Construction method of HPLC fingerprint of Wuziyanzong soft capsule and content determination method

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 medicine. The technical scheme adopts a Phenomenex Luna C18 (2) chromatographic column (250nm×4.6nm, 5μm), takes [acetonitrile-methanol (10:1)] (A)-0.4% phosphoric acid aqueous solution (B) as the mobile phase, performs gradient elution, establishes HPLC fingerprints of 10 batches of samples, applies similarity evaluation software for chromatographic fingerprints of traditional Chinese medicine to perform similarity evaluation, establishes the HPLC fingerprint of Wuzi Yanzong soft capsule and determines 15 common peaks, and the similarities of the 10 batches of samples are all greater than 0.9. The chromatographic information of the HPLC fingerprint of Wuzi Yanzong soft capsule established by the technical scheme is good, with high precision and good repeatability, which can provide a reference for its quality evaluation, and can more comprehensively reflect the information characteristics of the overall components of Wuzi Yanzong soft capsule samples, providing more scientific and reasonable theoretical support for its quality control.

Description

Technical Field

[0001] The invention relates to the technical field of quality detection of traditional Chinese medicines, and in particular to a method for constructing an HPLC fingerprint of Wuzi Yanzong soft capsules and a method for determining the content of the same. Background Art

[0002] Wuzi Yanzong soft capsules are composed of five medicinal materials: wolfberry, raspberry, dodder, plantain and schisandra. In the formula, wolfberry and dodder are the main medicines, supplemented by raspberry, schisandra and plantain, which have the effect of nourishing the kidney and replenishing essence. They are mainly used to treat impotence, infertility, low back pain, and residual urine 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.

[0003] Fingerprints are widely used as an important means of quality control for Chinese medicine prescriptions. They have the characteristics of large amount of information and strong characteristics, and can comprehensively reflect the quality stability and uniformity of Chinese medicinal materials and Chinese medicine preparations. At the same time, high performance liquid chromatography (HPLC) has the characteristics of high separation efficiency, fast analysis speed, high quantitative precision, multiple detector types, and good stability. Most components in the sample can be analyzed and detected on a high performance liquid chromatograph without being restricted by the volatility and thermal stability of the sample, which is one of the main methods for constructing fingerprints.

[0004] At present, there are literatures on the fingerprints of some dosage forms of Wuzi Yanzong prescription. The 2020 edition of the Chinese Pharmacopoeia also includes Wuzi Yanzong Pills and Wuzi Yanzong Tablets. However, there are few literatures on the quality evaluation of Wuzi Yanzong soft capsules, and there is a problem of difficulty in comprehensively controlling the quality of drugs. 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 efficacy of clinical medication, it is necessary to study and design a HPLC fingerprint determination method that can objectively, accurately and comprehensively detect the quality of Wuzi Yanzong soft capsules based on existing technologies. Summary of the invention

[0005] In response to 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 capsules. The established HPLC fingerprint of Wuzi Yanzong soft capsules has good chromatographic information such as peak intensity, number and overall separation of common peaks, which can reflect the information characteristics of the overall composition of Wuzi Yanzong soft capsule samples more comprehensively, providing more scientific and reasonable theoretical support for its quality control.

[0006] Another object of the present invention is to provide a method for determining the content of hyperoside in Wuzi Yanzong soft capsules, and the content of hyperoside in Wuzi Yanzong soft capsules is determined to be 80.65-208.02 μg / capsule.

[0007] Another object of the present invention is to provide a method for determining the content of schisandrin A in Wuzi Yanzong soft capsules, and the content of schisandrin A in Wuzi Yanzong soft capsules is determined to be 101.84-202.98 μg / capsule.

[0008] To achieve this object, the present invention adopts the following technical solutions:

[0009] A method for constructing an HPLC fingerprint of Wuzi Yanzong soft capsules comprises the following steps:

[0010] Step (1.1): taking the contents of Wuzi Yanzong soft capsules, adding methanol for extraction, and obtaining a first test solution;

[0011] Step (1.2): taking chlorogenic acid, hyperoside, verbascoside, gallic acid and schisandrin A reference substances, adding methanol to dissolve, and obtaining a mixed reference substance solution;

[0012] Step (1.3): respectively aspirate the first test solution and the mixed reference solution and inject them into a high performance liquid chromatograph for analysis to obtain the HPLC fingerprint of Wuzi Yanzong soft capsule; wherein the first chromatographic condition is as follows:

[0013] Chromatographic column: Phenomenex luna C18 chromatographic column, the filler particle size of the chromatographic column is 5μm, the column length is 250mm, and the column inner diameter is 4.6mm;

[0014] Detection wavelength: 254nm;

[0015] Mobile phase: Use acetonitrile-methanol mixture as mobile phase A and 0.4% phosphoric acid aqueous solution as mobile phase B. Perform gradient elution according to the following procedure:

[0016] 0~10min: 5%~10%A;

[0017] 10~30min: 10%~17%A;

[0018] 30~70min: 17%~47%A;

[0019] 70~80min: 47%~56%A;

[0020] 80~84min:56%A.

