CN104865320B - HPLC dual-wavelength fingerprint determination method for multiple components of Liuwei Dihuang preparation - Google Patents

Abstract

The invention discloses an HPLC dual-wavelength fingerprint determination method for multiple components of a six-ingredient glutinous rehmannia preparation. The method comprises the following steps: (1) taking the six-ingredient glutinous rehmannia preparation and dissolving the six-ingredient glutinous rehmannia preparation in methanol so as to obtain a six-ingredient glutinous rehmannia preparation solution with a concentration of 1 g/10-30 ml; (2) dissolving a standard substance in methanol so as to obtain a standard substance solution with a concentration of 20-30 [mu]g/ml; (3) respectively acquiring the chromatograms of the six-ingredient glutinous rehmannia preparation solution and the standard substance solution under same detection conditions by using HPLC, wherein the detection conditions are that octadecyl silane bonded silica glue is used as a filler for a chromatographic column, gradient elution is carried out, a mobile phase A is an aqueous phosphoric acid solution with a concentration of 0.08 ml/100 ml, a mobile phase B is acetonitrile, and detection wavelengths are in a range of 200 to 220 nm and a range of 238 to 250 nm, respectively; and (4) calculating the contents of components in the six-ingredient glutinous rehmannia preparation corresponding to components in the standard substance according to the concentration and chromatogram of the standard substance. The method provided by the invention can realize rapid and comprehensive detection of main components in the six-ingredient glutinous rehmannia preparation and has the advantages of easy availability of instruments, simplicity and good operationality.

Description

HPLC dual-wavelength fingerprint determination method for multiple components of Liuwei Dihuang preparation

technical field

The invention relates to the technical field of traditional Chinese medicine testing, and more specifically relates to a multi-component HPLC dual-wavelength fingerprint determination method of Liuwei Dihuang preparation.

Background technique

As a representative prescription of "nourishing kidney yin", Liuwei Dihuang's curative effect is unquestionable. There are many Liuwei Dihuang preparations on the market today, so only by ensuring its quality can this classic ancient prescription give full play to its therapeutic advantages. The content determination stipulated in the 2010 edition of the Chinese Pharmacopoeia: For the three dosage forms of Shuimiwan, Xiaomiwan and Damiwan, the content of Moutan cortex is calculated as paeonol, and Shuimiwan must not be less than 0.90mg per 1g; Xiaomiwan must not be less than 1g per 1g. Less than 0.70mg; each big honeyed pill should not be less than 6.3mg. Cornus cornus containing wine is calculated as loganin, water honeyed pills shall not be less than 0.70 mg per lg; small honeyed pills shall not be less than 0.50 mg per lg; large honeyed pills shall not be less than 4.5 mg per lg. For concentrated pills, every 1g of wine cornel should not be less than 1.4mg, calculated as loganin, and per 1g of Moutan cortex, calculated as paeonol, should not be less than 1.8mg. Each capsule contains Moutan cortex, calculated as paeonol, the specification (1) shall not be less than 3.0mg; specification (2) shall not be less than 1.5mg, and wine cornus shall be calculated as ursolic acid, and the specification (1) shall not be less than 0.60 mg; specification (2) shall not be less than 0.30mg. Among them, paeonol and loganin were detected by high-performance liquid chromatography, and the chromatographic conditions were different. The detection wavelengths were 274nm and 236nm, respectively. The content of ursolic acid was determined by thin-layer chromatography. The quality standard was simple, rough and unscientific. Usually a single component or several components are used to standardize and control the quality of compound preparations, which only controls a certain component of the drug, not the drug itself. If in the process of drug production, the quality of medicinal materials does not meet the requirements for medicinal use, but also meets the content requirements stipulated in the quality standards of finished medicines, chemical standards or other ingredients will be added to the product. The addition of chemical standards changes the original medicinal components, and its curative effect also changes, which brings unsafe or ineffective factors to people's medication.

Contents of the invention

The object of the present invention is to provide a high-performance liquid chromatography (HPLC) dual-wavelength fingerprint determination method for the multi-component Liuwei Dihuang preparation in view of the defects in the prior art. The method provided by the invention can quickly and comprehensively detect the main components in the Liuwei Dihuang preparation, has easy-to-obtain instruments, is simple and convenient, and has strong operability.

