A HPLC-QAMS method for determining the contents of multiple components in Prunella vulgaris and its preparations

Abstract

The present invention discloses an HPLC-QAMS method for determining the contents of multiple components of Prunella vulgaris and its preparations. When the content of danshensu in Prunella vulgaris and its preparations is known, the contents of protocatechuic acid, caffeic acid, rutin, hyperoside, isorosmarinic acid glycosides, rosmarinic acid, and luteolin can be directly calculated based on relative correction factors, thereby achieving the determination of the contents of eight major components in Prunella vulgaris and its preparations. While saving reference substances and reducing quality control costs, the present invention can simultaneously determine the contents of two major key components in Prunella vulgaris and its preparations, phenolic acids and flavonoids. The test results are more representative and can more accurately reflect the quality of the preparations, thus having broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug analysis, and particularly relates to a high performance liquid chromatography-quantitative analysis of multi-components by a single marker (HPLC-QAMS) method for determining the contents of multiple components of Prunella vulgaris and its preparations. Background Art

[0002] As one of the commonly used Chinese medicinal materials in clinical practice, Prunella vulgaris was first recorded in the "Shennong Bencao Jing" and was listed as a lower grade. It is the dried fruit spike of Prunella vulgaris L., a plant of the Lamiaceae family. It tastes pungent and bitter, is cold in nature, and enters the liver and gallbladder meridians. It has the effects of clearing the liver and purging fire, dispersing stagnation and reducing swelling. Prunella vulgaris mainly contains a variety of active ingredients such as terpenes, phenolic acids, flavonoids, polysaccharides, etc., and triterpenes, phenolic acids, and flavonoids have been studied more. Modern research shows that Prunella vulgaris has multiple pharmacological activities such as lowering blood pressure, lowering blood sugar, antibacterial, antiviral, and anti-tumor. Prunella vulgaris paste is a decoction made from processed Prunella vulgaris, which is included in the "Chinese Pharmacopoeia" (2020 edition) Volume 1. It has the effects of clearing fire and dispersing stagnation, and is often used to treat headaches, dizziness, scrofula, goiters, etc. caused by internal heat.

[0003] Studies have shown that phenolic acids and flavonoids in Prunella vulgaris have multiple pharmacological effects, including antioxidant, anti-inflammatory, and immunomodulatory effects, and are the main active ingredients of Prunella vulgaris. Regarding the detection of Prunella vulgaris and its preparations, the Chinese Pharmacopoeia only includes a content determination indicator for one component, rosmarinic acid. Other research reports also focus on the qualitative and quantitative analysis of one or several components using thin-layer chromatography or high-performance liquid chromatography (HPLC). For example, Yang Jie et al. determined the content of total triterpenes and total phenolic acids in Prunella vulgaris using UV spectrophotometry; Wang Jian et al. determined the content of caffeic acid, rosmarinic acid, and isorosmarinic acid glycosides, the main active phenolic acid components, in Prunella vulgaris and its single-ingredient granules and pastes using HPLC, and established corresponding HPLC fingerprints based on this determination; Bai Yubing et al. determined the content of four phenolic acids, chlorogenic acid, ferulic acid, protocatechuic acid, and protocatechuic aldehyde, in Prunella vulgaris using UPLC-MS / MS; and Fan Ya et al. determined the content of luteolin in Prunella vulgaris using RP-HPLC.

[0004] Therefore, there are few reports in the existing technology on the simultaneous determination of phenolic acids and flavonoids, two key components in Prunella vulgaris. This cannot fully reflect the changes in the chemical composition of Prunella vulgaris and its preparations, nor can it effectively control the quality of the products and ensure the consistency of the quality of the medicinal materials and preparations.