[0021] Furthermore, in step (1.3), the column temperature of the chromatographic column is 35°C.

[0022] Furthermore, in step (1.3), the flow rate of the mobile phase is 0.8 mL / min.

[0023] Furthermore, in the chromatographic conditions of step (1.3), the injection volume is 5 μL.

[0024] Furthermore, the preparation method of the first test solution in step (1.1) is as follows: take the contents of 4 Wuzi Yanzong soft capsules, accurately add 50 mL of 70% methanol, weigh the weight, ultrasonically treat for 60 minutes, take out and cool, then add 70% methanol to a volume of 50 mL, shake well, filter with a 0.22 μm microporous filter membrane, and take the filtrate to obtain the first test solution.

[0025] Furthermore, the preparation method of the mixed reference solution in step (1.2) is as follows: take chlorogenic acid, hyperoside, verbascoside, gallic acid, and schisandrin A reference substances, add 70% methanol to dissolve, vortex for 1 min to 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.

[0026] Furthermore, taking the hyperoside chromatographic peak as the reference peak, the HPLC fingerprint of Wuzi Yanzong soft capsules detected had 15 common peaks at 254 nm.

[0027] A method for determining the content of hyperoside in Wuzi Yanzong soft capsules comprises the following steps:

[0028] Step (2.1): taking the contents of Wuzi Yanzong soft capsules, adding methanol for extraction, and obtaining a second test solution of hyperoside;

[0029] Step (2.2): Take an appropriate amount of hyperoside reference substance, add methanol to dissolve, and obtain a hyperoside reference substance solution containing 500 μg / mL hyperoside;

[0030] Step (2.3): The second test solution and the hyperoside reference solution are respectively injected into a high performance liquid chromatograph, and the determination is performed according to the following second chromatographic conditions, and the content of hyperoside is calculated by a linear regression equation; the second chromatographic conditions are as follows:

[0031] Chromatographic column: Phenomenex luna C18 chromatographic column, the filler particle size of the chromatographic column is 5μm, the column length is 250mm, and the column inner diameter is 4.6mm;

[0032] 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 program: 0-10min: 5%-10% A; 10-30min: 10%-17% A; 30-70min: 17%-47% A; 70-80min: 47%-56% A; 80-84min: 56% A;

[0033] The flow rate of the mobile phase was 0.8 mL / min, the detection wavelength was 360 nm, the column temperature was 35° C., and the injection volume was 5 μL.

[0034] A method for determining the content of schisandrin A in Wuzi Yanzong soft capsules comprises the following steps:

[0035] Step (3.1): Take the contents of Wuzi Yanzong soft capsule, add chloroform to extract, filter and take the filtrate, heat the filtrate in a water bath and dry it under reduced pressure, then add methanol to dissolve it to obtain a third test solution;

[0036] 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 Schisandrin A;

[0037] Step (3.3): respectively aspirate the third test solution and the schisandrin A reference solution and inject them into a high performance liquid chromatograph, and measure according to the following third chromatographic condition, and calculate by a linear regression equation to obtain the content of schisandrin A; wherein the third chromatographic condition is as follows:

[0038] Chromatographic column: Phenomenex luna C18 chromatographic column, the filler particle size of the chromatographic column is 5μm, the column length is 250mm, and the column inner diameter is 4.6mm;

[0039] Mobile phase: Methanol-water with a mass volume ratio of 3:1 was used as the mobile phase for isocratic elution;

[0040] The flow rate of the mobile phase was 1.0 mL / min, the detection wavelength was 230 nm, the column temperature was 30°C, and the injection volume was 20 μL.

[0041] Furthermore, in step (3.1), the preparation method of the third test solution is as follows: take the contents of 10 Wuzi Yanzong soft capsules, mix them evenly, accurately weigh 0.6 g and place them in a 50 mL stoppered conical flask, add 20 mL of chloroform, seal it, ultrasonically treat it for 15 min, filter, place the filtrate in a 50 mL volumetric flask, add chloroform to the scale, shake it well, accurately measure 5-10 mL and place it in a stoppered graduated centrifuge tube, water bath until it is almost dry, transfer the centrifuge tube to an 80°C vacuum dryer and dry it under reduced pressure for 30 min, accurately add 2 mL of methanol, seal it, ultrasonically treat it for 10 min, oscillate to dissolve it, filter it using a 0.22 μm microporous filter membrane, and the filtrate is the third test solution.

[0042] The above technical solution has the following beneficial effects:

[0043] 1. This technical solution adopts a Phenomenex Luna C18(2) chromatographic column (250nm×4.6nm, 5μm), [acetonitrile-methanol (10:1)](A)-0.4% phosphoric acid aqueous solution (B) as the mobile phase, performs gradient elution, establishes HPLC fingerprints of 10 batches of samples, applies similarity evaluation software for chromatographic fingerprints of traditional Chinese medicine to perform similarity evaluation, establishes HPLC fingerprints of Wuzi Yanzong soft capsules and determines 15 common peaks, and the similarities of the 10 batches of samples are all greater than 0.9. The chromatographic information such as the peak intensity, number and overall separation of common peaks of the HPLC fingerprint of Wuzi Yanzong soft capsules established by this technical solution are all good, with high precision and good repeatability, which can provide a reference for its quality evaluation, and can more comprehensively reflect the information characteristics of the overall components of Wuzi Yanzong soft capsule samples, providing more scientific and reasonable theoretical support for its quality control.

