CN114660194A - HPLC fingerprint construction method and detection method of Yatouniu Hazhangbo prescription - Google Patents

Abstract

The invention discloses an HPLC fingerprint construction method and a detection method of a Yatou cattle Hakhapra prescription. The method combines HPLC fingerprint spectrum and multi-index content measurement for the first time, and has higher application value for better monitoring and evaluating the quality of the Jacobian hakubo particles and guiding standardized production through qualitative identification and content measurement of multiple batches of Jacobian hakubo particles; provides a research clue for establishing the quality standard of the Yatou cattle Hazhao Bobo granules with multiple components and indexes, and lays a solid foundation for the deep development of the Yatou cattle Hazhao Bobo granules. The fingerprint spectrum prepared by the method can be used for qualitatively and quantitatively detecting the Yatou cow Haemabo granule simultaneously, and can also be used for qualitatively and quantitatively detecting the Yatou cow Haemabo pills, extractum and other formulations.

Description

HPLC fingerprint spectrum construction method and detection method of Yatou cattle Hajabo prescription

Technical Field

The invention belongs to the technical field of chemical detection, and particularly relates to a method for constructing an HPLC fingerprint of a Yatou cattle Hakhart prescription and a detection method.

Background

Yaqiao cattle Hazhengbo is a Dai medical traditional menstruation prescription, is collected in ancient Dai medical menstruation book Shi Hayazhao book Wanna, and has more than 1300 years of history, the prescription consists of an inverted heart shield pterygus, clerodendranthus spicatus, a conical celastrus orbiculatus, coix lacryma-jobi root and liquorice, has the functions of clearing heat and promoting urination, and is used for treating acute and chronic nephritis, pyelonephritis, urinary stream infection, kidney, ureter and bladder calculus. The Xishuangbanna Dai medical hospital develops a medical institution preparation, namely the Wulin Huashi capsule (Yunnan medicine made with the Chinese character (Z) 20082254K) on the basis of the traditional meridian prescription and is applied clinically

Has definite curative effect and high safety in treating urinary tract infection and calculus for 20 years. Meanwhile, when a hospital researches the curative effect of the prescription preparation, the hospital discovers that the prescription preparation can reduce blood creatinine and urine creatinine and reduce the urea content besides the calculus removing effect, and the hospital is prompted to have a better renal function protecting effect. However, the Dai medicine is developed slowly at present, the Yaqiao cattle Hazhubao prescription lacks modern controllable quality control indexes, and the quality standard needs to be improved and perfected.

At present, the quality detection research on the Yatou cow Haemobo has fewer reports, and the invention combines HPLC fingerprint spectrum establishment

The multi-component synchronous content determination method develops the quality standard research of the Yaqian Hayabo prescription from qualitative and quantitative aspects, provides quality evaluation standard suggestions, provides clues for the quality standard research of the Yaqian Hayabo prescription, and provides references for the material basis explanation of other Dai medical traditional classical prescriptions.

Disclosure of Invention

The invention aims to provide a method for constructing an HPLC fingerprint of a Yatou cattle Hakholder prescription, and the invention aims to provide a method for detecting the Yatou cattle Hakholder prescription.

The first purpose of the invention is realized by the method for constructing the HPLC fingerprint of the Yatou bovine Hakhab prescription, which is realized by the following steps:

1) preparation of a test solution: dissolving Yatounihakha particle powder with an organic solvent, performing ultrasonic extraction, and filtering to obtain a test solution A;

2) preparation of control solutions:

taking 6 main active ingredients of the Yatou cow Hazhao Bo prescription, namely danshensu, protocatechuic acid, protocatechualdehyde, p-coumaric acid, rosmarinic acid and salvianolic acid B as reference substances, and preparing mixed reference substance solutions with different mass concentrations by using 40-70% methanol as a solvent;

3) constructing a fingerprint spectrum:

injecting the test solution A and the mixed reference solution into a liquid chromatograph for high performance liquid chromatography analysis to obtain an HPLC fingerprint of the Yatou cow Hamburg prescription; establishing a linear relation between the concentration or content of each substance in the mixed reference substance liquid and the peak area, injecting according to the chromatographic condition in the step 3 of claim 1, and detecting the peak areas of 6 effective components of the Jacobian Harbourne prescription to be detected, thereby obtaining the content of each substance, namely the content of the corresponding main active component.