[0005] The one-test-multiple-evaluation method is a method that utilizes the intrinsic functional relationship and proportional relationship of the effective ingredients of traditional Chinese medicine to achieve the simultaneous determination of multiple components by measuring one component. This method realizes the multi-component quality control of traditional Chinese medicine and its compound preparations, and greatly saves the detection time and cost. At present, the one-test-multiple-evaluation method has not been used to detect the content of Prunella vulgaris and its preparations, and the existing methods mostly only measure one or two components, which lack representativeness and are difficult to effectively reflect the overall quality of the preparation; or the use of multiple component reference substances is required. Based on this, the present invention develops a quality control method for Prunella vulgaris and its preparations based on the one-test-multiple-evaluation method to more effectively reflect the overall quality of the preparation and reduce the detection cost. Summary of the Invention

[0006] To overcome the deficiencies of the prior art, the present invention proposes an HPLC-QAMS method for determining the contents of multiple components in Prunella vulgaris and its preparations. This method can simultaneously determine the contents of two key components in Prunella vulgaris, phenolic acids and flavonoids. Specifically, eight compounds are involved. These compounds are highly representative and can more accurately reflect the quality of the medicinal material and preparations.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is:

[0008] A HPLC-QAMS method for determining the contents of multiple components of Prunella vulgaris and its preparations, wherein the components are danshensu, protocatechuic acid, caffeic acid, rutin, hyperoside, isorosmarinic acid, rosmarinic acid, and luteolin, comprising the following steps:

[0009] S1. Preparation of reference solution: Accurately weigh danshensu, protocatechuic acid, caffeic acid, rutin, hyperoside, isorosmarinic acid, rosmarinic acid, and luteolin reference substances, and add 70% methanol to prepare a mixed reference substance stock solution;

[0010] S2. Preparation of test solution: extracting Prunella vulgaris or its preparation with a solvent, evaporating the extract to dryness and preparing a test solution;

[0011] S3. HPLC detection: The reference solution and the test solution were detected separately on a high performance liquid chromatograph. The high performance liquid chromatograph contained a UV detector with a detection wavelength set at 190-400 nm. The stationary phase of the high performance liquid chromatograph was a C18 column. The mobile phase A was acetonitrile, and the mobile phase B was 0.4% formic acid solution. The elution was performed with a gradient elution rate of 1.0 mL min. -1 , column temperature was 30 °C, injection volume was 10 μL;

[0012] S4. Using danshensu as the internal reference, calculate the relative correction factors of protocatechuic acid, caffeic acid, rutin, hyperoside, isorosmarinic acid, rosmarinic acid, and luteolin based on the peak area and concentration of the mixed reference solution, and calculate the content of each component based on the peak area and relative correction factor of the test solution.

[0013] Preferably, the gradient elution program is: 0-0.1 min, 5%-5% A; 0.1-10 min, 5%-10% A; 10-20 min, 10%-15% A; 20-30 min, 15%-20% A; 30-35 min, 20%-23% A; 35-40 min, 23%-25% A; 40-50 min, 25%-30% A; 50-60 min, 30%-35% A; 60-65 min, 35% A, all by volume ratio.

[0014] Preferably, the detection wavelength of the ultraviolet detector is set to 290 nm.

[0015] Preferably, the C18 chromatographic column is of model Kromasil 100-5-C18, 250 mm×4.6 mm, 5 μm.

[0016] Preferably, in step S2, the solvent is 70% (volume) methanol.

[0017] Preferably, in the mixed reference substance stock solution, the contents of danshensu, protocatechuic acid, caffeic acid, rutin, hyperoside, isorosmarinic acid glycosides, rosmarinic acid, and luteolin are 2.600, 1.775, 1.815, 1.760, 1.250, 0.945, 5.095, and 1.535 mg / mL, respectively.

[0018] Furthermore, using danshensu as an internal reference, the contents of protocatechuic acid, caffeic acid, rutin, hyperoside, isorosmarinic acid, rosmarinic acid, and luteolin were calculated respectively, and the calculation formula was fsi=fs / fi=(AS / wS) / (Ai / wi), wherein fsi is the relative correction factor, AS is the peak area of ​​danshensu in the test sample, wS is the concentration of danshensu in the test sample, Ai is the peak area of ​​a certain component to be tested in the test sample, and wi is the concentration of a certain component to be tested in the test sample.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention utilizes a single-test, multiple-evaluation method to establish a method for detecting Prunella vulgaris and its preparations. When the danshensu content in the Prunella vulgaris and its preparations is known, the contents of protocatechuic acid, caffeic acid, rutin, hyperoside, isorosmarinic acid, rosmarinic acid, and luteolin can be directly calculated based on relative correction factors, thereby enabling the determination of the contents of eight major components (danshensu, protocatechuic acid, caffeic acid, rutin, hyperoside, isorosmarinic acid, rosmarinic acid, and luteolin) in the Prunella vulgaris and its preparations. While saving reference substances and reducing quality control costs, the present invention can simultaneously determine the contents of the eight major components in the Prunella vulgaris and its preparations, making it more representative and more accurately reflecting the quality of the preparations, thus having broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the HPLC chart of the mixed reference substance, in which 1-8 are: danshensu, protocatechuic acid, caffeic acid, rutin, hyperoside, isorosmarinic acid glycosides, rosmarinic acid, and luteolin.

