CN115575546A - Construction method and content determination method of HPLC (high Performance liquid chromatography) fingerprint spectrum of Wuzi Yanzong soft capsule - Google Patents

Abstract

The invention discloses a method for constructing an HPLC fingerprint of Wuzi Yanzong soft capsule and a method for determining its content, and relates to the technical field of quality detection of traditional Chinese medicines. This technical solution adopts Phenomenex Luna C18 (2) chromatographic column (250nm×4.6nm, 5μm), with [acetonitrile-methanol (10:1)] (A)-0.4% phosphoric acid aqueous solution (B) as the mobile phase, and carries out gradient After elution, the HPLC fingerprints of 10 batches of samples were established, and the similarity evaluation software was used to evaluate the similarity of Chinese medicine chromatographic fingerprints. The HPLC fingerprints of Wuzi Yanzong Soft Capsules were established and 15 common peaks were determined. The degree is greater than 0.9. The chromatographic information of the HPLC fingerprint of Wuzi Yanzong soft capsule established by this technical scheme is good, with high precision and good repeatability, which can provide a reference for its quality evaluation, and can compare the information characteristics of the overall composition of Wuzi Yanzong soft capsule samples The comprehensive reflection provides a more scientific and reasonable theoretical support for its quality control.

Description

Construction Method and Content Determination Method of HPLC Fingerprint of Wuzi Yanzong Soft Capsules

technical field

The invention relates to the technical field of quality detection of traditional Chinese medicines, in particular to a method for constructing an HPLC fingerprint of Wuzi Yanzong soft capsule and a method for determining its content.

Background technique

Wuzi Yanzong Soft Capsules are composed of 5 medicinal materials: Lycium barbarum, Raspberry, Cuscuta, Plantago and Schisandra. The effect of benefiting essence is mainly used to treat impotence, infertility, low back pain, urination and other diseases caused by kidney deficiency and essence deficiency. Other studies have shown that Wuzi Yanzong Soft Capsules can improve reproductive system diseases and increase the fertility of mice.

Fingerprint is widely used as an important means of quality control of traditional Chinese medicine prescriptions. It has the characteristics of large amount of information and strong characteristics, and can comprehensively reflect the quality stability and uniformity of Chinese medicinal materials and preparations. At the same time, high-performance liquid chromatography (HPLC) has the characteristics of high separation efficiency, fast analysis speed, high quantitative precision, many types of detectors, and good stability. Not limited by the volatility and thermal stability of the sample, most components in the sample can be analyzed and detected on the high performance liquid chromatography, which is one of the main methods for constructing fingerprints.

At present, there are literatures on the fingerprints of some dosage forms of Wuzi Yanzong Fang, and the 2020 edition of "Chinese Pharmacopoeia" also includes Wuzi Yanzong Pills and Wuzi Yanzong Tablets, but there are relatively few literatures on quality evaluation of Wuzi Yanzong Soft Capsules There is a problem that it is difficult to fully control the quality of medicines. Therefore, in order to more comprehensively and effectively control the quality of Wuzi Yanzong Soft Capsules, improve its quality control level, and ensure the safety and curative effect of clinical medication, it is necessary to research and design a drug based on the existing technology. The determination method of HPLC fingerprint that can objectively, accurately and comprehensively detect the quality of Wuzi Yanzong Soft Capsules.

Contents of the invention

In view of the problems raised by the background technology, the purpose of the present invention is to propose a method for constructing the HPLC fingerprint of Wuzi Yanzong soft capsule, and the chromatograms such as the peak intensity, the number and the overall separation of the common peaks of the established Wuzi Yanzong soft capsule HPLC fingerprint. The information is relatively good, which can comprehensively reflect the information characteristics of the overall composition of Wuzi Yanzong Soft Capsule samples, and provide a more scientific and reasonable theoretical support for its quality control.

Another object of the present invention is to propose a method for determining the content of hyperoside in Wuzi Yanzong soft capsules. The content of hyperin in Wuzi Yanzong soft capsules is determined to be 80.65-208.02 μg/capsule.

Another object of the present invention is to propose a method for determining the content of schisandrin in Wuzi Yanzong soft capsules. The content of schisandrin in Wuzi Yanzong soft capsules is 101.84-202.98 μg/capsule.

For reaching this purpose, the present invention adopts following technical scheme:

A method for constructing the HPLC fingerprint of Wuzi Yanzong soft capsule, comprising the following steps:

Step (1.1): Take the content of Wuzi Yanzong soft capsule, add methanol to extract, and obtain the first test solution;

Step (1.2): take chlorogenic acid, hyperin, verbascoside, gallic acid and schisandrin reference substances, add methanol to dissolve, and obtain a mixed reference substance solution;

Step (1.3): Draw the first need testing solution and the mixed reference solution respectively and inject them into the high-performance liquid chromatograph for analysis to obtain the HPLC fingerprint of Wuzi Yanzong Soft Capsules; wherein, the first chromatographic conditions are as follows:

Chromatographic column: Phenomenex luna C18 chromatographic column, the packing particle size of the chromatographic column is 5μm, the column length is 250mm, and the column inner diameter is 4.6mm;

Detection wavelength: 254nm;

Mobile phase: Acetonitrile-methanol mixture was used as mobile phase A, 0.4% phosphoric acid aqueous solution was used as mobile phase B, and gradient elution was carried out according to the following procedure:

0～10min: 5%～10%A;

10～30min: 10%～17%A;

30～70min: 17%～47%A;

70～80min: 47%～56%A;

80-84min: 56%A.

