CN115326984B - Method for measuring content of baicalin metal complex by HPLC (high Performance liquid chromatography) - Google Patents

Abstract

The invention discloses a method for determining the content of baicalin metal complex by HPLC, wherein the method uses a mixed solution of phosphoric acid aqueous solution and acetonitrile as an extracting solution, the extracting solution has good solubility for baicalin metal complex, can extract baicalin metal complex to the maximum extent and completely decompose it into baicalin; then a _C18 chromatographic column, phosphoric acid water as an aqueous phase, acetonitrile as an organic phase as a mobile phase, and baicalin as a reference substance are used for chromatographic analysis. The method is simple to operate, accurate in measurement, and has good repeatability, and can achieve quality control of baicalin metal complex and its preparation.

Description

A method for determining the content of baicalin metal complexes by HPLC

Technical Field

The invention relates to the technical field of drug detection, and in particular to a method for determining the content of baicalin metal complex by high performance liquid chromatography (HPLC).

Background technique

Baicalin aluminum is a baicalin metal complex drug synthesized from baicalin and aluminum salt. It mainly acts on the intestines and has antibacterial, anti-inflammatory, astringent and anti-astringent functions. Currently, there are dosage forms of dry suspension and premix for the treatment of intestinal diseases such as bacterial diarrhea in piglets. Baicalin aluminum is the main active ingredient of the currently commercially available drug baicalin aluminum capsule, which is mainly used to treat human enteritis and dysentery.

At present, there is no reported method for determining the aluminum content of baicalin. Baicalin aluminum capsules are in accordance with the seventh volume of the Ministry-issued Standard for Traditional Chinese Medicine Formula Preparations WS ₃ -B-1425-93, which only includes identification and inspection, and no content determination items. At present, only the determination method of baicalin zinc has been reported, which is the same as baicalin aluminum as baicalin metal complex. The main content determination methods include atomic absorption spectrophotometry, EDTA titration and liquid chromatography. Atomic absorption spectrophotometry and EDTA titration are used to determine the zinc content in baicalin zinc. Both methods require complex pretreatment (ashing after carbonization, long operation time, and serious sample loss), resulting in large errors in the determination results. The liquid chromatography method uses homemade baicalin zinc as a reference substance. After dissolving the sample in methanol under heating conditions, it is diluted and filtered. A LiChrospher (4.6mm × 250 mm, 5 μm) column is used, and methanol-water-0.10% phosphoric acid solution (47:53:0.2) is used as the mobile phase. The content of baicalin zinc is determined at a wavelength of 278 nm. However, under this condition, the elution time of baicalin and baicalin zinc differs by only 0.03 min, and they cannot be completely separated. The ultraviolet absorption of baicalin zinc is not strong and the sensitivity is low. The baicalin zinc reference substance is a homemade standard substance that is not commercially available. The calibration accuracy needs to be confirmed. This method is difficult to promote. All of the above will lead to inaccurate content determination results. Therefore, there is an urgent need for a method that is simple to operate, accurate in measurement, and has good repeatability to effectively determine the content of baicalin aluminum and its preparations. This method can be used to analyze the content of baicalin aluminum and drugs containing baicalin aluminum.

Summary of the invention

In view of the fact that the content of baicalin metal complexes and preparations thereof cannot be accurately determined during the current content determination of baicalin metal complexes and preparations thereof, the object of the present invention is to provide a method for determining the content of baicalin metal complexes by HPLC, so as to provide a detection method for the quality control of such compound products. The method has easy-to-obtain reference substances, simple operation, good specificity, high sensitivity, accurate measurement and good repeatability.