[0044] 2. This technical scheme established the HPLC fingerprint of Wuzi Yanzong soft capsules, and determined the contents of hyperoside and schisandrin A in Wuzi Yanzong soft capsules. The results showed that the contents of hyperoside and schisandrin A were 80.65-208.02 μg / capsule and 101.84-202.98 μg / capsule, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 HPLC fingerprints and reference spectra of 10 batches of Wuzi Yanzong soft capsule samples of the present invention, in which S1 to S10 refer to HPLC fingerprints of 10 batches of Wuzi Yanzong soft capsule samples, and R refers to the reference spectra;

[0046] Figure 2 is the chromatogram of the mixed reference substance, in which peak 1 is the gallic acid peak, peak 8 is the chlorogenic acid peak, peak 10 is the hyperoside peak, and peak 11 is the verbascoside peak;

[0047] Figure 3 This is an identification diagram of the characteristic spectrum of Wuzi Yanzong soft capsules based on the chromatographic peaks of wolfberry, (fried) dodder seed, raspberry, (salt) plantain seed and (steamed) schisandra chinensis. DETAILED DESCRIPTION

[0048] The following is combined with Figure 1-3 And specific implementation methods further illustrate the technical solution of the present invention.

[0049] A method for constructing an HPLC fingerprint of Wuzi Yanzong soft capsules comprises the following steps:

[0050] Step (1.1): taking the contents of Wuzi Yanzong soft capsules, adding methanol for extraction, and obtaining a first test solution;

[0051] Step (1.2): taking chlorogenic acid, hyperoside, verbascoside, gallic acid and schisandrin A reference substances, adding methanol to dissolve, and obtaining a mixed reference substance solution;

[0052] Step (1.3): The first test solution and the mixed reference solution are respectively injected into a high performance liquid chromatograph, and analyzed according to the following first chromatographic condition to obtain the HPLC fingerprint of Wuzi Yanzong soft capsule; wherein the first chromatographic condition is as follows:

[0053] Chromatographic column: Phenomenex luna C18 chromatographic column, the filler particle size of the chromatographic column is 5μm, the column length is 250mm, and the column inner diameter is 4.6mm;

[0054] Detection wavelength: 254nm;

[0055] Mobile phase: Use acetonitrile-methanol mixture as mobile phase A and 0.4% phosphoric acid aqueous solution as mobile phase B. Perform gradient elution according to the following procedure:

[0056] 0~10min: 5%~10%A;

[0057] 10~30min: 10%~17%A;

[0058] 30~70min: 17%~47%A;

[0059] 70~80min: 47%~56%A;

[0060] 80~84min:56%A.

[0061] It is worth noting that the present technical scheme adopts a Phenomenex Luna C18(2) chromatographic column (250nm×4.6nm, 5μm), and uses [acetonitrile-methanol (10:1)](A)-0.4% phosphoric acid aqueous solution (B) as the mobile phase for gradient elution, establishes HPLC fingerprints of 10 batches of samples, and uses the similarity evaluation software of Chinese medicine chromatographic fingerprints for similarity evaluation, establishes the HPLC fingerprint of Wuzi Yanzong soft capsules and determines 15 common peaks, and the similarities of the 10 batches of samples are all greater than 0.9. The chromatographic information such as the peak intensity, number and overall separation of common peaks of the HPLC fingerprint of Wuzi Yanzong soft capsules established by the present technical scheme are all good, and the precision and repeatability are good, which can provide a reference for its quality evaluation, and can more comprehensively reflect the information characteristics of the overall components of Wuzi Yanzong soft capsule samples, providing a more scientific and reasonable theoretical support for its quality control.

[0062] Specifically, the mass volume ratio of acetonitrile and methanol in the mobile phase A of the present technical solution is 10:1.

[0063] It is worth pointing out that in order to obtain a chromatogram with better separation, the present technical solution used a diode array detector (DAD) to examine the chromatograms obtained at different wavelengths when establishing the chromatographic method. The results showed that the chromatogram obtained at 254 nm contained the most peak information and the separation between the peaks was better, so 254 nm was selected as the detection wavelength. At the same time, the separation of the Phenomenex Luna C18 (2) (250nm × 4.6nm, 5μm) and Diamonsil C18 (2) (250nm × 4.6nm, 5μm) chromatographic columns was compared, and it was found that the Phenomenex Luna C18 (2) (250nm × 4.6nm, 5μm) chromatographic column had a better separation effect, so it was selected to measure the sample.

[0064] Further explanation: in step (1.3), the column temperature of the chromatographic column is 35°C.