The conditions of the liquid chromatography are as follows: a Diamonsil C18 chromatography column, specification: 4.6X 250 mm, 5 μm; the mobile phase is acetonitrile-0.5% glacial acetic acid water solution; the flow rate is 0.6-1.0 mL/min^-1The sample injection amount is 15-30 mu L, the detection wavelength is 260-330nm, and the column temperature is 20-45 ℃;

the gradient elution conditions were as follows: 0-7 min, 5% of A; 7-15 min, 5% -10% acetonitrile; 15-30 min, 10% -14% acetonitrile; 30-35 min, 14% -18% acetonitrile; 35-55 min, 18% -21% acetonitrile; 55-87 min, 21-32% acetonitrile; 87-90 min, 32% -95% acetonitrile; 90-100 min, 95% acetonitrile; 100-102 min, 95-5% acetonitrile; 102-110 min, 5% acetonitrile.

The second purpose of the invention is realized by the following steps:

1) dissolving, extracting and filtering a sample of the Jacobian Harbour wave prescription to be detected to obtain a test solution B, and injecting the test solution B into a high performance liquid chromatograph under the liquid chromatography condition for determination to obtain a spectrum of the Jacobian Harbour wave prescription to be detected;

2) and comparing the spectrum of the Yabuna-Happopoto prescription to be detected with the relative retention time of each peak and/or the peak area of each peak in the fingerprint spectrum constructed according to the claim 1, and finishing the qualitative and/or quantitative determination of the Yabuna-Happopoto prescription to be detected.

The invention has the beneficial effects that:

1) the invention optimizes the fingerprint spectrum condition of the Yapu Hazakh wave particles, determines the chromatographic condition and the extraction condition of the Yapu Hazakh wave particles, firstly establishes the HPLC fingerprint spectrum method of the Yapu Hazakh wave particles by combining methodology investigation, simultaneously marks 32 common peaks in a plurality of batches of samples under 280nm, and identifies 6 components, namely danshensu, protocatechuic acid, protocatechualdehyde, p-coumaric acid, rosmarinic acid and salvianolic acid B as characteristic peaks, thereby realizing the qualitative detection of the Yapu Hazakh wave particles and having good stability and reproducibility.

2) The invention simultaneously realizes the synchronous quantitative analysis of 6 main active ingredients in the Yatou nihan particles by utilizing the chromatographic separation condition of the fingerprint.

3) The method combines HPLC fingerprint and multi-index content measurement for the first time, and has higher application value for better monitoring and evaluating the quality of the Yakuwa particles and guiding standardized production through qualitative identification and content measurement of multiple batches of Yakuwa particles; provides a research clue for establishing the quality standard of the Yatou cattle Hazhao Bobo granules with multiple components and indexes, and lays a solid foundation for the deep development of the Yatou cattle Hazhao Bobo granules.

4) The fingerprint spectrum prepared by the method can be used for qualitatively and quantitatively detecting the Yatou cow Haemabo granule simultaneously, and can also be used for qualitatively and quantitatively detecting the Yatou cow Haemabo pills, extractum and other formulations.

Drawings

FIG. 1 is an overlay of HPLC fingerprints (280 nm) for 11 batches of Yakuwa bovine Haematococcus particles;

FIG. 2 is an HPLC fingerprint common pattern (R, 280 nm) of Yakuwa Boehringer's particles;

FIG. 3 is an HPLC chromatogram (280 nm) of a mixed control (A), Yatou bovine Haemabo granules and a prescription drug taste (B);

FIG. 411 Cluster analysis plot of batched bovine Hardpoe particle samples (S1-S11);

FIG. 511 OPLS-DA score plots for the batch bovine Hardpoe particle samples (S1-S11);

FIG. 611 OPLS-DA load plot for the batch bovine Hardpoe particle sample (S1-S11);

FIG. 711 sets of OPLS-DA variable importance projection plots (red: VIP > 1; green: VIP < 1) for bovine Hartazapte samples (S1-S11).

Detailed Description

The invention is further described in detail below with reference to the drawings and examples, but the invention is not limited in any way, and any changes or modifications made based on the teachings of the invention fall within the scope of the invention.

The invention relates to a method for constructing an HPLC fingerprint of a Yatou cattle Hakhapra prescription, which is realized by the following steps:

1) preparation of a test solution: dissolving Yatou cow Harbour particle powder with an organic solvent, performing ultrasonic extraction, and filtering to obtain a test solution A;

2) preparation of control solutions:

taking 6 main active ingredients of the Yatou cow Hazhao Bo prescription, namely danshensu, protocatechuic acid, protocatechualdehyde, p-coumaric acid, rosmarinic acid and salvianolic acid B as reference substances, and preparing mixed reference substance solutions with different mass concentrations by using 40-70% methanol as a solvent;

3) constructing a fingerprint spectrum:

injecting the test solution A and the mixed reference solution into a liquid chromatograph for high performance liquid chromatography analysis to obtain an HPLC fingerprint of the Yatou cow Hamburg prescription; establishing a linear relation between the concentration or content of each substance in the mixed reference substance liquid and the peak area, injecting according to the chromatographic condition in the step 3 of claim 1, and detecting the peak areas of 6 effective components of the Jacobian Harbourne prescription to be detected, thereby obtaining the content of each substance, namely the content of the corresponding main active component.