Further, in step (1.3), the column temperature of the chromatographic column is 35°C.

Further, in step (1.3), the flow rate of the mobile phase is 0.8mL/min.

Further, in the chromatographic conditions of step (1.3), the injection volume is 5 μL.

Further, the preparation method of the first test solution in step (1.1) is as follows: take the contents of 4 Wuzi Yanzong soft capsules, add 50mL of 70% methanol accurately, weigh them, ultrasonically treat for 60min, take out and let cool Use 70% methanol to make up to a volume of 50mL, shake well, filter through a 0.22μm microporous membrane, and take the subsequent filtrate to obtain the first test solution.

Further, the preparation method of the mixed reference solution in step (1.2) is as follows: take chlorogenic acid, hyperin, verbascoside, gallic acid, and schisandrin reference substances, add 70% methanol to dissolve, vortex for 1min to make Mix evenly, and prepare a mixed reference solution containing 79.20 μg/mL of chlorogenic acid, 40.00 μg/mL of hyperoside, 44.60 μg/mL of verbascoside, and 76.80 μg/mL of gallic acid.

Further, taking the hyperin chromatographic peak as the reference peak, the detected HPLC fingerprint of Wuzi Yanzong Soft Capsules has 15 common fronts at 254nm.

A method for determining the content of hyperin in Wuzi Yanzong soft capsules, comprising the following steps:

Step (2.1): Take the contents of Wuzi Yanzong soft capsules, add methanol to extract, and obtain the second hyperoside test solution;

Step (2.2): Take an appropriate amount of the hyperin reference substance, add methanol to dissolve, and obtain a hyperin reference substance solution containing 500 μg/mL of hyperin;

Step (2.3): Draw the second test product solution and the hyperoside reference substance solution respectively and inject them into the high-performance liquid chromatograph, measure according to the following second chromatographic conditions, and obtain the content of hyperin through linear regression equation calculation ; The second chromatographic condition is as follows:

Chromatographic column: Phenomenex luna C18 chromatographic column, the packing particle size of the chromatographic column is 5μm, the column length is 250mm, and the column inner diameter is 4.6mm;

Mobile phase: acetonitrile-methanol mixture as mobile phase A, 0.4% phosphoric acid aqueous solution as mobile phase B, gradient elution according to the following procedure: 0～10min: 5%～10%A; 10～30min: 10% ～17%A; 30～70min: 17%～47%A; 70～80min: 47%～56%A; 80～84min: 56%A;

The flow rate of the mobile phase is 0.8mL/min, the detection wavelength is 360nm, the column temperature of the chromatographic column is 35°C; the injection volume is 5 μL.

A method for determining the content of schisandrin in Wuzi Yanzong soft capsules, comprising the following steps:

Step (3.1): Take the contents of Wuzi Yanzong soft capsules, add chloroform to extract, filter and take the filtrate, heat the filtrate in a water bath and dry under reduced pressure in turn, add methanol and dissolve it to obtain the third test sample solution;

Step (3.2): Take an appropriate amount of Schisandrin A reference substance, add methanol to dissolve, and obtain a Schisandrin A reference substance solution containing 10 μg/mL of Schizandrin A;

Step (3.3): Draw the third test product solution and the Schisandrin reference substance solution respectively and inject it into the high-performance liquid chromatograph, measure according to the following third chromatographic conditions, and calculate the content of Schisandrin A by linear regression equation; Wherein, the third chromatographic condition is as follows:

Chromatographic column: Phenomenex luna C18 chromatographic column, the packing particle size of the chromatographic column is 5μm, the column length is 250mm, and the column inner diameter is 4.6mm;

Mobile phase: Use methanol-water with a mass volume ratio of 3:1 as the mobile phase for isocratic elution;

The flow rate of the mobile phase was 1.0 mL/min, the detection wavelength was 230 nm, the column temperature of the chromatographic column was 30° C., and the injection volume was 20 μL.

Further, the preparation method of the third test product solution in step (3.1) is as follows: take the contents of 10 Wuzi Yanzong soft capsules, mix them uniformly and accurately weigh 0.6g and place them in a 50mL conical In the bottle, add 20mL of chloroform, seal tightly, sonicate for 15min, filter, put the filtrate in a 50mL volumetric flask, add chloroform to the mark, shake well, accurately measure 5-10mL and put it in a stoppered graduated centrifuge tube, bath in water until fast When dry, transfer the centrifuge tube to a vacuum desiccator at 80°C for 30 minutes under reduced pressure, add 2 mL of methanol precisely, seal it up, ultrasonicate for 10 minutes, oscillate to dissolve, and filter with a 0.22 μm microporous membrane, the filtrate is the third The test solution.

The above technical solution has the following beneficial effects:

1. This technical solution adopts Phenomenex Luna C18 (2) chromatographic column (250nm×4.6nm, 5μm), with [acetonitrile-methanol (10:1)] (A)-0.4% phosphoric acid aqueous solution (B) as mobile phase, Gradient elution was carried out to establish the HPLC fingerprints of 10 batches of samples, and the similarity evaluation software was used to evaluate the similarity of Chinese medicine chromatographic fingerprints. The HPLC fingerprints of Wuzi Yanzong Soft Capsules were established and 15 common peaks were determined. 10 batches of samples The similarities are greater than 0.9. The chromatographic information of the HPLC fingerprint of Wuzi Yanzong Soft Capsules established by this technical scheme is good, such as peak intensity, number, and overall resolution of common peaks, and has high precision and good repeatability, which can provide a reference for its quality evaluation. It can comprehensively reflect the information characteristics of the overall composition of Wuzi Yanzong soft capsule samples, and provide more scientific and reasonable theoretical support for its quality control.