[0065] Further explanation: in step (1.3), the flow rate of the mobile phase is 0.8 mL / min.

[0066] Further explanation, in the chromatographic conditions of step (1.3), the injection volume is 5 μL.

[0067] When the column temperature was 35℃ and the flow rate was 0.8mL / min, the chromatographic peak had good peak shape, large number of peaks and good separation, so this condition was selected as the optimal chromatographic condition.

[0068] 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, accurately add 50 mL of 70% methanol, weigh the weight, ultrasonically treat for 60 minutes, take out and cool, then add 70% methanol to a volume of 50 mL, shake well, filter with a 0.22 μm microporous filter membrane, and take the filtrate to obtain the first test solution.

[0069] Further explanation, the preparation method of the mixed reference solution in step (1.2) is as follows: take chlorogenic acid, hyperoside, verbascoside, gallic acid, and schisandrin A reference substances, add 70% methanol to dissolve, vortex for 1 min to 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.

[0070] Further explanation, taking the hyperoside chromatographic peak as the reference peak, the HPLC fingerprint of Wuzi Yanzong soft capsules detected had 15 common peaks at 254 nm.

[0071] A method for determining the content of hyperoside in Wuzi Yanzong soft capsules comprises the following steps:

[0072] Step (2.1): taking the contents of Wuzi Yanzong soft capsules, adding methanol for extraction, and obtaining a second test solution;

[0073] Step (2.2): Take an appropriate amount of hyperoside reference substance, add methanol to dissolve, and obtain a hyperoside reference substance solution containing 500 μg / mL hyperoside;

[0074] Step (2.3): The second test solution and the hyperoside reference solution are respectively injected into a high performance liquid chromatograph, and the determination is performed according to the following second chromatographic conditions, and the content of hyperoside is calculated by a linear regression equation; the second chromatographic conditions are as follows:

[0075] Chromatographic column: Phenomenex luna C18 chromatographic column, the filler particle size of the chromatographic column is 5μm, the column length is 250mm, and the column inner diameter is 4.6mm;

[0076] 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 program: 0-10min: 5%-10% A; 10-30min: 10%-17% A; 30-70min: 17%-47% A; 70-80min: 47%-56% A; 80-84min: 56% A;

[0077] The flow rate of the mobile phase was 0.8 mL / min, the detection wavelength was 360 nm, the column temperature was 35° C., and the injection volume was 5 μL.

[0078] It is worth noting that the preparation method of the second test solution in this technical solution is the same as the preparation method of the first test solution.

[0079] A method for determining the content of schisandrin A in Wuzi Yanzong soft capsules comprises the following steps:

[0080] Step (3.1): Take the contents of Wuzi Yanzong soft capsule, add chloroform to extract, filter and take the filtrate, heat the filtrate in a water bath and dry it under reduced pressure, then add methanol to dissolve it to obtain a third test solution;

[0081] 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 Schisandrin A;

[0082] Step (3.3): respectively aspirate the third test solution and the schisandrin A reference solution and inject them into a high performance liquid chromatograph, and measure according to the following third chromatographic condition, and calculate by a linear regression equation to obtain the content of schisandrin A; wherein the third chromatographic condition is as follows:

[0083] Chromatographic column: Phenomenex luna C18 chromatographic column, the filler particle size of the chromatographic column is 5μm, the column length is 250mm, and the column inner diameter is 4.6mm;

[0084] Mobile phase: Methanol-water with a mass volume ratio of 3:1 was used as the mobile phase for isocratic elution;

[0085] The flow rate of the mobile phase was 1.0 mL / min, the detection wavelength was 230 nm, the column temperature was 30°C, and the injection volume was 20 μL.

[0086] This technical solution established the HPLC fingerprint of Wuzi Yanzong soft capsules, and determined the contents of hyperoside and schisandrin A in Wuzi Yanzong soft capsules. The results showed that the contents of hyperoside and schisandrin A were 80.65-208.02 μg / capsule and 101.84-202.98 μg / capsule, respectively.

[0087] Further explanation, in step (3.1), the preparation method of the third test solution is as follows: take the contents of 10 Wuzi Yanzong soft capsules, mix them evenly, accurately weigh 0.6 g and place them in a 50 mL stoppered conical flask, add 20 mL of chloroform, seal it, ultrasonically treat it for 15 minutes, filter, place the filtrate in a 50 mL volumetric flask, add chloroform to the scale, shake it well, accurately measure 5-10 mL and place it in a stoppered graduated centrifuge tube, water bath until it is almost dry, transfer the centrifuge tube to an 80°C vacuum dryer and dry it under reduced pressure for 30 minutes, accurately add 2 mL of methanol, seal it, ultrasonically treat it for 10 minutes, oscillate to dissolve it, filter it using a 0.22 μm microporous filter membrane, and the filtrate is the third test solution.

[0088] The present technical solution is further described below in conjunction with embodiments.