To achieve the above object, the present invention provides a technical solution, which specifically comprises the following steps: weighing a baicalin metal complex or a preparation thereof, adding an extraction solution for ultrasonic dissolution and filtering to obtain a main component solution of the baicalin metal complex or the preparation thereof, and then subjecting the solution to chromatographic analysis using a _C18 chromatographic column, a solution of 0.05% phosphoric acid solution as an aqueous phase and acetonitrile as an organic phase as a mobile phase, a flow rate of 1.0 ml/min, a column temperature of 30°C, and a detection wavelength of 278 nm; the extraction solution is a mixed solution of phosphoric acid aqueous solution and acetonitrile in a volume ratio of 7:3, the phosphoric acid volume fraction of the phosphoric acid aqueous solution is 0.05%, and the specifications of the _C18 chromatographic column are 4.6 mm × 150 mm, 5 μm.

Preferably, the extract is 0.05% phosphoric acid water: acetonitrile in a volume ratio of 7:3.

Preferably, the dissolving method is ultrasonic dissolving until the dissolution is complete.

Preferably, the ultrasonic dissolution time is 20 to 30 min, the power is 40 KHz, and the temperature is room temperature.

Preferably, the detection wavelength is 278 nm.

Preferably, the volume ratio of aqueous phase to organic phase in the mobile phase is 7:3 to 6:4, wherein the aqueous phase is a 0.05% phosphoric acid aqueous solution and the organic phase is acetonitrile.

Preferably, the chromatographic column is a _C18 column, and its length is 150 mm, or 250 mm.

Preferably, the baicalin metal complex is baicalin aluminum, and it may also be baicalin zinc, baicalin copper, and the like.

Preferably, the preparation containing baicalin metal complex is baicalin aluminum dry suspension and premix, and can also be a preparation of other complexes such as baicalin zinc.

Technical Effects

1. The method uses a mixture of aqueous phosphoric acid solution and acetonitrile as an extracting solution. The extracting solution has good solubility for baicalin metal complex. Under the action of ultrasound, the baicalin metal complex can be extracted to the maximum extent and completely decomposed into baicalin. Then, a _C18 chromatographic column, aqueous phosphoric acid solution as an aqueous phase and acetonitrile as an organic phase are used as mobile phases for HPLC detection. Baicalin is used as a reference substance. The amount of baicalin measured by the external standard method is multiplied by a coefficient to calculate the content of the baicalin metal complex, thereby realizing accurate content determination of the baicalin metal complex and its preparation.

2. This method is simple to operate, the reference material is easily available, the measurement is accurate, the repeatability is good, and the cost is low.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

Figure 1, solution spectrum of the patent system applicability;

Figure 2, the reference solution spectrum of this patent;

Figure 3, the sample solution spectrum of this patent;

Figure 4. UV absorption spectra of baicalin and baicalin aluminum of this patent after being treated with extraction solvent.

Detailed ways

The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

The HPLC conditions for content detection in the present invention are as follows:

Chromatographic column: C ₁₈ ; detection wavelength: 278 nm; flow rate: 1 ml/min; column temperature: 30°C.

Preparation of mixed extraction solution: a mixed solution of 0.05% phosphoric acid solution and acetonitrile in a ratio of 7:3. The specific preparation method is to take 0.35 ml of phosphoric acid, dissolve it in 700 ml of water, mix well, then add 300 ml of acetonitrile and mix well.

Preparation of mobile phase: Take 0.35 ml of phosphoric acid, dissolve it in 700 ml of water, mix well, then add 300 ml of acetonitrile, mix well, filter, and degas by ultrasonication.

The spectrum of the system suitability solution is shown in Figure 1. The results show that the parameters such as the number of theoretical plates, resolution, tailing factor and repeatability all meet the standards.

The spectra of the test solution and the reference are shown in Figures 2 and 3. The results show that the main peak and the reference peak appear at the same time, the impurity peak and the main peak have good separation, and do not interfere with the main peak detection; the main peak theoretical plate number, tailing factor and other parameters meet the acceptance criteria.

