CN103969372A - Content determination and identification method for Xinkeshu capsules - Google Patents
Content determination and identification method for Xinkeshu capsules Download PDFInfo
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- CN103969372A CN103969372A CN201410215955.9A CN201410215955A CN103969372A CN 103969372 A CN103969372 A CN 103969372A CN 201410215955 A CN201410215955 A CN 201410215955A CN 103969372 A CN103969372 A CN 103969372A
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Abstract
The invention discloses a method for carrying out content determination on four effective components including salvianic acid A sodium, protocatechualdehyde, danshinolic acid B, puerarin and the like in Xinkeshu capsules and carrying out specific chromatogram identification onsalvia miltiorrhiza and radix puerariae, and a method for identifying root of lobed kudzuvine and the radix puerariae. The method has the characteristics of high sensitivity, good repeatability, good stability and the like; the checking efficiency is greatly improved; the method is convenient and rapid and is economical and environmentally friendly, and can be used for effectively controlling the quality of the Xinkeshu capsules.
Description
Technical field
The present invention relates to a kind of discrimination method that plurality of active ingredients in capsule for protecting heart is carried out to assay and characteristic spectrum, specifically, be to utilize Ultra Performance Liquid Chromatography method (UPLC) method the content of 4 kinds of effective constituents such as Sodium Danshensu, protocatechualdehyde, tanshin polyphenolic acid B, Puerarin in capsule for protecting heart to be measured and the red sage root, the root of kudzu vine are carried out a kind of method of 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 medicines, has effect promoting blood circulation and removing blood stasis, promoting qi circulation and relieving pain.The disease such as uncomfortable in chest, angina pectoris, hypertension, dizziness, headache, neck pain and arrhythmia cordis, high fat of blood causing for qi stagnation and blood stasis type coronary heart disease.The 15 of 1998 annual incomes " the Sanitation Ministry medicine standard " Traditional Chinese medicine historical preparation.At present, the whole nation has 7 pharmaceutical producing enterprises.
Capsule for protecting heart act.std is sent out literary composition [2004] No. 389 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, discriminating and routine inspection (referring to table 1 capsule for protecting heart act.std Interventions Requested), Interventions Requested are less, only have and differentiate and check item, lack the projects such as assay, and ursolic acid is that non-exclusive composition in hawthorn, protocatechualdehyde are the hydrolysing component in the red sage root, carry out thin layers with this two kinds of compositions contrast and differentiate, specificity is poor, so the quality that act.std can not fine control medicine.
The present invention is exactly in order to overcome above-mentioned defect, sets up a kind of detection method of quick environmental protection the content of plurality of active ingredients in capsule for protecting heart is controlled, and monarch drug in a prescription and the ministerial drug red sage root, the root of kudzu vine are differentiated, thereby ensure drug quality.
Summary of the invention
A kind of method of characteristic differentiation is measured and the red sage root, the root of kudzu vine are carried out to 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, the method utilizes Ultra Performance Liquid Chromatography method (UPLC) method to measure, the method is quick, environmental protection, can effectively 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 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, to put in tool plug conical flask, precision adds 50%-80% methyl alcohol 50ml, close plug, weighed weight, ultrasonic processing 10-60 minute, take out, let cool, more weighed weight, the weight of supplying less loss, shakes up, and filters, get subsequent filtrate, as need testing solution;
C, liquid phase chromatogram condition: C
18chromatographic column, taking acetonitrile as mobile phase A, taking the trifluoroacetic acid solution of 0.05-0.2% as 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; Detection wavelength is 270-310nm;
D, mensuration: accurate reference substance solution and the each 1-10 μ of the need testing solution l of drawing, injection liquid chromatography, calculates respectively each component content with external standard method.
Content assaying method is preferably:
The preparation of A, reference substance solution: get Sodium Danshensu reference substance, protocatechualdehyde reference substance, tanshin polyphenolic acid B reference substance, Puerarin reference substance 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 0.1g, accurately weighed, to put in tool plug conical flask, precision adds 50% methyl alcohol 50ml, close plug, weighed weight, ultrasonic processing 10 minutes, take out, let cool, more weighed weight, 50% methyl alcohol is supplied the weight of less loss, shakes up, and filters, get subsequent filtrate, as need testing solution;
C, liquid phase chromatogram condition: C
18chromatographic column, taking acetonitrile as mobile phase A, the trifluoroacetic acid solution taking 0.05% is Mobile phase B, condition of gradient elution: 0 minute to 2 minutes, the ratio of mobile phase A was 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; Detection wavelength is 270nm;
D, mensuration: accurate reference substance solution and the each 10 μ l of need testing solution of drawing, injection liquid chromatography, calculates respectively each component content with external standard method.