[0089] Example 1

[0090] Method for constructing HPLC fingerprint of Wuzi Yanzong soft capsule

[0091] 1. Materials

[0092] 1-1 Instruments: KQ5200E ultrasonic cleaner, Kunshan Ultrasonic Instrument Co., Ltd.; Sarturius-BS124S electronic balance, Beijing Sartorius Balance Co., Ltd.; MettlerToledo XA105 electronic balance, Mettler-Toledo Group, Switzerland; Shimadzu LC-20AT high performance liquid chromatograph, Shimadzu Corporation, Japan; Phenomenex Luna C18(2) chromatographic column (250 nm×4.6 nm, 5 μm).

[0093] 1-2 Medicinal materials and reagents: Methanol (analytical grade), acetonitrile (chromatographic grade), and methanol (chromatographic grade) were all purchased from Shandong Yuwang Industrial Co., Ltd.; chlorogenic acid reference substance, hyperoside reference substance, verbascoside reference substance, gallic acid reference substance, and schisandra chinensis reference substance were all purchased from China Food and Drug Inspection Institutes; wolfberry reference medicinal materials, dodder reference medicinal materials, rubus idaeus reference medicinal materials, plantago reference medicinal materials, schisandra chinensis reference medicinal materials, and Wuzi Yanzong soft capsules (batch numbers: 2102609, 2107888, 2107889, 2107890, 2107891, 2107892, 2107893, 2107894, 2107895, 2107896, numbers S1-S10) were all purchased from Guangdong Foshan Shouxin Pharmaceutical Co., Ltd.

[0094] 2. Chromatographic conditions

[0095] 2-1 Establish the chromatographic conditions (i.e., the first chromatographic conditions) for 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℃; injection volume: 5μL.

[0096] 2-2 Chromatographic conditions for determination of hyperoside 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.

[0097] 2-3 Chromatographic conditions for the determination of Schisandrin A content (i.e., the third chromatographic condition): Chromatographic column: PhenomenexLuna C 18 (2) (250nm×4.6nm, 5μm); mobile phase: methanol-water (3:1); isocratic elution; flow rate: 1.0mL / min; detection wavelength: 230nm; column temperature: 30℃; injection volume: 20μL.

[0098] 3. Solution preparation

[0099] 3-1 Establishment of HPLC fingerprint of Wuzi Yanzong soft capsules and preparation of test solution for determination of hyperoside content: take the contents of 4 Wuzi Yanzong soft capsules and place them in a 50mL stoppered conical flask, accurately add 50mL of 70% methanol, weigh the weight, ultrasonically treat for 60min, take out and cool, then add 70% methanol to the top, shake well, filter through a 0.22μm microporous filter membrane, and take the filtrate to obtain the test solution (the first test solution or the second test solution).

[0100] 3-2 Preparation of test solution for determination of schisandrin A content: Take the contents of 10 Wuzi Yanzong soft capsules and put them in a small beaker. After mixing evenly, accurately weigh 0.6 g and put them in a 50 mL stoppered conical flask, add 20 mL of chloroform, seal it tightly, treat it ultrasonically for 15 min, filter it, put the filtrate in a 50 mL volumetric flask, extract it with filter paper and filter residue twice, filter the filtrate, combine it, add chloroform to the scale, shake it well, accurately measure 5-10 mL and put it in a stoppered graduated centrifuge tube, bathe it in water until it is almost dry, then transfer the centrifuge tube to a vacuum desiccator at 80°C and dry it under reduced pressure for 30 min, accurately add 2 mL of methanol, seal it tightly, treat it ultrasonically for 10 min, oscillate it to dissolve it, filter it through a 0.22 μm microporous filter membrane, and the filtrate is the schisandrin A test solution (i.e., the third test solution).

[0101] 3-3 Preparation of control medicinal material solution: Accurately weigh 2.0 g of each of the five medicinal material powders and place them in 5 50 mL stoppered conical flasks, accurately add 50 mL of 70% methanol, weigh the weight, ultrasonically treat for 60 min, take out and cool, then add 70% methanol to the top, shake well, filter through a 0.22 μm microporous filter membrane, and take the filtrate to obtain the control medicinal material solution.

[0102] 3-4 Preparation of reference solution: Accurately weigh appropriate amount of chlorogenic acid reference, hyperoside reference, verbascoside reference, gallic acid reference, and schisandrin reference, add 70% methanol to dissolve, vortex for 1 min to 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, as well as a hyperoside reference solution containing 500 μg / mL of hyperoside and a schisandrin reference solution containing 10 μg / mL of schisandrin.

[0103] 4. Investigation of fingerprint methodology

[0104] 4-1 Precision investigation: Take a sample of Wuzi Yanzong soft capsules with batch number 2107894, prepare the test solution according to the method in 3-1 above, and continuously inject 6 times according to the chromatographic conditions of the HPLC fingerprint of Wuzi Yanzong soft capsules in 2-1 above (i.e., the first chromatographic conditions), and record the chromatogram; select the hyperoside chromatographic peak (peak 10) as the reference peak (S), and measure the relative peak area RSD of each common peak to be less than 3.0% (as shown in Table 1 below), and the relative retention time RSD is less than 0.17% (as shown in Table 2 below), indicating that the instrument has good precision.

