CN103969372B

CN103969372B - A kind of assay of capsule for protecting heart and discrimination method

Info

Publication number: CN103969372B
Application number: CN201410215955.9A
Authority: CN
Inventors: 刘永利; 赵振霞; 王敏; 冯丽; 王钰宁
Original assignee: Hebei Province Food And Medicine Check Institute
Current assignee: Hebei Province Food And Medicine Check Institute
Priority date: 2014-05-22
Filing date: 2014-05-22
Publication date: 2016-03-02
Anticipated expiration: 2034-05-22
Also published as: CN103969372A

Abstract

The invention discloses one and assay is carried out to 4 kinds of effective constituents such as Sodium Danshensu, protocatechualdehyde, tanshin polyphenolic acid B, Puerarins in capsule for protecting heart, the red sage root, the root of kudzu vine are carried out to characteristic spectrum mirror method for distinguishing and carry out mirror method for distinguishing to Pachyrhizua angulatus and the root of kudzu vine.The inventive method has highly sensitive, the feature such as reappearance and good stability, and substantially increase checkability, convenient and swift, economic environmental protection, effectively can control the quality of capsule for protecting heart.

Description

A kind of assay of capsule for protecting heart and discrimination method

Technical field

The present invention relates to a kind of discrimination method plurality of active ingredients in capsule for protecting heart being carried out to assay and characteristic spectrum, specifically, be a kind of method utilizing the content of ultra-performance liquid chromatography (UPLC) method to 4 kinds of effective constituents such as Sodium Danshensu, protocatechualdehyde, tanshin polyphenolic acid B, Puerarins in capsule for protecting heart to measure and the red sage root, the root of kudzu vine are carried out to characteristic differentiation.

Background technology

Capsule for protecting heart is made up of the red sage root, the root of kudzu vine, pseudo-ginseng, the banksia rose and hawthorn 5 taste medicine, has effect that is promoting blood circulation and removing blood stasis, promoting qi circulation and relieving pain.Uncomfortable in chest, angina pectoris, the hypertension that cause for qi stagnation and blood stasis type coronary heart disease, dizziness, headache, neck pain and the disease such as arrhythmia cordis, high fat of blood.1998 annual incomes " the Sanitation Ministry medicine standard " Traditional Chinese medicine historical preparation the 15.At present, the whole nation has 7 pharmaceutical producing enterprises.

Capsule for protecting heart act.std sends out [2004] No. 389 literary compositions for " the Sanitation Ministry medicine standard " Chinese traditional patent formulation system the 15 (standard No. is WS3-B-2856-98) and pharmacopeia industry word.This product quality standard has mainly been recorded proterties, has been differentiated and routine inspection (referring to table 1 capsule for protecting heart act.std Interventions Requested), Interventions Requested are less, only have discriminating and check item, lack the projects such as assay, and ursolic acid be non-exclusive composition in hawthorn, protocatechualdehyde is hydrolysing component in the red sage root, carry out TLC distinguish with these two kinds of composition contrasts, specificity is poor, so act.std can not control the quality of medicine very well.

The present invention is exactly that the detection method setting up a kind of quick environmental protection controls the content of plurality of active ingredients in capsule for protecting heart, and differentiates monarch drug in a prescription and the ministerial drug red sage root, the root of kudzu vine, thus ensures drug quality in order to overcome above-mentioned defect.

Summary of the invention

The content that the object of the invention is to provide 4 kinds of effective constituents such as Sodium Danshensu, protocatechualdehyde, tanshin polyphenolic acid B, Puerarin in a kind of capsule for protecting heart carries out measuring and the red sage root, the root of kudzu vine is carried out to a kind of method of characteristic differentiation, the method utilizes ultra-performance liquid chromatography (UPLC) method to measure, the method is quick, environmental protection, effectively can control drug quality.

The invention provides a kind of content assaying method of capsule for protecting heart, this content assaying method is made up of following steps:

The preparation of A, reference substance solution: get Sodium Danshensu reference substance, protocatechualdehyde reference substance, tanshin polyphenolic acid B reference substance, Puerarin reference substance are appropriate, accurately weighed, add 70% methyl alcohol and make the mixed solution of every 1ml containing Sodium Danshensu 50 μ g, protocatechualdehyde 20 μ g, tanshin polyphenolic acid B 100 μ g, Puerarin 150 μ g, product solution in contrast;

The preparation of B, need testing solution: get the capsule for protecting heart content under content uniformity item, porphyrize, gets 0.1-1g, accurately weighed, put in tool plug conical flask, precision adds 50%-80% methyl alcohol 50ml, close plug, weighed weight, ultrasonic process 10-60 minute, take out, let cool, more weighed weight, supply the weight of less loss, shake up, filter, get subsequent filtrate, as need testing solution;

C, liquid phase chromatogram condition: C ₁₈chromatographic column take acetonitrile as mobile phase A, and with the trifluoroacetic acid solution of 0.05-0.2% for Mobile phase B, condition of gradient elution is 0 minute to 2 minutes, and the ratio of mobile phase A is 1%-10%, and the ratio of Mobile phase B is 99%-90%; 2 minutes to 30 minutes, the ratio of mobile phase A was increased to 20%-40%, and the ratio of Mobile phase B is reduced to 80%-60%; Column temperature is 20-50 DEG C; Flow velocity: 0.2-1.0ml/min; Determined wavelength is 270-310nm;

D, mensuration: accurate absorption reference substance solution and need testing solution each 1-10 μ l, injection liquid chromatography, calculates each component content respectively with external standard method.

Content assaying method is preferably:

The preparation of B, need testing solution: get the capsule for protecting heart content under content uniformity item, porphyrize, gets 0.1g, accurately weighed, put in tool plug conical flask, precision adds 50% methyl alcohol 50ml, close plug, weighed weight, ultrasonic process 10 minutes, take out, let cool, more weighed weight, 50% methyl alcohol supplies the weight of less loss, shakes up, and filters, get subsequent filtrate, as need testing solution;

C, liquid phase chromatogram condition: C ₁₈chromatographic column, take acetonitrile as mobile phase A, the trifluoroacetic acid solution with 0.05% is Mobile phase B, condition of gradient elution: 0 minute to 2 minutes, and the ratio of mobile phase A is 1%, and the ratio of Mobile phase B is 99%; 2 minutes to 30 minutes, the ratio of mobile phase A was increased to 20%, and the ratio of Mobile phase B is reduced to 80%; Column temperature is 20 DEG C; Flow velocity: 0.2ml/min; Determined wavelength is 270nm;

D, mensuration: accurate absorption reference substance solution and each 10 μ l of need testing solution, injection liquid chromatography, calculates each component content respectively with external standard method.