Example 1

The method for detecting the content of baicalin aluminum raw material comprises the following steps:

(1) Preparation of reference solution: Accurately weigh an appropriate amount of baicalin reference substance into a 100 mL volumetric flask, dissolve it with extraction solvent, sonicate for 30 min, cool to room temperature, add mixed extraction solution to make up to the mark; shake well, filter, and prepare a reference solution containing approximately 50 μg of baicalin per 1 mL;

(2) Preparation of test solution: Accurately weigh an appropriate amount of dry baicalin aluminum powder into a 100 mL volumetric flask, add 20 mL of mixed extraction solution, sonicate for 30 min to dissolve, cool to room temperature, add mixed extraction solution to the mark; shake well, filter, and prepare a test solution containing approximately 50 μg of baicalin aluminum per 1 mL;

(3) Determination of UV wavelength: Add extraction solvent to baicalin and baicalin aluminum respectively to prepare a sample with a concentration of 20 mmol/L, ultrasonicate for 30 min, and cool to constant volume. Take the extraction solvent as the blank control and perform full wavelength scanning in the range of 250-800 nm. The UV full wavelength scanning of the sample is shown in Figure 4. The maximum absorption wavelength of both samples is 278 nm. The absorbance of baicalin aluminum is about twice that of baicalin at the same molar concentration, indicating that under the action of the extraction solvent, baicalin aluminum is completely converted into baicalin after 30 min of ultrasonication. Therefore, the content of baicalin aluminum can be determined under this condition with baicalin as the control.

(4) Chromatographic analysis: The reference substance and test solution prepared in steps (1) and (2) were respectively injected into a liquid chromatography system for detection using a _C18 column as a chromatographic column; a 0.05% phosphoric acid aqueous solution as the aqueous phase and acetonitrile as the organic phase as the mobile phase. The flow rate was 1 ml/min, the column temperature was 30°C, the detection wavelength was 278 nm, and the injection volume was 10 μl. The chromatogram was recorded. The content of baicalin was calculated by the external standard one-point method and then converted to the content of baicalin aluminum, which was 99.42%.

(5) Repeatability test: Prepare 6 test solutions according to the preparation method of the test solution in step (2). Record the chromatogram and calculate the content of baicalin aluminum using the method in (4). The RSD value of the content determination results of the 6 test solutions was 0.9%, indicating that the preparation method of the test solution has good repeatability.

(6) Recovery test: Take the baicalin aluminum test solution with known content and place it in 100 ml volumetric bottles. Add 3 portions of baicalin reference substance at 80%, 100% and 120% of the baicalin content respectively. Prepare the test solution according to the preparation method of the test solution in step (2). Inject it into the liquid chromatography system for detection according to the chromatography method in step (4). Record the chromatogram and calculate the content of baicalin aluminum by external standard method. The recovery rates at three concentration levels are in the range of 99.43% to 101.4%, with an average recovery rate of 100.28% and a relative standard deviation of < 1.0%, indicating that the detection method can accurately determine the content of baicalin aluminum.

Example 2

The method for detecting the content of baicalin aluminum raw material comprises the following steps:

(1) Accurately weigh an appropriate amount of dried baicalin aluminum sample and place it in a crucible. Place the crucible on a discharge furnace and slowly drip concentrated sulfuric acid into the crucible for low-temperature carbonization.

(2) Place the carbonized sample in a muffle furnace for ash treatment (at 550 °C for about 5 h). After cooling to room temperature, slowly add 10 mL of 10% hydrochloric acid solution into the crucible along the wall of the vessel, cover the crucible mouth with a watch glass, and heat it in a water bath for 20 min. Rinse the watch glass carefully with 5 mL of hot distilled water, then wash it with 25 mL of water, and combine the filtrate and washing solution in a conical flask.

(3) Add 1-2 drops of methyl red indicator to the solution in step (2), mix well, add ammonia test solution until the solution changes from red to yellow, and then add 25 mL of acetic acid-ammonium acetate buffer. Accurately take 30 mL of EDTA titrant and add it to the color-developing solution, then place the beaker on an electric furnace and boil for about 5 minutes. After the solution in the beaker is cooled to room temperature, add 4-5 drops of xylenol orange indicator, and continue to add the prepared zinc solution (0.05 mol/L) to the color-developing solution in the beaker until the solution turns orange-red, stop adding, and record the amount of zinc solution added. Calculate the aluminum content and convert it into baicalin aluminum content of 72.7%.