Content assaying method is also preferably:
The preparation of A, reference substance solution: get Sodium Danshensu reference substance, protocatechualdehyde reference substance, tanshin polyphenolic acid B reference substance, Puerarin reference substance 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 1g, accurately weighed, to put in tool plug conical flask, precision adds 80% methyl alcohol 50ml, close plug, weighed weight, ultrasonic processing 60 minutes, take out, let cool, more weighed weight, the weight of supplying less loss, shakes up, and filters, get subsequent filtrate, as need testing solution;
C, liquid phase chromatogram condition: C
18chromatographic column, taking acetonitrile as mobile phase A, the trifluoroacetic acid solution taking 0.2% is Mobile phase B, condition of gradient elution: 0 minute to 2 minutes, the ratio of mobile phase A was 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; Detection wavelength is 310nm;
D, mensuration: accurate reference substance solution and the each 1 μ l of need testing solution of drawing, injection liquid chromatography, calculates respectively each component content with external standard method.
Content assaying method is also preferably:
The preparation of A, reference substance solution: get Sodium Danshensu reference substance, protocatechualdehyde reference substance, tanshin polyphenolic acid B reference substance, Puerarin reference substance 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 0.5g, accurately weighed, to 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 is supplied the weight of less loss, shakes up, filter, get subsequent filtrate, as need testing solution;
C, liquid phase chromatogram condition: chromatographic column is Acquity BEH C
18(2.1 × 100mm, 1.7 μ are m); Taking acetonitrile as mobile phase A, the trifluoroacetic acid solution taking 0.1% is Mobile phase B, condition of gradient elution: 0 minute to 2 minutes, the ratio of mobile phase A was 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; Detection wavelength is 287nm;
D, mensuration: accurate reference substance solution and the each 3 μ l of need testing solution of drawing, injection liquid chromatography, calculates respectively each component content with external standard method.
The present invention also provides the characteristic spectrum discrimination method of the root of kudzu vine in a kind of capsule for protecting heart, and 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 the not negative sample containing the root of kudzu vine, porphyrize, gets 0.1-1g, accurately weighed, to 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, the weight of supplying less loss, shakes up, and filters, 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, put in tool plug conical flask, add 50-80% methyl alcohol 50ml, ultrasonic 10-60 minute, taking-up, lets cool, more weighed weight, supplies the weight of less loss, shake up, filter, get subsequent filtrate as root of kudzu vine control medicinal material solution;
(4) measure: accurate reference substance solution, need testing solution, root of kudzu vine negative control solution, the each 1-10 μ of the root of kudzu vine control medicinal material solution l of drawing, injection liquid chromatography, contrast colors spectrogram, the Characteristic chromatographic peak of determining 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:
(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 the not negative sample containing the root of kudzu vine, porphyrize, gets 0.5g, accurately weighed, to 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 is supplied 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, put in tool plug conical flask, add 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 reference substance solution, need testing solution, root of kudzu vine negative control solution, the each 3 μ l of root of kudzu vine control medicinal material solution of drawing, injection liquid chromatography, contrast colors spectrogram, the Characteristic chromatographic peak of determining 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:
(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 red sage root negative control solution: get the not negative sample containing the red sage root, porphyrize, gets 0.1-1g, accurately weighed, to 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, the weight of supplying less loss, shakes up, and filters, get subsequent filtrate, as red sage root negative control solution;
(3) measure: accurate reference substance solution, need testing solution, the each 1-10 μ of the red sage root negative control solution l of drawing, injection liquid chromatography, contrast colors spectrogram, the Characteristic chromatographic peak of determining 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:
(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 red sage root negative control solution: get the not negative sample containing the red sage root, porphyrize, gets 0.5g, accurately weighed, to 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 is supplied the weight of less loss, shakes up, filter, get subsequent filtrate, as red sage root negative control solution;
(3) measure: accurate reference substance solution, need testing solution, the each 3 μ l of red sage root negative control solution of drawing, injection liquid chromatography, contrast colors spectrogram, the Characteristic chromatographic peak of determining the red sage root is 1,2, No. 8 peak.