The invention provides a HPLC dual-wavelength fingerprint assay method for the multi-components of Liuwei Dihuang preparation, said method comprising the following steps:

(1) Take the Liuwei Dihuang preparation, pulverize it, weigh it accurately, dissolve it in methanol at a concentration of 1g/10-30ml, extract it by ultrasonic, centrifuge, take the supernatant and filter it, and obtain the Liuwei Dihuang preparation solution, which is set aside;

(2) Accurately weigh the standard substance, dissolve it in methanol at a concentration of 20-30 μg/ml, and obtain the standard substance solution, and set aside;

(3) Adopt high-performance liquid chromatography to obtain the chromatograms of Liuwei Dihuang preparation solution and standard solution respectively under the same detection conditions; the detection conditions include:

The column filler is octadecylsilane bonded silica gel;

Gradient elution is adopted; among them, the mobile phase A is phosphoric acid aqueous solution with a concentration of 0.08ml/100ml, and the mobile phase B is acetonitrile; the elution procedure is: 0～15min, 2～15% mobile phase B; 15～25min, 15～25 % mobile phase B; 25 ~ 40min, 25 ~ 70% mobile phase B;

The detection wavelength is 200-220nm and 238-250nm;

(4) According to the concentration of the standard substance, the peak area of the standard substance in the chromatogram and the peak area of the corresponding component in the Liuwei Dihuang preparation in the chromatogram, calculate the content of the corresponding component in the Liuwei Dihuang preparation and the standard substance .

The Liuwei Dihuang preparation of the present invention is Liuwei Dihuang water honeyed pills, large honeyed pills, concentrated pills or capsules.

The step (1) of the present invention optimizes the extraction of the Liuwei Dihuang preparation and the preparation method of the solution, thereby increasing the detection accuracy. Specifically, the ultrasonic frequency of the ultrasonic extraction is preferably 35000-45000 Hz, and the extraction time is preferably 20-40 min. Under the above extraction conditions, the centrifugation speed is preferably 5000-20000 rpm, and the centrifugation time is 5-15 min. After centrifugation, the supernatant can be filtered using a filter membrane with a pore size of 0.2-0.3 μm. By optimizing the above conditions, the accuracy of detection in subsequent steps can be improved.

As a preferred solution, the step (1) of the present invention is specifically: take Liuwei Dihuang preparation, crush it and weigh it accurately, dissolve it in methanol at a concentration of 1g/10ml, extract it with an ultrasonic wave with a frequency of 40000Hz for 30min, and extract it with a frequency of 40000Hz for 30min. Centrifuge at a high speed for 10 minutes, take the supernatant, and filter it with a filter membrane with a pore size of 0.22 μm to obtain the Liuwei Dihuang preparation solution.

The standard product described in the step (2) of the present invention is selected according to the active ingredients in the Liuwei Dihuang preparation and the detection ingredients specified in the Pharmacopoeia, including gallic acid, protocatechuic acid, 1,2,3,4,6-pentagalloyl glucose , Paeonol, 5-Hydroxymethylfurfural, Morroniside, Loganin, Swaticurin, Paeoniflorin, Benzoic Acid, and Benzoyl Paeoniflorin. In the present invention, the concentration of the standard substance refers to the concentration of each standard substance. In actual operation, various standard substances can be accurately weighed respectively, combined, dissolved in a specific amount of methanol, and mixed Uniformly, a mixed standard solution is obtained. The concentration of the standard is preferably: gallic acid, protocatechuic acid, 1,2,3,4,6-pentagalloyl glucose, paeonol, 5-hydroxymethylfurfural, morroniside, loganin , swertigin, paeoniflorin, benzoic acid and benzoylpaeoniflorin concentrations were 25μg/ml.

Since different components have different peaks at different wavelengths, the present invention preferably detects gallic acid, protocatechuic acid, and 1,2,3,4,6-pentagalloyl under the condition that the detection wavelength is 200-220nm. Glucose, paeonol; under the detection wavelength of 238-250nm, detect 5-hydroxymethylfurfural, morroniside, loganin, swertigin, paeoniflorin, benzoic acid, benzoylpaeoniflorin.

The wavelengths detected in step (3) of the present invention are preferably 210 nm and 238 nm. Using these two wavelengths to detect the above components can achieve the best analysis results.

The meaning of the phosphoric acid aqueous solution concentration of 0.08ml/100ml in the step (3) of the present invention is: every 100ml solution contains 0.08ml phosphoric acid. In the gradient elution procedure, the percentage of mobile phase B refers to the volume percentage of mobile phase B in the sum of the volumes of the two phases.