2. This technical scheme establishes the HPLC fingerprint of Wuzi Yanzong soft capsule, and measures the content of hyperin and schisandrin in Wuzi Yanzong soft capsule, and determines the content of hyperoside and schisandrin The contents are 80.65～208.02μg/grain and 101.84～202.98μg/grain respectively.

Description of drawings

Fig. 1 is the HPLC fingerprint of 10 batches of Wuzi Yanzong soft capsule samples of the present invention and contrast collection of illustrative plates, among the figure, S1 to S10 refer to the HPLC fingerprint collection of samples of 10 batches of Wuzi Yanzong soft capsules, R is contrast collection of illustrations;

Fig. 2 is the chromatogram of mixed reference substance, among the figure No. 1 peak is gallic acid peak, No. 8 peak is chlorogenic acid peak, No. 10 peak is hyperoside peak, No. 11 peak is verbascoside peak;

Fig. 3 is the identification chart of the characteristic spectra of Wuzi Yanzong soft capsules by the chromatographic peaks of wolfberry fruit, (fried) dodder fruit, raspberry, (salt) plantago fruit and (steamed) schisandra fruit.

detailed description

The technical solution of the present invention will be further described below in conjunction with accompanying drawings 1-3 and specific embodiments.

A method for constructing the HPLC fingerprint of Wuzi Yanzong soft capsule, comprising the following steps:

Step (1.1): Take the content of Wuzi Yanzong soft capsule, add methanol to extract, and obtain the first test solution;

Step (1.2): take chlorogenic acid, hyperin, verbascoside, gallic acid and schisandrin reference substances, add methanol to dissolve, and obtain a mixed reference substance solution;

Step (1.3): Draw the first test solution and the mixed reference solution respectively and inject them into the high-performance liquid chromatograph, analyze according to the following first chromatographic conditions, and obtain the HPLC fingerprint of Wuzi Yanzong Soft Capsules; wherein, the first chromatographic The conditions are as follows:

Chromatographic column: Phenomenex luna C18 chromatographic column, the packing particle size of the chromatographic column is 5μm, the column length is 250mm, and the column inner diameter is 4.6mm;

Detection wavelength: 254nm;

Mobile phase: Acetonitrile-methanol mixture was used as mobile phase A, 0.4% phosphoric acid aqueous solution was used as mobile phase B, and gradient elution was carried out according to the following procedure:

0～10min: 5%～10%A;

10～30min: 10%～17%A;

30～70min: 17%～47%A;

70～80min: 47%～56%A;

80-84min: 56%A.

It is worth noting that this technical solution uses a Phenomenex Luna C18 (2) chromatographic column (250nm×4.6nm, 5μm), with [acetonitrile-methanol (10:1)] (A) - 0.4% phosphoric acid aqueous solution (B) as The mobile phase was carried out with gradient elution, and the HPLC fingerprints of 10 batches of samples were established, and the similarity evaluation software was used to evaluate the similarity of the chromatographic fingerprints of traditional Chinese medicine. The HPLC fingerprints of Wuzi Yanzong Soft Capsules were established and 15 common peaks were determined. The similarities of the 10 batches of samples were all greater than 0.9. The chromatographic information of the HPLC fingerprint of Wuzi Yanzong Soft Capsules established by this technical scheme is good, such as peak intensity, number, and overall resolution of common peaks, and has high precision and good repeatability, which can provide a reference for its quality evaluation. It can comprehensively reflect the information characteristics of the overall composition of Wuzi Yanzong soft capsule samples, and provide more scientific and reasonable theoretical support for its quality control.

Specifically, the mass volume ratio of acetonitrile and methanol in mobile phase A of the technical solution is 10:1.

It is worth pointing out that, in order to obtain better chromatograms of resolution, this technical scheme inspects the chromatograms obtained under different wavelengths by a diode array detector (DAD) when establishing a chromatographic method. It was found that the chromatograms obtained at 254nm The graph contains the most peak information and the separation between the peaks is good, so 254nm is selected as the detection wavelength. At the same time, compared the separation of Phenomenex Luna C18(2) (250nm×4.6nm, 5μm) and Diamonsil C18(2) (250nm×4.6nm, 5μm) chromatographic columns, it was found that Phenomenex Luna C18(2) (250nm×4.6nm ,5μm) chromatographic column has a better separation effect, so it was selected for the determination of the sample.

Further explanation, in step (1.3), the column temperature of the chromatographic column is 35°C.

Further description, in step (1.3), the flow rate of the mobile phase is 0.8mL/min.

Further explanation, in the chromatographic conditions of step (1.3), the injection volume is 5 μL.

When the column temperature is 35°C and the flow rate is 0.8mL/min, the chromatographic peak shape is good, the number of peaks is large, and the resolution is good, so this condition is selected as the best chromatographic condition.

Further explanation, the preparation method of the first test solution in step (1.1) is as follows: Take the contents of 4 Wuzi Yanzong soft capsules, add 50mL of 70% methanol precisely, weigh them, ultrasonically treat for 60min, take out and let cool Afterwards, make up to 50 mL with 70% methanol, shake well, filter with a 0.22 μm microporous membrane, and take the subsequent filtrate to obtain the first test solution.