The conditions of the liquid chromatography are as follows: a Diamonsil C18 chromatography column, specification: 4.6X 250 mm, 5 μm; the mobile phase is acetonitrile-0.5% glacial acetic acid water solution; the flow rate is 0.6-1.0 mL/min^-1The sample injection amount is 15-30 mu L, the detection wavelength is 260-330nm, and the column temperature is 20-45 ℃;

the gradient elution conditions were as follows: 0-7 min, 5% A; 7-15 min, 5% -10% acetonitrile; 15-30 min, 10% -14% acetonitrile; 30-35 min, 14% -18% acetonitrile; 35-55 min, 18-21% acetonitrile; 55-87 min, 21-32% acetonitrile; 87-90 min, 32% -95% acetonitrile; 90-100 min, 95% acetonitrile; 100-102 min, 95-5% acetonitrile; 102-110 min, 5% acetonitrile.

In the step 1, the organic solvent is 40-70% methanol, 2-4g of Jacob's cow-Hadambo granules are dissolved in 25-50 mL of methanol, ultrasonic extraction is carried out for 25-35min, weighing is carried out after cooling to room temperature, methanol is used for complementing lost weight, and filtering is carried out through a 0.45-micron filter membrane.

In step 2, the reference solution contains danshensu, protocatechuic acid, protocatechualdehyde, p-coumaric acid, rosmarinic acid, and salvianolic acid B38-42 μ g^-1，15-16μg•mL^-1，3.20-3.25μg•mL^-1，8.5-9.0μg•mL^-1，36.5-37.0μg•mL^-1，27.5-28.0μg•mL^-1。

The HPLC fingerprint spectrum of the Yatou cattle Hazhangbo formula specifies 32 common peaks under 280nm, takes the No. 17 peak as a reference peak and takes the No. 4, 5, 8, 17, 24 and 30 peaks which can represent main active ingredients in the formula as characteristic peaks; wherein peak 4 is danshensu, peak 5 is protocatechuic acid, peak 8 is protocatechualdehyde, peak 17 is p-coumaric acid, peak 24 is rosmarinic acid, and peak 30 is salvianolic acid B.

According to ultraviolet spectrum information and retention time, the related peaks of the finger print of the Yakuaihazhao particles and the single medicine are compared, and the source attribution of characteristic peaks is determined, wherein the chromatographic peaks 5, 8, 11, 14 and 18 belong to the soulier pteris, the chromatographic peaks 4, 7, 8, 15, 23, 24, 25, 26, 28, 29, 30 and 32 belong to the clerodendranthus spicatus, the chromatographic peaks 12 and 14 belong to the curare, the chromatographic peak 17 belongs to the coix seed root, and the chromatographic peaks 18 and 19 belong to the liquorice.

The Yatouniu Hazhangbo formulation comprises granules, decoction, pills, tablets and extract.

The invention also provides a detection method of the Yatou cattle Haemabo prescription, which is realized according to the following steps:

1) dissolving, extracting and filtering a sample of the Jacobian Harbour wave prescription to be detected to obtain a test solution B, and injecting the test solution B into a high performance liquid chromatograph under the liquid chromatography condition for determination to obtain a spectrum of the Jacobian Harbour wave prescription to be detected;

2) and comparing the spectrum of the formulation of the Jacobian Harbour wave to be detected with the relative retention time of each peak and/or the peak area of each peak in the fingerprint constructed according to the claim 1, and finishing the qualitative and/or quantitative determination of the formulation of the Jacobian Harbour wave to be detected.

When the quantitative measurement is carried out, when 6 main active ingredients are synchronously and quantitatively analyzed, the detection wavelength is 330nm at 63-78min, and the detection wavelength is 280nm at the rest time.

Example 1 formulation of HPLC fingerprint of Yatou Niuhaheau granules

First, experimental material

The instrument comprises the following steps: LC-20A high performance liquid chromatograph (DAD detector, Shimadzu); KQ-300B ultrasonic cleaner (Kunshan ultrasonic Instrument Co., Ltd.); analytical balance model XS105DU (mettler-toledo, switzerland).

