, the molar concentration of the phosphate contained in the phosphate solution is 10-50 mmol / L; the mobile phase B is acetonitrile; gradient elution is adopted, the program of the gradient elution includes: 0-8 min, maintaining the volume percentage of the mobile phase A at 85%, and the volume percentage of the mobile phase B at 15%; 8-15 min, reducing the volume percentage of the mobile phase A from 85% to 76%, and increasing the volume percentage of the mobile phase B from 15% to 24%; 15-20 min, reducing the volume percentage of the mobile phase A from 76% to 74%, and increasing the volume percentage of the mobile phase B from 24% to 26%; 20-36 min, reducing the volume percentage of the mobile phase A from 74% to 70%, and increasing the volume percentage of the mobile phase B from 26% to 30%; 36-40 min, reducing the volume percentage of the mobile phase A from 70% to 60%, and increasing the volume percentage of the mobile phase B from 30% to 40%; 40-42 min, increasing the volume percentage of the mobile phase A from 60% to 85%, and reducing the volume percentage of the mobile phase B from 40% to 15%.

[0247] The technical scheme of the present application realizes the synchronous determination of the contents of the seven index components in the traditional Chinese medicine compound preparation, reduces the complex operation of the existing standard for detecting the same by using separate liquid phase conditions, improves the inspection efficiency, and reduces the amount of organic reagents.

[0248] As a non-limiting example, the molar concentration of the phosphate contained in the phosphate solution can be, but is not limited to, 10 mmol / L, 15 mmol / L, 20 mmol / L, 25 mmol / L, 30 mmol / L, 35 mmol / L, 40 mmol / L, 45 mmol / L, 50 mmol / L, or a range between any two of the above values.

[0249] In some embodiments, the phosphate contained in the phosphate solution includes one or more of sodium phosphate and potassium phosphate.

[0250] As a possible implementation, the phosphate contained in the phosphate solution includes one or more of sodium dihydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, and dipotassium hydrogen phosphate.

[0251] In some optional implementations, the packing of the chromatographic column is octadecylsilane bonded silica gel.

[0252] As a possible implementation, the length of the chromatographic column is 250 mm, the inner diameter is 4.6 mm, and the packing particle size is 5 μm.

[0253] In some exemplary embodiments, the column temperature is 25-40℃; for example, it can be but is not limited to 25℃, 30℃, 35℃, 40℃, or a range between any two of the above temperatures, etc.

[0254] In some of the embodiments, the detection wavelength is 250-260nm; for example, it can be but is not limited to 250nm, 255nm, 260nm, or a range between any two of the above wavelengths, etc.

[0255] As a possible embodiment, the flow rate is 0.5-1.5mL / min; for example, it can be but is not limited to 0.5mL / min, 0.6mL / min, 0.7mL / min, 0.8mL / min, 0.9mL / min, 1mL / min, 1.1mL / min, 1.2mL / min, 1.3mL / min, 1.4mL / min, 1.5mL / min, or a range between any two of the above flow rates, etc.

[0256] In some alternative embodiments, the sample injection volume is 10-20μL; for example, it can be but is not limited to 10μL, 12μL, 14μL, 16μL, 18μL, 20μL, or a range between any two of the above values, etc.

[0257] In some embodiments, the step of preparing the solution of the Chinese medicine compound preparation to be tested includes:

[0258] The Chinese medicine compound preparation to be tested is mixed with anhydrous ethanol, ultrasonically extracted, centrifuged, and the supernatant after centrifugation is taken to prepare the solution of the preparation to be tested in the first step; the residue after centrifugation is dried to a constant weight, then mixed with an acid-containing solution to perform a hydrolysis reaction, an alkaline solution is used to adjust the pH of the solution after the hydrolysis reaction to neutral, water is added for dilution to prepare a dilution reaction liquid C; the dilution reaction liquid C is mixed with a sodium hydroxide aqueous solution and a 1-phenyl-3-methyl-5-pyrazolone methanol solution, heated, mixed with an acidic aqueous solution after cooling, and diluted with water to prepare a dilution reaction D; the dilution reaction liquid D is mixed with chloroform, shaken, extracted, and the upper aqueous phase layer is taken to prepare the solution of the preparation to be tested in the second step; and the solution of the preparation to be tested in the first step and the solution of the preparation to be tested in the second step are mixed, diluted with an ethanol aqueous solution, to prepare the sample solution.

[0259] In some alternative embodiments, the mass-volume ratio of the Chinese medicine compound preparation to be tested to anhydrous ethanol is (0.3-0.5)g:(25-40)mL; for example, it can be but is not limited to 0.3g:40mL, 0.4g:40mL, 0.5g:40mL, 0.4g:30mL, 0.5g:30mL, 0.3g:25mL, 0.4g:25mL, 0.5g:25mL, or a range between any two of the above mass-volume ratios, etc.

[0260] As a possible implementation, the acid solution includes one or more of trifluoroacetic acid aqueous solution, hydrochloric acid aqueous solution and sulfuric acid aqueous solution.

[0261] In some optional embodiments, the acid solution is trifluoroacetic acid aqueous solution, and the molar concentration of trifluoroacetic acid in the trifluoroacetic acid aqueous solution is 2-4 mol / L; for example, it can be but is not limited to 2 mol / L, 2.5 mol / L, 3 mol / L, 3.5 mol / L, 4 mol / L or a range between any two of the above molar concentrations.