The results show that this method is time-consuming, has a complex pre-treatment process and causes large sample loss.

Example 3

The method for detecting the content of baicalin aluminum raw material comprises the following steps:

(1) Preparation of reference solution: Accurately weigh an appropriate amount of aluminum standard solution into a volumetric flask to prepare a series of aluminum standard solutions of different concentrations.

(2) Preparation of test solution: Accurately weigh an appropriate amount of dried baicalin aluminum sample and place it in a crucible. Place the crucible on a discharge furnace and slowly drip concentrated sulfuric acid into the crucible for low-temperature carbonization. Place the carbonized sample in a muffle furnace for ashing (at 550 °C for about 5 h). After cooling to room temperature, slowly add 10 mL of 10% hydrochloric acid solution into the crucible along the wall of the vessel, cover the crucible mouth with a watch glass and place it in a water bath and heat for 20 min. Rinse the watch glass carefully with 5 mL of hot distilled water, then wash it with 25 mL of water, combine the filtrate and washing solution in a 100 mL volumetric flask, and dilute to the mark with 1% nitric acid. Take 1 mL of the above baicalin aluminum solution and dilute to the mark in a 100 mL volumetric flask. Prepare a blank solution in the same way.

(3) Determination conditions: With the blank solution as the reference solution, an atomic absorption spectrophotometer (graphite furnace atomizer) with a slit of 0.7 nm, an ashing temperature of 1400°C, an atomization temperature of 2300°C, and a matrix modifier of 5% magnesium nitrate solution of 5 μL was used to measure the absorbance of the solution at a wavelength of 309.3 nm. Finally, the corresponding concentration was obtained according to the standard curve, and the aluminum content was calculated and converted to 66.66% aluminum content in baicalin. The results show that this method is time-consuming, the pretreatment process is complicated, the sample loss is large, and the graphite furnace atomic absorption spectrophotometer is not common, so this content determination method is not easy to promote.

Example 4

The method for detecting the content of baicalin aluminum raw material comprises the following steps:

(1) Accurately weigh an appropriate amount of dried baicalin aluminum into a crucible, add 5 mL of hydrochloric acid solution and heat on a hot plate. To prevent the sample from precipitating, add a small amount of aqua regia to further dissolve it. After the sample is completely dissolved, cool the crucible to room temperature. Transfer the solution in the crucible to a conical flask, rinse the crucible carefully with 10% hydrochloric acid solution and 5 mL of hot distilled water, then wash it with 25 mL of water, and combine the filtrate and washing solution in a conical flask.

(2) Add 1-2 drops of methyl red indicator to the solution of step (1), mix well, add ammonia test solution until the solution changes from red to yellow, and then add 25 mL of acetic acid-ammonium acetate buffer. Accurately take 30 mL of EDTA titrant and add it to the color-developing solution, then place the beaker on an electric furnace and boil for about 5 minutes. After the solution in the beaker is cooled to room temperature, add 4-5 drops of xylenol orange indicator, and continue to add the prepared zinc solution (0.05 mol/L) to the color-developing solution in the beaker until the solution turns orange-red, stop adding, and record the amount of zinc solution added. Calculate the aluminum content and convert it into baicalin aluminum content of 72.70%. The results show that this method has a large sample loss and requires the use of highly corrosive liquids such as aqua regia.

Example 5

The method for detecting the content of baicalin aluminum raw material comprises the following steps:

(1) Preparation of reference solution: Accurately weigh an appropriate amount of aluminum standard solution into a volumetric flask to prepare a series of aluminum standard solutions of different concentrations.