[0022] Figure 2 It is the HPLC chart of Prunella vulgaris and its preparation samples.

[0023] Figure 3 Determination results of 8 components in Prunella vulgaris by different methods (mg / g).

[0024] Figure 4 Determination results of 8 components in Prunella vulgaris extract by different methods (mg / g).

[0025] Figure 5 Determination results of 8 components in Prunella Vulgaris Paste by different methods (mg / g).

[0026] Figure 6 Determination results of 8 components in Prunella Vulgaris paste using different methods (mg / g).

[0027] Figure 7 Ultraviolet absorption spectrum of Prunella vulgaris sample.

[0028] Figure 8 HPLC profiles of Prunella vulgaris samples at different detection wavelengths.

[0029] Figure 9 Information on the components to be measured at different detection wavelengths.

[0030] Figure 10 HPLC profiles of Prunella vulgaris samples under different elution programs.

[0031] Figure 11 Information on the components to be tested under different elution systems.

[0032] Figure 2 、 7In 10, G represents Prunella Vulgaris paste, Q represents Prunella Vulgaris paste, T represents freeze-dried powder of extract, and Y represents Prunella Vulgaris raw material. DETAILED DESCRIPTION

[0033] The technical solutions of the present invention are further described in detail below through specific examples. The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are all available through conventional commercial channels unless otherwise specified.

[0034] Example 1: Detection method and methodological investigation

[0035] 1. Experimental Materials

[0036] (1) Instruments: High performance liquid chromatograph (Agilent 1200), chromatographic column Kromasil 100-5-C18 (250 mm × 4.6 mm, 5 μm), 1 / 100,000 analytical balance.

[0037] (2) Reagents: Isorosmarinic acid glycosides (Batch No. WP23091203) were purchased from Sichuan Weikeqi Biotechnology Co., Ltd.; rosmarinic acid (Batch No. B20862), danshensu (Batch No. B20254), protocatechuic acid (Batch No. B21614), caffeic acid (Batch No. B20660), rutin (Batch No. B20771), hyperoside (Batch No. B20631), and luteolin (Batch No. B20888) were all purchased from Shanghai Yuanye Biotechnology Co., Ltd.; the mass fraction of each reference substance was ≥98.00%. Water was purified water; acetonitrile and phosphoric acid were chromatographic grade; all other reagents were analytical grade. Prunella vulgaris (230210-0, 230577-0, and 230503-0) were purchased from Henan, Hubei, and Anhui, respectively.

[0038] 2. Preparation of test solution

[0039] (1) Preparation of reference solution: Accurately weigh appropriate amounts of danshensu, protocatechuic acid, caffeic acid, rutin, hyperoside, isorosmarinic acid, rosmarinic acid, and luteolin reference substances, dissolve them in 70% methanol to prepare mixed reference substance stock solutions with mass concentrations of 5.0, 0.5, 1.0, 2.0, 1.0, 1.5, 15.0, and 1.0 mg / mL, respectively, and store at 4°C for later use;

[0040] (2) Preparation of test solution:

[0041] Take Prunella vulgaris, add water to decoct, combine the decoctions, filter, take 1 / 100 of the filtrate, concentrate and freeze-dry to obtain a freeze-dried powder sample of the extract; concentrate the remaining filtrate to obtain a clear paste sample; take part of the clear paste, add sucrose, heat and dissolve, mix, and concentrate to obtain a Prunella vulgaris paste sample;