Content assaying method is also preferably:

The preparation of B, need testing solution: get the capsule for protecting heart content under content uniformity item, porphyrize, gets 1g, accurately weighed, put in tool plug conical flask, precision adds 80% methyl alcohol 50ml, close plug, weighed weight, ultrasonic process 60 minutes, take out, let cool, more weighed weight, supply the weight of less loss, shake up, filter, get subsequent filtrate, as need testing solution;

C, liquid phase chromatogram condition: C ₁₈chromatographic column, take acetonitrile as mobile phase A, the trifluoroacetic acid solution with 0.2% is Mobile phase B, condition of gradient elution: 0 minute to 2 minutes, and the ratio of mobile phase A is 10%, and the ratio of Mobile phase B is 90%; 2 minutes to 30 minutes, the ratio of mobile phase A was increased to 40%, and the ratio of Mobile phase B is reduced to 60%; Column temperature is 50 DEG C; Flow velocity: 1.0ml/min; Determined wavelength is 310nm;

D, mensuration: accurate absorption reference substance solution and each 1 μ l of need testing solution, injection liquid chromatography, calculates each component content respectively with external standard method.

Content assaying method is also preferably:

The preparation of B, need testing solution: get the capsule for protecting heart content under content uniformity item, porphyrize, gets 0.5g, accurately weighed, put in tool plug conical flask, precision adds 70% methyl alcohol 50ml, close plug, weighed weight, ultrasonic (power 250W, frequency 40kHz) 30 minutes, take out, let cool, weighed weight again, 70% methyl alcohol supplies the weight of less loss, shakes up, filter, get subsequent filtrate, as need testing solution;

C, liquid phase chromatogram condition: chromatographic column is AcquityBEHC ₁₈(2.1 × 100mm, 1.7 μm); Take acetonitrile as mobile phase A, the trifluoroacetic acid solution with 0.1% is Mobile phase B, condition of gradient elution: 0 minute to 2 minutes, and the ratio of mobile phase A is 3%, and the ratio of Mobile phase B is 97%; 2 minutes to 30 minutes, the ratio of mobile phase A was increased to 28%, and the ratio of Mobile phase B is reduced to 72%; Column temperature is 25 DEG C; Flow velocity: 0.4ml/min; Determined wavelength is 287nm;

D, mensuration: accurate absorption reference substance solution and each 3 μ l of need testing solution, injection liquid chromatography, calculates each component content respectively with external standard method.

Present invention also offers the characteristic spectrum discrimination method of the root of kudzu vine in a kind of capsule for protecting heart, this discrimination method is made up of following steps:

(1) preparation of the preparation of reference substance solution, need testing solution, liquid phase chromatogram condition are with the steps A in the content assaying method of capsule for protecting heart, step B, step C;

(2) preparation of root of kudzu vine negative control solution: get not containing the negative sample of the root of kudzu vine, porphyrize, gets 0.1-1g, accurately weighed, put in tool plug conical flask, precision adds 50-80% methyl alcohol 50ml, close plug, weighed weight, ultrasonic 10-60 minute, take out, let cool, more weighed weight, supply the weight of less loss, shake up, filter, get subsequent filtrate, as root of kudzu vine negative control solution;

(3) preparation of root of kudzu vine control medicinal material solution: get root of kudzu vine control medicinal material 0.5g, puts in tool plug conical flask, adds 50-80% methyl alcohol 50ml, ultrasonic 10-60 minute, take out, let cool, more weighed weight, supply the weight of less loss, shake up, filter, get subsequent filtrate as root of kudzu vine control medicinal material solution;

(4) measure: accurate absorption reference substance solution, need testing solution, root of kudzu vine negative control solution, root of kudzu vine control medicinal material solution each 1-10 μ l, injection liquid chromatography, contrast colors spectrogram, determines that the Characteristic chromatographic peak of the root of kudzu vine is 3,4,5,6, No. 7 peaks;

In capsule for protecting heart, root of kudzu vine characteristic differentiation method is preferably:

(2) preparation of root of kudzu vine negative control solution: get not containing the negative sample of the root of kudzu vine, porphyrize, gets 0.5g, accurately weighed, put in tool plug conical flask, precision adds 70% methyl alcohol 50ml, close plug, weighed weight, ultrasonic (power 250W, frequency 40kHz) 30 minutes, take out, let cool, weighed weight again, 70% methyl alcohol supplies the weight of less loss, shakes up, filter, get subsequent filtrate, as root of kudzu vine negative control solution;

(3) preparation of root of kudzu vine control medicinal material solution: get root of kudzu vine control medicinal material 0.5g, puts in tool plug conical flask, adds 70% methyl alcohol 50ml, ultrasonic (power 250W, frequency 40kHz) 30 minutes, shakes up, filter, get subsequent filtrate, as root of kudzu vine control medicinal material solution;

(4) measure: accurate absorption reference substance solution, need testing solution, root of kudzu vine negative control solution, each 3 μ l of root of kudzu vine control medicinal material solution, injection liquid chromatography, contrast colors spectrogram, determines that the Characteristic chromatographic peak of the root of kudzu vine is 3,4,5,6, No. 7 peaks;

The present invention also provides the characteristic spectrum discrimination method of the red sage root in a kind of capsule for protecting heart, and this discrimination method is made up of following steps:

(2) preparation of red sage root negative control solution: get not containing the negative sample of the red sage root, porphyrize, gets 0.1-1g, accurately weighed, put in tool plug conical flask, precision adds 50-80% methyl alcohol 50ml, close plug, weighed weight, ultrasonic 10-60 minute, take out, let cool, more weighed weight, supply the weight of less loss, shake up, filter, get subsequent filtrate, as red sage root negative control solution;

(3) measure: accurate absorption reference substance solution, need testing solution, red sage root negative control solution each 1-10 μ l, injection liquid chromatography, contrast colors spectrogram, determines that the Characteristic chromatographic peak of the red sage root is 1,2, No. 8 peak.