(2) Preparation of test solution: Accurately weigh an appropriate amount of dried baicalin aluminum into a clean 150 mL small beaker and add 5 mL of hydrochloric acid solution. Heat on a hot plate for a short time. To prevent the sample from precipitating, add a small amount of aqua regia to further dissolve it. After it is completely dissolved, remove the beaker and let it cool. Dilute the baicalin aluminum solution with water and make up to volume in a 100 mL volumetric flask for later use. Take 1 mL of the above baicalin aluminum solution and make up to volume in a 50 mL volumetric flask. Prepare blank reagent in the same way.

(3) Determination conditions: blank reagent was used as reference solution, atomic absorption spectrophotometer (graphite furnace atomizer) was used with slit: 0.7 nm, ashing temperature: 1400°C, atomization temperature: 2300°C, matrix modifier: 5% magnesium nitrate solution 5 μL, and the absorbance of the solution was measured at a wavelength of 309.3 nm. Finally, the corresponding concentration was calculated based on the standard curve, and the aluminum content was calculated and converted to 125.82% aluminum content in baicalin. The results show that the results of this method have large deviations and require the use of highly corrosive liquids such as aqua regia.

Example 6

The method for detecting the content of baicalin aluminum raw material comprises the following steps:

(1) Preparation of reference solution: Accurately weigh an appropriate amount of baicalin reference substance into a 100 mL volumetric flask, dissolve it with extraction solvent, sonicate for 30 min, cool to room temperature, add mixed extraction solution to make up to the mark; shake well, filter, and prepare a reference solution containing approximately 50 μg of baicalin per 1 mL;

(2) Preparation of test solution: Accurately weigh an appropriate amount of dried baicalin aluminum powder into a 100 mL volumetric flask, add 20 mL of extraction solution a, sonicate for 30 min, cool to room temperature, add extraction solution a to the mark; shake well, filter, and prepare a baicalin aluminum test solution of a certain concentration;

(3) Chromatographic analysis: The reference substance and the test solution prepared in steps (1) and (2) were respectively chromatographically analyzed using C18 columns; 0.05% phosphoric acid aqueous solution was used as the aqueous phase, acetonitrile was used as the organic phase mobile phase, the flow rate was 1 ml/min, the column temperature was 30°C, the detection wavelength was 278 nm, and the injection volume was 10 μl. The chromatogram was recorded, and the content of baicalin was calculated by the external standard one-point method and then converted into the content of baicalin aluminum, which was 65.03%. The results showed that when preparing the test solution, the baicalin aluminum powder could not be completely dissolved, resulting in a low content determination result.

Example 7

The method for detecting the content of baicalin aluminum raw material comprises the following steps:

(1) Preparation of reference solution: Accurately weigh an appropriate amount of baicalin reference substance into a 100 mL volumetric flask, dissolve it with extraction solvent, sonicate for 30 min, cool to room temperature, add mixed extraction solution to make up to the mark; shake well, filter, and prepare a reference solution containing approximately 50 μg of baicalin per 1 mL;

(2) Preparation of test solution: Accurately weigh the dried baicalin aluminum powder into a 100 mL volumetric flask, add 20 mL of extraction solution b, sonicate for 30 min, cool to room temperature, add extraction solution b to the mark; shake well, filter, and prepare a test solution of a certain concentration;

(3) Chromatographic analysis: The reference substance and the test solution prepared in steps (1) and (2) were respectively used as chromatographic columns with C18 as the chromatographic column; 0.05% phosphoric acid aqueous solution was used as the aqueous phase, acetonitrile was used as the organic phase mobile phase, the flow rate was 1 ml/min, the column temperature was 30°C, the detection wavelength was 278 nm, and the injection volume was 10 μl. The chromatogram was recorded, and the content of baicalin was calculated by the external standard one-point method and then converted into the content of baicalin aluminum, which was 32.10%. The results showed that when preparing the test solution, the baicalin aluminum powder could not be completely dissolved, resulting in inaccurate content determination results.