[0042] Preparation of the Prunella Vulgaris test solution: Take 1.0 g of Prunella Vulgaris powder (passed through a No. 3 sieve), add 50 ml of water, heat and reflux to extract for 30 min, filter, and evaporate the filtrate; add 25 ml of 70% methanol to the residue, ultrasonically extract for 10 min, filter, evaporate the filtrate, re-dissolve the residue in 70% methanol, make the volume to 5 ml, mix well, centrifuge, take the supernatant, and filter with a microporous filter to obtain the test solution;

[0043] Preparation of extract, clear paste and paste test solution: Take appropriate amount of extract lyophilized powder, clear paste and paste respectively, add 70% methanol and ultrasonically extract for 30 minutes, filter, evaporate the filtrate, add 70% methanol to dissolve the residue, make the volume to 10 ml, mix well, centrifuge, take the supernatant, filter with a microporous filter membrane to obtain the test solution.

[0044] 3. HPLC chromatography analysis

[0045] The test solution and the reference solution were injected separately for HPLC analysis under the following chromatographic conditions: chromatographic column: Kromasil 100-5-C18 (250 mm × 4.6 mm, 5 μm); mobile phase: acetonitrile (A)-0.4% formic acid solution (B); gradient elution (0-0.1 min, 5%-5% A; 0.1-10 min, 5%-10% A; 10-20 min, 10%-15% A; 20-30 min, 15%-20% A; 30-35 min, 20%-23% A; 35-40 min, 23%-25% A; 40-50 min, 25%-30% A; 50-60 min, 30%-35% A; 60-65 min, 35% A); volume flow rate: 1.0 mL min -1 ; Column temperature: 30℃; Detection wavelength: 290nm; Injection volume: 10μL.

[0046] 4. Methodological Investigation

[0047] (1) Specificity

[0048] Inject the mixed reference substance according to the HPLC chromatographic analysis conditions to obtain a chromatogram, such as Figure 1 As shown, after comparison with the chromatograms of each single reference substance, it was confirmed that peaks 1-8 in the figure are: danshensu, protocatechuic acid, caffeic acid, rutin, hyperoside, isorosmarinic acid glycosides, rosmarinic acid, and luteolin.

[0049] (2) Linear

[0050] Take 75mL, 150mL, 375mL, 750mL, 900mL, and 1500mL of the above mixed reference substance stock solution respectively, place it in a 2mL volumetric flask, add methanol to dilute to the scale, mix well, and prepare mixed reference substance solutions of different concentrations. After filtering through a microporous filter membrane, HPLC detection is performed. With the mass concentration of each reference substance as the abscissa (X) and the peak area as the ordinate (Y), a standard curve is drawn, and linear regression is performed to obtain a linear regression equation. The results show that the mass concentrations of the eight components have a good linear relationship within the corresponding range. The results are shown in the table below.

[0051] Table 1 Linear regression equations of eight components in Prunella vulgaris and its preparations

[0052]

[0053] (3) Precision

[0054] Six injections of the Prunella Vulgaris paste test solution were performed according to the chromatographic conditions. The RSDs of the peak areas were determined and calculated. The RSDs of the peak areas of the components were 2.41%, 1.43%, 1.51%, 0.91%, 1.84%, 1.28%, 0.63%, and 1.71%, respectively. This indicates good instrument precision.

[0055] (4) Stability

[0056] A solution of the Prunella Vulgaris paste sample was injected and measured at 0, 2, 4, 8, 12, and 24 hours after preparation. The RSDs of the chromatographic peak areas were calculated. The RSDs of the peak areas of the individual components were 2.24%, 2.31%, 0.77%, 0.46%, 1.66%, 2.57%, 0.76%, and 0.46%, respectively. This indicates that the sample obtained by this method is stable within 24 hours.

[0057] (5) Repeatability

[0058] Six parallel aliquots of the test solution were prepared from Prunella Vulgaris paste. The samples were injected according to the chromatographic conditions, and the relative standard deviations (RSDs) of the chromatographic peak areas were determined and calculated. The RSDs of the peak areas of the individual components were 2.28%, 1.51%, 2.36%, 2.05%, 2.26%, 2.49%, 2.51%, and 1.86%, respectively. This demonstrates good reproducibility of the method.