[0262] As a possible implementation, the temperature of the hydrolysis reaction is 90-120℃; for example, it can be but is not limited to 90℃, 100℃, 110℃, 120℃ or a range between any two of the above temperatures.

[0263] In some optional embodiments, the time of the hydrolysis reaction is 0.5-6 h; for example, it can be but is not limited to 0.5 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h or a range between any two of the above times.

[0264] As a possible implementation, the alkaline solution includes one or more of sodium hydroxide aqueous solution and ammonia aqueous solution, and the molar concentration of the alkaline substance in the alkaline solution is 2-4 mol / L; for example, it can be but is not limited to 2 mol / L, 3 mol / L, 4 mol / L or a range between any two of the above values.

[0265] In some optional embodiments, the molar concentration of sodium hydroxide in the sodium hydroxide aqueous solution is 0.2-0.5 mol / L; for example, it can be but is not limited to 0.2 mol / L, 0.3 mol / L, 0.4 mol / L, 0.5 mol / L or a range between any two of the above values. The volume ratio of the diluted reaction solution C to the sodium hydroxide aqueous solution is 1:(1-3); for example, it can be but is not limited to 1:1, 1:2, 1:3 or a range between any two of the above values.

[0266] In some embodiments, the molar concentration of 1-phenyl-3-methyl-5- pyrazolone in the 1-phenyl-3-methyl-5-pyrazolone methanol solution is 0.5 mol / L to 1 mol / L; for example, it can be, but is not limited to, 0.5 mol / L, 0.6 mol / L, 0.7 mol / L, 0.8 mol / L, 0.9 mol / L, 1 mol / L, or a range between any two of the above molar concentrations. The volume ratio of the diluted reaction solution C to the 1-phenyl-3-methyl-5-pyrazolone methanol solution is 1:(1-2); for example, it can be, but is not limited to, 1:1, 1:1.5, 1:2, or a range between any two of the above values.

[0267] As a possible embodiment, the heating temperature is 60°C to 90°C; for example, it can be, but is not limited to, 60°C, 70°C, 80°C, 90°C, or a range between any two of the above temperatures. The heating time is 60 min to 90 min; for example, it can be, but is not limited to, 60 min, 70 min, 80 min, 90 min, or a range between any two of the above times.

[0268] In some alternative embodiments, the acidic aqueous solution comprises one or more of acetic acid and hydrochloric acid.

[0269] In some embodiments, the molar concentration of the acidic substance contained in the acidic aqueous solution is 0.2 mol / L to 0.5 mol / L; for example, it can be, but is not limited to, 0.2 mol / L, 0.3 mol / L, 0.4 mol / L, 0.5 mol / L, or a range between any two of the above molar concentrations. The volume ratio of the diluted reaction solution C to the acidic aqueous solution is 1:(1-2); for example, it can be, but is not limited to, 1:1, 1:1.5, 1:2, or a range between any two of the above values.

[0270] In some embodiments, the volume ratio of the diluted reaction solution D to the chloroform is (2-4):(4-6); for example, it can be, but is not limited to, 2:6, 3:6, 4:6, 4:5, 4:4, or a range between any two of the above ratios.

[0271] As a possible embodiment, the volume ratio of the first-step test substance solution to the second-step test substance solution is (2-5):(0.5-1); for example, it can be, but is not limited to, 2:1, 2:0.5, 3:1, 5:1, 5:0.9, 5:0.8, 5:0.7, 5:0.6, 5:0.5, or a range between any two of the above ratios.

[0272] In some embodiments, the step of preparing the control solution comprises: mixing D(+)-glucose, D(-)-galactose and water to prepare a mixture A; mixing the mixture A with an aqueous sodium hydroxide solution and a 1-phenyl-3-methyl-5-pyrazolone methanol solution, heating, mixing with an acidic aqueous solution after cooling, diluting with water to obtain a mixture B; mixing the mixture B with chloroform, shaking, extracting, and absorbing the upper aqueous phase layer to obtain a first control stock solution; dissolving puerarin, doriden A, doriden B, doriden C and icariin in anhydrous ethanol, then diluting with an aqueous ethanol solution to obtain a second control stock solution; mixing the first control stock solution and the second control stock solution, and diluting with an aqueous ethanol solution to obtain the control solution.

[0273] In some embodiments, the aqueous sodium hydroxide solution has a molar concentration of sodium hydroxide of 0.2 mol / L-0.5 mol / L, and the volume ratio of the mixture A to the aqueous sodium hydroxide solution is 1:(1-3).

[0274] As a possible embodiment, the 1-phenyl-3-methyl-5-pyrazolone methanol solution has a molar concentration of 1-phenyl-3-methyl-5-pyrazolone of 0.5 mol / L-1 mol / L, and the volume ratio of the mixture A to the 1-phenyl-3-methyl-5-pyrazolone methanol solution is 1:(1-2).

[0275] In some alternative embodiments, the heating temperature is 60°C-90°C, and the heating time is 60 min-90 min.

[0276] In some of the embodiments, the acidic aqueous solution comprises one or more of acetic acid and hydrochloric acid.