Further explanation, the preparation method of the mixed reference substance solution in step (1.2) is as follows: take chlorogenic acid, hyperin, verbascoside, gallic acid, schisandrin reference substance, add 70% methanol to dissolve, vortex for 1min to make Mix them evenly, and prepare a mixed reference solution containing 79.20 μg/mL of chlorogenic acid, 40.00 μg/mL of hyperoside, 44.60 μg/mL of verbascoside, and 76.80 μg/mL of gallic acid.

Further explanation, taking the hyperin chromatographic peak as the reference peak, the detected HPLC fingerprint of Wuzi Yanzong Soft Capsules has 15 common fronts at 254nm.

A method for determining the content of hyperin in Wuzi Yanzong soft capsules, comprising the following steps:

Step (2.1): Take the content of Wuzi Yanzong soft capsule, add methanol to extract, and obtain the second test solution;

Step (2.2): Take an appropriate amount of the hyperin reference substance, add methanol to dissolve, and obtain a hyperin reference substance solution containing 500 μg/mL of hyperin;

Step (2.3): Draw the second test product solution and the hyperoside reference substance solution respectively and inject them into the high-performance liquid chromatograph, measure according to the following second chromatographic conditions, and obtain the content of hyperin through linear regression equation calculation ; The second chromatographic condition is as follows:

Chromatographic column: Phenomenex luna C18 chromatographic column, the packing particle size of the chromatographic column is 5μm, the column length is 250mm, and the column inner diameter is 4.6mm;

Mobile phase: acetonitrile-methanol mixture as mobile phase A, 0.4% phosphoric acid aqueous solution as mobile phase B, gradient elution according to the following procedure: 0～10min: 5%～10%A; 10～30min: 10% ～17%A; 30～70min: 17%～47%A; 70～80min: 47%～56%A; 80～84min: 56%A;

The flow rate of the mobile phase is 0.8mL/min, the detection wavelength is 360nm, the column temperature of the chromatographic column is 35°C; the injection volume is 5 μL.

It is worth noting that the preparation method of the second need testing solution in this technical solution is the same as the preparation method of the first need testing solution.

A method for determining the content of schisandrin in Wuzi Yanzong soft capsules, comprising the following steps:

Step (3.1): Take the contents of Wuzi Yanzong soft capsules, add chloroform to extract, filter and take the filtrate, heat the filtrate in a water bath and dry under reduced pressure in turn, add methanol and dissolve it to obtain the third test sample solution;

Step (3.2): Take an appropriate amount of Schisandrin A reference substance, add methanol to dissolve, and obtain a Schisandrin A reference substance solution containing 10 μg/mL of Schizandrin A;

Step (3.3): Draw the third test product solution and the Schisandrin reference substance solution respectively and inject it into the high-performance liquid chromatograph, measure according to the following third chromatographic conditions, and calculate the content of Schisandrin A by linear regression equation; Wherein, the third chromatographic condition is as follows:

Chromatographic column: Phenomenex luna C18 chromatographic column, the packing particle size of the chromatographic column is 5μm, the column length is 250mm, and the column inner diameter is 4.6mm;

Mobile phase: Use methanol-water with a mass volume ratio of 3:1 as the mobile phase for isocratic elution;

The flow rate of the mobile phase was 1.0 mL/min, the detection wavelength was 230 nm, the column temperature of the chromatographic column was 30° C., and the injection volume was 20 μL.

This technical scheme establishes the HPLC fingerprint of Wuzi Yanzong Soft Capsules, and measures the contents of hyperin and schisandrin in Wuzi Yanzong Soft Capsules, and determines the contents of hyperoside and schisandrin respectively 80.65-208.02 μg/grain and 101.84-202.98 μg/grain.

Further explanation, the preparation method of the third need testing solution in step (3.1) is as follows: take the contents of the contents of 10 Wuzi Yanzong soft capsules, mix them uniformly and accurately weigh 0.6g and place them in a 50mL stopper cone In a shaped flask, add 20mL of chloroform, seal it tightly, sonicate for 15min, filter, put the filtrate in a 50mL volumetric flask, add chloroform to the mark, shake well, accurately measure 5-10mL and place it in a centrifuge tube with a stoppered scale, bath in water until When drying quickly, transfer the centrifuge tube to a vacuum desiccator at 80°C for 30 minutes under reduced pressure, add 2 mL of methanol precisely, seal it tightly, treat it with ultrasound for 10 minutes, shake it to dissolve, and filter it with a 0.22 μm microporous membrane. The filtrate is the first Three test solution.

The technical solution will be further described below in conjunction with the examples.

Example 1

Construction Method of HPLC Fingerprint of Wuzi Yanzong Soft Capsules

1. Materials

1-1 Instruments: KQ5200E ultrasonic cleaner, Kunshan Ultrasonic Instrument Co., Ltd.; Sarturius-BS124S electronic balance, Beijing Sartorius Balance Co., Ltd.; MettlerToledo XA105 electronic balance, Swiss Mettler-Toledo Group; Shimadzu LC-20AT efficient Liquid chromatograph, Shimadzu Corporation, Japan; Phenomenex Luna C18 (2) chromatographic column (250nm×4.6nm, 5μm).

