HPLC-QAMS (High Performance Liquid Chromatography-Quantitative Amplification Mass Spectrometry) method for measuring contents

Abstract

The invention discloses an HPLC-QAMS (High Performance Liquid Chromatography-Quadrature Amplification Mass Spectrometry) method for determining the contents of various components of Under the condition that the content of tanshinol in the selfheal and the preparation thereof is known, the content of protocatechuic acid, caffeic acid, rutin, hyperoside, isorosmarinic acid glycoside, rosmarinic acid and luteolin can be directly calculated according to relative correction factors, so that the content determination of eight main components in the selfheal and the preparation thereof is realized. According to the method, reference substances are saved, the quality control cost is reduced, the content of phenolic acid and flavone in the selfheal and the preparation of the selfheal can be obtained at the same time, the detection result is more representative, the quality of the preparation can be reflected more accurately, and the method has a wide application prospect.

Description

Technical Field

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

[0002] As one of the commonly used Chinese medicinal materials in clinical practice, Prunella vulgaris L. was first recorded in Shennong's Herbal Classic 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 spicy 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 nodules 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 studies have shown 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 Prunella vulgaris, which is included in the Chinese Pharmacopoeia (2020 edition). It has the effects of clearing fire and dispersing nodules. It 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 such as antioxidant, anti-inflammatory, and immunomodulatory, and are the main active ingredients of Prunella vulgaris. For the detection of Prunella vulgaris and its preparations, the Chinese Pharmacopoeia only includes the content determination index of one component, rosmarinic acid. Other research reports also mainly focus on the qualitative and quantitative determination of one or several components by thin layer chromatography or high performance liquid chromatography (HPLC). For example, Yang Jie et al. used ultraviolet spectrophotometry to determine the content of total triterpenes and total phenolic acids in Prunella vulgaris; Wang Jian et al. used HPLC to determine the content of caffeic acid, rosmarinic acid and isorosmarinic acid glycosides, the main phenolic acid active ingredients in Prunella vulgaris and its single granules and pastes, and established the corresponding HPLC fingerprint on this basis; Bai Yubing et al. used UPLC-MS / MS to determine the content of chlorogenic acid, ferulic acid, protocatechuic acid, and protocatechuic aldehyde in Prunella vulgaris; Fan Ya et al. used RP-HPLC to determine the content of luteolin in Prunella vulgaris, etc.

[0004] Therefore, there are few reports in the prior art on the simultaneous determination of phenolic acid and flavonoids, two key components in Prunella vulgaris, which cannot fully reflect the changing patterns of the chemical components in 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 simultaneous determination of multiple ingredients by determining one ingredient. This method realizes multi-component quality control of traditional Chinese medicine and its compound preparations, and greatly saves 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 ingredients, which lack representativeness and are difficult to effectively reflect the overall quality of the preparations; or multiple component reference substances are 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 preparations and reduce detection costs. Summary of the invention

[0006] In order to overcome the deficiencies of the above-mentioned prior art, the present invention proposes a HPLC-QAMS method for determining the contents of multiple components of Prunella vulgaris and its preparations. The method can simultaneously determine the contents of two major key components in Prunella vulgaris, phenolic acids and flavonoids, specifically involving eight compounds, which are highly representative and can more accurately reflect the quality of medicinal materials 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 glycosides, rosmarinic acid, and luteolin, and the method comprises 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 the test solution: extracting the herb or its preparation with a solvent, evaporating the extract to dryness and preparing the test solution;

[0011] S3. HPLC detection: The reference solution and the test solution were respectively detected by a high performance liquid chromatograph, wherein the high performance liquid chromatograph contained an ultraviolet detector, the detection wavelength of the ultraviolet detector was set to 190-400 nm, the stationary phase of the high performance liquid chromatograph was a C18 chromatographic column, the mobile phase A was acetonitrile, the mobile phase B was 0.4% formic acid solution, the gradient elution was performed, and the elution flow rate was 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 according to the peak area and concentration of the mixed reference solution, and calculate the content of each component according to 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 model of the C18 chromatographic column is 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, taking danshensu as the 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 uses a one-test-multiple-evaluation method to establish a method for detecting Prunella vulgaris and its preparations. When the content of danshensu in Prunella vulgaris and its preparations is known, the content of protocatechuic acid, caffeic acid, rutin, hyperoside, isorosmarinic acid glycosides, rosmarinic acid, and luteolin can be directly calculated according to the relative correction factor, thereby realizing the content determination of 8 main components (danshensu, protocatechuic acid, caffeic acid, rutin, hyperoside, isorosmarinic acid glycosides, rosmarinic acid, and luteolin) in Prunella vulgaris and its preparations. The present invention can simultaneously obtain the contents of 8 main components in Prunella vulgaris and its preparations while saving reference substances and reducing quality control costs, is more representative, can more accurately reflect the quality of the preparations, and has 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 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 by different methods (mg / g).