In capsule for protecting heart, the characteristic differentiation method of the red sage root is preferably:

(2) preparation of red sage root negative control solution: get not containing the negative sample of the red sage root, porphyrize, gets 0.5g, accurately weighed, put in tool plug conical flask, precision adds 70% methyl alcohol 50ml, close plug, weighed weight, ultrasonic (power 250W, frequency 40kHz) 30 minutes, take out, let cool, weighed weight again, 70% methyl alcohol supplies the weight of less loss, shakes up, filter, get subsequent filtrate, as red sage root negative control solution;

(3) measure: accurate absorption reference substance solution, need testing solution, each 3 μ l of red sage root negative control solution, injection liquid chromatography, contrast colors spectrogram, determines that the Characteristic chromatographic peak of the red sage root is 1,2, No. 8 peak.

Present invention also offers the discrimination method of a kind of root of kudzu vine medicinal material and Pachyrhizua angulatus medicinal material, the method is made up of following steps:

(1) liquid phase chromatogram condition is with the step step C in the content assaying method of capsule for protecting heart;

(2) preparation of root of kudzu vine control medicinal material solution: get root of kudzu vine control medicinal material 0.1-1g, puts in tool plug conical flask, and add 50-80% methyl alcohol 50ml, ultrasonic 10-60 minute, shakes up, and filters, gets subsequent filtrate, as root of kudzu vine control medicinal material solution;

(3) preparation of Pachyrhizua angulatus control medicinal material solution: get Pachyrhizua angulatus control medicinal material 0.1-1g, puts in tool plug conical flask, and add 50-80% methyl alcohol 50ml, ultrasonic 10-60 minute, shakes up, and filters, gets subsequent filtrate, as Pachyrhizua angulatus control medicinal material solution;

(4) preparation of root of kudzu vine medicinal material solution: get root of kudzu vine medicinal material 0.1-1g, puts in tool plug conical flask, and add 50-80% methyl alcohol 50ml, ultrasonic 10-60 minute, shakes up, and filters, gets subsequent filtrate, as root of kudzu vine medicinal material solution;

(5) preparation of Pachyrhizua angulatus medicinal material solution: get Pachyrhizua angulatus medicinal material 0.1-1g, puts in tool plug conical flask, and add 50-80% methyl alcohol 50ml, ultrasonic 10-60 minute, shakes up, and filters, gets subsequent filtrate, as Pachyrhizua angulatus medicinal material solution;

(6) measure: accurate absorption root of kudzu vine control medicinal material, Pachyrhizua angulatus control medicinal material, root of kudzu vine medicinal material, Pachyrhizua angulatus medicinal material solution each 1-10 μ l, injection liquid chromatography, contrast colors spectrogram, root of kudzu vine control medicinal material and root of kudzu vine medicinal material all have 3,4,5,6, No. 7 peaks; Pachyrhizua angulatus control medicinal material has 4,5,6, No. 7 peaks; Pachyrhizua angulatus medicinal material only has 4, No. 7 peaks.

The discrimination method of root of kudzu vine medicinal material and Pachyrhizua angulatus medicinal material is preferably:

(2) preparation of root of kudzu vine control medicinal material solution: get root of kudzu vine control medicinal material 0.5g, puts in tool plug conical flask, adds 70% methyl alcohol 50ml, ultrasonic 30 minutes, shakes up, and filters, gets subsequent filtrate, as root of kudzu vine control medicinal material solution;

(3) preparation of Pachyrhizua angulatus control medicinal material solution: get Pachyrhizua angulatus control medicinal material 0.5g, puts in tool plug conical flask, adds 70% methyl alcohol 50ml, ultrasonic 30 minutes, shakes up, and filters, gets subsequent filtrate, as Pachyrhizua angulatus control medicinal material solution;

(4) preparation of root of kudzu vine medicinal material solution: get root of kudzu vine medicinal material 0.5g, puts in tool plug conical flask, adds 70% methyl alcohol 50ml, ultrasonic 30 minutes, shakes up, and filters, gets subsequent filtrate, as root of kudzu vine medicinal material solution;

(5) preparation of Pachyrhizua angulatus medicinal material solution: get Pachyrhizua angulatus medicinal material 0.5g, puts in tool plug conical flask, adds 70% methyl alcohol 50ml, ultrasonic 30 minutes, shakes up, and filters, gets subsequent filtrate, as Pachyrhizua angulatus medicinal material solution;

In order to verify the stability, accuracy, specificity and the system flexibility that measure content method, following methods confirmatory experiment has been carried out to method, and confirm with the characteristic spectrum of the method to the root of kudzu vine, the red sage root, and distinguish the root of kudzu vine and Pachyrhizua angulatus by the method, and set up distinguishing characteristics, to guarantee that this assay and discrimination method can as the method for quality control of capsule for protecting heart.

One, the content method of capsule for protecting heart is investigated

1, chromatographic condition and system suitability

1.1 chromatographic columns: AcquityBEHC ₁₈(2.1 × 100mm, 1.7 μm)

1.2 mobile phases: acetonitrile is mobile phase A, 0.1% trifluoroacetic acid solution is Mobile phase B, and gradient elution, gradient table is in table 1.

Table 1 eluent gradient wash-out table

1.3 determined wavelength: 287nm.

1.4 column temperatures: 25 DEG C.

1.5 flow velocitys: 0.4ml/min.

2, solution preparation and assay method

The preparation of 2.1 need testing solutions: get the capsule for protecting heart content under content uniformity item, porphyrize, gets 0.5g, accurately weighed, put in tool plug conical flask, precision adds 70% methyl alcohol 50ml, close plug, weighed weight, ultrasonic (power 250W, frequency 40kHz) 30 minutes, take out, let cool, weighed weight again, 70% methyl alcohol supplies the weight of less loss, shakes up, filter, get subsequent filtrate, as need testing solution;

The preparation of 2.2 reference substance solution: get Sodium Danshensu reference substance, protocatechualdehyde reference substance, tanshin polyphenolic acid B reference substance, Puerarin reference substance are appropriate, accurately weighed, add 70% methyl alcohol and make the mixed solution of every 1ml containing Sodium Danshensu 50 μ g, protocatechualdehyde 20 μ g, tanshin polyphenolic acid B 100 μ g, Puerarin 150 μ g, to obtain final product.

2.3 assay methods: accurate absorption reference substance solution and each 3 μ l of need testing solution, injection liquid chromatography, measures, utilizes reference substance chromatogram and test sample chromatographic peak area to calculate each component content respectively.

3, specificity test: do not contain the negative sample 1 of the red sage root in prescription ratio and method for making preparation and do not contain the negative sample 2 of the root of kudzu vine, negative sample solution is prepared by the preparation method of need testing solution, accurate absorption reference substance solution respectively, need testing solution and two kinds of each 3 μ l of negative control solution, inject Ultra Performance Liquid Chromatography instrument, record chromatographic peak.Result shows, in side, other flavour of a drug do not disturb the mensuration of Sodium Danshensu, protocatechualdehyde, Puerarin, tanshin polyphenolic acid B, see Fig. 1.