1-2 Medicinal materials and reagents: Methanol (analytically pure), acetonitrile (chromatographically pure), and methanol (chromatographically pure) were purchased from Shandong Yuwang Industrial Co., Ltd.; chlorogenic acid reference substance, hyperin reference substance, verbascoside reference substance Herbal medicine, gallic acid reference substance and Schisandrin reference substance were all purchased from China National Institutes for Food and Drug Control; Chinese wolfberry reference material, Cuscuta fruit reference material, raspberry reference material, Plantago seed reference material, Schisandra reference material and Wuzi Yanzong Soft Capsules (batch numbers: 2102609, 2107888, 2107889, 2107890, 2107891, 2107892, 2107893, 2107894, 2107895, 2107896, number S1-S10) were purchased from Guangdong Foshan Hand Heart Pharmaceutical Co., Ltd.

2. Chromatographic conditions

2-1 Establish the chromatographic conditions (i.e. the first chromatographic conditions) of the HPLC fingerprint of Wuzi Yanzong Soft Capsules: Chromatographic column: Phenomenex Luna C18 (2) (250nm×4.6nm, 5μm); mobile phase: [acetonitrile-methanol (10 : 1)] (A)-0.4% phosphoric acid water (B); Gradient elution: 0～10min, 5%～10%A; 10～30min, 10%～17%A; 30～70min, 17%～47 %A; 70-80min, 47%-56%A; 80-84min, 56%A; flow rate: 0.8mL/min; detection wavelength: 254nm; column temperature: 35°C; injection volume: 5μL.

2-2 Chromatographic conditions for the determination of hyperin content (i.e. the second chromatographic conditions): Phenomenex Luna C18 (2) (250nm×4.6nm, 5μm); mobile phase: [acetonitrile-methanol (10:1)] (A )-0.4% phosphoric acid water (B); Gradient elution: 0~10min, 5%~10%A; 10~30min, 10%~17%A; 30~70min, 17%~47%A; 70~80min , 47%～56%A; 80～84min, 56%A; flow rate: 0.8mL/min; detection wavelength: 360nm; column temperature: 35°C; injection volume: 5μL.

2-3 Chromatographic conditions for determination of schisandrin A content (that is, the third chromatographic conditions): Chromatographic column: Phenomenex Luna C ₁₈ (2) (250nm×4.6nm, 5μm); mobile phase: methanol-water (3:1); etc. Speed elution; flow rate: 1.0mL/min; detection wavelength: 230nm; column temperature: 30°C; injection volume: 20μL.

3. Solution preparation

3-1 Establishment of HPLC fingerprints of Wuzi Yanzong Soft Capsules and determination of hyperin content Preparation of the test solution: Take the contents of 4 capsules of Wuzi Yanzong Soft Capsules and place them in a 50mL conical flask with a stopper, and precisely add 70% Methanol 50mL, weighed, sonicated for 60min, taken out and allowed to cool, supplemented with 70% methanol, shaken evenly, filtered through a 0.22 μm microporous membrane, and the filtrate was taken to obtain the test solution (the first test solution Or the second test solution).

3-2 Determination of content of schisandrin A Preparation of the test solution: Take the contents of 10 Wuzi Yanzong soft capsules, put them in a small beaker, mix them evenly, weigh 0.6g precisely, put them in a 50mL conical flask with a stopper, add 20mL of chloroform , densely plugged, sonicated for 15 minutes, filtered, the filtrate was placed in a 50mL volumetric flask, the filter paper and filter residue were extracted twice, the obtained filtrate was filtered, combined, added chloroform to the scale, shaken, and accurately measured 5-10mL was placed on a stoppered scale In the centrifuge tube, when the water bath is dry, transfer the centrifuge tube to a vacuum dryer at 80°C for 30 minutes under reduced pressure, add 2 mL of methanol precisely, seal it tightly, treat it with ultrasound for 10 minutes, oscillate to dissolve it, and filter through a 0.22 μm microporous membrane , the filtrate is Schisandrin A test solution (ie the third test solution).

3-3 Preparation of the control medicinal material solution: Accurately weigh 2.0g of each of the five medicinal medicinal material powders and place them in five 50mL conical flasks with stoppers, accurately add 50mL of 70% methanol, weigh, ultrasonically treat for 60min, take it out and let it cool Make up with 70% methanol, shake well, filter through a 0.22 μm microporous membrane, and take the subsequent filtrate to obtain the reference medicinal material solution.

3-4 Preparation of reference substance solution: Accurately weigh an appropriate amount of chlorogenic acid reference substance, hyperoside reference substance, verbascoside reference substance, gallic acid reference substance, and schisandrin reference substance, add 70% methanol to dissolve, vortex 1min to make it mix evenly, and prepare a mixed reference solution containing 79.20 μg/mL of chlorogenic acid, 40.00 μg/mL of hyperoside, 44.60 μg/mL of verbascoside, 76.80 μg/mL of gallic acid and a mixed reference solution containing hyperin 500 μg/mL hyperin reference substance solution, Schizandrin A reference substance solution containing 10 μg/mL Schizandrin A.

4. Research on Fingerprint Methodology

4-1 Precision investigation: take the Wuzi Yanzong soft capsule sample whose batch number is 2107894, prepare the test solution according to the method in the above-mentioned 3-1, and press the chromatographic conditions of the HPLC fingerprint of the Wuzi Yanzong soft capsule in the above-mentioned 2-1 (i.e. The first chromatographic condition) continuous sample injection 6 times, record chromatogram; Select hyperin chromatographic peak (No. 10 peak) as reference peak (S), record the relative peak area RSD of each common peak all less than 3.0% (as follows As shown in Table 1), the relative retention time RSDs are all less than 0.17% (as shown in Table 2 below), indicating that the precision of the instrument is good.