[0059] (6) Sample recovery experiment

[0060] Six portions of Prunella vulgaris paste with known content (1.5 g each) were accurately weighed. Eight reference solutions equivalent to 1.5 g of sample were accurately added to each portion to prepare sample recovery solutions. Samples were injected and analyzed according to chromatographic conditions, and the recoveries and RSDs were calculated. Results: The average recoveries of danshensu, protocatechuic acid, caffeic acid, rutin, hyperoside, isorosmarinic acid, rosmarinic acid, and luteolin in Prunella vulgaris paste samples were 100.69%, 101.07%, 99.93%, 98.89%, 98.64%, 98.78%, 100.07%, and 99.68%, respectively, with RSDs of 0.82%, 1.79%, 2.16%, 2.08%, 1.51%, 2.29%, 0.31%, and 1.87%, respectively, indicating that the method has good accuracy.

[0061] Table 2 Sample recovery test results (n=6)

[0062]

[0063]

[0064] 5. Determination of relative correction factor

[0065] The linear relationship (Table 1) was used to investigate the relative correction factors of the mixed standard at 6 different injection volumes. Using danshensu as the internal reference, the relative correction factors fsi of protocatechuic acid, caffeic acid, rutin, hyperoside, isorosmarinic acid, rosmarinic acid, and luteolin were calculated respectively. The calculation formula is as follows:

[0066] fs / i=fs / fi=(As×Ci) / (Ai×Cs)

[0067] Wherein, As is the peak area of ​​danshensu, Ci is the concentration of the component i to be tested, Cs is the concentration of danshensu, and Ai is the peak area of ​​the component i to be tested. The results are shown in Table 3.

[0068] Table 3 Calculation results of relative correction factors of 7 components with Danshensu as internal reference

[0069]

[0070]

[0071] 6. Durability investigation of relative correction factor

[0072] 6.1 Effect of different chromatographic columns on relative correction factors

[0073] Using danshensu as the internal reference, the effects of the chromatographic columns Kromasil 100-5-C18 (250 mm × 4.6 mm, 5 μm), Diamonsil 5 μm C18 (250 mm × 4.6 mm, 5 μm), and Hypersil GOLD (250 mm × 4.6 mm, 5 μm) on the relative correction factors of the analytes were investigated. The results are shown in Table 4. No significant effects were observed (RSD < 3%).

[0074] Table 4 Effect of different chromatographic columns on relative correction factors

[0075]

[0076] 6.2 Effect of different column temperatures on relative correction factors

[0077] Using danshensu as an internal reference, the effects of column temperatures of 25, 30, and 35°C on the relative correction factors were investigated. The results are shown in Table 5. The relative correction factors of protocatechuic acid, caffeic acid, rutin, hyperoside, isorosmarinic acid, rosmarinic acid, and luteolin were well-matched with the internal reference danshensu at different column temperatures.

[0078] Table 5 Effect of different column temperatures on relative correction factors

[0079]

[0080] 6.3 Effect of different flow rates on relative correction factors

[0081] Comparing the effects of different flow rates (0.8, 1.0, 1.2 mL / min) on the relative correction factors of each component, the RSDs of the relative correction factors of each component were all <3%, indicating that different flow rates had no significant effect on the relative correction factors of each component. The results are shown in Table 6.

[0082] Table 6 Effect of different flow rates on the relative correction factors of each component

[0083]

[0084] 6.4 Positioning of chromatographic peaks of the components to be measured

[0085] The relative retention time positioning method was used to locate the chromatographic peaks of the analytes. Knowing the retention time of the internal standard peak, the seven analytes were located based on their relative retention times. The relative retention time of danshensu was selected as the positioning standard for the analytes, and the relative retention times of different chromatographic columns were calculated (see Table 7). The results showed that different chromatographic columns significantly affected the relative retention values ​​of each chromatographic peak. Therefore, to ensure accurate positioning of the chromatographic peaks, the required chromatographic column model and specifications should be specified when developing the standard. The Kromasil 100-5-C18 column is recommended.