4, linear relationship is investigated: precision measures concentration Sodium Danshensu, and (concentration is followed successively by: 0.00369mg/ml, 0.01845mg/ml, 0.07380mg/ml, 0.1476mg/ml, 0.3690mg/ml), (concentration is followed successively by protocatechualdehyde: 0.000487mg/ml, 0.001948mg/ml, 0.009741mg/ml, 0.01948mg/ml, 0.04871mg/ml), (concentration is followed successively by Puerarin: 0.04654mg/ml, 0.1164mg/ml, 0.2327mg/ml, 0.5817mg/ml, 1.1635mg/ml), (concentration is followed successively by tanshin polyphenolic acid B: 0.00396mg/ml, 0.01981mg/ml, 0.03961mg/ml, 0.07923mg/ml, 0.3961) 3 μ l, injection liquid chromatography respectively, measure by the chromatographic condition drafted, with reference substance sample size (μ g) for horizontal ordinate, integrating peak areas value is ordinate, drawing standard curve.Result shows, Sodium Danshensu is in good linear relationship between 0.01107 ~ 1.107 μ g, and regression equation is: y=13.269x-0.0061 (r=0.9999); Protocatechualdehyde is in good linear relationship between 0.001461 ~ 0.146121 μ g, and regression equation is: y=96.504x-0.0035 (r=0.9999); Puerarin is good linear relationship between 0.13962 ~ 3.49056 μ g, regression equation is: y=32.542x+0.0983 (r=0.9999), tanshin polyphenolic acid B is in good linear relationship between 0.01188 ~ 1.18845 μ g, and regression equation is: y=31.244x-0.0165 (r=0.9999).Experimental result is in table 2.

Table 2 capsule for protecting heart four-component linear relationship investigates result

5, replica test: get same batch sample (lot number 110606), porphyrize, gets each three parts of 0.1g, 0.5g, 1.0g, accurately weighed, measures by text method.Result shows, this method repeatability good (the results are shown in Table 3).

Table 3 replica test result

6, recovery test: the sample (lot number 110606 getting known content, Sodium Danshensu content: 4.7599mg/g, protocatechualdehyde content: 0.5890mg/g, puerarin content: l580mg/g, content of danshinolic acid B: 4.6732mg/g) 9 parts, powder, every part of about 0.25g, accurately weighed, every three parts is one group, add the reference substance solution 50ml of basic, normal, high three concentration with 70% methanol solution preparation respectively, by legal system available test sample solution below need testing solution preparation.Measure by the chromatographic condition drafted, calculate the recovery.The results are shown in Table 4-7.Show that this method recovery is better.

Table 4 Sodium Danshensu recovery test result

Table 5 protocatechualdehyde recovery test result

Table 6 Puerarin recovery test result

Table 7 tanshin polyphenolic acid B recovery test result

7, stability test: get same need testing solution and started in 0 hour to measure, measure 1 time later at interval of certain hour, result shows that need testing solution is at least stable in 24 hours.

8, sample size measures: the 17 batches of hearts getting four different manufacturing enterprises respectively can relax sample, measure as stated above, the results are shown in Table 8:

Table 8: 17 batches of capsule for protecting heart assay results (unit: mg/ grain) of different enterprise

Known by testing above, the content assaying method of the Sodium Danshensu that the present invention sets up, protocatechualdehyde, tanshin polyphenolic acid B, Puerarin, highly sensitive, specificity is strong, accuracy is high, reproducible, objectively can reflect the situations such as whether enterprise feeds intake by prescription and whether production technology stable, can be used as the reliable method of Quality control quality.

Two, the confirmation of root of kudzu vine characteristic peak

1, chromatographic condition and system suitability

1.1 chromatographic columns: AcquityBEHC ₁₈(2.1 × 100mm, 1.7 μm)

1.2 mobile phases: acetonitrile is mobile phase A, 0.1% trifluoroacetic acid solution is Mobile phase B, gradient elution, and gradient table is in table 9:

Table 9 eluent gradient wash-out table

1.3 determined wavelength: 287nm.

1.4 column temperatures: 25 DEG C.

1.5 flow velocitys: 0.4ml/min.

2, the preparation of solution and assay method

The preparation of 2.1 need testing solutions: get the capsule for protecting heart content under content uniformity item, porphyrize, gets 0.5g, accurately weighed, put in tool plug conical flask, precision adds 70% methyl alcohol 50ml, close plug, weighed weight, ultrasonic (power 250W, frequency 40kHz) 30 minutes, take out, let cool, weighed weight again, 70% methyl alcohol supplies the weight of less loss, shakes up, filter, get subsequent filtrate, as need testing solution.

The preparation of 2.2 reference substance solution: get Sodium Danshensu reference substance, protocatechualdehyde reference substance, tanshin polyphenolic acid B reference substance, Puerarin reference substance are appropriate, accurately weighed, add 70% methyl alcohol and make the mixed solution of every 1ml containing Sodium Danshensu 50 μ g, protocatechualdehyde 20 μ g, tanshin polyphenolic acid B 100 μ g, Puerarin l50 μ g, to obtain final product.

The preparation of 2.3 root of kudzu vine negative control solutions: get not containing the negative sample of the root of kudzu vine, porphyrize, gets 0.5g, accurately weighed, put in tool plug conical flask, precision adds 70% methyl alcohol 50ml, close plug, weighed weight, ultrasonic (power 250W, frequency 40kHz) 30 minutes, take out, let cool, weighed weight again, 70% methyl alcohol supplies the weight of less loss, shakes up, filter, get subsequent filtrate, as root of kudzu vine negative control solution.

The preparation of 2.4 root of kudzu vine control medicinal material solution: get root of kudzu vine control medicinal material 0.5g, puts in tool plug conical flask, adds 70% methyl alcohol 50ml, and ultrasonic (power 250W, frequency 40kHz) 30 minutes, shakes up, and filters, gets subsequent filtrate, as root of kudzu vine control medicinal material solution.

3, the confirmation of root of kudzu vine characteristic peak

Accurate absorption reference substance solution, control medicinal material solution, need testing solution, each 3 μ l of negative control solution, injection liquid chromatography, carries out chromatographic peak comparison, establishes the characteristic spectrum of the root of kudzu vine.