The present invention also provides the discrimination method of a kind of root of kudzu vine medicinal material and Pachyrhizua angulatus medicinal material, and 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, put in tool plug conical flask, add 50-80% methyl alcohol 50ml, ultrasonic 10-60 minute, shakes up, filters, and 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, put in tool plug conical flask, add 50-80% methyl alcohol 50ml, ultrasonic 10-60 minute, shakes up, filters, and 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, put in tool plug conical flask, add 50-80% methyl alcohol 50ml, ultrasonic 10-60 minute, shakes up, filters, and 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, put in tool plug conical flask, add 50-80% methyl alcohol 50ml, ultrasonic 10-60 minute, shakes up, filters, and gets subsequent filtrate, as Pachyrhizua angulatus medicinal material solution;
(6) measure: accurate root of kudzu vine control medicinal material, Pachyrhizua angulatus control medicinal material, root of kudzu vine medicinal material, the each 1-10 μ of the Pachyrhizua angulatus medicinal material solution l of drawing, injection liquid chromatography, contrast colors spectrogram, root of kudzu vine control medicinal material and root of kudzu vine medicinal material all have peak 3,4,5,6, No. 7; Pachyrhizua angulatus control medicinal material has peak 4,5,6, No. 7; Pachyrhizua angulatus medicinal material only has peak 4, No. 7.
The discrimination method of root of kudzu vine medicinal material and Pachyrhizua angulatus medicinal material is preferably:
(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 0.5g, put in tool plug conical flask, add 70% methyl alcohol 50ml, ultrasonic 30 minutes, shake up, filter, get 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, put in tool plug conical flask, add 70% methyl alcohol 50ml, ultrasonic 30 minutes, shake up, filter, get 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, put in tool plug conical flask, add 70% methyl alcohol 50ml, ultrasonic 30 minutes, shake up, filter, get 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, put in tool plug conical flask, add 70% methyl alcohol 50ml, ultrasonic 30 minutes, shake up, filter, get subsequent filtrate, as Pachyrhizua angulatus medicinal material solution;
(6) measure: accurate root of kudzu vine control medicinal material, Pachyrhizua angulatus control medicinal material, root of kudzu vine medicinal material, the each 1-10 μ of the Pachyrhizua angulatus medicinal material solution l of drawing, injection liquid chromatography, contrast colors spectrogram, root of kudzu vine control medicinal material and root of kudzu vine medicinal material all have peak 3,4,5,6, No. 7; Pachyrhizua angulatus control medicinal material has peak 4,5,6, No. 7; Pachyrhizua angulatus medicinal material only has peak 4, No. 7.
In order to verify stability, accuracy, specificity and the system flexibility of measuring content method, method is carried out to following methods and learned confirmatory experiment, and the characteristic spectrum of the root of kudzu vine, the red sage root is confirmed by the method, and with the method difference root of kudzu vine and Pachyrhizua angulatus, and set up distinguishing characteristics, to guarantee that this assay and discrimination method can be used as the method for quality control of capsule for protecting heart.
One, the content method of capsule for protecting heart is learned and is investigated
1, chromatographic condition and system suitability
1.1 chromatographic columns: Acquity BEH C
18(2.1 × 100mm, 1.7 μ m)
1.2 mobile phases: acetonitrile is mobile phase A, 0.1% trifluoroacetic acid solution is Mobile phase B, gradient elution, gradient table is in table 1.
Table 1 eluent gradient wash-out table
1.3 detect 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, to 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 is supplied 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 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 reference substance solution and the each 3 μ l of need testing solution of drawing, injection liquid chromatography, measures, and utilizes reference substance chromatogram and test sample chromatographic peak area to calculate respectively each component content.