[0105] Table 1 Precision study of fingerprint of Wuzi Yanzong soft capsules Common peak relative peak area RSD

[0106]

[0107]

[0108] Table 2 Precision study of fingerprint of Wuzi Yanzong soft capsules Common peak relative retention time RSD

[0109]

[0110]

[0111] 4-2 Repeatability study: Take Wuzi Yanzong soft capsule sample with batch number 2107894, prepare 6 portions of the first test solution in parallel according to the method in 3-1 above, and analyze the sample according to the chromatographic conditions (i.e., the first chromatographic conditions) of the HPLC fingerprint of Wuzi Yanzong soft capsule in 2-1 above, and record the chromatogram. The hyperoside chromatographic peak (peak 10) was selected as the reference peak (S), and the relative peak area RSD of each common peak was measured to be less than 3.0% (as shown in Table 3 below), and the relative retention time RSD was less than 0.12% (as shown in Table 4 below), indicating that the repeatability of this method is good.

[0112] Table 3 Relative peak area RSD of common peaks in repeatability study of Wuzi Yanzong soft capsule fingerprint

[0113]

[0114] Table 4 Relative retention time RSD of common peaks in repeatability study of fingerprint of Wuzi Yanzong soft capsule

[0115]

[0116] 4-3 Stability study: Take the Wuzi Yanzong soft capsule sample with batch number 2107894, prepare the test solution (i.e., the first test solution) according to the method in 3-1 above, and perform sample analysis according to the chromatographic conditions of the HPLC fingerprint of Wuzi Yanzong soft capsule in 2-1 above at 0, 2, 4, 6, 12, and 24 hours, and record the chromatogram. The peak (10) of the hyperoside chromatographic peak was selected as the reference peak (S), and the relative peak area RSD of each common peak was measured to be less than 3.0% (as shown in Table 5 below), and the relative retention time RSD was less than 0.22% (as shown in Table 6 below), indicating that the Wuzi Yanzong soft capsule test solution has good stability within 24 hours.

[0117] Table 5 Common peak relative peak area RSD of Wuzi Yanzong soft capsule fingerprint stability study

[0118]

[0119]

[0120] Table 6 Common peak relative retention time RSD of Wuzi Yanzong soft capsule fingerprint stability study

[0121]

[0122] 5. Fingerprint establishment and similarity evaluation

[0123] 5-1 Establishment of the fingerprint of Wuzi Yanzong soft capsule: Prepare 10 batches of test solution of Wuzi Yanzong soft capsule samples (S1-S10) in parallel according to the method in 3-1 above, and measure the 10 batches of test solution and mixed reference solution according to the chromatographic conditions of the HPLC fingerprint of Wuzi Yanzong soft capsule in 2-1 above, record the corresponding chromatograms, and the results are attached. Figure 1 .

[0124] 5-2 Identification of characteristic peaks and attribution of common peaks: Based on the chromatograms of the Wuzi Yanzong soft capsule test solution and the mixed reference solution, the results of the 10 batches of samples were analyzed, and 4 chromatographic peaks (such as the attached Figure 2 The peaks of the control herbal medicines were compared with the Wuzi Yanzong soft capsules, and 15 common peaks were identified (see Figure 1). Figure 3 Peaks 2, 6, 7, and 9 are from wolfberry fruit, peaks 4, 5, 7, 8, 10, 11, 12, 13, and 14 are from dodder seed, peaks 2, 3, 7, 8, 9, and 14 are from rubus idaeus, peak 11 is from plantain seed, and peaks 1, 3, 4, 5, and 6 are from schisandra chinensis. In addition, peak 15 has no corresponding medicinal ingredient, and the content of this ingredient in the compound is relatively large. It is speculated that it may be a new ingredient after decoction.

[0125] 5-3 Fingerprint similarity evaluation: The fingerprints of the 10 batches of samples obtained in 5-1 above were imported into the 2012 version of the official "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" software of the Pharmacopoeia Committee to obtain the reference spectrum (see Appendix Figure 1 R in), fingerprints of 10 batches of samples ( Figure 1 S1~S10 in the above table) and the generated reference map ( Figure 1 The similarities between the R) in the samples were 0.996, 0.973, 0.994, 0.983, 0.994, 0.995, 0.984, 0.995, 0.976 and 0.976, respectively, all of which were greater than 0.900, indicating that the quality of the Wuzi Yanzong soft capsules produced by this manufacturer was stable and there was no obvious fluctuation in the chemical composition.

[0126] Example 2

[0127] Determination of hyperoside and schisandrin in Wuziyanzong soft capsules

[0128] Linear range investigation: Accurately measure the hyperoside reference solution, dilute it step by step, prepare a series of reference solutions of different concentrations, and analyze them according to the chromatographic conditions (i.e., the second chromatographic conditions) for the determination of the hyperoside content in 2-2 above. Regress the peak area integral value (Y) against the injection volume (X), and the standard curve of hyperoside is Y=12579X-4450.5, r=0.9999, and the linear range is 3.91-500 μg / mL.