Sample has 8 characteristic peaks, warp and reference substance comparison, 1,2,4, No. 8 peaks are defined as Sodium Danshensu, protocatechualdehyde, Puerarin, tanshin polyphenolic acid B four compositions respectively, adopt TOF to belong to all the other 4 peaks, 3,5,6, No. 7 peaks are respectively 3'-hydroxypuerarin, 3 '-methoxy puerarin, Puerarin-7-xyloside and daidzin as a result.Therefore can determine that 3,4,5,6, No. 7 peaks in test sample chromatogram are composition in the root of kudzu vine, the characteristic peak (see Fig. 2) of the root of kudzu vine can be defined as.

For investigating characteristic peak situation in Different sources root of kudzu vine medicinal material, the root of kudzu vine medicinal material that we have collected Different sources (Anhui, Beijing, Shanghai) measures, 3,4,5,6, No. 7 peaks (see Fig. 3) are had in the medicinal material test sample chromatogram of result Different sources, the characteristic peak selecting 3,4,5,6, No. 7 peaks in test sample chromatogram as the root of kudzu vine is described, accurately, objectively can reflect the situations such as whether enterprise feeds intake by prescription and whether production technology stable, can be used as the reliable method of Quality control quality.

Three, the confirmation of red sage root characteristic peak

1, chromatographic condition and system suitability

1.1 chromatographic columns: AcquityBEHC ₁₈(2.1 × 100mm, 1.7 μm)

1.2 mobile phases: acetonitrile is mobile phase A, 0.1% trifluoroacetic acid solution is Mobile phase B, gradient elution, and gradient table is in table 10:

Table 10 eluent gradient wash-out table

1.3 determined wavelength: 287nm.

1.4 column temperatures: 25 DEG C.

1.5 flow velocitys: 0.4ml/min.

2, the preparation of solution and assay method

The preparation of 2.3 red sage root negative control solutions: get not containing the negative sample of the red sage root, porphyrize, gets 0.5g, accurately weighed, put in tool plug conical flask, precision adds 70% methyl alcohol 50ml, close plug, weighed weight, ultrasonic (power 250W, frequency 40kHz) 30 minutes, take out, let cool, weighed weight again, 70% methyl alcohol supplies the weight of less loss, shakes up, filter, get subsequent filtrate, as red sage root negative control solution.

3, the confirmation of red sage root characteristic peak: accurate absorption reference substance solution, need testing solution, each 3 μ 1 of negative control solution, injection liquid chromatography, carries out chromatographic peak comparison, establishes the characteristic spectrum of the red sage root.Can find out that from collection of illustrative plates sample has 8 characteristic peaks, warp and reference substance comparison, 1,2, No. 8 peaks are defined as Sodium Danshensu, protocatechualdehyde, tanshin polyphenolic acid B three compositions respectively, namely determine that 1,2, No. 8 peak is the characteristic peak of the red sage root in capsule for protecting heart (see Fig. 4).

Above-mentioned test is known, 1,2, No. 8 peak that this method can be determined in test sample chromatogram is the characteristic peak of the red sage root in capsule for protecting heart, the situations such as whether enterprise feeds intake by prescription and whether production technology stable can be reflected strictly according to the facts, can be used as the reliable method of Quality control quality.

Four, the discriminating of the root of kudzu vine and Pachyrhizua angulatus

L, chromatographic condition and system suitability

1.1 chromatographic columns: AcquityBEHC ₁₈(2.1 × 100mm, 1.7 μm)

1.2 mobile phases: acetonitrile is mobile phase A, 0.1% trifluoroacetic acid solution is Mobile phase B, gradient elution, and gradient table is in table 11:

Table 11 eluent gradient wash-out table

1.3 determined wavelength: 287nm.

1.4 column temperatures: 25 DEG C.

1.5 flow velocitys: 0.4ml/min.

2, the preparation of solution and assay method

The preparation of 2.1 root of kudzu vine control medicinal material solution: get root of kudzu vine control medicinal material 0.5g, puts in tool plug conical flask, adds 70% methyl alcohol 50ml, and ultrasonic (power 250W, frequency 40kHz) 30 minutes, shakes up, and filters, gets subsequent filtrate, as root of kudzu vine control medicinal material solution.

The preparation of 2.2 root of kudzu vine medicinal material solution: get root of kudzu vine medicinal material 0.5g, puts in tool plug conical flask, adds 70% methyl alcohol 50ml, and ultrasonic (power 250W, frequency 40kHz) 30 minutes, shakes up, and filters, gets subsequent filtrate, as root of kudzu vine medicinal material solution.

The preparation of 2.3 Pachyrhizua angulatus control medicinal material solution: get Pachyrhizua angulatus control medicinal material 0.5g, puts in tool plug conical flask, adds 70% methyl alcohol 50ml, and ultrasonic (power 250W, frequency 40kHz) 30 minutes, shakes up, and filters, gets subsequent filtrate, as Pachyrhizua angulatus control medicinal material solution.

The preparation of 2.4 Pachyrhizua angulatus medicinal material solution: get Pachyrhizua angulatus medicinal material 0.5g, puts in tool plug conical flask, adds 70% methyl alcohol 50ml, and ultrasonic (power 250W, frequency 40kHz) 30 minutes, shakes up, and filters, gets subsequent filtrate, as Pachyrhizua angulatus medicinal material solution.

3, measure: each 3 μ l of the accurate absorption root of kudzu vine, Pachyrhizua angulatus control medicinal material and medicinal material, injection liquid chromatography, measure, the peak area recording 3,4,5,6, No. 7 peaks is respectively as follows:

Table 12 root of kudzu vine and Pachyrhizua angulatus peak area

From testing above, the root of kudzu vine and Pachyrhizua angulatus not only principal ingredient Puerarin (No. 4 peaks) differ greatly, content in the root of kudzu vine is probably 8 times of content in Pachyrhizua angulatus, all the other 4 characteristic peak Pachyrhizua angulatus content are also general lower, and No. 3 peaks disappearance in Pachyrhizua angulatus control medicinal material, 3,5, No. 6 peaks disappearance (see Fig. 5) in Pachyrhizua angulatus medicinal material, therefore, the inventive method can be distinguished the root of kudzu vine and Pachyrhizua angulatus well, guarantee the accuracy fed intake, the source that can feed intake from medicinal material controls quality and the safety of capsule for protecting heart.