The step (3) of the present invention may also include the following detection conditions: the specification of the chromatographic column is 250mm×4.6mm. The particle size of the column filler is 5 μm. The filler can be selected from Agilent C ₁₈ purchased from Agilent Technologies. The column temperature of the chromatographic column is 25-35°C, preferably 30°C. The flow rate of the mobile phase is 0.5-1.5 ml/min, preferably 1.0 ml/min. The injection volume is 10-30 μl, preferably 20 μl.

Because the detection conditions of the standard solution and the Liuwei Dihuang preparation solution are the same, in the chromatogram, the retention time and peak shape of the peaks corresponding to the standard components in the Liuwei Dihuang preparation should be basically the same as the standard product. The retention time judges the corresponding components in the two chromatograms, and the confirmation is assisted by the shape of the peak. Step (4) of the present invention is by comparing the peak area of the corresponding component in the standard solution chromatogram and the Liuwei Dihuang preparation solution chromatogram, according to the known concentration of the standard product, calculates the corresponding component in the Liuwei Dihuang preparation solution and the standard product. Concentration, and further calculate the content of the components corresponding to the standard in the Liuwei Dihuang preparation.

The invention selects two wavelengths to simultaneously detect multiple components in the Liuwei Dihuang preparation, comprehensively quantifies Liuwei Dihuang pills and capsule preparations, and is beneficial to evaluate the quality of the Liuwei Dihuang preparation as a whole and ensure its clinical efficacy.

Description of drawings

Fig. 1 is the comparison diagram of the first batch of Liuwei Dihuang Concentrated Pill samples and the mixed standard in Example 1 at a wavelength of 210nm; the marks in the figure: a, Liuwei Dihuang sample, b, mixed standard, 1, gallic acid, 2, Protocatechuic acid, 3, 1,2,3,4,6-pentagalloyl glucose, 4, paeonol.

Fig. 2 is a comparison chart of the first batch of Liuwei Dihuang Concentrated Pill samples and the mixed standard in Example 1 at a wavelength of 238nm; the marks in the figure: a, Liuwei Dihuang sample, b, mixed standard; 5, 5-hydroxymethyl Furfural, 6, morroniside, 7, loganin, 8, swertigin, 9, paeoniflorin, 10, benzoic acid, 11, benzoylpaeoniflorin.

Fig. 3 is the chromatogram of another five different batches of Liuwei Dihuang concentrated pill samples in the dual wavelength (210nm and 238nm) mode in Example 1; marks in the figure: a, detection wavelength is 210nm, b, detection wavelength is 238nm ; 1. The second batch, 2. The third batch, 3. The fourth batch, 4. The fifth batch, 5. The sixth batch.

Fig. 4 is the chromatograms of Liuwei Dihuang preparation samples in four different dosage forms in Example 1 under dual wavelength (210nm and 238nm) mode; Marks in the figure: a, detection wavelength is 210nm, b, detection wavelength is 238nm; 1. Liuwei Dihuang Concentrated Pills, 2. Liuwei Dihuang Water Honey Pills, 3. Liuwei Dihuang Big Honey Pills, 4. Liuwei Dihuang Capsules.

detailed description

Embodiments of the present invention will be further described in detail below in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but should not be used to limit the scope of the present invention.

The instruments and reagents used in the embodiments of the present invention include: Agilent 1260 high performance liquid chromatograph, diode array detector, electronic balance of 1/10,000 (ME204E, Mettler-Toledo), ultrapure water (Milli-Q, Millipore), Acetonitrile (chromatographically pure, Merk), phosphoric acid (chromatographically pure, DikmaPure). Standard products: gallic acid (Beijing Saibaicao Co., Ltd., 130718), protocatechuic acid (Beijing Saibaicao Co., Ltd., 130106), 1,2,3,4,6-pentagalloyl glucose (Beijing Saibaicao Co., Ltd., 130508), paeonol (China Food and Drug Administration, 110708-200506), 5-Hydroxymethylfurfural (Beijing Saibaicao Co., Ltd., 131124), morroniside (Beijing Saibaicao Co., Ltd., 130912), horse money Glycoside (Beijing Saibaicao Co., Ltd., 1201204), swertigin (Beijing Saibaicao Co., Ltd., 140425), paeoniflorin (Beijing Saibaicao Co., Ltd., 130726), benzoic acid (Beijing Saibaicao Co., Ltd.), benzoyl Paeoniflorin (Beijing Saibaicao Co., Ltd., 131108).