[0277] In some exemplary embodiments, the acidic aqueous solution has a molar concentration of the acidic substance of 0.2 mol / L-0.5 mol / L, and the volume ratio of the mixture A to the acidic aqueous solution is 1:(1-2).

[0278] In some embodiments, the volume ratio of the mixture B to chloroform is (2-4):(4-6).

[0279] In some of the embodiments, the volume ratio of the first control stock solution to the second control stock solution is (2-5):(0.5-1). The mass concentration of puerarin, doriden A, doriden B, doriden C and icariin in the second control stock solution is 0.077 mg / mL, 1.0 mg / mL, 1.0 mg / mL, 1.0 mg / mL and 0.16 mg / mL, respectively.

[0280] The technical solutions of the present application are described in detail below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. If the specific conditions are not indicated in the following examples, the experimental methods are preferred to be referred to the indications given in the present application, and can also be carried out according to the experimental manuals or conventional conditions in the art, or according to the conditions suggested by the manufacturers, or by referring to the experimental methods known in the art.

[0281] In the following specific examples, the measurement parameters of the raw material components may have slight deviations within the weighing accuracy range if not specifically stated. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operation accuracy are allowed.

[0282] 1. Example

[0283] Example 1

[0284] A method for determining the content of 7 index components in a traditional Chinese medicine compound preparation, the traditional Chinese medicine compound preparation is Shenge Bushen Capsules, and the 7 index components are, in turn, puerarin, glucose, galactose, desmodium A, desmodium B, desmodium C and icariin; the content determination method is determined by high performance liquid chromatography, and the specific process is as follows:

[0285] 1.1. Experimental medicinal materials

[0286] Shenge Bushen Capsules (batch number: 23004, Xinjiang Huachun Biomedicine Co., Ltd.)

[0287] 1.2. Experimental reagents and materials

[0288] Acetonitrile (chromatographic grade, Fisher Company); anhydrous ethanol (analytical pure, Tianjin Zhiyuan Chemical Reagent Co., Ltd.); sodium dihydrogen phosphate (chromatographic pure, Shanghai Aladdin Biochemical Technology Co., Ltd.); Shenge Bushen Capsules (batch number: 23004, Xinjiang Huachun Biomedicine Co., Ltd.); water is a laboratory ultrapure water system. Desmodium A (batch number: wp24040111, purity: 98%) and desmodium B (batch number: wp24051610, purity: 98%) are purchased from Sichuan Weikeqi Biotechnology Co., Ltd., puerarin (batch number: 110752-201313, purity: 95.5%), desmodium C (batch number: 111780-201905, purity: 94.3%), icariin (batch number: 110737-202017, purity: 98.1%) and anhydrous glucose (batch number: 110833-202109, purity 99.9%) are purchased from China Institute for Drug Control, and D-galactose (batch number: FY47288, purity ≥ 99%) is purchased from Henan Standard Material Research Center.

[0289] 1.3. Instruments and equipment

[0290] Instrument: Thermo Vanquish™ (Thermo Fisher Scientific) high performance liquid chromatograph;

[0291] Column: Thermo Hypersil GOLD™ - C18 250 x 4.6 mm, 5 μm;

[0292] Analytical balance: MS205DU analytical balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.);

[0293] Ultrasonic cleaner: AS2310193 ultrasonic cleaner (Tianjin Autesen Instruments Co., Ltd.);

[0294] Pure water instrument: UPL-II-40RZ pure water instrument (Sichuan Youpu Superpure Technology Co., Ltd.).

[0295] 1.4. Experimental steps

[0296] 1.4.1 Preparation of test solution

[0297] Take about 0.3 g of the contents of Shenji Bushen Capsules, accurately weigh and add to a 50 ml centrifuge tube. Accurately pipette 25 ml of anhydrous ethanol, ultrasonically extract for 10 min, and low-speed centrifuge for 5 min at 4000 rpm. Transfer the supernatant after centrifugation to a 50 ml beaker to obtain the first-step test solution.

[0298] Take the remaining residue and centrifuge tube, dry in a 40°C air oven to constant weight to obtain the residue.

[0299] Take the residue and place it in a screw-capped glass bottle. Add 4 mL of 4M trifluoroacetic acid, seal the bottle, and hydrolyze at 100°C for 2 h. Cool, adjust the pH of the solution to neutral with 4M sodium hydroxide solution, and then add water to make up to 10 mL. Derive by taking 1 mL of the above solution, adding 1 mL of 0.3 mol / L sodium hydroxide aqueous solution, and 1 mL of 0.5 mol / L 1-phenyl-3-methyl-5-pyrazolone methanol solution. Place in a 70°C water bath for 70 min, cool, add 1 mL of 0.3 mol / L acetic acid aqueous solution, and then add water to make up to 10 mL. Take 2 mL in a 10 mL stoppered test tube, add 4 mL of chloroform, shake and extract, and then pass the upper aqueous layer through an organic filter membrane to obtain the second-step test solution.

[0300] Take 5 mL of the first-step test solution and 0.5 mL of the second-step test solution, and place them in a 10 mL volumetric flask. Add 50% ethanol to make up to the mark to obtain the test solution.