Reagent: methanol (batch No. 10044118, national drug group chemical Co., Ltd.), acetonitrile (batch No. 193502, Sammer Feishell technology (China) Co., Ltd.), glacial acetic acid (batch No. 20190530, Wako pure Water Co., Ltd.), and purified water (batch No. 3212TJ, Wawa Haha purified Water Co., Ltd.).

A sample to be detected: 11 batches of the Yakuaibuhabo particles (S1-S11, with the batch numbers being 200514, 200610, 200612, 200613, 200614, 200710, 200711, 200712, 200713, 200714, 200810 and 10 g/bag in sequence) and the ampelopsis grossedentata (the plant of the Kimuramidata is)Aspidopterys obcordataCaulis et folium Ampelopsis Grossdentata), and herba Clerodendranthi Spicati (Clerodendranthus spicatus (Thunb.) nakai of Labiatae)Clerodendranthus spicatusWhole plant of Celastrus paniculatus (L.) Makino of Celastraceae), and Celastrus paniculatus (L.) Makino of CelastraceaeCelastrus paniculatusRoot of Coix lacryma-joli (Coix lacryma-joli) of Gramineae family, and root of Coix lacryma-joli (Coix lacryma-joli) of Coix lacryma-joliCoix lacryma-jobiRoot of Glycyrrhiza uralensis Fisch, Glycyrrhiza uralensis Fisch (Glycyrrhiza uralensis Fisch of Leguminosae)Glycyrrhiza uralensisThe dried roots and rhizomes) and other medicinal materials are provided by Xishuangbanna pharmaceutical industry, Limited liability company, and 5 medicinal materials with the prescription meet the requirements of related standards through Zhaoxianghong identification by the chief pharmacist of Dai medical hospital in the Xishuangbanna Dai nationality. The samples are crushed and screened by a No. 4 sieve for experimental research.

And (3) standard substance: salvianic acid sodium (lot number: CHB180731, purity: 98% or more), protocatechuic acid (lot number: CHB180929, purity: 98% or more), protocatechualdehyde (lot number: CHB180928, purity: 98% or more), p-coumaric acid (lot number: CHB180223, purity: 98% or more), salvianolic acid B (lot number: CHB180108, purity: 98% or more) were provided by Chengdu Klomao Biotech Co., Ltd, and rosmarinic acid (lot number: PS012101, purity: 98% or more) was provided by Chengdu Pus Biotech Co., Ltd.

Second, test methods and results

1. Preparation of test solution

A. Preparing a finished product medicine test solution: 2g of Yatou Niuhahanbo granule S5 (batch number: 200614) powder is precisely weighed, 25 mL of 50% methanol is precisely added, weighing is carried out, ultrasonic extraction is carried out for 30 min, after cooling to room temperature, weight supplement is carried out, and filtration is carried out through a 0.45-micron filter membrane, thus obtaining the test solution A.

B. Preparing a single medicine test solution: according to the proportion of the prescription, respectively taking the Birdish peltate yam rhizome, the clerodendranthus spicatus, the celastrus paniculatus stem and the like,

Appropriate amount of the root of Job's tears, liquorice and the like are respectively prepared into the test solution B of each single medicine according to the method.

2. Preparation of control solutions

Precisely weighing appropriate amount of each reference substance, and preparing with 50% methanol to obtain extract containing danshensu, protocatechuic acid, protocatechualdehyde, p-coumaric acid, rosmarinic acid, and salvianolic acid B with concentration of 40 μ g^-1，15.6 μg•mL^-1，3.22 μg•mL^-1，8.8 μg•mL^-1，36.8 μg•mL^-1，27.8 μg•mL^-1Mixed control solution of (4).

3. Chromatographic conditions

Diamonsil C₁₈Chromatography column (4.6X 250 mm, 5 μm) with mobile phase acetonitrile (A) -0.5% glacial acetic acid in water (B) and gradient elution conditions as follows: 0-7 min, 5% A; 7-15 min, 5% -10% A; 15-30 min, 10% -14% A; 30-35 min, 14% -18% A; 35-55 min, 18% -21% A; 55-87 min, 21-32% A; 87-90 min, 32% -95% A; 90-100 min, 95% A; 100-102 min, 95% -5% A; 102-110 min, 5% A. The flow rate was 0.8 mL/min^-1The column temperature was 40 ℃, the sample injection amount was 20 μ L, and the detection wavelength was 280 nm.