[0086] Table 7 Relative retention times measured on different chromatographic columns

[0087]

[0088]

[0089] 6.5 Comparison of test results between the single-test, multiple-evaluation method and the external standard method

[0090] External standard method (ESM): Prepare the test solutions of Prunella vulgaris and its preparations respectively according to the above method, inject the samples for determination, record the peak area of ​​each component, substitute it into the linear equation to calculate Ci, and calculate the contents of danshensu, protocatechuic acid, caffeic acid, rutin, hyperoside, isorosmarinic acid, rosmarinic acid, and luteolin in Prunella vulgaris and its preparations according to the formula Mi = (Ci × Vs) / Ms (Mi is the content of component i to be tested, Ci is the mass concentration of component i to be tested, Vs is the volume of the sample to be tested, and Ms is the mass of the sample to be tested).

[0091] One-test multiple-evaluation method (QAMS): 5. Using danshensu as the internal reference, the formula fs / i = fs / fi = (As × Ci) / (Ai × Cs) (As is the peak area of ​​danshensu, Ci is the concentration of the component i to be tested, Cs is the concentration of danshensu, Ai is the peak area of ​​the component i to be tested), the relative correction factors of protocatechuic acid, caffeic acid, rutin, hyperoside, isorosmarinic acid, rosmarinic acid and luteolin were calculated to be 0.1931, 0.0894, The contents of protocatechuic acid, caffeic acid, rutin, hyperoside, isorosmarinic acid, rosmarinic acid and luteolin in the samples were calculated according to the formula Ci = (fs / i × Cs × Ai) / As (fs / i is the relative correction factor of component i, Cs is the concentration of danshensu standard, Ai is the peak area of ​​component i, and As is the peak area of ​​danshensu standard).

[0092] The results of the determination of danshensu, protocatechuic acid, caffeic acid, rutin, hyperoside, isorosmarinic acid, rosmarinic acid and luteolin in 15 batches of Prunella vulgaris and its preparations obtained by the external standard method (ESM) and the one-measurement-multiple-evaluation method (QAMS) were compared to verify the accuracy and reliability of the one-measurement-multiple-evaluation method for the determination of the eight components in Prunella vulgaris and its preparations. The results of the two methods are shown in Table 1. Figures 3 to 6 There was no significant difference in the results of the two tests, indicating that the one-test-multiple-evaluation method can be used to determine the contents of the eight components in Prunella vulgaris and its preparations.

[0093] Example 2 Screening and Optimization of Detection Conditions

[0094] 1. Screening of detection wavelength

[0095] In this experiment, we first scanned the four Prunella vulgaris samples using a UV-visible spectrophotometer at full wavelengths, recording the UV absorption spectra from 200 to 800 nm. We found that the overall absorption of the samples was very low at 400 to 800 nm, so we selected the UV absorption spectra from 200 to 400 nm to reflect the characteristics of the main UV-absorbing substances in the Prunella vulgaris samples. Using UV Win6.0 software, we monitored the samples within 200 to 400 nm with a resolution of 1 nm, obtaining 200 point sample data. The data was imported into Origin 2017, as shown in the following example. Figure 7 As shown in the figure, the four Prunella vulgaris samples have strong ultraviolet absorption signals at 200-250nm, 250-300nm, and 300-400nm. Combined with the ultraviolet spectral characteristics of various components of Prunella vulgaris in the literature, it is speculated that they may be triterpenes, flavonoids, and phenolic acids. The solvent absorption area has a large interference at 190-210nm. After removing this wavelength range, the interference is small at 280-300nm. All four samples have maximum absorption, and this wavelength range is mainly composed of flavonoids and phenolic acids. Therefore, 280-300nm is selected as the detection wavelength reference range of HPLC fingerprint.

[0096] Then, the high performance liquid phase diode array detector (DAD) was used to scan the full wavelength of the UV spectrum of the Prunella vulgaris sample in the wavelength range of 190-400nm, focusing on comparing the effect of the wavelength range of 280-300nm on the fingerprint spectrum. Figure 8 、 9 It was found that under the wavelength of 290nm, the number of chromatographic peaks of the Prunella vulgaris samples was large, the amount of information was large, the baseline was stable, and the separation of the content determination index components was good, so 290nm was selected as the detection wavelength.