[0301] 1.4.2 Preparation of control substance stock solution

[0302] Take D (+) -glucose, D (-) -galactose, add water to dissolve, prepare a solution with a concentration of 1 mg / ml, and use the derivation method in the above test solution preparation method, take 1 ml for derivation, extraction, and membrane filtration, to obtain a first control stock solution with a glucose and galactose concentration of 0.1 mg / ml and 0.1 mg / ml respectively.

[0303] Take puerarin, epimedin A, epimedin B, epimedin C, and icariin, add ethanol to dissolve, then dilute with 50% ethanol to the mark, to prepare a solution with a concentration of 0.077 mg / ml, 1.0 mg / ml, 1.0 mg / ml, 1.0 mg / ml, and 0.16 mg / ml respectively, to obtain a second control stock solution.

[0304] 1.4.3 Preparation of mixed control solution

[0305] Take 2 ml of the first control stock solution and 1 ml of the second control stock solution, and place them in a 10 ml volumetric flask, add 50% ethanol to the mark, to obtain a control solution.

[0306] 1.4.4 Blank solution

[0307] Take a glass bottle with a screw cap, add 4 mL of 4M trifluoroacetic acid, seal with a cap, hydrolyze at 100°C for 2h, take it out and cool it, adjust the solution pH to neutral with 4M sodium hydroxide solution, and then add water to 10 mL. Derivation, take 1 mL of the above solution, add 1 mL of 0.3 mol / L sodium hydroxide aqueous solution, add 1 mL of 0.5 mol / L 1-phenyl-3-methyl-5-pyrazolone methanol solution, and place it in a 70°C water bath for 70 min, then add 1 mL of 0.3 mol / L acetic acid aqueous solution after cooling, add water to 10 mL, and take 2 mL in a 10 mL test tube with a stopper, add 4 ml of chloroform, shake and extract, take the upper aqueous phase through an organic filter membrane, to obtain a first step blank solution.

[0308] Take 0.5 ml of the first step blank solution, place it in a 10 ml volumetric flask, add 50% ethanol to the mark, to obtain a blank solution.

[0309] 1.4.5 Chromatographic conditions

[0310] Chromatographic column: Thermo Hypersll GOLDTM C18 (250 x 4.6 mm, 5 μm);

[0311] Mobile phase A: 50 mmol / L NaH2PO4, mobile phase B: acetonitrile;

[0312] The elution gradient is as follows:

[0313] 0-2min, the volume percentage of mobile phase A was maintained at 85%, and the volume percentage of mobile phase B was 15%; 2min-8min, the volume percentage of mobile phase A was decreased from 85% to 83%, and the volume percentage of mobile phase B was increased from 15% to 17%; 8min-20min, the volume percentage of mobile phase A was decreased from 83% to 82%, and the volume percentage of mobile phase B was increased from 17% to 18%; 20min-36min, the volume percentage of mobile phase A was decreased from 82% to 60%, and the volume percentage of mobile phase B was increased from 18% to 40%; 36min-40min, the volume percentage of mobile phase A was maintained at 60%, and the volume percentage of mobile phase B was 40%; 40min-42min, the volume percentage of mobile phase A was increased from 60% to 85%, and the volume percentage of mobile phase B was decreased from 40% to 15%.

[0314] Flow rate: 1 mL / min; column temperature: 40℃; injection volume: 10μL; detection wavelength: 254nm.

[0315] The blank solution, blank excipient solution, mixed reference solution and test sample solution were injected into the high performance liquid chromatograph, and the contents of puerarin, glucose, galactose, desmodium A, desmodium B, desmodium C and icariin were calculated by external standard method.

[0316] Example 2

[0317] Compared with Example 1, the elution gradient was adjusted, and other chromatographic conditions were unchanged. The elution gradient of Example 2 was as follows:

[0318] 0-8min, the volume percentage of mobile phase A was maintained at 85%, and the volume percentage of mobile phase B was 15%; 8min-15min, the volume percentage of mobile phase A was decreased from 85% to 76%, and the volume percentage of mobile phase B was increased from 15% to 24%; 15min-20min, the volume percentage of mobile phase A was decreased from 76% to 74%, and the volume percentage of mobile phase B was increased from 24% to 26%; 20min-36min, the volume percentage of mobile phase A was decreased from 74% to 70%, and the volume percentage of mobile phase B was increased from 26% to 30%; 36min-40min, the volume percentage of mobile phase A was decreased from 70% to 60%, and the volume percentage of mobile phase B was increased from 30% to 40%; 40min-42min, the volume percentage of mobile phase A was increased from 60% to 85%, and the volume percentage of mobile phase B was decreased from 40% to 15%.

[0319] Comparative Example 1

[0320] The mobile phase was different from Example 1, and the elution gradient was different, and other chromatographic conditions were unchanged. The mobile phase and elution gradient of Comparative Example 1 were as follows:

[0321] Acetonitrile was used as mobile phase A, and water was used as mobile phase B.

[0322] The gradient elution program included:

[0323] 0 min-10 min, maintaining the volume percentage of the mobile phase A at 12%;

[0324] 10 min-11 min, the volume percentage of the mobile phase A was increased from 12% to 24%;

[0325] 11 min-30 min, the volume percentage of the mobile phase A was increased from 24% to 26%;

[0326] 30 min-31 min, the volume percentage of the mobile phase A was increased from 26% to 45%;

[0327] 31 min-45 min, the volume percentage of the mobile phase A was increased from 45% to 47%.