3, specificity test: prepare the negative sample 2 that does not contain the negative sample 1 of the red sage root and do not contain the root of kudzu vine in prescription ratio and method for making, prepare negative sample solution by the preparation method of need testing solution, the accurate reference substance solution of drawing 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 of drafting, taking reference substance sample size, (μ is g) as horizontal ordinate, peak area integrated value is ordinate, drawing standard curve.Result shows, Sodium Danshensu is good linear relationship between 0.01107～1.107 μ g, and regression equation is: y=13.269x-0.0061 (r=0.9999); Protocatechualdehyde is 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 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 is investigated 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 of 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 respectively the reference substance solution 50ml by basic, normal, high three concentration of 70% methanol solution preparation, by need testing solution preparation below legal system available test sample solution.Measure calculate recovery rate by the chromatographic condition of drafting.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 is measured: 17 batches of hearts getting respectively four different manufacturing enterprises sample that can relax, measure as stated above, and the results are shown in Table 8:
Table 8: 17 batches of capsule for protecting heart assay results (unit: mg/ grain) of different enterprises
Known by above experiment, the content assaying method of Sodium Danshensu, protocatechualdehyde that the present invention sets up, tanshin polyphenolic acid B, Puerarin, highly sensitive, specificity is strong, accuracy is high, reproducible, the situation such as can objectively reflect 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: Acquity BEH C
18(2.1 × 100mm, 1.7 μ m)
1.2 mobile phases: acetonitrile is mobile phase A, 0.1% trifluoroacetic acid solution is Mobile phase B, gradient elution, gradient table is in table 9:
Table 9 eluent gradient wash-out table
1.3 detect 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, to 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 is supplied 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 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 the not negative sample containing the root of kudzu vine, porphyrize, gets 0.5g, accurately weighed, to 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 is supplied 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, put in tool plug conical flask, add 70% methyl alcohol 50ml, ultrasonic (power 250W, frequency 40kHz) 30 minutes, shakes up, filters, and gets subsequent filtrate, as root of kudzu vine control medicinal material solution.
3, the confirmation of root of kudzu vine characteristic peak
Accurate reference substance solution, control medicinal material solution, need testing solution, the each 3 μ l of negative control solution of drawing, injection liquid chromatography, carries out chromatographic peak comparison, has set up the characteristic spectrum of the root of kudzu vine.
Sample has 8 characteristic peaks, through comparing with reference substance, 1,2,4, No. 8 peaks are defined as respectively Sodium Danshensu, protocatechualdehyde, Puerarin, four compositions of tanshin polyphenolic acid B, 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, can be defined as the characteristic peak (seeing Fig. 2) of the root of kudzu vine.
For investigating characteristic peak situation in the root of kudzu vine medicinal material of the different places of production, we have collected the root of kudzu vine medicinal material in the different places of production (Anhui, Beijing, Shanghai) and have measured, in the medicinal material test sample chromatogram in the different places of production of result, there are 3,4,5,6, No. 7 peaks (seeing Fig. 3), 3,4,5,6, No. 7 peaks selecting in test sample chromatogram characteristic peak as the root of kudzu vine is described, the situation such as can accurately, objectively reflect 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: Acquity BEH C
18(2.1 × 100mm, 1.7 μ m)
1.2 mobile phases: acetonitrile is mobile phase A, 0.1% trifluoroacetic acid solution is Mobile phase B, gradient elution, gradient table is in table 10:
Table 10 eluent gradient wash-out table
1.3 detect 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, to 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 is supplied 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 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.
The preparation of 2.3 red sage root negative control solutions: get the not negative sample containing the red sage root, porphyrize, gets 0.5g, accurately weighed, to 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 is supplied 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 reference substance solution, need testing solution, each 3 μ 1 of negative control solution of drawing, injection liquid chromatography, carries out chromatographic peak comparison, has set up the characteristic spectrum of the red sage root.From collection of illustrative plates, can find out that sample has 8 characteristic peaks, through comparing with reference substance, 1,2, No. 8 peaks are defined as respectively Sodium Danshensu, protocatechualdehyde, three compositions of tanshin polyphenolic acid B, the characteristic peak (seeing Fig. 4) that definite 1,2, No. 8 peak is the red sage root in capsule for protecting heart.
Above-mentioned test is known, this method can be determined the characteristic peak that 1,2, No. 8 peak in test sample chromatogram is the red sage root in capsule for protecting heart, the situation such as can reflect strictly according to the facts whether enterprise feeds intake by prescription and whether production technology stable, 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: Acquity BEH C
18(2.1 × 100mm, 1.7 μ m)
1.2 mobile phases: acetonitrile is mobile phase A, 0.1% trifluoroacetic acid solution is Mobile phase B, gradient elution, gradient table is in table 11:
Table 11 eluent gradient wash-out table
1.3 detect 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, put in tool plug conical flask, add 70% methyl alcohol 50ml, ultrasonic (power 250W, frequency 40kHz) 30 minutes, shakes up, filters, and 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, put in tool plug conical flask, add 70% methyl alcohol 50ml, ultrasonic (power 250W, frequency 40kHz) 30 minutes, shakes up, filters, and 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, put in tool plug conical flask, add 70% methyl alcohol 50ml, ultrasonic (power 250W, frequency 40kHz) 30 minutes, shakes up, filters, and 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, put in tool plug conical flask, add 70% methyl alcohol 50ml, ultrasonic (power 250W, frequency 40kHz) 30 minutes, shakes up, filters, and gets subsequent filtrate, as Pachyrhizua angulatus medicinal material solution.