4. HPLC fingerprint establishment methodology investigation of Yatou cattle Hakheau granules

1) Precision test

Taking the sample solution A, continuously sampling for 6 times according to the chromatographic conditions, recording the chromatogram, and calculating the relative retention time of each chromatogram peak and the RSD value of the relative peak area by taking the No. 17 peak as a reference peak. Results RSD values for relative retention times of 32 peaks <3% and RSD values for relative peak areas < 5%. Indicating that the precision of the instrument is good.

2) Stability test

Taking the test solution A, respectively measuring the test solution A at 0, 2, 4, 8, 10 and 24 h according to the chromatographic conditions, taking the No. 17 peak as a reference peak, and calculating the relative retention time of each chromatographic peak and the RSD value of the relative peak area, wherein the RSD value of the relative retention time of 32 peaks is less than 3 percent, and the RSD value of the relative peak area is less than 5 percent. Indicating that the test solution is basically stable within 24 h.

3) Repeatability test

Taking a Yakuwa Boehringer particle sample S5, preparing 6 parts of test sample solution (CFX-1-CFX-6) in parallel according to the method of the test sample solution A, measuring according to the chromatographic condition, taking a No. 17 peak as a reference peak, calculating the relative retention time of each chromatographic peak and the RSD value of the relative peak area, and as a result, the RSD value of the relative retention time of 32 peaks is less than 3 percent, and the RSD value of the relative peak area is less than 5 percent. The method is shown to have good repeatability.

5. Establishment of Yatou cow Hazhan wave particle fingerprint

1) 11 batches of Yakuwa boehrpe samples (S1-S11) were sampled to prepare a sample solution A, and HPLC analysis was performed under the above chromatographic conditions. Exporting 11 batches of sample chromatograms into a CDF format by using an Shimadzu workstation, importing the files into software traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2004A edition), taking the Jacobian Hakka particles S1 as a reference spectrum, setting the time window width to be 0.2 min, carrying out multi-point correction and then automatically matching to obtain the HPLC fingerprint of the Jacobian Hakka particles (figure 1), and generating a comparison spectrum.

2) And (3) taking the generated reference map as a reference map (R), carrying out similarity evaluation on 11 batches of samples, and calculating the relative retention time and the RSD value of the relative peak area of each spectrum peak by taking the No. 17 peak as the reference peak, wherein the fingerprint chromatogram is shown in a figure 1 and a figure 2. The results show that the similarity of the samples of the batch S1-S11 Yakuwa particles to the control map is more than 0.980, which indicates that the difference between 11 batches of samples is small and the chemical components are basically consistent. The relative retention time RSD values of the 32 common peaks are all less than 1%, and the RSD of the relative peak area is 0% -17.21%, which indicates that the whole fingerprint spectrums of different batches of samples have no difference, but the chromatographic peak height or the peak area has a certain difference, and the method can be used for qualitative identification of Yawa bovine halbo particles.

3) Peak assignment of chromatograms

A total of 32 peaks were detected at 280nm, and 6 peaks were identified by control comparison. Wherein peak 4 is danshensu, peak 5 is protocatechuic acid, peak 8 is protocatechuic aldehyde, peak 17 is p-coumaric acid, peak 24 is rosmarinic acid, and peak 30 is salvianolic acid B. See figure 3A for the mixed control solution.

4) The single medicine has chromatogram peak attribution

According to the ultraviolet spectrum information and the retention time, the related peaks of the finger-print of the Yaqian Hazhuanbo particles and the single medicine are compared, the source attribution of the characteristic peaks is determined, and the result is shown in figure 3B. Wherein chromatographic peaks 5 (protocatechuic acid), 8 (protocatechuic aldehyde), 11, 14 and 18 are from radix Stephaniae Cepharanthae, chromatographic peaks 4 (tanshinol), 7, 8 (protocatechuic aldehyde), 15, 23 and 24 (rosmarinic acid), 25, 26, 28, 29, 30 (salvianolic acid B) and 32 are from herba Clerodendranthi Spicati, chromatographic peaks 12 and 14 are from caulis Celastri Turcticae, chromatographic peak 17 (p-coumaric acid) is from radix Coicis, and chromatographic peaks 18 and 19 are from radix Glycyrrhizae. Wherein, chromatographic peaks 1, 2, 3 and 31 are not appeared in each single medicine and may be new components generated after the compatibility.