[0328] Comparative Example 2

[0329] The mobile phase was different from Example 1, and other chromatographic conditions were unchanged. The mobile phase of Comparative Example 2 was as follows:

[0330] Acetonitrile was used as mobile phase A, and water was used as mobile phase B.

[0331] Comparative Example 3

[0332] The elution gradient was different from Example 1, and other chromatographic conditions were unchanged. The elution gradient of Comparative Example 3 was as follows:

[0333] 0-8 min, maintaining the volume percentage of the mobile phase A at 85% and the volume percentage of the mobile phase B at 15%; 8 min-15 min, decreasing the volume percentage of the mobile phase A from 85% to 70% and increasing the volume percentage of the mobile phase B from 15% to 30%; 15 min-36 min, decreasing the volume percentage of the mobile phase A from 70% to 60% and increasing the volume percentage of the mobile phase B from 30% to 40%; 36 min-40 min, maintaining the volume percentage of the mobile phase A at 60% and the volume percentage of the mobile phase B at 40%; 40 min-42 min, increasing the volume percentage of the mobile phase A from 60% to 85% and decreasing the volume percentage of the mobile phase B from 40% to 15%.

[0334] The test sample detection results of the above examples and comparative examples are shown in Table 1.

[0335] Table 1

[0336]

[0337] Note: The "minimum separation from adjacent peaks" in Table 1 refers to the minimum value in the separation of puerarin, glucose, galactose, DAE, DAF, DAC, and icariin from their respective adjacent peaks.

[0338] From the results in Table 1, it can be seen that the minimum separation of each main peak from adjacent peaks is 2.27, which is greater than 1.5, indicating that the peaks of puerarin, glucose, galactose, DAE, DAF, DAC, and icariin can be effectively separated by the liquid chromatography detection conditions of Examples 1-2.

[0339] 2. Methodology investigation

[0340] 2.1. Specificity: 10 μL of test solution, reference solution, and blank solution were injected into the HPLC chromatograph, respectively, and determined according to the chromatographic conditions in item 1.4.5 of Example 1. The chromatogram is shown in Figure 1 The results show that the separation of puerarin, glucose, galactose, DAE, DAF, DAC, and icariin from other adjacent components is greater than 1.5, and the theoretical plate number is not less than 4000. The blank solution has no interference, indicating that the determination method has good specificity. Among them, Figure 1 peak 1 is puerarin, peak 2 is glucose, peak 3 is galactose, peak 4 is DAE, peak 5 is DAF, peak 6 is DAC, and peak 7 is icariin.

[0341] 2.2. Precision

[0342] The mixed reference solution was injected 6 times continuously according to the chromatographic conditions in item 1.4.5 of Example 1, the peak area was recorded, and the relative standard deviation (RSD) of each peak area was calculated. The results are shown in Table 2, indicating that the determination method has good precision.

[0343] Table 2

[0344]

[0345] 2.3. Reproducibility

[0346] The same batch of samples was taken, and 6 test product solutions were prepared in parallel according to the method in item 1.4.1 of Example 1. The peak area was determined and recorded according to the chromatographic conditions in item 1.4.5 of Example 1. The results are shown in Table 3, indicating that the determination method has good reproducibility.

[0347] Table 3

[0348]

[0349] 2.4. Linear relationship

[0350] The standard solution was precisely pipetted, and the chromatogram data were recorded under the chromatographic conditions in 1.4.5 of Example 1 with the injection volume of 2, 4, 6, 8, and 10 μL, respectively. The standard curve was plotted with the peak area of the 7 components to be detected as the vertical coordinate and the injection amount (μg) as the horizontal coordinate, and the results were shown in Table 4, indicating that the linear relationship was good within a certain range.

[0351] Table 4

[0352] Ingredients Regression equation r Linear range (pg) Puerarin y = 63.455x - 0.0144 0.9998 0.015～0.077 Glucose y = 8.2942x + 0.0766 0.9991 0.041～0.205 Galactose y = 27.884x + 0.1698 0.9996 0.041～0.207 Davalliformin A y = 18.115x - 0.1319 1.0000 0.208～1.038 Davalliformin B y = 16.082x - 0.0981 1.0000 0.202～1.012 Davalliformin C y = 17.154x - 0.1046 1.0000 0.200～0.998 Icaritin y = 22.251x - 0.0295 1.0000 0.031～0.157

[0353] In summary, the specificity, precision, repeatability, linearity, and range of the detection method of the present application all meet the requirements, and the puerarin, glucose, galactose, damianin A, damianin B, damianin C, and icariin in the Shenge Bushen Capsules can be effectively separated and accurately quantified at the same time. The method has good specificity, good separation degree, and good accuracy. Therefore, the detection method of the present application can be used for the routine detection of the content of puerarin, glucose, galactose, damianin A, damianin B, damianin C, and icariin in the Shenge Bushen Capsules, and the preparation process detection of the test sample.

[0354] The technical features of the above-described embodiments can be combined arbitrarily. To make the description concise, all possible combinations of the technical features in the above-described embodiments are not described, however, as long as the combinations of the technical features do not exist contradictions, they should be considered as the scope of the present disclosure.