3, measure: the accurate root of kudzu vine, Pachyrhizua angulatus control medicinal material and the each 3 μ l of medicinal material of drawing, injection liquid chromatography, measures, and the peak area that records respectively 3,4,5,6, No. 7 peaks is 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 generally lower, and 3,5, No. 6 peaks disappearances (seeing Fig. 5) in No. 3 peaks disappearance, Pachyrhizua angulatus medicinal material in Pachyrhizua angulatus control medicinal material, therefore, the inventive method can be distinguished the root of kudzu vine and Pachyrhizua angulatus well, guarantee the accuracy feeding intake, quality and the safety of capsule for protecting heart is controlled in the source that can feed intake from medicinal material.
Brief description of the drawings
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, the characteristic spectrum peak that 4,5,6, No. 7 peaks are the root of kudzu vine, is respectively 3'-hydroxypuerarin, Puerarin, 3 '-methoxy puerarin, Puerarin-7-xyloside and daidzin;
Fig. 3: 3,4,5,6, No. 7 peak characteristic spectrums of root of kudzu vine medicinal material in Beijing, Anhui, the place of production, Shanghai;
Fig. 4: red rooted salvia characteristic spectrum in capsule for protecting heart; 1, the characteristic spectrum that 2, No. 8 peaks are the red sage root, is 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: Acquity BEH C
18(2.1 × 100mm, 1.7 μ m)
1.2 mobile phases: acetonitrile is mobile phase A, 0.1% trifluoroacetic acid solution is Mobile phase B, gradient elution, gradient table is in table 13.
Table 13 eluent gradient wash-out table
1.3 detect 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, to 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 is supplied 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 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.
The preparation of 2.3 root of kudzu vine control medicinal material solution: get root of kudzu vine control medicinal material 0.5g, put in tool plug conical flask, add 70% methyl alcohol 50ml, ultrasonic (power 250W, frequency 40kHz) 30 minutes, shakes up, filters, and gets subsequent filtrate, as root of kudzu vine control medicinal material solution.
2.4 assay methods: accurate reference substance solution and the each 3 μ l of need testing solution of drawing, injection liquid chromatography, measures, and utilizes appearance two-point method, calculates respectively each component content with reference substance chromatogram and test sample chromatographic peak area.
2.5 methodological studies:
2.5.1 specificity test: prepare the negative sample 2 that does not contain the negative sample 1 of the red sage root and do not contain the root of kudzu vine in prescription ratio and method for making, prepare negative sample solution by the preparation method of need testing solution, the accurate reference substance solution of drawing 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 of drafting, taking reference substance sample size, (μ is g) as horizontal ordinate, peak area integrated value is ordinate, drawing standard curve.Result shows, Sodium Danshensu is good linear relationship between 0.01107～1.107 μ g, and regression equation is: y=13.269x-0.0061 (r=0.9999); Protocatechualdehyde is 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 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 is investigated 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 of 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 respectively the reference substance solution 50ml by basic, normal, high three concentration of 70% methanol solution preparation, by need testing solution preparation below legal system available test sample solution.Measure calculate recovery rate by the chromatographic condition of drafting.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 is measured: 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 with the relevant position of root of kudzu vine control medicinal material collection of illustrative plates on there is corresponding chromatographic peak, 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: Acquity HSS T3C
18(2.1 × 100mm, 1.8 μ m)
1.2 mobile phases: acetonitrile is mobile phase A, 0.05% trifluoroacetic acid solution is Mobile phase B, gradient elution, gradient table is in table 20.
Table 20 eluent gradient wash-out table
1.3 detect 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, to 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 is supplied the weight of less loss, shakes up, and filters, 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 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 control medicinal material solution: get root of kudzu vine control medicinal material 0.5g, put in tool plug conical flask, add 70% methyl alcohol 50ml, ultrasonic (power 250W, frequency 40kHz) 30 minutes, shakes up, filters, and gets subsequent filtrate, as root of kudzu vine control medicinal material solution.