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

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

[0029] Fig. 9 Information about the components to be measured at different detection wavelengths.

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

[0031] Fig.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 extract paste, T represents freeze-dried powder of extract, and Y represents Prunella vulgaris medicinal material. DETAILED DESCRIPTION

[0033] The technical scheme of the present invention is 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 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 number WP23091203) were purchased from Sichuan Weikeqi Biotechnology Co., Ltd.; rosmarinic acid (batch number B20862), danshensu (batch number B20254), protocatechuic acid (batch number B21614), caffeic acid (batch number B20660), rutin (batch number B20771), hyperoside (batch number B20631), and luteolin (batch number 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 chromatographically pure, and other reagents were analytically pure. 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 substance solution: accurately weigh appropriate amounts of danshensu, protocatechuic acid, caffeic acid, rutin, hyperoside, isorosmarinic acid, rosmarinic acid, and luteolin reference substances, dissolve 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 future use;

[0040] (2) Preparation of test solution:

[0041] Take Prunella vulgaris, add water to decoct, combine the decoction, filter, take 1 / 100 of the filtrate, concentrate and freeze-dry, and 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 to dissolve, mix well, and concentrate to obtain a Prunella vulgaris paste sample;

[0042] Preparation of the test solution of Prunella vulgaris: Take 1.0g of Prunella vulgaris powder (passed through No. 3 sieve), add 50ml of water, heat and reflux to extract for 30min, filter, and evaporate the filtrate; add 25ml of 70% methanol to the residue, ultrasonically extract for 10min, filter, evaporate the filtrate, re-dissolve the residue in 70% methanol, make the volume to 5ml, mix, centrifuge, take the supernatant, filter with a microporous filter membrane, and 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 to ultrasonically extract for 30 minutes, filter, evaporate the filtrate, add 70% methanol to dissolve the residue, make up 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 into the sample solution and analyzed by HPLC according to the following chromatographic conditions: chromatographic column: Kromasil100-5-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) Exclusivity

[0048] Inject the mixed reference substance according to the HPLC chromatographic analysis conditions to obtain a chromatogram such as Figure 1 As shown, after comparing 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 mixed reference substance stock solution respectively, place 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, perform HPLC detection, and use the mass concentration of each reference substance as the abscissa (X) and the peak area as the ordinate (Y) to draw a standard curve, perform linear regression, and 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] Take the test solution of Prunella Vulgaris paste, inject and measure 6 times according to the chromatographic conditions, and measure and calculate the RSD of the chromatographic peak area. The RSD of the peak area of ​​each component is 2.41%, 1.43%, 1.51%, 0.91%, 1.84%, 1.28%, 0.63%, and 1.71%, respectively. It shows that the instrument has good precision.

[0055] (4) Stability

[0056] The test solution of Prunella Vulgaris paste was taken and sampled at 0, 2, 4, 8, 12, and 24 hours after preparation, and the RSD of the chromatographic peak area was calculated. The RSD of the peak area of ​​each component was 2.24%, 2.31%, 0.77%, 0.46%, 1.66%, 2.57%, 0.76%, and 0.46%, respectively. This shows that the test sample obtained by this method is stable within 24 hours.

[0057] (5) Repeatability

[0058] Take Prunella Vulgaris paste, prepare 6 test solutions in parallel, inject and measure according to the chromatographic conditions, and measure and calculate the RSD of the chromatographic peak area. The RSD of the peak area of ​​each component is 2.28%, 1.51%, 2.36%, 2.05%, 2.26%, 2.49%, 2.51%, and 1.86%, respectively. This shows that the method has good repeatability.

[0059] (6) Sample recovery experiment

[0060] Six portions of Prunella vulgaris paste with known content were accurately weighed, each portion was 1.5 g, and 8 reference solutions equivalent to 1.5 g sample content were accurately added to each portion to prepare the sample recovery solution, and the sample was injected and analyzed according to the chromatographic conditions, and the recovery rate and RSD were calculated. Results The average recovery rates of danshensu, protocatechuic acid, caffeic acid, rutin, hyperoside, isorosmarinic acid glycosides, 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, and the RSDs were 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 relative correction factors of the mixed standard at 6 different injection volumes were investigated using the linear relationship (Table 1). Using danshensu as the internal reference, the relative correction factors fsi between 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 study of relative correction factor

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

[0073] Using Danshensu as the internal reference, the effects of chromatographic columns Kromasil 100-5-C18 (250mm×4.6mm, 5μm), Diamonsil 5μm C18 (250mm×4.6mm, 5μm), and Hypersil GOLD (250mm×4.6mm, 5μm) on the relative correction factors of the components to be measured were investigated, and the results are shown in Table 4. It can be seen that there is no significant effect (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] Danshensu was used as the internal reference, and the influence of column temperature on relative correction factor at 25, 30, and 35°C was investigated. The results are shown in Table 5. The relative correction factors of protocatechuic acid, caffeic acid, rutin, hyperoside, isorosmarinic acid, rosmarinic acid, luteolin and the internal reference Danshensu at different column temperatures have good applicability.