[0097] 2. Elution program optimization

[0098] In the experiment, the elution procedure of acetonitrile (A)-0.4% formic acid solution (B) was optimized as follows:

[0099] Procedure 1: Isocratic elution (0-30 min, 35% A; 30-60 min, 35% A);

[0100] Procedure 2: gradient elution (0-0.1 min, 10%-10% A; 0.1-30 min, 10%-25% A; 30-50 min, 25%-35% A; 50-60 min, 35% A);

[0101] Program 3: gradient elution (0-0.1 min, 5%-5% A; 0.1-10 min, 5%-10% A; 10-20 min, 10%-15% A; 20-30 min, 15%-25% A; 30-40 min, 25%-30% A; 40-50 min, 30%-35% A; 50-60 min, 35% A);

[0102] Program 4: gradient elution (0-0.1 min, 5%-5% A; 0.1-10 min, 5%-10% A; 10-20 min, 10%-15% A; 20-30 min, 15%-20% A; 30-35 min, 20%-23% A; 35-40 min, 23%-25% A; 40-50 min, 25%-30% A; 50-60 min, 30%-35% A; 60-65 min, 35% A).

[0103] See the results Figure 10 、 11 , and finally determined that program 4 was the best elution program.

Claims

1. A HPLC-QAMS method for determining the contents of multiple components of Prunella vulgaris and its preparations, characterized in that: The ingredients are danshensu, protocatechuic acid, caffeic acid, rutin, hyperoside, isorosmarinic acid glycosides, rosmarinic acid, and luteolin, and the method comprises the following steps: S1. Preparation of reference solution: Accurately weigh danshensu, protocatechuic acid, caffeic acid, rutin, hyperoside, isorosmarinic acid, rosmarinic acid, and luteolin reference substances, and add 70% methanol to prepare a mixed reference substance stock solution; S2. Preparation of test solution: Extract Prunella vulgaris or its preparation with 70% methanol, evaporate the extract to dryness and prepare the test solution; S3. HPLC detection: The reference solution and the test solution were detected separately on a high performance liquid chromatograph. The high performance liquid chromatograph contained a UV detector with a detection wavelength of 290 nm. The stationary phase of the high performance liquid chromatograph was a C18 column. The mobile phase A was acetonitrile, and the mobile phase B was 0.4% formic acid solution. The elution was performed with a gradient elution rate of 1.0 mL min. −1 , column temperature was 30 °C, injection volume was 10 μL; S4. Using danshensu as the internal reference, calculate the relative correction factors of protocatechuic acid, caffeic acid, rutin, hyperoside, isorosmarinic acid glycosides, rosmarinic acid, and luteolin based on the peak area and concentration of the mixed reference solution, and calculate the content of each component based on the peak area and relative correction factor of the test solution. The gradient elution program is as follows: 0-0.1 min, 5%-5% A; 0.1-10 min, 5%-10% A; 10-20 min, 10%-15% A; 20-30 min, 15%-20% A; 30-35 min, 20%-23% A; 35-40 min, 23%-25% A; 40-50 min, 25%-30% A; 50-60 min, 30%-35% A; 60-65 min, 35% A; The model of the C18 chromatographic column is Kromasil 100-5-C18, 250 mm×4.6 mm, 5 μm.

2. The HPLC-QAMS method for determining the contents of various components of Prunella vulgaris and its preparations as claimed in claim 1, characterized in that: In the mixed reference substance stock solution, the contents of danshensu, protocatechuic acid, caffeic acid, rutin, hyperoside, isorosmarinic acid glycosides, rosmarinic acid, and luteolin are 2.600, 1.775, 1.815, 1.760, 1.250, 0.945, 5.095, and 1.535 mg / mL, respectively.

3. The HPLC-QAMS method for determining the contents of multiple components of Prunella vulgaris and its preparations as claimed in claim 1, characterized in that: Taking danshensu as the internal reference, the contents of protocatechuic acid, caffeic acid, rutin, hyperoside, isorosmarinic acid, rosmarinic acid, and luteolin were calculated respectively. The calculation formula was fsi=fs / fi=(AS / wS) / (Ai / wi), where fsi is the relative correction factor, AS is the peak area of ​​danshensu in the test sample, wS is the concentration of danshensu in the test sample, Ai is the peak area of ​​a certain component to be tested in the test sample, and wi is the concentration of a certain component to be tested in the test sample.

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