5) Chemometric analysis

The common peak area of 11 batches of Jacobian herbeck Harbour particles was introduced into SIMCA 13.0 data analysis software for cluster analysis (HCA). According to the results of the cluster analysis chart (fig. 4), 11 samples can be classified into 2 types, wherein S6, S5, S3 and S4 can be grouped into one type; s1, S2, S8, S10, S9, S7, S11 can be grouped into a second group, indicating that there is some difference in the contents of 32 components. This may be related to the Yatou Niuhaabo granule manufacturing process and the batch-to-batch variation of the medicinal material.

Based on HCA, orthogonal partial least squares discriminant analysis (OPLS-DA) is established, and a scoring matrix diagram is shown in FIG. 5. Wherein the index R²Y is 0.963, which can reflect the stability of the model; index Q²0.903, which reflects the model's predictability; both values are greater than 0.5, indicating good stability and predictability of the model.

In addition, an OPLS-DA load map (FIG. 6) is established to further predict the difference of each parameter, wherein the more distant a parameter on the load map from the origin represents the more weight of the change of the component. Meanwhile, a Variable Importance Projection (VIP) method is combined to screen components with larger component content difference among batches, wherein the VIP value of a variable larger than 1 (figure 7) can be used as a difference marker. As can be seen from fig. 7, there are 14 chemical components in 11 batches of samples that have large changes, including chromatographic peaks, which indicates that these 14 components can be used as the difference markers of the quality of the japuhaba particles.

Example 2 determination of the content of active ingredients in Yatou bovine Haemabo particle 6

Methodology investigation

1. Investigation of linear relationships

Preparing mixed reference substance solution according to the term of 2.2, respectively injecting 2 μ L, 4 μ L, 8 μ L, 10 μ L, 12 μ L, 16 μ L and 20 μ L, taking peak area as ordinate and reference substance sample amount as abscissa, performing linear regression, drawing standard curve, fitting linear equation, calculating linear range and R²The value is obtained. The results of the linear relationship are shown in Table 1. The content of the salvianolic acid B is 8.0-80 μ g/mL respectively^-1，3.2~32 μg•mL^-1，0.6~6.0 μg•mL^-1，1.8~18 μg•mL^-1，7.4~74 μg•mL^-1，5.6~56 μg•mL^-1In a range of good linear relation with the peak area, R²Are all greater than 0.999.

Table 16 linear relationship examination of the components

2. Precision test

Taking a Yakuaihakha particle sample S5, preparing a sample solution according to the method under the item 2.1, and continuously feeding samples for 6 times according to the chromatographic condition under the item 2.3, so that the RSD values of the peak areas of 6 components such as danshensu, protocatechuic acid, protocatechuic aldehyde, p-coumaric acid, rosmarinic acid, salvianolic acid B and the like are all less than 3 percent. Indicating that the precision of the instrument is good.

3. Stability test

Taking a Yakuaihakha particle sample S5, preparing a test sample solution according to the method under the item 2.1, and respectively carrying out sample injection analysis for 0, 2, 4, 6, 8, 10 and 24 h under the chromatographic condition under the item 2.3, wherein the RSD values of the peak areas of 6 components such as danshensu, protocatechuic acid, protocatechualdehyde, p-coumaric acid, rosmarinic acid, salvianolic acid B and the like in the test sample are all less than 3 percent. The stability of the test article in 24 hours is good.

4. Repeatability test

Taking a Yakuaihazhao particle sample S5, preparing 6 parts of test sample solutions in parallel according to the method under the item 2.1, and respectively injecting samples according to the chromatographic condition under the item 2.3, so that the contents of danshensu, protocatechuic acid, protocatechualdehyde, p-coumaric acid, rosmarinic acid and salvianolic acid B in the test sample are respectively 0.245 mg/g, 0.072 mg/g, 0.015 mg/g, 0.058 mg/g, 0.255 mg/g and 0.188 mg/g. RSD is 0.31%, 1.04%, 0.00%, 2.77%, 0.20%, 2.94%, respectively. The method is shown to have good repeatability.

5. Sample application recovery test

Taking 6 parts of Yakuaihakha particle samples (S5), weighing 1 g of the samples respectively, adding equivalent danshensu, protocatechuic acid, protocatechualdehyde, p-coumaric acid, rosmarinic acid and salvianolic acid B reference substances into each sample, preparing 6 parts of sample solutions in parallel according to the method under the item 2.1, injecting samples according to the chromatographic condition under the item 2.3, recording the peak areas of the samples and calculating the sample injection recovery rate, which is shown in Table 2. The experimental result shows that the average sample adding recovery rate of the danshensu, the protocatechuic acid, the protocatechuic aldehyde, the p-coumaric acid, the rosmarinic acid and the salvianolic acid B is 100.66-101.99%, and the RSD is less than 3%, which shows that the sample adding recovery rate results of the danshensu, the protocatechuic acid, the protocatechuic aldehyde, the p-coumaric acid, the rosmarinic acid and the salvianolic acid B are good.