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

[0079]

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

[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 relative correction factors of each component

[0083]

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

[0085] The relative retention time positioning method is used to locate the chromatographic peak of the component to be tested. The retention time of the internal standard peak is known, and it can be located according to the relative retention time of the seven components to be tested. The relative retention time value of Danshensu was selected as the positioning standard of the component to be tested, and the relative retention time results of different chromatographic columns were calculated, as shown in Table 7. The results show that different chromatographic columns have a great influence on the relative retention value of each chromatographic peak, and the difference is significant. Therefore, in order to ensure the accurate positioning of the chromatographic peak to be tested, the model and specifications of the chromatographic column required for the experiment should be specified when formulating the standard. It is recommended to use Kromasil 100-5-C18 chromatographic column.

[0086] Table 7 Relative retention time measured by different chromatographic columns

[0087]

[0088]

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

[0090] External standard method (ESM): Prepare the test solutions of Prunella vulgaris medicinal materials and their preparations respectively according to the above method, inject the samples for determination, record the peak area of ​​each component, substitute 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 medicinal materials and their 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): In 5, danshensu was used 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) was used to calculate the relative correction factors of protocatechuic acid, caffeic acid, rutin, hyperoside, isorosmarinic acid, rosmarinic acid and luteolin, which were 0.1931, 0.0894, Then, according to the formula Ci=(fs / i×Cs×Ai) / As (fs / i is the relative correction factor of the component i to be tested, Cs is the concentration of danshensu standard, Ai is the peak area of ​​the component i to be tested, As is the peak area of ​​danshensu standard), the contents of protocatechuic acid, caffeic acid, rutin, hyperoside, isorosmarinic acid, rosmarinic acid and luteolin in the sample were calculated.

[0092] The results of determination of danshensu, protocatechuic acid, caffeic acid, rutin, hyperoside, isorosmarinic acid, rosmarinic acid and luteolin in 15 batches of Prunella vulgaris and its preparations by external standard method (ESM) and QAMS were compared to verify the accuracy and reliability of QAMS for determination of 8 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, the UV-visible spectrophotometer was used to scan the full wavelength of the four Prunella vulgaris samples to be tested, and the UV absorption spectra from 200 to 800 nm were recorded. It was found that the overall absorption of the samples at 400 to 800 nm was very low, so the UV absorption spectrum at 200 to 400 nm was selected to reflect the characteristics of the main UV absorbing substances in the Prunella vulgaris samples. Combined with UV Win6.0 software, monitoring was performed within 200 to 400 nm, with a resolution of 1 nm, and 200 point sample data were obtained. The obtained data was imported into Origin2017, as shown in Figure 7 As shown, 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 flavonoids and phenolic acids. Therefore, 280-300nm is selected as the detection wavelength reference range of the 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 influence of the wavelength range of 280-300nm on the fingerprint spectrum. Figure 8 , 9 It was found that under the condition of 290nm wavelength, the number of chromatographic peaks of 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] Results Fig.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 the test solution: extracting the herb or its preparation with a solvent, evaporating the extract to dryness and preparing the test solution; S3. HPLC detection: The reference solution and the test solution were respectively detected by a high performance liquid chromatograph, wherein the high performance liquid chromatograph contained an ultraviolet detector, the detection wavelength of the ultraviolet detector was set to 190-400 nm, the stationary phase of the high performance liquid chromatograph was a C18 chromatographic column, the mobile phase A was acetonitrile, the mobile phase B was 0.4% formic acid solution, the gradient elution was performed, and the elution flow rate was 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, rosmarinic acid, and luteolin according to the peak area and concentration of the mixed reference solution, and calculate the content of each component according to the peak area and relative correction factor of the test solution.

2. 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: 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.

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: The detection wavelength of the ultraviolet detector is set to 290 nm.

4. 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: The model of the C18 chromatographic column is Kromasil 100-5-C18, 250 mm×4.6 mm, 5 μm.

5. 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: In step S2, the solvent is 70% methanol.

6. 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: 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.

7. 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 was the relative correction factor, AS was the peak area of ​​danshensu in the test sample, wS was the concentration of danshensu in the test sample, Ai was the peak area of ​​a certain component to be tested in the test sample, and wi was the concentration of a certain component to be tested in the test sample.

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