[0355] The above-described embodiments only express several implementation manners of the present application, and the description is relatively specific and detailed, however, it should not be understood as the limitation on the scope of the patent. It should be pointed out that, for the ordinary skilled in the art, several modifications and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. A method for constructing a characteristic spectrum of a traditional Chinese medicine compound preparation, characterized in that, The traditional Chinese medicine compound preparation is a mixture of extracts from kudzu root, codonopsis root, and epimedium; the method for constructing the characteristic spectrum includes the following steps: The test solution was prepared from the tested traditional Chinese medicine compound preparation; and, The test solution was subjected to liquid chromatography to obtain the characteristic chromatogram of the traditional Chinese medicine compound preparation. The conditions for the liquid chromatography detection include: The mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is a phosphate solution containing phosphate at a molar concentration of 10 mmol / L to 50 mmol / L; and mobile phase B is acetonitrile. Gradient elution is employed, and the gradient elution procedure includes: From 0 to 2 minutes, maintain the volume percentage of mobile phase A at 85% and the volume percentage of mobile phase B at 15%. From 2 to 8 minutes, reduce the volume percentage of mobile phase A from 85% to 83% and increase the volume percentage of mobile phase B from 15% to 17%. From 8 to 20 minutes, reduce the volume percentage of mobile phase A from 83% to 82% and increase the volume percentage of mobile phase B from 17% to 18%. From 20 to 36 minutes, reduce the volume percentage of mobile phase A from 82% to 60% and increase the volume percentage of mobile phase B from 18% to 40%. From 36 to 40 minutes, maintain the volume percentage of mobile phase A at 60% and the volume percentage of mobile phase B at 40%. From 40 to 42 minutes, increase the volume percentage of mobile phase A from 60% to 85% and reduce the volume percentage of mobile phase B from 40% to 15%.

2. A method for constructing a characteristic spectrum of a traditional Chinese medicine compound preparation, characterized in that, The traditional Chinese medicine compound preparation is a mixture of extracts from kudzu root, codonopsis root, and epimedium; the method for constructing the characteristic spectrum includes the following steps: The test solution was prepared from the tested traditional Chinese medicine compound preparation; and, The test solution was subjected to liquid chromatography to obtain the characteristic chromatogram of the traditional Chinese medicine compound preparation. The conditions for the liquid chromatography detection include: The mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is a phosphate solution containing phosphate at a molar concentration of 10 mmol / L to 50 mmol / L; and mobile phase B is acetonitrile. Gradient elution is employed, and the gradient elution procedure includes: From 0 to 8 min, maintain the volume percentage of mobile phase A at 85% and the volume percentage of mobile phase B at 15%; from 8 to 15 min, reduce the volume percentage of mobile phase A from 85% to 76% and increase the volume percentage of mobile phase B from 15% to 24%; from 15 to 20 min, reduce the volume percentage of mobile phase A from 76% to 74% and increase the volume percentage of mobile phase B from 24% to 26%; from 20 to 36 min, reduce the volume percentage of mobile phase A from 74% to 70% and increase the volume percentage of mobile phase B from 26% to 30%; from 36 to 40 min, reduce the volume percentage of mobile phase A from 70% to 60% and increase the volume percentage of mobile phase B from 30% to 40%; from 40 to 42 min, increase the volume percentage of mobile phase A from 60% to 85% and reduce the volume percentage of mobile phase B from 40% to 15%.

3. The method for constructing the characteristic spectrum of the traditional Chinese medicine compound preparation as described in claim 1 or 2, characterized in that, The phosphate solution contains one or more of sodium phosphate and potassium phosphate. Optionally, the phosphate solution contains one or more of sodium dihydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, and dipotassium hydrogen phosphate.

4. The method for constructing the characteristic spectrum of the traditional Chinese medicine compound preparation as described in claim 1 or 2, characterized in that, The conditions for liquid chromatography detection also include at least one of the following: (1) The chromatographic column packing material is octadecylsilane bonded silica gel; (2) The column temperature is 25℃-40℃; (3) The detection wavelength is 250nm-260nm; (4) The flow rate is 0.5 mL / min - 1.5 mL / min; and, (5) The injection volume is 10 μL-20 μL; Optionally, the chromatographic column has a length of 250 mm, an inner diameter of 4.6 mm, and a packing particle size of 5 μm.