2.4 assay methods: accurate reference substance solution and the each 10 μ l of need testing solution of drawing, injection liquid chromatography, measures, and utilizes appearance two-point method, calculates respectively each component content with reference substance chromatogram and test sample chromatographic peak area,
2.5 methodological studies:
2.5.1 specificity test: prepare the negative sample 2 that does not contain the negative sample 1 of the red sage root and do not contain the root of kudzu vine in prescription ratio and method for making, prepare negative sample solution by the preparation method of need testing solution, the accurate reference substance solution of drawing 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 of drafting, taking reference substance sample size, (μ is g) as horizontal ordinate, peak area integrated value is ordinate, drawing standard curve.Result shows, Sodium Danshensu is good linear relationship between 0.01107～1.107 μ g, and regression equation is: y=13.269x-0.0061 (r=0.9999); Protocatechualdehyde is 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 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, accurately weighed, measures by text method, and the RSD of content is all less than 1.7%, shows that this method repeatability is good.
2.5.4 recovery test: 9 parts of the sample powder of getting known content, every part of about 0.25g, accurately weighed, every three parts is one group, add respectively the reference substance solution 50ml by basic, normal, high three concentration of 70% methanol solution preparation, by need testing solution preparation below legal system available test sample solution.Measure calculate recovery rate by the chromatographic condition of drafting.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 later at interval of certain hour, the RSD of content is less than 0.56%, shows that need testing solution is stable in 24 hours.
2.5.6 sample size is measured: 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 with the relevant position of root of kudzu vine control medicinal material collection of illustrative plates on there is corresponding chromatographic peak, 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: Shiseido C
18(2.1 × 100mm, 2.7 μ m)
1.2 mobile phases: acetonitrile is mobile phase A, 0.2% trifluoroacetic acid solution is Mobile phase B, gradient elution, gradient table is in table 22.
Table 22 eluent gradient wash-out table
1.3 detect 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, to 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 is supplied the weight of less loss, shakes up, and filters, 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 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.
The preparation of 2.3 root of kudzu vine control medicinal material solution: get root of kudzu vine control medicinal material 0.5g, put in tool plug conical flask, add 70% methyl alcohol 50ml, ultrasonic (power 250W, frequency 40kHz) 30 minutes, shakes up, filters, and gets subsequent filtrate, as root of kudzu vine control medicinal material solution.
2.4 assay methods: accurate reference substance solution and the each 1 μ l of need testing solution of drawing, injection liquid chromatography, measures, and utilizes appearance two-point method, calculates respectively each component content with reference substance chromatogram and test sample chromatographic peak area
2.5 methodological studies:
2.5.1 specificity test: prepare the negative sample 2 that does not contain the negative sample 1 of the red sage root and do not contain the root of kudzu vine in prescription ratio and method for making, prepare negative sample solution by the preparation method of need testing solution, the accurate reference substance solution of drawing 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 of drafting, taking reference substance sample size, (μ is g) as horizontal ordinate, peak area integrated value is ordinate, drawing standard curve.Result shows, Sodium Danshensu is good linear relationship between 0.01107～1.107 μ g, and regression equation is: y=13.269x-0.0061 (r=0.9999); Protocatechualdehyde is 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 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, accurately weighed, measures by text method, and the RSD of content is all less than 1.25%, shows that this method repeatability is good.
2.5.4 recovery test: 9 parts of the sample powder of getting known content, every part of about 0.25g, accurately weighed, every three parts is one group, add respectively the reference substance solution 50ml by basic, normal, high three concentration of 70% methanol solution preparation, by need testing solution preparation below legal system available test sample solution.Measure by the chromatographic condition of drafting, calculate recovery rate, 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 later at interval of certain hour, the RSD of content is less than 0.73%, 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 with the relevant position of root of kudzu vine control medicinal material collection of illustrative plates on there is corresponding chromatographic peak, 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 (10)
1. a content assaying method for capsule for protecting heart, is characterized in that described 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 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, to put in tool plug conical flask, precision adds 50%-80% methyl alcohol 50ml, close plug, weighed weight, ultrasonic processing 10-60 minute, take out, let cool, more weighed weight, the weight of supplying less loss, shakes up, and filters, get subsequent filtrate, as need testing solution;
C, liquid phase chromatogram condition: C
18chromatographic column, taking acetonitrile as mobile phase A, taking the trifluoroacetic acid solution of 0.05-0.2% as 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; Detection wavelength is 270-310nm;
D, mensuration: accurate reference substance solution and the each 1-10 μ of the need testing solution l of drawing, injection liquid chromatography, calculates respectively each component content with external standard method.