Example 8

The method for detecting the content of baicalin aluminum raw material comprises the following steps:

(1) Preparation of reference solution: Accurately weigh an appropriate amount of baicalin reference substance into a 100 mL volumetric flask, dissolve it with extraction solvent, sonicate for 30 min, cool to room temperature, add mixed extraction solution to make up to the mark; shake well, filter, and prepare a reference solution containing approximately 50 μg of baicalin per 1 mL;

(2) Preparation of test solution: Accurately weigh an appropriate amount of dried baicalin aluminum powder into a 100 mL volumetric flask, add the extraction solvent and shake until no obvious particles are visible, then dilute to the mark (about 3 h), shake well, filter, and prepare the test solution;

(3) Chromatographic analysis: The reference substance and the test solution prepared in step (1) and (2) were respectively chromatographically analyzed using C18 columns; 0.05% phosphoric acid aqueous solution was used as the aqueous phase, acetonitrile was used as the organic phase mobile phase, the flow rate was 1 ml/min, the column temperature was 30°C, the detection wavelength was 278 nm, and the injection volume was 10 μl. The chromatogram was recorded, and the content of baicalin was calculated by the external standard one-point method and then converted into the content of baicalin aluminum, which was 63.10%. The results showed that the baicalin aluminum powder was not completely dissolved without ultrasound, and the content determination result was low.

Example 9

(1) Preparation of reference solution: Accurately weigh an appropriate amount of baicalin reference substance into a 100 mL volumetric flask, dissolve it with extraction solvent, sonicate for 30 min, cool to room temperature, add mixed extraction solution to make up to the mark; shake well, filter, and prepare a reference solution containing approximately 50 μg of baicalin per 1 mL;

(2) Preparation of test solution: Accurately weigh an appropriate amount of dried baicalin aluminum powder into a 100 mL volumetric flask, add 20 mL of mixed extraction solution, sonicate for 15 min, cool to room temperature, add mixed extraction solution to the mark; shake well, filter, and prepare a test solution of a certain concentration;

(3) Chromatographic analysis: The reference substance and the test solution prepared in steps (1) and (2) were respectively chromatographically analyzed using C18 columns; 0.05% phosphoric acid aqueous solution was used as the aqueous phase, acetonitrile was used as the organic phase mobile phase, the flow rate was 1 ml/min, the column temperature was 30°C, the detection wavelength was 278 nm, and the injection volume was 10 μl. The chromatogram was recorded, and the content of baicalin was calculated by the external standard one-point method and then converted into the content of baicalin aluminum, which was 62.10%. The results showed that when preparing the test solution, the ultrasonic time was too short, resulting in the inability of baicalin aluminum powder to be completely dissolved, and the content determination result was too low.

Example 10

(1) Take an appropriate amount of baicalin aluminum powder in a 100 mL volumetric flask, add 20 mL of mixed extraction solution, sonicate for 30 min to dissolve, cool to room temperature, add mixed extraction solution to the mark, shake well, and filter;

(2) Place the filter membrane in a beaker, add 10 ml of the mixed extraction solution, sonicate for 30 min to dissolve, and cool to room temperature;

(3) Take the solution from step (2) and use C18 as the chromatographic column; use 0.05% phosphoric acid aqueous solution as the aqueous phase, acetonitrile as the organic phase as the mobile phase, the flow rate is 1 ml/min, the column temperature is 30°C, the detection wavelength is 278 nm, the injection volume is 10 μl, and the liquid chromatography system is injected for detection. Record the chromatogram. No chromatographic peak appears.