Accompanying drawing explanation

Fig. 1: capsule for protecting heart assay specificity experimental patterns; Peak in test sample chromatogram successively label is No. 1-8, peak 1: Sodium Danshensu, peak 2: protocatechualdehyde, peak 3:3 '-hydroxyl Puerarin, peak 4: Puerarin, peak 5:3 '-methoxy puerarin, peak 6: Puerarin-7-xyloside, peak 7: daidzin, peak 8: tanshin polyphenolic acid B;

Fig. 2: root of kudzu vine medicinal material characteristic spectrum in capsule for protecting heart; 3,4,5,6, No. 7 peaks are the characteristic spectrum peak of the root of kudzu vine, are respectively 3'-hydroxypuerarin, Puerarin, 3 '-methoxy puerarin, Puerarin-7-xyloside and daidzin;

Fig. 3: root of kudzu vine medicinal material 3,4,5,6, No. 7 peak characteristic spectrums in Beijing, Anhui, the place of production, Shanghai;

Fig. 4: red rooted salvia characteristic spectrum in capsule for protecting heart; 1,2, No. 8 peaks are the characteristic spectrum of the red sage root, are respectively Sodium Danshensu, protocatechualdehyde, tanshin polyphenolic acid B;

Fig. 5: the root of kudzu vine and Pachyrhizua angulatus control medicinal material and medicinal material collection of illustrative plates.

embodiment 1

1, chromatographic condition and system suitability

1.1 chromatographic columns: AcquityBEHC ₁₈(2.1 × 100mm, 1.7 μm)

1.2 mobile phases: acetonitrile is mobile phase A, 0.1% trifluoroacetic acid solution is Mobile phase B, and gradient elution, gradient table is in table 13.

Table 13 eluent gradient wash-out table

1.3 determined wavelength: 287nm.

1.4 column temperatures: 25 DEG C.

1.5 flow velocitys: 0.4ml/min.

2, solution preparation and assay method

The preparation of 2.3 root of kudzu vine control medicinal material solution: get root of kudzu vine control medicinal material 0.5g, puts in tool plug conical flask, adds 70% methyl alcohol 50ml, and ultrasonic (power 250W, frequency 40kHz) 30 minutes, shakes up, and filters, gets subsequent filtrate, as root of kudzu vine control medicinal material solution.

2.4 assay methods: accurate absorption reference substance solution and each 3 μ l of need testing solution, injection liquid chromatography, measures, utilizes appearance two-point method, calculate each component content respectively with reference substance chromatogram and test sample chromatographic peak area.

2.5 methodological studies:

2.5.1 specificity test: do not contain the negative sample 1 of the red sage root in prescription ratio and method for making preparation and do not contain the negative sample 2 of the root of kudzu vine, negative sample solution is prepared by the preparation method of need testing solution, accurate absorption reference substance solution respectively, need testing solution and two kinds of each 3 μ l of negative control solution, inject Ultra Performance Liquid Chromatography instrument, record chromatographic peak.Result shows, in side, other flavour of a drug do not disturb the mensuration of Sodium Danshensu, protocatechualdehyde, Puerarin, tanshin polyphenolic acid B, see Fig. 1.

2.5.2 linear relationship is investigated: precision measures concentration Sodium Danshensu, and (concentration is followed successively by: 0.00369mg/ml, 0.01845mg/ml, 0.07380mg/ml, 0.1476mg/ml, 0.3690mg/ml), (concentration is followed successively by protocatechualdehyde: 0.000487mg/ml, 0.001948mg/ml, 0.009741mg/ml, 0.01948mg/ml, 0.04871mg/ml), (concentration is followed successively by Puerarin: 0.04654mg/ml, 0.1164mg/ml, 0.2327mg/ml, 0.5817mg/ml, 1.1635mg/ml), (concentration is followed successively by tanshin polyphenolic acid B: 0.00396mg/ml, 0.01981mg/ml, 0.03961mg/ml, 0.07923mg/ml, 0.3961) 3 μ l, injection liquid chromatography respectively, measure by the chromatographic condition drafted, with reference substance sample size (μ g) for horizontal ordinate, integrating peak areas value is ordinate, drawing standard curve.Result shows, Sodium Danshensu is in good linear relationship between 0.01107 ~ 1.107 μ g, and regression equation is: y=13.269x-0.0061 (r=0.9999); Protocatechualdehyde is in good linear relationship between 0.001461 ~ 0.146121 μ g, and regression equation is: y=96.504x-0.0035 (r=0.9999); Puerarin is good linear relationship between 0.13962 ~ 3.49056 μ g, regression equation is: y=32.542x+0.0983 (r=0.9999), tanshin polyphenolic acid B is in good linear relationship between 0.01188 ~ 1.18845 μ g, and regression equation is: y=31.244x-0.0165 (r=0.9999).Experimental result is in table 14.

Table 14 capsule for protecting heart four-component linear relationship investigates result

2.5.3 replica test: get same batch sample (lot number 110606), porphyrize, gets each three parts of 0.1g, 0.5g, 1.0g, accurately weighed, measures by text method.Result shows, this method repeatability good (the results are shown in Table 15).

Table 15 replica test result

2.5.4 recovery test: the sample (lot number 110606 getting known content, Sodium Danshensu content: 4.7599mg/g, protocatechualdehyde content: 0.5890mg/g, puerarin content: 1580mg/g, content of danshinolic acid B: 4.6732mg/g) 9 parts, powder, every part of about 0.25g, accurately weighed, every three parts is one group, add the reference substance solution 50ml of basic, normal, high three concentration with 70% methanol solution preparation respectively, by legal system available test sample solution below need testing solution preparation.Measure by the chromatographic condition drafted, calculate the recovery.The results are shown in Table 15-18.Show that this method recovery is better.

Table 15 Sodium Danshensu recovery test result

Table 16 protocatechualdehyde recovery test result

Table 17 Puerarin recovery test result

Table 18 tanshin polyphenolic acid B recovery test result

2.5.5 stability test: get same need testing solution and started in 0 hour to measure, measure 1 time later at interval of certain hour, result shows that need testing solution is at least stable in 24 hours.

2.5.6 sample size measures: assay result, sees the following form 19:

Table 19 assay result

2.5.7 root of kudzu vine characteristic spectrum result, test sample collection of illustrative plates has corresponding chromatographic peak on the relevant position of root of kudzu vine control medicinal material collection of illustrative plates, i.e. 3,4,5,6, No. 7 peaks, and degree of separation is all greater than 2.0.