Table 1 The relative peak area RSD of common peaks in Wuzi Yanzong Soft Capsule fingerprint precision inspection

Table 2 Wuzi Yanzong Soft Capsule Fingerprint Precision Inspection Common Peak Relative Retention Time RSD

4-2 Repeatability investigation: Take the Wuzi Yanzong soft capsule sample with batch number 2107894, prepare 6 parts of the first test solution in parallel according to the above 3-1 method, and perform the HPLC fingerprint of Wuzi Yanzong soft capsule according to the above 2-1 Wuzi Yanzong soft capsule The chromatographic conditions (that is, the first chromatographic conditions) are injected and analyzed, and the chromatograms are recorded. Select hyperin chromatographic peak (No. 10 peak) as reference peak (S), record that the relative peak area RSD of each common peak is all less than 3.0% (as shown in table 3 below), and relative retention time RSD is all less than 0.12% ( As shown in Table 4 below), it shows that the repeatability of the method is good.

Table 3 Wuzi Yanzong Soft Capsule Fingerprint Reproducibility Investigation of Common Peak Relative Peak Area RSD

Table 4 Wuzi Yanzong Soft Capsule Fingerprint Reproducibility Investigation Common Peak Relative Retention Time RSD

4-3 Stability investigation: Take the Wuzi Yanzong soft capsule sample with batch number 2107894, prepare the test solution (ie the first test solution) according to the method in the above 3-1, and prepare the test solution at 0, 2, 4, 6 , 12, and 24 hours were injected and analyzed according to the chromatographic conditions of the HPLC fingerprint of the 2-1 Wuzi Yanzong soft capsule described above, and the chromatogram was recorded. Choose hyperin chromatographic peak (10) No. peak as reference peak (S), record the relative peak area RSD of each common peak all less than 3.0% (as shown in table 5 below), relative retention time RSD is all less than 0.22% ( As shown in the following table 6), it is illustrated that the Wuzi Yanzong soft capsule need testing solution has good stability in 24 hours.

Table 5 Relative peak area RSD of common peaks in Wuzi Yanzong soft capsule fingerprint stability investigation

Table 6 Wuzi Yanzong Soft Capsule Fingerprint Stability Investigation Common Peak Relative Retention Time RSD

5. Fingerprint establishment and similarity evaluation

5-1 Establishment of fingerprints of Wuzi Yanzong Soft Capsules: Prepare 10 batches of Wuzi Yanzong Soft Capsule samples (S1-S10) in parallel according to the method 3-1 above, The chromatographic condition of capsule HPLC fingerprint is measured to 10 batches of need testing solution and mixed reference substance solution, record corresponding chromatogram, accompanying drawing 1 of result.

5-2 Identification of characteristic peaks and attribution of common peaks: Combining the chromatograms of Wuzi Yanzong soft capsule test solution and mixed reference solution to analyze the measurement results of 10 batches of samples, 4 chromatographic peaks can be identified (as shown in Figure 2 shown), are gallic acid (peak No. 1), chlorogenic acid (peak No. 8), hyperoside (peak No. 10) and verbascoside (peak No. 11), respectively. Comparing the test solution of each control medicinal material with the test solution of Wuzi Yanzong Soft Capsule, 15 common peaks can be determined (as shown in Figure 3), wherein No. 2, 6, 7, and 9 peaks come from Lycium barbarum , peaks 4, 5, 7, 8, 10, 11, 12, 13, and 14 come from dodder, peaks 2, 3, 7, 8, 9, and 14 come from raspberry, peak 11 comes from plantago, 1, Peaks 3, 4, 5, and 6 come from Schisandra chinensis, and peak 15 has no medicinal ingredients corresponding to it, and the content of this ingredient in the compound is relatively large. It is speculated that it may be a new ingredient after decoction.

5-3 Fingerprint similarity evaluation: Import the fingerprints of the 10 batches of samples obtained in the above 5-1 into the official 2012 edition of the Pharmacopoeia Commission "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" software to obtain a comparison spectrum (see R in the accompanying drawing 1) ), the similarities between the fingerprints of 10 batches of samples (S1～S10 in Figure 1) and the generated control spectrum (R in Figure 1) were 0.996, 0.973, 0.994, 0.983, 0.994, 0.995, 0.984, 0.995 .

Example 2

Determination method of hyperin and schisandrin in Wuzi Yanzong soft capsules

Linear range inspection: Accurately measure the hyperin reference substance solution, dilute step by step, prepare a series of reference substance solutions with different concentrations, and measure the chromatographic conditions according to the above-mentioned 2-2 hyperin content (ie the second chromatographic condition) Injection analysis, regression analysis is performed on the injection volume (X) with the peak area integral value (Y), and the standard curve of hyperin is Y=12579X-4450.5, r=0.9999, and the linear range is 3.91-500 μg/mL .

Precision investigation: take the Wuzi Yanzong soft capsule sample whose batch number is 2107894, prepare the test solution (i.e. the second test solution) according to the method of 3-1 above, and measure the chromatogram according to the content of hyperin in 2-2 The conditions were continuously injected 6 times, and the chromatograms were recorded. The RSD of the measured hyperoside content is 0.26%, which is less than 1%, which meets the verification requirements of the methodology, indicating that the precision of the instrument is good.

Repeatability investigation: Take the Wuzi Yanzong soft capsule sample with batch number 2107894, prepare 6 parts of the second test solution in parallel according to the method of 3-1, inject the sample according to the chromatographic conditions of 2-2 hyperin content determination, and record Chromatogram. The RSD of the measured hyperoside content was 0.74%, which was less than 1%, which met the validation requirements of the methodology, indicating that the method had good repeatability.