Embodiment 11

(1) Preparation of reference solution: Accurately weigh an appropriate amount of baicalin reference substance into a 100 mL volumetric flask, dissolve it with extraction solvent, sonicate for 30 min, cool to room temperature, add mixed extraction solution to make up to the mark; shake well, filter, and prepare a reference solution containing approximately 50 μg of baicalin per 1 mL;

(2) Preparation of test solution: Take an appropriate amount of the dried baicalin aluminum suspension and place it in a 100 mL volumetric flask, add 20 mL of the mixed extraction solution, sonicate for 30 min to dissolve, cool to room temperature, add the mixed extraction solution to the mark; shake well, filter, and prepare a test solution containing approximately 50 μg of baicalin aluminum per 1 mL;

(3) Chromatographic analysis: The reference substance and test solution prepared in steps (1) and (2) were respectively injected into a liquid chromatography system for detection using a C18 column as a chromatographic column; a 0.05% phosphoric acid aqueous solution as the aqueous phase and acetonitrile as the organic phase as the mobile phase. The flow rate was 1 ml/min, the column temperature was 30°C, the detection wavelength was 278 nm, and the injection volume was 10 μl. The baicalin content was calculated by the external standard one-point method and then converted to the labeled percentage of baicalin aluminum, which was 100.07%.

(4) Specificity test: According to the prescription ratio and the chromatographic conditions in step (3), 20 µL of blank solvent, mixed excipient solution, baicalin reference solution and test solution were injected respectively and the chromatograms were recorded. The excipients had no interference with the determination of the samples, indicating that the method had good specificity.

(5) Repeatability test: Prepare 6 test solutions according to the preparation method of the test solution in step (2). Record the chromatograms according to the method in (4) and calculate the content of baicalin aluminum by external standard method. The RSD value of the content determination results of the 6 test solutions was 0.97%, indicating that the preparation method of the test solution has good repeatability.

(6) Recovery test: Perform sample recovery test at 80%, 100%, and 120% of the labeled concentration of the product. Accurately weigh 3 portions of baicalin reference substance at 80%, 100%, and 120% of the labeled concentration of the product, place them in 100 ml volumetric flasks, add blank mixed excipients according to the prescribed amount, prepare the test solution according to the preparation method of the test solution in step (2), use C18 as the chromatographic column; use 0.05% phosphoric acid solution as the aqueous phase and acetonitrile as the organic phase as the mobile phase, the flow rate is 1.0 ml/min, the column temperature is 30℃, the detection wavelength is 278 nm, and the injection volume is 20 μl. Inject into the liquid chromatography system for detection, record the chromatogram, and calculate the content of baicalin aluminum by external standard method. The recoveries at three concentration levels were in the range of 100.91% to 102.78%, with an average recovery of 101.82% and a relative standard deviation of < 1.0%. The detection method can accurately determine the content of baicalin aluminum.

Claims (2)

1. A method for determining the content of baicalin metal complex by HPLC, characterized in that it comprises the following steps: (1) Weighing baicalin metal complex or its preparation, adding extraction solution to ultrasonically dissolve and filter to obtain a main component solution of baicalin metal complex or its preparation; the ultrasonic dissolution time is 20 to 30 min, the power is 40 kHz, and the temperature is room temperature; (2) The above solution was subjected to chromatographic analysis using a _C18 chromatographic column, with a solution of 0.05% phosphoric acid solution as an aqueous phase and acetonitrile as an organic phase as a mobile phase, a flow rate of 1.0 ml/min, a column temperature of 30°C, and a detection wavelength of 278 nm; the volume ratio of the aqueous phase: the organic phase in the mobile phase was 7:3 to 6:4; baicalin was used as a reference substance, and the amount of baicalin was measured by an external standard method and multiplied by a coefficient to calculate the content of the baicalin metal complex; the extraction solution was a mixture of aqueous phosphoric acid solution and acetonitrile in a volume ratio of 7:3, the volume fraction of phosphoric acid in the aqueous phosphoric acid solution was 0.05%, and the specifications of the _C18 chromatographic column were 4.6 mm × 150 mm, 5 μm; and the baicalin metal complex was baicalin aluminum. 2. The method for determining the content of baicalin metal complex by HPLC according to claim 1, characterized in that the preparation of the baicalin metal complex is baicalin aluminum dry suspension and premix.