[0129] Precision investigation: Take Wuzi Yanzong soft capsule sample with batch number 2107894, prepare the test solution (i.e., the second test solution) according to the method in 3-1 above, inject 6 times continuously according to the chromatographic conditions of 2-2 hyperoside content determination, and record the chromatogram. 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.

[0130] Repeatability study: Take Wuzi Yanzong soft capsule sample with batch number 2107894, prepare 6 portions of the second test solution in parallel according to method 3-1, inject according to the chromatographic conditions of 2-2 hyperoside content determination, and record the chromatogram. The RSD of the hyperoside content was measured to be 0.74%, less than 1%, which meets the verification requirements of the methodology, indicating that the method has good repeatability.

[0131] Stability study: Take Wuzi Yanzong soft capsule sample with batch number 2107894, prepare the test solution according to method 3-1, and analyze the sample according to the chromatographic conditions of 2-2 hyperoside content determination at 0, 2, 4, 8, 12, and 24 hours, and record the chromatogram. The RSD of the hyperoside content was 1.04%, which was less than 2%, meeting the verification requirements of the methodology, indicating that the test solution had good stability within 24 hours.

[0132] Sample recovery experiment: Take Wuzi Yanzong soft capsule sample with batch number 2107894, add hyperoside reference substance at a ratio of 1:1, prepare 6 test sample solutions in parallel according to the preparation method of 3-1, inject and analyze according to the chromatographic conditions of 2-2 hyperoside content determination, and record the chromatogram. The average sample recovery of hyperoside in 6 samples was measured to be 104.64%, and the RSD was 8.94%, which met the verification requirements of the methodology.

[0133] Determination of hyperoside content in Wuzi Yanzong soft capsules: Take 10 batches of Wuzi Yanzong soft capsule samples, prepare the second test solution according to the preparation method 3-1, and determine the content of hyperoside according to the chromatographic conditions of 2-2. The hyperoside content is measured to be between 80.65 and 208.02 μg / capsule. The results are shown in Table 1 below.

[0134] Determination of the content of schisandrin in Wuzi Yanzong soft capsules: 10 batches of Wuzi Yanzong soft capsule samples were taken, and schisandrin test solution (i.e., the third test solution) was prepared according to the preparation method of 3-2 schisandrin content determination test solution, and the chromatographic conditions of 2-3 schisandrin content determination (i.e., the third chromatographic conditions) were used for determination. The detection results showed that the content of schisandrin in Wuzi Yanzong soft capsules was between 101.84 and 202.98 μg / capsule. The results are shown in Table 7 below.

[0135] Table 7 Content determination results of 10 batches of Wuzi Yanzong soft capsules

[0136] Sample No. Hyperoside content (μg / grain) Schisandrin 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

[0137] In order to obtain a chromatogram with better separation, the present technical solution used a diode array detector (DAD) to examine the chromatograms obtained at different wavelengths when establishing the chromatographic method. The results showed that the chromatogram obtained at 254 nm contained the most peak information and the separation between the peaks was good, so 254 nm was selected as the detection wavelength. At the same time, the separation of the Phenomenex Luna C18 (2) (250nm × 4.6nm, 5μm) and Diamonsil C18 (2) (250nm × 4.6nm, 5μm) chromatographic columns was compared, and it was found that the Phenomenex Luna C18 (2) (250nm × 4.6nm, 5μm) chromatographic column had a better separation effect, so it was selected to measure the sample. In addition, the effects of different column temperatures and different flow rates on the separation effect were investigated. The results showed that the chromatographic peaks had good peak shape, more peaks, and better separation under the conditions of column temperature of 35°C and flow rate of 0.8mL / min, so this condition was selected as the optimal chromatographic condition.

[0138] This technical solution established the HPLC-DAD fingerprint method of Wuzi Yanzong soft capsules by measuring 10 batches of Wuzi Yanzong soft capsules, calibrated 15 common peaks, and identified 4 chromatographic peaks, namely gallic acid (peak 1), chlorogenic acid (peak 8), hyperoside (peak 10) and verbascoside (peak 11). The chromatograms of the 10 batches of samples were compared with those of the reference solution, and the similarities were all greater than 0.900, indicating that the similarity between the ten batches of samples was good. On the basis of the original fingerprint method, combined with the 2020 edition of the "Chinese Pharmacopoeia" and the company's internal standards, the contents of hyperoside and schisandrin A in 10 batches of Wuzi Yanzong soft capsules were quantitatively investigated. The results showed that the content of hyperoside was between 80.65 and 208.02 μg / capsule, and the content of schisandrin A was between 101.84 and 202.98 μg / capsule.

[0139] The technical principle of the present invention is described above in conjunction with specific embodiments. These descriptions are only for explaining the principle of the present invention and cannot be interpreted as limiting the scope of protection of the present invention in any way. Based on the explanations herein, those skilled in the art can associate other specific implementations of the present invention without paying creative labor, and these methods will fall within the scope of protection of the present invention.