2. content assaying method according to claim 1, is characterized in that described 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 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 0.1g, accurately weighed, to put in tool plug conical flask, precision adds 50% methyl alcohol 50ml, close plug, weighed weight, ultrasonic processing 10 minutes, take out, let cool, more weighed weight, 50% methyl alcohol is supplied the weight of less loss, shakes up, and filters, get subsequent filtrate, as need testing solution;
C, liquid phase chromatogram condition: C
18chromatographic column, taking acetonitrile as mobile phase A, the trifluoroacetic acid solution taking 0.05% is Mobile phase B, condition of gradient elution: 0 minute to 2 minutes, the ratio of mobile phase A was 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; Detection wavelength is 270nm;
D, mensuration: accurate reference substance solution and the each 10 μ l of need testing solution of drawing, injection liquid chromatography, calculates respectively each component content with external standard method.
3. content assaying method according to claim 1, is characterized in that described 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 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 1g, accurately weighed, to put in tool plug conical flask, precision adds 80% methyl alcohol 50ml, close plug, weighed weight, ultrasonic processing 60 minutes, take out, let cool, more weighed weight, the weight of supplying less loss, shakes up, and filters, get subsequent filtrate, as need testing solution;
C, liquid phase chromatogram condition: C
18chromatographic column, taking acetonitrile as mobile phase A, the trifluoroacetic acid solution taking 0.2% is Mobile phase B, condition of gradient elution: 0 minute to 2 minutes, the ratio of mobile phase A was 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; Detection wavelength is 310nm;
D, mensuration: accurate reference substance solution and the each 1 μ l of need testing solution of drawing, injection liquid chromatography, calculates respectively each component content with external standard method.
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 A, reference substance solution: get Sodium Danshensu reference substance, protocatechualdehyde reference substance, tanshin polyphenolic acid B reference substance, Puerarin reference substance 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 0.5g, accurately weighed, to 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 is supplied the weight of less loss, shakes up, filter, get subsequent filtrate, as need testing solution;
C, liquid phase chromatogram condition: chromatographic column is Acquity BEH C
18(2.1 × 100mm, 1.7 μ are m); Taking acetonitrile as mobile phase A, the trifluoroacetic acid solution taking 0.1% is Mobile phase B, condition of gradient elution: 0 minute to 2 minutes, the ratio of mobile phase A was 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; Detection wavelength is 287nm;
D, mensuration: accurate reference substance solution and the each 3 μ l of need testing solution of drawing, injection liquid chromatography, calculates respectively each component content with external standard method.
5. a discrimination method for capsule for protecting heart, is characterized in that described discrimination method is the characteristic spectrum discrimination method of the root of kudzu vine, and 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;
(2) preparation of root of kudzu vine negative control solution: get the not negative sample containing the root of kudzu vine, porphyrize, gets 0.1-1g, accurately weighed, to 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, the weight of supplying less loss, shakes up, and filters, 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, put in tool plug conical flask, add 50-80% methyl alcohol 50ml, ultrasonic 10-60 minute, taking-up, lets cool, more weighed weight, supplies the weight of less loss, shake up, filter, get subsequent filtrate as root of kudzu vine control medicinal material solution;
(4) measure: accurate reference substance solution, need testing solution, root of kudzu vine negative control solution, the each 1-10 μ of the root of kudzu vine control medicinal material solution l of drawing, injection liquid chromatography, contrast colors spectrogram, the Characteristic chromatographic peak of determining 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 the not negative sample containing the root of kudzu vine, porphyrize, gets 0.5g, accurately weighed, to 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 is supplied 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, put in tool plug conical flask, add 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 reference substance solution, need testing solution, root of kudzu vine negative control solution, the each 3 μ l of root of kudzu vine control medicinal material solution of drawing, injection liquid chromatography, contrast colors spectrogram, the Characteristic chromatographic peak of determining 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, is characterized in that described discrimination method is the characteristic spectrum discrimination method of the red sage root, and 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;
(2) preparation of red sage root negative control solution: get the not negative sample containing the red sage root, porphyrize, gets 0.1-1g, accurately weighed, to 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, the weight of supplying less loss, shakes up, and filters, get subsequent filtrate, as red sage root negative control solution;
(3) measure: accurate reference substance solution, need testing solution, the each 1-10 μ of the red sage root negative control solution l of drawing, injection liquid chromatography, contrast colors spectrogram, the Characteristic chromatographic peak of determining 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:
(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 red sage root negative control solution: get the not negative sample containing the red sage root, porphyrize, gets 0.5g, accurately weighed, to 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 is supplied the weight of less loss, shakes up, filter, get subsequent filtrate, as red sage root negative control solution;
(3) measure: accurate reference substance solution, need testing solution, the each 3 μ l of red sage root negative control solution of drawing, injection liquid chromatography, contrast colors spectrogram, the Characteristic chromatographic peak of determining the red sage root is Sodium Danshensu, protocatechualdehyde, tanshin polyphenolic acid B.