TABLE 2 sample recovery test results

II, 6 active ingredient content determination

Taking 11 batches of Yakuniuhahanbo particle samples (S1-S11), preparing a test solution according to the method in the embodiment 1, carrying out sample injection analysis by a method, selecting 280nm as the detection wavelength for measuring the contents of danshensu, protocatechuic acid, protocatechuic aldehyde and p-coumaric acid, and selecting 330nm as the detection wavelength of rosmarinic acid and salvianolic acid B. The results of the content determination of Yatou niuhava granules are shown in Table 3.

TABLE 3 measurement of content of Yatou cow Hakhbo granules (mg/g, n = 11)

And (4) analyzing results:

as can be seen from Table 1, the average content of rosmarinic acid in the Yakuaihaha particles is 0.257 mg/g at most, and the contents of the other components are 0.249 mg/g of danshensu, 0.183 mg/g of danshinolic acid B, 0.073 mg/g of protocatechuic acid, 0.057 mg/g of p-coumaric acid and 0.014 mg/g of protocatechuic aldehyde in sequence from high to low.

Wherein the content of rosmarinic acid is the highest and is consistent with the previous research result; the content of the danshensu is 0.249 mg/g and the content of the salvianolic acid B is 0.183 mg/g, and the danshensu and the salvianolic acid B can be brought into the quality control standard of the Yaqian Hazhao particles according to the measurement results of more than 10 batches of samples. Because the content of protocatechuic acid and p-coumaric acid is low, the total amount of the protocatechuic acid and p-coumaric acid, rosmarinic acid and salvianolic acid B in the Yatou Haja particles can reach 0.570 mg/g, and the total amount of the organic acid components can be considered as a quality control index.

Example 3

The finger-print prepared in example 1 was used to detect the yatou cow hakhart decoction:

preparation of a test solution: precisely weighing 2g of Yatou Niuhaangbo decoction lyophilized powder, precisely adding 30mL of 50% methanol, weighing, ultrasonically extracting for 30 min, cooling to room temperature, supplementing weight, and filtering with 0.45 μm filter membrane to obtain test solution A. The rest of the operation was the same as in example 1.

The fingerprint result shows that compared with the control map, 32 common peaks appear, and the similarity is more than 0.95. The result shows that the fingerprint constructed in the embodiment 1 can also be used for detecting the Yatou cow Haemabo decoction.

Example 4

The finger-print prepared in example 1 is used to detect the yawa bohambo extract:

preparation of a test solution: precisely weighing 3g of Yatou cow Haemabo extract, precisely adding 25 mL of 70% methanol, weighing, ultrasonically extracting for 30 min, cooling to room temperature, supplementing weight, and filtering with 0.45 μm filter membrane to obtain test solution A. The rest of the operation was the same as in example 1.

The fingerprint result shows that compared with the control map, 32 common peaks appear, and the similarity is more than 0.95. The result shows that the fingerprint constructed in the embodiment 1 can also be used for detecting the Jacob cattle Haematococcus extractum.

Example 5

The finger-print prepared in example 1 was used to detect the yawa bohama waveplate agent:

preparation of a test solution: precisely weighing 4g of the Yatou Hayazakh wave plate agent, precisely adding 50mL of 60% methanol, weighing, ultrasonically extracting for 35min, cooling to room temperature, supplementing weight, and filtering with 0.45 μm filter membrane to obtain a sample solution A. The rest of the operation was the same as in example 1.

The fingerprint result shows that compared with the control map, 32 common peaks appear, and the similarity is more than 0.95. The result shows that the fingerprint constructed in the example 1 can also be used for detecting the Yatou bovine Haemabo tablet.

Example 6

The Yatou bovine Haemabo bolus was tested using the fingerprint prepared in example 1:

preparation of a test solution: precisely weighing 3g of Yatou Niuhahanbo pill, precisely adding 40mL of 50% methanol, weighing, ultrasonically extracting for 25 min, cooling to room temperature, supplementing weight, and filtering with 0.45 μm filter membrane to obtain test solution A. The rest of the operation was the same as in example 1.

The fingerprint spectrum result shows that compared with the contrast spectrum, 32 common peaks all appear, and the similarity is more than 0.95. The result shows that the fingerprint constructed in the example 1 can also be used for detecting the Yatou cow Haemabo pill.