5. The method for constructing the characteristic spectrum of the traditional Chinese medicine compound preparation according to any one of claims 1 to 4, characterized in that, The steps for preparing the test solution from the tested traditional Chinese medicine compound preparation include: The tested traditional Chinese medicine compound preparation was mixed with anhydrous ethanol, extracted by ultrasonication, centrifuged, and the supernatant was collected to prepare the first step sample solution. After centrifugation, the residue was dried to constant weight and then mixed with acid solution for hydrolysis. The pH of the hydrolysis solution was adjusted to neutral using an alkaline solution and diluted with water to prepare diluted reaction solution A. The diluted reaction solution A was mixed with an aqueous sodium hydroxide solution and a methanol solution of 1-phenyl-3-methyl-5-pyrazolone, heated, cooled, and then mixed with an acidic aqueous solution. The mixture was then diluted with water to prepare diluted reaction B. The diluted reaction solution B was mixed with chloroform, shaken, extracted, and the upper aqueous phase was collected to prepare the second-step sample solution; and... The first step sample solution and the second step sample solution are mixed and diluted with an ethanol-water solution to prepare a sample solution; Optionally, the mass-to-volume ratio of the tested traditional Chinese medicine compound preparation to anhydrous ethanol is (0.3-0.5) g:(25-40) mL; Optionally, the acid solution includes one or more of the following: an aqueous solution of trifluoroacetic acid, an aqueous solution of hydrochloric acid, and an aqueous solution of sulfuric acid; Optionally, the acid solution is an aqueous solution of trifluoroacetic acid, wherein the molar concentration of trifluoroacetic acid in the aqueous solution is 2 mol / L-4 mol / L; Optionally, the hydrolysis reaction is carried out at a temperature of 90℃-120℃ for a time of 0.5h-6h. Optionally, the alkaline solution includes one or more of sodium hydroxide aqueous solution and ammonia aqueous solution, and the molar concentration of the alkaline substance in the alkaline solution is 2 mol / L-4 mol / L; Optionally, the molar concentration of sodium hydroxide in the sodium hydroxide aqueous solution is 0.2 mol / L-0.5 mol / L, and the volume ratio of the diluted reaction solution A to the sodium hydroxide aqueous solution is 1:(1-3). Optionally, the molar concentration of 1-phenyl-3-methyl-5-pyrazolone in the 1-phenyl-3-methyl-5-pyrazolone methanol solution is 0.5 mol / L to 1 mol / L, and the volume ratio of the diluted reaction solution A to the 1-phenyl-3-methyl-5-pyrazolone methanol solution is 1:(1-2). Optionally, the heating temperature is 60℃-90℃ and the heating time is 60min-90min; Optionally, the acidic aqueous solution comprises one or more of acetic acid and hydrochloric acid; Optionally, the molar concentration of the acidic substance contained in the acidic aqueous solution is 0.2 mol / L-0.5 mol / L, and the volume ratio of the diluted reaction solution A to the acidic aqueous solution is 1:(1-2). Optionally, the volume ratio of the diluted reaction solution B to the trichloromethane is (2-4):(4-6); Optionally, the volume ratio of the first step sample solution to the second step sample solution is (2-5):(0.5-1).

6. A method for determining the content of seven indicator components in a traditional Chinese medicine compound preparation, characterized in that, The traditional Chinese medicine compound preparation is a mixture of extracts from kudzu root, codonopsis root, and epimedium; the seven indicator components are, in order, puerarin, glucose, galactose, icariin A, icariin B, icariin C, and epimedin; the content determination method includes the following steps: A reference solution containing all seven index components was prepared, and liquid chromatography was performed with different injection volumes of the reference solution to detect the components and construct standard curves for the mass and peak area of ​​puerarin, glucose, galactose, astragalin A, astragalin B, astragalin C and icariin. Take the traditional Chinese medicine compound preparation to be tested and prepare the test solution; and, The test solution was subjected to liquid chromatography to obtain the peak areas of puerarin, glucose, galactose, astragalin A, astragalin B, astragalin C and icariin in the test solution, and the peak areas were respectively substituted into the corresponding standard curves for calculation. The conditions for the liquid chromatography detection include: The mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is a phosphate solution containing phosphate at a molar concentration of 10 mmol / L to 50 mmol / L; and mobile phase B is acetonitrile. Gradient elution is employed, and the gradient elution procedure includes: From 0 to 2 minutes, maintain the volume percentage of mobile phase A at 85% and the volume percentage of mobile phase B at 15%. From 2 to 8 minutes, reduce the volume percentage of mobile phase A from 85% to 83% and increase the volume percentage of mobile phase B from 15% to 17%. From 8 to 20 minutes, reduce the volume percentage of mobile phase A from 83% to 82% and increase the volume percentage of mobile phase B from 17% to 18%. From 20 to 36 minutes, reduce the volume percentage of mobile phase A from 82% to 60% and increase the volume percentage of mobile phase B from 18% to 40%. From 36 to 40 minutes, maintain the volume percentage of mobile phase A at 60% and the volume percentage of mobile phase B at 40%. From 40 to 42 minutes, increase the volume percentage of mobile phase A from 60% to 85% and reduce the volume percentage of mobile phase B from 40% to 15%.