2.5.8 red sage root characteristic spectrum result, test sample collection of illustrative plates has corresponding chromatographic peak with Sodium Danshensu, protocatechualdehyde, tanshin polyphenolic acid B reference substance, i.e. 1,2, No. 8 peak, and degree of separation is all greater than 2.8.

embodiment 2

L, chromatographic condition and system suitability

1.1 chromatographic columns: AcquityHSST3C ₁₈(2.1 × 100mm, 1.8 μm)

1.2 mobile phases: acetonitrile is mobile phase A, 0.05% trifluoroacetic acid solution is Mobile phase B, and gradient elution, gradient table is in table 20.

Table 20 eluent gradient wash-out table

1.3 determined wavelength: 270nm.

1.4 column temperatures: 20 DEG C.

1.5 flow velocitys: 0.2ml/min.

2, solution preparation and assay method

The preparation of 2.1 need testing solutions: get the capsule for protecting heart content under content uniformity item, porphyrize, gets 0.1g, accurately weighed, put in tool plug conical flask, precision adds 50% methyl alcohol 50ml, close plug, weighed weight, ultrasonic 10 minutes, take out, let cool, more weighed weight, 50% methyl alcohol supplies the weight of less loss, shakes up, and filters, get subsequent filtrate, as need testing solution;

2.4 assay methods: accurate absorption reference substance solution and each 10 μ l of need testing solution, injection liquid chromatography, measures, utilizes appearance two-point method, calculate each component content respectively with reference substance chromatogram and test sample chromatographic peak area,

2.5 methodological studies:

2.5.1 specificity test: do not contain the negative sample 1 of the red sage root in prescription ratio and method for making preparation and do not contain the negative sample 2 of the root of kudzu vine, negative sample solution is prepared by the preparation method of need testing solution, accurate absorption reference substance solution respectively, need testing solution and two kinds of each 3 μ l of negative control solution, inject Ultra Performance Liquid Chromatography instrument, record chromatographic peak.Result shows, in side, other flavour of a drug do not disturb the mensuration of Sodium Danshensu, protocatechualdehyde, Puerarin, tanshin polyphenolic acid B.

2.5.2 linear relationship is investigated: precision measures concentration Sodium Danshensu, and (concentration is followed successively by: 0.00369mg/ml, 0.01845mg/ml, 0.07380mg/ml, 0.1476mg/ml, 0.3690mg/ml), (concentration is followed successively by protocatechualdehyde: 0.000487mg/ml, 0.001948mg/ml, 0.009741mg/ml, 0.01948mg/ml, 0.04871mg/ml), (concentration is followed successively by Puerarin: 0.04654mg/ml, 0.1164mg/ml, 0.2327mg/ml, 0.5817mg/ml, 1.1635mg/ml), (concentration is followed successively by tanshin polyphenolic acid B: 0.00396mg/ml, 0.01981mg/ml, 0.03961mg/ml, 0.07923mg/ml, 0.3961) 3 μ l, injection liquid chromatography respectively, measure by the chromatographic condition drafted, with reference substance sample size (μ g) for horizontal ordinate, integrating peak areas value is ordinate, drawing standard curve.Result shows, Sodium Danshensu is in good linear relationship between 0.01107 ~ 1.107 μ g, and regression equation is: y=13.269x-0.0061 (r=0.9999); Protocatechualdehyde is in good linear relationship between 0.001461 ~ 0.146121 μ g, and regression equation is: y=96.504x-0.0035 (r=0.9999); Puerarin is good linear relationship between 0.13962 ~ 3.49056 μ g, regression equation is: y=32.542x+0.0983 (r=0.9999), tanshin polyphenolic acid B is in good linear relationship between 0.01188 ~ 1.18845 μ g, and regression equation is: y=31.244x-0.0165 (r=0.9999).

2.5.3 replica test: get same batch sample, porphyrize, gets each three parts of 0.1g, 0.5g, 1.0g, and accurately weighed, measure by text method, the RSD of content is all less than 1.7%, shows that this method repeatability is good.

2.5.4 recovery test: the sample powder 9 parts of getting known content, every part of about 0.25g, accurately weighed, every three parts is one group, add the reference substance solution 50ml of basic, normal, high three concentration with 70% methanol solution preparation respectively, by legal system available test sample solution below need testing solution preparation.Measure by the chromatographic condition drafted, calculate the recovery.The RSD of the result recovery is less than 1.56%, shows that this method recovery is better.

2.5.5 stability test: get same need testing solution and started in 0 hour to measure, measure 1 time at interval of certain hour, the RSD of content is less than 0.56% later, shows that need testing solution is stable in 24 hours.

2.5.6 sample size measures: assay result, sees the following form and the results are shown in following table 21:

Table 21 assay result

2.5.7 root of kudzu vine characteristic spectrum result, test sample collection of illustrative plates has corresponding chromatographic peak on the relevant position of root of kudzu vine control medicinal material collection of illustrative plates, i.e. 3,4,5,6, No. 7 peaks, and degree of separation is all greater than 1.8.

2.5.8 red sage root characteristic spectrum result, test sample collection of illustrative plates has corresponding chromatographic peak with Sodium Danshensu, protocatechualdehyde, tanshin polyphenolic acid B reference substance, i.e. 1,2, No. 8 peak, and degree of separation is all greater than 2.6.

embodiment 3

1, chromatographic condition and system suitability

1.1 chromatographic columns: ShiseidoC ₁₈(2.1 × 100mm, 2.7 μm)

1.2 mobile phases: acetonitrile is mobile phase A, 0.2% trifluoroacetic acid solution is Mobile phase B, and gradient elution, gradient table is in table 22.

Table 22 eluent gradient wash-out table

1.3 determined wavelength: 310nm.

1.4 column temperatures: 50 DEG C.

1.5 flow velocitys: 1ml/min.

2, solution preparation and assay method

The preparation of 2.1 need testing solutions: get the capsule for protecting heart content under content uniformity item, porphyrize, gets lg, accurately weighed, put in tool plug conical flask, precision adds 80% methyl alcohol 50ml, close plug, weighed weight, ultrasonic 60 minutes, take out, let cool, more weighed weight, 80% methyl alcohol supplies the weight of less loss, shakes up, and filters, get subsequent filtrate, as need testing solution;

2.4 assay methods: accurate absorption reference substance solution and each 1 μ l of need testing solution, injection liquid chromatography, measures, utilizes appearance two-point method, calculate each component content respectively with reference substance chromatogram and test sample chromatographic peak area

2.5 methodological studies:

2.5.3 replica test: get same batch sample, porphyrize, gets each three parts of 0.1g, 0.5g, 1.0g, and accurately weighed, measure by text method, the RSD of content is all less than 1.25%, shows that this method repeatability is good.