9. a discrimination method for the root of kudzu vine and Pachyrhizua angulatus, is characterized in that described discrimination method is made up of following steps:
(1) liquid phase chromatogram condition is with the step step C in claim 1;
(2) preparation of root of kudzu vine control medicinal material solution: get root of kudzu vine control medicinal material 0.1-1g, put in tool plug conical flask, add 50-80% methyl alcohol 50ml, ultrasonic 10-60 minute, shakes up, filters, and 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, put in tool plug conical flask, add 50-80% methyl alcohol 50ml, ultrasonic 10-60 minute, shakes up, filters, and 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, put in tool plug conical flask, add 50-80% methyl alcohol 50ml, ultrasonic 10-60 minute, shakes up, filters, and 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, put in tool plug conical flask, add 50-80% methyl alcohol 50ml, ultrasonic 10-60 minute, shakes up, filters, and gets subsequent filtrate, as Pachyrhizua angulatus medicinal material solution;
(6) measure: accurate root of kudzu vine control medicinal material, Pachyrhizua angulatus control medicinal material, root of kudzu vine medicinal material, the each 1-10 μ of the Pachyrhizua angulatus medicinal material solution l of drawing, injection liquid chromatography, contrast colors spectrogram, root of kudzu vine control medicinal material and root of kudzu vine medicinal material all have 3'-hydroxypuerarin, Puerarin, 3 '-methoxy puerarin, Puerarin-7-xyloside and daidzin; Pachyrhizua angulatus control medicinal material has Puerarin, 3 '-methoxy puerarin, Puerarin-7-xyloside and daidzin; Pachyrhizua angulatus medicinal material only has Puerarin, daidzin.
10. discrimination method according to claim 9, is characterized in that this discrimination method is made up of following steps:
(1) liquid phase chromatogram condition is with the step step C in claim 2,3 or 4;
(2) preparation of root of kudzu vine control medicinal material solution: get root of kudzu vine control medicinal material 0.5g, put in tool plug conical flask, add 70% methyl alcohol 50ml, ultrasonic 30 minutes, shake up, filter, get 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, put in tool plug conical flask, add 70% methyl alcohol 50ml, ultrasonic 30 minutes, shake up, filter, get 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, put in tool plug conical flask, add 70% methyl alcohol 50ml, ultrasonic 30 minutes, shake up, filter, get 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, put in tool plug conical flask, add 70% methyl alcohol 50ml, ultrasonic 30 minutes, shake up, filter, get subsequent filtrate, as Pachyrhizua angulatus medicinal material solution;
(6) measure: accurate root of kudzu vine control medicinal material, Pachyrhizua angulatus control medicinal material, root of kudzu vine medicinal material, the each 1-10 μ of the Pachyrhizua angulatus medicinal material solution l of drawing, injection liquid chromatography, contrast colors spectrogram, root of kudzu vine control medicinal material and root of kudzu vine medicinal material all have 3'-hydroxypuerarin, Puerarin, 3 '-methoxy puerarin, Puerarin-7-xyloside and daidzin; Pachyrhizua angulatus control medicinal material has Puerarin, 3 '-methoxy puerarin, Puerarin-7-xyloside and daidzin; Pachyrhizua angulatus medicinal material only has Puerarin, daidzin.
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| CN109239250A (en) * | 2018-09-27 | 2019-01-18 | 无锡济民可信山禾药业股份有限公司 | The measuring method and its standard finger-print of sharp brain lamination finger-print |
| CN113406241A (en) * | 2021-07-14 | 2021-09-17 | 鲁南制药集团股份有限公司 | Quality detection method of Xintong granules |
| CN113406241B (en) * | 2021-07-14 | 2022-09-13 | 鲁南制药集团股份有限公司 | Detection method of Xintong granules |
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