Stability investigation: Take the Wuzi Yanzong soft capsule sample with batch number 2107894, prepare the test solution according to the method of 3-1, and prepare the test solution according to the method of 2-2 hyperin at 0, 2, 4, 8, 12, and 24 hours respectively. Chromatographic conditions for content determination Injection analysis, record chromatograms. The measured RSD of the hyperin content is 1.04%, which is less than 2%, which meets the validation requirements of the methodology, indicating that the test solution has good stability within 24 hours.

Sample recovery rate experiment: Take the Wuzi Yanzong soft capsule sample with batch number 2107894, add hyperoside reference substance at a ratio of 1:1, prepare 6 parts of the test solution in parallel according to the preparation method of 3-1, and press 2 -2 Chromatographic conditions for determination of hyperin content Injection analysis, record chromatograms. The average recovery rate of hyperin in 6 samples was 104.64%, and the RSD was 8.94%, which met the validation requirements of the methodology.

Determination of the content of hyperin in Wuzi Yanzong soft capsules: take 10 batches of samples of Wuzi Yanzong soft capsules, prepare the second test solution according to the preparation method of 3-1, and measure the content of hyperoside according to 2-2 The chromatographic conditions were determined, and the measured hyperoside content was between 80.65 and 208.02 μg/grain, and the results are shown in Table 1 below.

Determination of Schizandrin A in Wuzi Yanzong Soft Capsules: Get 10 batches of Wuzi Yanzong Soft Capsule samples, prepare Schizandrin A test solution according to the preparation method of 3-2 Schizandrin A content determination test solution (i.e. Three need testing solution), and measure by the chromatographic condition (being the third chromatographic condition) of 2-3 schisandrin content determination, detect and obtain the content of schisandrin in Wuzi Yanzong soft capsule at 101.84～202.98 μ g/ The results are shown in Table 7 below.

Table 7 Determination results of 10 batches of Wuzi Yanzong Soft Capsules

Sample serial number Hyperin content (μg/grain) Schizandrin A content (μg/grain) S1 89.21 132.78 S2 181.02 121.69 S3 155.97 135.42 S4 80.65 101.84 S5 123.15 202.98 S6 177.98 114.79 S7 208.02 201.71 S8 98.82 174.40 S9 125.43 179.62 S10 100.96 128.74

In order to obtain a chromatogram with a better resolution, this technical scheme uses a diode array detector (DAD) to investigate the chromatograms obtained at different wavelengths when establishing a chromatographic method. It is found that the chromatogram obtained at 254nm contains the most peak information And the separation between the peaks is good, so 254nm is selected as the detection wavelength. At the same time, compared the separation of Phenomenex Luna C18(2) (250nm×4.6nm, 5μm) and Diamonsil C18(2) (250nm×4.6nm, 5μm) chromatographic columns, it was found that Phenomenex Luna C18(2) (250nm×4.6nm, 5μm) chromatographic column has a better separation effect, so it was selected for the determination of the sample. And the influence of different column temperatures and different flow rates on the separation effect was investigated. The results showed that the column temperature was 35°C and the flow rate was 0.8mL/min. Chromatographic conditions.

This technical solution established the HPLC-DAD fingerprint method of Wuzi Yanzong soft capsules by measuring 10 batches of samples of Wuzi Yanzong soft capsules, calibrated 15 common peaks, and identified 4 chromatographic peaks, which were gallic acid (peak 1) , chlorogenic acid (peak No. 8), hyperoside (peak No. 10) and verbascoside (peak No. 11), compare the chromatograms of 10 batches of samples with the reference solution, and the similarities are all greater than 0.900, indicating that ten The similarity between batch samples is good. On the basis of the original fingerprint method, combined with the 2020 edition of "Chinese Pharmacopoeia" and the internal standards of the enterprise, the contents of hyperin and schisandrin in 10 batches of Wuzi Yanzong soft capsules were quantitatively investigated, and the results showed that the content of hyperoside The content of schisandrin A is between 80.65-208.02 μg/grain, and the content of schisandrin is 101.84-202.98 μg/grain.

The above describes the technical principles of the present invention in conjunction with specific embodiments. These descriptions are only for explaining the principles of the present invention, and cannot be construed as limiting the protection scope of the present invention in any way. Based on the explanations herein, those skilled in the art can think of other specific implementation modes of the present invention without creative efforts, and these modes will all fall within the protection scope of the present invention.

Claims (10)