2.5.4 recovery test: the sample powder 9 parts of getting known content, every part of about 0.25g, accurately weighed, every three parts is one group, add the reference substance solution 50ml of basic, normal, high three concentration with 70% methanol solution preparation respectively, by legal system available test sample solution below need testing solution preparation.Measure by the chromatographic condition drafted, calculate the recovery, the RSD of the result recovery is less than 1.32%, shows that this method recovery is better.

2.5.5 stability test: get same need testing solution and started in 0 hour to measure, measure 1 time at interval of certain hour, the RSD of content is less than 0.73% later, shows that need testing solution is stable in 24 hours.

2.5.6 assay result, sees the following form and the results are shown in Table 23:

Table 23 assay result

2.5.7 root of kudzu vine characteristic spectrum result, test sample collection of illustrative plates has corresponding chromatographic peak on the relevant position of root of kudzu vine control medicinal material collection of illustrative plates, i.e. 3,4,5,6, No. 7 peaks, and degree of separation is all greater than 1.9.

2.5.8 red sage root characteristic spectrum result, test sample collection of illustrative plates has corresponding chromatographic peak with Sodium Danshensu, protocatechualdehyde, tanshin polyphenolic acid B reference substance, i.e. 1,2, No. 8 peak, and degree of separation is all greater than 2.3.

Claims

1. a content assaying method for capsule for protecting heart, is characterized in that described content assaying method is made up of following steps:

2. content assaying method according to claim 1, is characterized in that described content assaying method is made up of following steps:

3. content assaying method according to claim 1, is characterized in that described content assaying method is made up of following steps:

4. content assaying method according to claim 1, is characterized in that described content assaying method is made up of following steps:

The preparation of B, need testing solution: get the capsule for protecting heart content under content uniformity item, porphyrize, gets 0.5g, accurately weighed, put in tool plug conical flask, precision adds 70% methyl alcohol 50ml, close plug, weighed weight, ultrasonic 30 minutes, ultrasonic power 250W, frequency 40kHz, takes out, lets cool, weighed weight again, 70% methyl alcohol supplies the weight of less loss, shakes up, filter, get subsequent filtrate, as need testing solution;

C, liquid phase chromatogram condition: chromatographic column is AcquityBEHC ₁₈, specification is: 2.1 × 100mm, 1.7 μm; Take acetonitrile as mobile phase A, the trifluoroacetic acid solution with 0.1% is Mobile phase B, condition of gradient elution: 0 minute to 2 minutes, and the ratio of mobile phase A is 3%, and the ratio of Mobile phase B is 97%; 2 minutes to 30 minutes, the ratio of mobile phase A was increased to 28%, and the ratio of Mobile phase B is reduced to 72%; Column temperature is 25 DEG C; Flow velocity: 0.4ml/min; Determined wavelength is 287nm;

5. a discrimination method for capsule for protecting heart, it is characterized in that described discrimination method is the characteristic spectrum discrimination method of the root of kudzu vine, this discrimination method is made up of following steps:

(1) preparation of reference substance solution with the preparation of the steps A in claim 1, need testing solution with the step B in claim 1, liquid phase chromatogram condition with the step C in claim 1;

(4) measure: accurate absorption reference substance solution, need testing solution, root of kudzu vine negative control solution, root of kudzu vine control medicinal material solution each 1-10 μ l, injection liquid chromatography, contrast colors spectrogram, determines that the Characteristic chromatographic peak of the root of kudzu vine is hydroxyl Puerarin, Puerarin, 3 '-methoxy puerarin, Puerarin-7-xyloside and daidzin.

6. discrimination method according to claim 5, is characterized in that described discrimination method is made up of following steps:

(1) preparation of reference substance solution with the preparation of the steps A in claim 2,3 or 4, need testing solution with the step B in claim 2,3 or 4, liquid phase chromatogram condition with the step C in claim 2,3 or 4;

(2) preparation of root of kudzu vine negative control solution: get not containing the negative sample of the root of kudzu vine, porphyrize, gets 0.5g, accurately weighed, put in tool plug conical flask, precision adds 70% methyl alcohol 50ml, close plug, weighed weight, ultrasonic 30 minutes, ultrasonic power 250W, frequency 40kHz, takes out, lets cool, weighed weight again, 70% methyl alcohol supplies the weight of less loss, shakes up, filter, get subsequent filtrate, as root of kudzu vine negative control solution;

(3) preparation of root of kudzu vine control medicinal material solution: get root of kudzu vine control medicinal material 0.5g, puts in tool plug conical flask, adds 70% methyl alcohol 50ml, and ultrasonic 30 minutes, ultrasonic power 250W, frequency 40kHz, shook up, and filters, gets subsequent filtrate, as root of kudzu vine control medicinal material solution;

(4) measure: accurate absorption reference substance solution, need testing solution, root of kudzu vine negative control solution, each 3 μ l of root of kudzu vine control medicinal material solution, injection liquid chromatography, contrast colors spectrogram, determines that the Characteristic chromatographic peak of the root of kudzu vine is hydroxyl Puerarin, Puerarin, 3 '-methoxy puerarin, Puerarin-7-xyloside and daidzin.

7. a discrimination method for capsule for protecting heart, it is characterized in that described discrimination method is the characteristic spectrum discrimination method of the red sage root, this discrimination method is made up of following steps:

(3) measure: accurate absorption reference substance solution, need testing solution, red sage root negative control solution each 1-10 μ l, injection liquid chromatography, contrast colors spectrogram, determines that the Characteristic chromatographic peak of the red sage root is Sodium Danshensu, protocatechualdehyde, tanshin polyphenolic acid B.

8. discrimination method according to claim 7, is characterized in that described discrimination method is made up of following steps:

(2) preparation of red sage root negative control solution: get not containing the negative sample of the red sage root, porphyrize, gets 0.5g, accurately weighed, put in tool plug conical flask, precision adds 70% methyl alcohol 50ml, close plug, weighed weight, ultrasonic 30 minutes, ultrasonic power 250W, frequency 40kHz, takes out, lets cool, weighed weight again, 70% methyl alcohol supplies the weight of less loss, shakes up, filter, get subsequent filtrate, as red sage root negative control solution;

(3) measure: accurate absorption reference substance solution, need testing solution, each 3 μ l of red sage root negative control solution, injection liquid chromatography, contrast colors spectrogram, determines that the Characteristic chromatographic peak of the red sage root is Sodium Danshensu, protocatechualdehyde, tanshin polyphenolic acid B.