Example 1

Follow the steps below to test the Liuwei Dihuang preparation:

(1) Take the Liuwei Dihuang preparation, crush it, weigh it accurately, dissolve it in methanol at a concentration of 1g/10ml, extract it with ultrasonic waves with a frequency of 40000Hz for 30min, centrifuge at a speed of 10000rpm for 10min, take the supernatant, and filter it with a pore size of 0.22 Filter through a filter membrane of μm to obtain the Liuweidihuang preparation solution, which is set aside;

(2) Accurately weigh gallic acid, protocatechuic acid, 1,2,3,4,6-pentagalloyl glucose, paeonol, 5-hydroxymethylfurfural, morroniside, loganin, and swerve Brain, paeoniflorin, benzoic acid, and benzoylpaeoniflorin were prepared into standard solutions with a concentration of 25 μg/mL, respectively, using methanol as a solvent, and set aside;

(3) Adopt high-performance liquid chromatography to obtain the chromatograms of Liuwei Dihuang preparation solution and standard solution respectively under the same detection conditions; the detection conditions include:

The column filler is octadecylsilane bonded silica gel Agilent C ₁₈ with a particle size of 5 μm; the column specification is 250mm×4.6mm;

Gradient elution is adopted; among them, the mobile phase A is phosphoric acid aqueous solution with a concentration of 0.08ml/100ml, and the mobile phase B is acetonitrile; the elution procedure is: 0～15min, 2～15% mobile phase B; 15～25min, 15～25 % mobile phase B; 25 ~ 40min, 25 ~ 70% mobile phase B;

The detection wavelength is 210nm and 238nm;

The column temperature is 30°C;

The mobile phase flow rate is 1.0mL/min;

The injection volume is 20 μL;

(4) According to the concentration of the standard substance, the peak area of the standard substance in the chromatogram and the peak area of the corresponding component in the Liuwei Dihuang preparation in the chromatogram, calculate the content of the corresponding component in the Liuwei Dihuang preparation and the standard substance .

Using the method of this example, six batches of four formulations of commercially available Liuwei Dihuang Concentrated Pills, Liuwei Dihuang Water Honey Pills, Liuwei Dihuang Honey Pills, and Liuwei Dihuang Capsules were tested. Among them, see Figure 1 for the chromatogram of the first batch of Liuwei Dihuang Concentrated Pills at a wavelength of 210nm, and Figure 2 for the chromatogram at a wavelength of 238nm, and the chromatograms of the other five batches of Liuwei Dihuang Concentrated Pills at a wavelength of 210nm and 238nm See Figure 3; see Figure 4 for the chromatograms of one batch of Liuwei Dihuang Concentrated Pills, Liuwei Dihuang Honey Pills, Liuwei Dihuang Honey Pills, and Liuwei Dihuang Capsules at wavelengths of 210nm and 238nm.

Example 2

Follow the steps below to test the Liuwei Dihuang preparation:

(1) Take the Liuwei Dihuang preparation, crush it and weigh it accurately, dissolve it in methanol at a concentration of 1g/15ml, extract it with an ultrasonic wave with a frequency of 35000Hz for 40min, centrifuge it at a speed of 5000rpm for 15min, take the supernatant, and filter it with a pore size of 0.2 Filter through a filter membrane of μm to obtain the Liuweidihuang preparation solution, which is set aside;

(2) Accurately weigh gallic acid, protocatechuic acid, 1,2,3,4,6-pentagalloyl glucose, paeonol, 5-hydroxymethylfurfural, morroniside, loganin, and swerve Brain, paeoniflorin, benzoic acid, and benzoylpaeoniflorin were prepared into standard solutions with a concentration of 25 μg/mL, respectively, using methanol as a solvent, and set aside;

(3) Using ultra-high performance liquid chromatography, obtain the chromatograms of Liuwei Dihuang concentrated pill solution and standard solution respectively under the same detection conditions; the detection conditions include:

The column filler is octadecylsilane bonded silica gel Agilent C ₁₈ with a particle size of 5 μm; the column specification is 250mm×4.6mm;

Gradient elution is adopted; among them, the mobile phase A is phosphoric acid aqueous solution with a concentration of 0.08ml/100ml, and the mobile phase B is acetonitrile; the elution procedure is: 0～15min, 2～15% mobile phase B; 15～25min, 15～25 % mobile phase B; 25 ~ 40min, 25 ~ 70% mobile phase B;

The detection wavelength is 200nm and 238nm;

The column temperature is 25°C;

The mobile phase flow rate is 0.5mL/min;

The injection volume is 10 μL;

(4) According to the concentration of the standard substance, the peak area of the standard substance in the chromatogram and the peak area of the corresponding component in the Liuwei Dihuang preparation in the chromatogram, calculate the content of the corresponding component in the Liuwei Dihuang preparation and the standard substance .