7. A method for determining the content of seven indicator components in a traditional Chinese medicine compound preparation, characterized in that, The traditional Chinese medicine compound preparation is a mixture of extracts from kudzu root, codonopsis root, and epimedium; the seven indicator components are, in order, puerarin, glucose, galactose, icariin A, icariin B, icariin C, and epimedin; the content determination method includes the following steps: A reference solution containing all seven index components was prepared, and liquid chromatography was performed with different injection volumes of the reference solution to detect the components and construct standard curves for the mass and peak area of ​​puerarin, glucose, galactose, astragalin A, astragalin B, astragalin C and icariin. Take the traditional Chinese medicine compound preparation to be tested and prepare the test solution; and, The test solution was subjected to liquid chromatography to obtain the peak areas of puerarin, glucose, galactose, astragalin A, astragalin B, astragalin C and icariin in the test solution, and the peak areas were respectively substituted into the corresponding standard curves for calculation. The conditions for the liquid chromatography detection include: The mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is a phosphate solution containing phosphate at a molar concentration of 10 mmol / L to 50 mmol / L; and mobile phase B is acetonitrile. Gradient elution is employed, and the gradient elution procedure includes: From 0 to 8 min, maintain the volume percentage of mobile phase A at 85% and the volume percentage of mobile phase B at 15%; from 8 to 15 min, reduce the volume percentage of mobile phase A from 85% to 76% and increase the volume percentage of mobile phase B from 15% to 24%; from 15 to 20 min, reduce the volume percentage of mobile phase A from 76% to 74% and increase the volume percentage of mobile phase B from 24% to 26%; from 20 to 36 min, reduce the volume percentage of mobile phase A from 74% to 70% and increase the volume percentage of mobile phase B from 26% to 30%; from 36 to 40 min, reduce the volume percentage of mobile phase A from 70% to 60% and increase the volume percentage of mobile phase B from 30% to 40%; from 40 to 42 min, increase the volume percentage of mobile phase A from 60% to 85% and reduce the volume percentage of mobile phase B from 40% to 15%.

8. The method for determining the content of seven indicator components in a traditional Chinese medicine compound preparation as described in claim 6 or 7, characterized in that, The phosphate solution contains one or more of sodium phosphate and potassium phosphate. Optionally, the phosphate solution contains one or more of sodium dihydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, and dipotassium hydrogen phosphate.

9. The method for determining the content of seven indicator components in a traditional Chinese medicine compound preparation as described in claim 6 or 7, characterized in that, The conditions for liquid chromatography detection also include at least one of the following: (1) The chromatographic column packing material is octadecylsilane bonded silica gel; (2) The column temperature is 25℃-40℃; (3) The detection wavelength is 250nm-260nm; (4) The flow rate is 0.5 mL / min - 1.5 mL / min; and, (5) The injection volume is 10 μL-20 μL; Optionally, the chromatographic column has a length of 250 mm, an inner diameter of 4.6 mm, and a packing particle size of 5 μm.

10. The method for determining the content of seven indicator components in a traditional Chinese medicine compound preparation as described in any one of claims 6 to 9, characterized in that, The steps for preparing the test solution from the traditional Chinese medicine compound preparation to be tested include: The traditional Chinese medicine compound preparation to be tested was mixed with anhydrous ethanol, extracted by ultrasonication, centrifuged, and the supernatant after centrifugation was taken to prepare the first step test solution. After centrifugation, the residue was dried to constant weight and then mixed with acid solution for hydrolysis. The pH of the hydrolysis solution was adjusted to neutral using an alkaline solution and diluted with water to prepare diluted reaction solution C. The diluted reaction solution C was mixed with an aqueous sodium hydroxide solution and a methanol solution of 1-phenyl-3-methyl-5-pyrazolone, heated, cooled, and then mixed with an acidic aqueous solution. The mixture was then diluted with water to prepare the diluted reaction solution D. The diluted reaction solution D was mixed with chloroform, shaken, extracted, and the upper aqueous phase was collected to prepare the second step test solution; and... The test solution from the first step and the test solution from the second step are mixed and diluted with an aqueous ethanol solution to prepare the test solution. Optionally, the mass-to-volume ratio of the traditional Chinese medicine compound preparation to be tested to anhydrous ethanol is (0.3-0.5) g:(25-40) mL; Optionally, the acid solution includes one or more of the following: an aqueous solution of trifluoroacetic acid, an aqueous solution of hydrochloric acid, and an aqueous solution of sulfuric acid; Optionally, the acid solution is an aqueous solution of trifluoroacetic acid, wherein the molar concentration of trifluoroacetic acid in the aqueous solution is 2 mol / L-4 mol / L; Optionally, the hydrolysis reaction is carried out at a temperature of 90℃-120℃ for a time of 0.5h-6h. Optionally, the alkaline solution includes one or more of sodium hydroxide aqueous solution and ammonia aqueous solution, and the molar concentration of the alkaline substance in the alkaline solution is 2 mol / L-4 mol / L; Optionally, the molar concentration of sodium hydroxide in the sodium hydroxide aqueous solution is 0.2 mol / L-0.5 mol / L, and the volume ratio of the diluted reaction solution C to the sodium hydroxide aqueous solution is 1:(1-3). Optionally, the molar concentration of 1-phenyl-3-methyl-5-pyrazolone in the 1-phenyl-3-methyl-5-pyrazolone methanol solution is 0.5 mol / L to 1 mol / L, and the volume ratio of the diluted reaction solution C to the 1-phenyl-3-methyl-5-pyrazolone methanol solution is 1:(1-2). Optionally, the heating temperature is 60℃-90℃ and the heating time is 60min-90min; Optionally, the acidic aqueous solution comprises one or more of acetic acid and hydrochloric acid; Optionally, the molar concentration of the acidic substance contained in the acidic aqueous solution is 0.2 mol / L-0.5 mol / L, and the volume ratio of the diluted reaction solution C to the acidic aqueous solution is 1:(1-2). Optionally, the volume ratio of the diluted reaction solution D to the chloroform is (2-4):(4-6); Optionally, the volume ratio of the first step test solution to the second step test solution is (2-5):(0.5-1).

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