1. a construction method of Wuzi Yanzong soft capsule HPLC fingerprint, is characterized in that, comprises the following steps: Step (1.1): Take the content of Wuzi Yanzong soft capsule, add methanol to extract, and obtain the first test solution; Step (1.2): take chlorogenic acid, hyperin, verbascoside, gallic acid and schisandrin reference substances, add methanol to dissolve, and obtain a mixed reference substance solution; Step (1.3): Draw the first need testing solution and the mixed reference solution respectively and inject them into the high-performance liquid chromatograph for analysis to obtain the HPLC fingerprint of Wuzi Yanzong Soft Capsules; wherein, the first chromatographic conditions are as follows: Chromatographic column: Phenomenex luna C18 chromatographic column, the packing particle size of the chromatographic column is 5μm, the column length is 250mm, and the column inner diameter is 4.6mm; Detection wavelength: 254nm; Mobile phase: Acetonitrile-methanol mixture was used as mobile phase A, 0.4% phosphoric acid aqueous solution was used as mobile phase B, and gradient elution was carried out according to the following procedure: 0～10min: 5%～10%A; 10～30min: 10%～17%A; 30～70min: 17%～47%A; 70～80min: 47%～56%A; 80-84min: 56%A. 2. The method for constructing the HPLC fingerprint of Wuzi Yanzong soft capsule according to claim 1, characterized in that, in step (1.3), the column temperature of the chromatographic column is 35°C. 3. the construction method of Wuzi Yanzong soft capsule HPLC fingerprint according to claim 2, is characterized in that, in step (1.3), the flow velocity of described mobile phase is 0.8mL/min. 4. The method for constructing the HPLC fingerprint of Wuzi Yanzong soft capsule according to claim 3, characterized in that, in the first chromatographic condition of step (1.3), the injection volume is 5 μL. 5. the construction method of Wuzi Yanzong soft capsule HPLC fingerprint according to claim 1, is characterized in that, the preparation method of the first need testing solution in step (1.1) is as follows: get 4 Wuzi Yanzong soft capsules Add 50mL of 70% methanol to the content, weigh it, and ultrasonically treat it for 60min. Take it out and let it cool, and make up to 50mL with 70% methanol. Obtain the first test solution. 6. the construction method of Wuzi Yanzong soft capsule HPLC fingerprint according to claim 1, is characterized in that, the preparation method of mixing reference substance solution in step (1.2) is as follows: get chlorogenic acid, hyperin, mullein Glycosides, gallic acid, and schisandrin reference substances were dissolved in 70% methanol, vortexed for 1 min to mix evenly, and prepared to contain 79.20 μg/mL of chlorogenic acid, 40.00 μg/mL of hyperoside, and 44.60 μg/mL of verbascoside. mL, gallic acid 76.80μg/mL mixed reference solution. 7. the construction method of HPLC fingerprint of Wuzi Yanzong soft capsule according to claim 4, is characterized in that, with hyperin chromatographic peak as reference peak, the HPLC fingerprint of Wuzi Yanzong soft capsule that detects is at 254nm There are 15 common fronts. 8. A method for determining the content of hyperin in Wuzi Yanzong soft capsules, characterized in that it comprises the following steps: Step (2.1): Take the content of Wuzi Yanzong soft capsule, add methanol to extract, and obtain the second test solution; Step (2.2): Take an appropriate amount of the hyperin reference substance, add methanol to dissolve, and obtain a hyperin reference substance solution containing 500 μg/mL of hyperin; Step (2.3): Draw the second test product solution and the hyperoside reference substance solution respectively and inject them into the high-performance liquid chromatograph, measure according to the following second chromatographic conditions, and obtain the content of hyperin through linear regression equation calculation ; The second chromatographic condition is as follows: Chromatographic column: Phenomenex luna C18 chromatographic column, the packing particle size of the chromatographic column is 5μm, the column length is 250mm, and the column inner diameter is 4.6mm; Mobile phase: acetonitrile-methanol mixture as mobile phase A, 0.4% phosphoric acid aqueous solution as mobile phase B, gradient elution according to the following procedure: 0～10min: 5%～10%A; 10～30min: 10% ～17%A; 30～70min: 17%～47%A; 70～80min: 47%～56%A; 80～84min: 56%A; The flow rate of the mobile phase is 0.8mL/min, the detection wavelength is 360nm, the column temperature of the chromatographic column is 35°C; the injection volume is 5 μL. 9. A method for determining the content of schisandrin in Wuzi Yanzong soft capsules, comprising the following steps: Step (3.1): Take the contents of Wuzi Yanzong soft capsules, add chloroform to extract, filter and take the filtrate, heat the filtrate in a water bath and dry under reduced pressure in turn, add methanol and dissolve it to obtain the third test sample solution; Step (3.2): Take an appropriate amount of Schisandrin A reference substance, add methanol to dissolve, and obtain a Schisandrin A reference substance solution containing 10 μg/mL of Schizandrin A; Step (3.3): Draw the third test product solution and the Schisandrin reference substance solution respectively and inject it into the high-performance liquid chromatograph, measure according to the following third chromatographic conditions, and calculate the content of Schisandrin A by linear regression equation; Wherein, the third chromatographic condition is as follows: Chromatographic column: Phenomenex luna C18 chromatographic column, the packing particle size of the chromatographic column is 5μm, the column length is 250mm, and the column inner diameter is 4.6mm; Mobile phase: Use methanol-water with a mass volume ratio of 3:1 as the mobile phase for isocratic elution; The flow rate of the mobile phase was 1.0 mL/min, the detection wavelength was 230 nm, the column temperature of the chromatographic column was 30° C., and the injection volume was 20 μL. 10. the content assay method of Schizandrin A in Wuzi Yanzong soft capsule according to claim 4, is characterized in that, in step (3.1), the preparation method of the third need testing solution is as follows: get 10 Wuzi Yanzong The content of the soft capsule, after mixing evenly, accurately weigh 0.6g and place it in a 50mL Erlenmeyer flask with a stopper, add 20mL of chloroform, seal it up, ultrasonicate for 15min, filter, put the filtrate in a 50mL volumetric flask, add chloroform to the scale, shake well, accurately measure 5-10mL and place it in a graduated centrifuge tube with a stopper. When the water bath is dry, transfer the centrifuge tube to a vacuum desiccator at 80°C for 30min under reduced pressure, add 2mL of methanol precisely, and seal it tightly. Sonicate for 10 minutes, shake to dissolve, and filter with a 0.22 μm microporous membrane, and the filtrate is the third test solution.

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