Example 3

Follow the steps below to test the Liuwei Dihuang preparation:

(1) Take Liuwei Dihuang preparation, crush it and weigh it accurately, dissolve it in methanol at a concentration of 1g/10ml, extract it with ultrasonic waves with a frequency of 45000Hz for 20min, centrifuge at a speed of 20000r/min for 5min, take the supernatant, and filter it with a pore size of 0.3 Filter through a filter membrane of μm to obtain the Liuweidihuang preparation solution, which is set aside;

(2) Accurately weigh gallic acid, protocatechuic acid, 1,2,3,4,6-pentagalloyl glucose, paeonol, 5-hydroxymethylfurfural, morroniside, loganin, and swerve Brain, paeoniflorin, benzoic acid, and benzoylpaeoniflorin were prepared into standard solutions with a concentration of 25 μg/mL, respectively, using methanol as a solvent, and set aside;

(3) Using ultra-high performance liquid chromatography, obtain the chromatograms of Liuwei Dihuang concentrated pill solution and standard solution respectively under the same detection conditions; the detection conditions include:

The column filler is octadecylsilane bonded silica gel Agilent C ₁₈ with a particle size of 5 μm; the column specification is 250mm×4.6mm;

Gradient elution is adopted; among them, the mobile phase A is phosphoric acid aqueous solution with a concentration of 0.08ml/100ml, and the mobile phase B is acetonitrile; the elution procedure is: 0～15min, 2～15% mobile phase B; 15～25min, 15～25 % mobile phase B; 25 ~ 40min, 25 ~ 70% mobile phase B;

The detection wavelength is 220nm and 250nm;

The column temperature is 35°C;

The mobile phase flow rate is 1.5mL/min;

The injection volume is 30 μL;

(4) According to the concentration of the standard substance, the peak area of the standard substance in the chromatogram and the peak area of the corresponding component in the Liuwei Dihuang preparation in the chromatogram, calculate the content of the corresponding component in the Liuwei Dihuang preparation and the standard substance .

After comparison, among the three embodiments provided by the present invention, embodiment 1 has the best comprehensive effects such as accuracy.

Experimental example: methodological verification

1. Linear relationship investigation: Take the standard product, prepare five different concentrations of the standard product solution from high to low, draw the standard curve with the standard product concentration as the abscissa, and the chromatographic peak area as the ordinate to obtain the regression equation. The results showed that gallic acid, protocatechuic acid, 1,2,3,4,6-pentagalloyl glucose, paeonol, 5-hydroxymethylfurfural, morroniside, loganin, swertigin, Paeoniflorin, benzoic acid and benzoylpaeoniflorin all have good linear relationship at the above-mentioned concentrations, and the concentration of the standard substance provided by the present invention is within the linear range of 20-30 μg/ml.

2. Precision test: Take the first batch of samples of Liuwei Dihuang Concentrated Pills, prepare 1 part of Liuwei Dihuang preparation solution according to the method provided in Example 1, and test the solution for 6 times according to the method provided in Example 1. According to the results of 6 tests, the relative standard deviation (RSD) values of the 11 components are not more than 2%, which shows that the precision of the instrument is good.

3. Repeatability experiment: Take the first batch of samples of Liuwei Dihuang Concentrated Pills, prepare 6 parts of Liuwei Dihuang preparation solutions according to the method provided in Example 1, and test the 6 parts of the solutions according to the method provided in Example 1. According to the test results, gallic acid, protocatechuic acid, 1,2,3,4,6-pentagalloyl glucose, paeonol, 5-hydroxymethylfurfural, morroniside, loganin, and swertigin The RSD values of paeoniflorin, paeoniflorin, benzoic acid and benzoylpaeoniflorin were all less than 2%, indicating that the method has good repeatability.

4. Stability test: Take the first batch of samples of Liuwei Dihuang Concentrated Pills, and prepare 1 part of Liuwei Dihuang preparation solution according to the method provided in Example 1. After preparation, 0, 2, 4, 6, 8, 12 and 24 hours respectively Take an equal amount of solution, and detect according to the method provided in Example 1. According to the test results, gallic acid, protocatechuic acid, 1,2,3,4,6-pentagalloyl glucose, paeonol, 5-hydroxymethylfurfural, morroniside, loganin, and swertigin The RSD values of paeoniflorin, paeoniflorin, benzoic acid and benzoylpaeoniflorin were all less than 2.5%, which proved that the sample was stable within 24 hours.