Claims (7)

1. The method for constructing the HPLC fingerprint of the Yatou cattle Hazhangbo formula is characterized by comprising the following steps of:

1) preparing a test solution: dissolving Yatou cow Harbour particle powder with an organic solvent, performing ultrasonic extraction, and filtering to obtain a test solution A;

2) preparation of control solutions:

taking 6 main active ingredients of the Yatou cow Hazhao Bo prescription, namely danshensu, protocatechuic acid, protocatechualdehyde, p-coumaric acid, rosmarinic acid and salvianolic acid B as reference substances, and preparing mixed reference substance solutions with different mass concentrations by using 40-70% methanol as a solvent;

3) constructing a fingerprint spectrum:

injecting the test solution A and the mixed reference solution into a liquid chromatograph for high performance liquid chromatography analysis to obtain an HPLC fingerprint of the Yatou cow Hamburg prescription; establishing a linear relation between the concentration or content of each substance in the mixed reference substance liquid and the peak area, injecting according to the chromatographic condition in the step 3 of claim 1, and detecting the peak areas of 6 effective components of the Jacobian Harbourne prescription to be detected, so as to obtain the content of each substance, namely the content of the corresponding main active component;

the conditions of the liquid chromatography are as follows: a Diamonsil C18 chromatography column, specification: 4.6X 250 mm, 5 μm; the mobile phase is acetonitrile-0.5% glacial acetic acid water solution; the flow rate is 0.6-1.0 mL/min^-1The sample injection amount is 15-30 mu L, and the detection wavelength is260 ℃ and 330nm, and the column temperature is 20-45 ℃;

the gradient elution conditions were as follows: 0-7 min, 5% A; 7-15 min, 5% -10% acetonitrile; 15-30 min, 10% -14% acetonitrile; 30-35 min, 14% -18% acetonitrile; 35-55 min, 18-21% acetonitrile; 55-87 min, 21-32% acetonitrile; 87-90 min, 32% -95% acetonitrile; 90-100 min, 95% acetonitrile; 100-102 min, 95-5% acetonitrile; 102-110 min, 5% acetonitrile.

2. The method according to claim 1, wherein in step 1, the organic solvent is 40-70% methanol, 2-4g of the Jacob's Harbin particles are dissolved in 25-50 mL of methanol, ultrasonic extraction is carried out for 25-35min, weighing is carried out after cooling to room temperature, the lost weight is made up by methanol, and filtration is carried out by a 0.45 μm filter membrane.

3. The method according to claim 1, wherein in step 2, the concentrations of danshensu, protocatechuic acid, protocatechuic aldehyde, p-coumaric acid, rosmarinic acid, and salvianolic acid B in the reference solution are 38-42 μ g^-1，15-16μg•mL^-1，3.20-3.25μg•mL^-1，8.5-9.0μg•mL^-1，36.5-37.0μg•mL^-1，27.5-28.0μg•mL^-1。

4. The construction method according to claim 1, wherein the HPLC fingerprint of the Yatou Niuhaheau prescription specifies 32 common peaks under 280nm, takes the No. 17 peak as a reference peak, and takes the No. 4, 5, 8, 17, 24 and 30 peaks which can represent the main active ingredients in the prescription as characteristic peaks; wherein peak 4 is danshensu, peak 5 is protocatechuic acid, peak 8 is protocatechualdehyde, peak 17 is p-coumaric acid, peak 24 is rosmarinic acid, and peak 30 is salvianolic acid B.

5. The method of claim 1, wherein the Jacobian Haematoco formulation comprises granules, decoctions, pills, tablets, and extracts.

6. The detection method of the Yatou cattle Hazhangbo prescription is characterized by being realized according to the following steps:

1) dissolving, extracting and filtering a sample of the Jacobian Harbour wave prescription to be detected to obtain a test solution B, and injecting the test solution B into a high performance liquid chromatograph under the liquid chromatography condition of claim 1 for determination to obtain a map of the Jacobian Harbour wave prescription to be detected;

2) and comparing the spectrum of the formulation of the Jacobian Harbour wave to be detected with the relative retention time of each peak and/or the peak area of each peak in the fingerprint constructed according to the claim 1, and finishing the qualitative and/or quantitative determination of the formulation of the Jacobian Harbour wave to be detected.

7. The detection method according to claim 6, wherein in the quantitative determination, when 6 main active ingredients are simultaneously quantitatively analyzed, the detection wavelength is 330nm for 63-78min, and the detection wavelength is 280nm for the rest of time.

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