Claims

1. A method for constructing an HPLC fingerprint of Wuzi Yanzong soft capsule, characterized in that: The following steps are involved: Step (1.1): Take the contents of 4 Wuzi Yanzong soft capsules, accurately add 50 mL of 70% methanol, weigh the weight, and ultrasonically treat for 60 min. Take out and let cool, then add 70% methanol to a volume of 50 mL, shake well, filter with a 0.22 μm microporous filter membrane, and take the filtrate to obtain the first test solution; Step (1.2): Take chlorogenic acid, hyperoside, verbascoside, gallic acid and schisandrin A reference substances, add methanol to dissolve, and obtain a mixed reference substance solution; Step (1.3): respectively aspirate the first test solution and the mixed reference solution and inject them into a high performance liquid chromatograph for analysis to obtain the HPLC fingerprint of Wuzi Yanzong soft capsule; wherein the first chromatographic condition is as follows: Chromatographic column: Phenomenex luna C18 chromatographic column, the filler particle size of the chromatographic column is 5μm, the column length is 250mm, and the column inner diameter is 4.6mm; Detection wavelength: 254 nm; Mobile phase: Use acetonitrile-methanol mixture as mobile phase A and 0.4% phosphoric acid aqueous solution as mobile phase B. Perform 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.

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 method for constructing the HPLC fingerprint of Wuzi Yanzong soft capsule according to claim 2, characterized in that: In step (1.3), the flow rate of the mobile phase is 0.8 mL / 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 was 5 μL.

5. The method for constructing the HPLC fingerprint of Wuzi Yanzong soft capsule according to claim 1, characterized in that: The preparation method of the mixed reference solution in step (1.2) is as follows: take chlorogenic acid, hyperoside, verbascoside, gallic acid, and schisandrin reference substances, add 70% methanol to dissolve, vortex for 1 min to mix evenly, and prepare a mixed reference solution containing 79.20 μg / mL chlorogenic acid, 40.00 μg / mL hyperoside, 44.60 μg / mL verbascoside, and 76.80 μg / mL gallic acid.

6. The method for constructing the HPLC fingerprint of Wuzi Yanzong soft capsule according to claim 4, characterized in that: Taking the hyperoside chromatographic peak as the reference peak, the HPLC fingerprint of Wuzi Yanzong soft capsules detected had 15 common peaks at 254 nm.

7. A method for determining the content of hyperoside in Wuzi Yanzong soft capsules, characterized in that: The following steps are involved: Step (2.1): Take the contents of 4 Wuzi Yanzong soft capsules, accurately add 50 mL of 70% methanol, weigh the weight, and ultrasonically treat for 60 min. Take out and let cool, then add 70% methanol to a volume of 50 mL, shake well, filter with a 0.22 μm microporous filter membrane, and take the filtrate to obtain the first test solution; Step (2.2): Take an appropriate amount of hyperoside reference substance, add methanol to dissolve, and obtain a hyperoside reference substance solution containing 500 μg / mL hyperoside; Step (2.3): respectively aspirate the second test solution and the hyperoside reference solution and inject them into the high performance liquid chromatograph, perform the determination according to the following second chromatographic conditions, and calculate the content of hyperoside by the linear regression equation; the second chromatographic conditions are as follows: Chromatographic column: Phenomenex luna C18 chromatographic column, the filler 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 program: 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 was 0.8 mL / min, the detection wavelength was 360 nm, the column temperature was 35°C, and the injection volume was 5 μL.

8. A method for determining the content of schisandrin in Wuzi Yanzong soft capsules, characterized in that: The following steps are involved: Step (3.1): Take the contents of 10 Wuzi Yanzong soft capsules, mix them evenly, weigh 0.6 g accurately and place them in a 50 mL stoppered conical flask, add 20 mL of chloroform, seal it tightly, treat it ultrasonically for 15 min, filter, place the filtrate in a 50 mL volumetric flask, add chloroform to the mark, shake well, accurately measure 5-10 mL and place it in a stoppered graduated centrifuge tube, bathe it in water until it is almost dry, transfer the centrifuge tube to a vacuum dryer at 80℃ and dry it under reduced pressure for 30 min, accurately add 2 mL of methanol, seal it tightly, treat it ultrasonically for 10 min, shake it to dissolve it, filter it with a 0.22 μm microporous filter membrane, and the filtrate is the third test 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 Schisandrin A; Step (3.3): respectively aspirate the third test solution and the schisandrin A reference solution and inject them into the high performance liquid chromatograph, and measure them according to the following third chromatographic conditions, and calculate the content of schisandrin A by the linear regression equation; wherein the third chromatographic conditions are as follows: Chromatographic column: Phenomenex luna C18 chromatographic column, the filler particle size of the chromatographic column is 5μm, the column length is 250mm, and the column inner diameter is 4.6mm; Mobile phase: Methanol-water with a mass volume ratio of 3:1 was used 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 was 30°C, and the injection volume was 20 μL.

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