5. Accuracy test: Take a sample of Liuwei Dihuang Concentrated Pills with known content of each component, add a standard substance equal to the corresponding component in the sample, and then detect the content of each component in the sample after adding the sample, and add a sample for each standard substance 6 times, and detected according to the method provided in Example 1. The sample addition recoveries of each standard product were all between 96.5-103.8%, indicating that the content determination method was accurate and reliable.

The above experimental results show that the method provided by the present invention has good repeatability, stability, precision and accuracy, and it is feasible to use the HPLC dual-wavelength multi-index content determination method provided by the present invention to carry out quality control of the Liuwei Dihuang Pill preparation.

The above embodiments are only used to illustrate the present invention, but not to limit the present invention. Although the present invention has been described in detail with reference to the embodiments, those skilled in the art should understand that various combinations, modifications or equivalent replacements of the technical solutions of the present invention do not depart from the spirit and scope of the technical solutions of the present invention, and all should cover Within the scope of the claims of the present invention.

Claims (7)

1. a HPLC dual-wavelength fingerprint assay method of Liuwei Dihuang preparation multi-component, is characterized in that, described method comprises the following steps: (1) Take the Liuwei Dihuang preparation, pulverize it, weigh it accurately, dissolve it in methanol at a concentration of 1g/10-30ml, extract it by ultrasonic, centrifuge, take the supernatant and filter it, and obtain the Liuwei Dihuang preparation solution, which is set aside; (2) Accurately weigh the standard substance, dissolve it in methanol at a concentration of 20-30 μg/ml, and obtain the standard substance solution, and set aside; The standard products include gallic acid, protocatechuic acid, 1,2,3,4,6-pentagalloyl glucose, paeonol, 5-hydroxymethylfurfural, morroniside, loganin, swertia Brain, paeoniflorin, benzoic acid and benzoylpaeoniflorin; (3) Adopt high-performance liquid chromatography to obtain the chromatograms of Liuwei Dihuang preparation solution and standard solution respectively under the same detection conditions; the detection conditions include: The chromatographic column filler is octadecylsilane bonded silica gel; the specification of the chromatographic column is 250mm×4.6mm; the particle size of the filler is 5 μm; Gradient elution is adopted; among them, the mobile phase A is phosphoric acid aqueous solution with a concentration of 0.08ml/100ml, and the mobile phase B is acetonitrile; the elution procedure is: 0～15min, 2～15% mobile phase B; 15～25min, 15～25 % mobile phase B; 25 ~ 40min, 25 ~ 70% mobile phase B; The detection wavelength is 200-220nm and 238-250nm; under the condition of detection wavelength of 200-220nm, gallic acid, protocatechuic acid, 1,2,3,4,6-pentagalloyl glucose and paeonol are detected; Under the condition of detection wavelength of 238-250nm, detect 5-hydroxymethylfurfural, morroniside, loganin, swertiroside, paeoniflorin, benzoic acid, benzoylpaeoniflorin; (4) According to the concentration of the standard substance, the peak area of the standard substance in the chromatogram and the peak area of the corresponding component in the Liuwei Dihuang preparation in the chromatogram, calculate the content of the corresponding component in the Liuwei Dihuang preparation and the standard substance . 2. The method according to claim 1, wherein the detection conditions in step (3) also include: the column temperature of the chromatographic column is 25-35°C; the flow rate of the mobile phase is 0.5-1.5ml/min; The volume is 10-30 μl. 3. The method according to claim 1 or 2, characterized in that the detection wavelengths in step (3) are 210nm and 238nm. 4. The method according to claim 1 or 2, characterized in that, in the ultrasonic extraction in step (1), the ultrasonic frequency is 35000-45000 Hz, and the extraction time is 20-40 min. 5. The method according to claim 4, characterized in that, in step (1), the centrifugal speed is 5000-20000 rpm, and the centrifugation time is 5-15 minutes. 6 . The method according to claim 5 , characterized in that, the filtering in step (1) adopts a membrane filter with a pore size of 0.2-0.3 μm. 7. The method according to claim 1, characterized in that the dosage form of the Liuwei Dihuang preparation is: honeyed water pills, honeyed pills, concentrated pills or capsules.