CN104374844A - Method for detecting content of water-soluble ingredients in salvia miltiorrhiza medicinal material and application of method - Google Patents
Method for detecting content of water-soluble ingredients in salvia miltiorrhiza medicinal material and application of method Download PDFInfo
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Abstract
The invention discloses a method for detecting the content of water-soluble ingredients in a salvia miltiorrhiza medicinal material and an application of the method. The method is an ultra-performance liquid chromatography method by using reference substances of tanshinol, protocatechualdehyde, rosmarinic acid, salvianolic acid B and salvianolic acid A as reference and 0.026% phosphoric acid aqueous solution-acetonitrile in a ratio being (85-10):(15-90) as a moving phase for gradient elution. The method for detecting the content of water-soluble ingredients in a salvia miltiorrhiza medicinal material can be used for simultaneously detecting the content of multiple water-soluble ingredients in a salvia miltiorrhiza medicinal material, and can be applied to detection of salvia miltiorrhiza medicinal material fingerprint spectrum. The method has a good effect for separating water-soluble ingredients in the salvia miltiorrhiza medicinal material, and has the characteristics of quickness, accuracy, high reproducibility and high recovery rate, and has strong specificity, high precision, good repeatability, high stability and accurate measurement result. By applying the method, multiple peaks of the fingerprint spectrum can be detected, and reflected information is relatively complete; and the peaks have good shapes, can be easily identified, have high similarity, and are accurate and reliable.
Description
Technical field
The present invention relates to water soluble ingredient content assaying method and application thereof in red rooted salvia, belong to Chinese medicine detection technique field.
Background technology
The red sage root (Classification system: Salvia miltiorrhiza Bunge) has another name called red ginseng, radix salviae miltiorrhizae, red etc.Root is used as medicine, and containing tanshinone, is robustness emmenagogue, the stasis of blood of dispelling, raw new, invigorate blood circulation, the effectiveness such as menstruation regulating, be gynaecology's key medicine, cure mainly uterine hemorrhage, irregular menstruation, blood stasis, stomachache, dysmenorrhoea, through closing, mausoleum pain.Good result is had to treatment coronary heart disease.In addition also control the weak insomnia of nerve, arthralgia, anaemia, mastitis, adenolymphitis, arthritis, sore furuncle pain is swollen, erysipelas, acute, chronic hepatitis, nephropyelitis, traumatic injury, advanced schistosomiasis hepatosplenomegaly ， Dian Epilepsy.External application can wash dermatitis rhus again.
The main chemical compositions of the red sage root is divided into fat-soluble and water-soluble two large classes: the liposoluble constituent of the red sage root is mostly conjugation quinone, ketone compounds, and the water soluble ingredient of the red sage root is mainly phenolic acid.The main pharmacological of the red sage root is to reduce the M & M of pallasiomy and infarction area after cerebral middle artery occlusion in rats; the encephaledema caused after alleviating ischemic, improves coronary circulation, to myocardial ischemia and myocardial infarction protection; improve microcirculation; improve hemorheological property, suppress blood coagulation, activate fibrinolytic, suppress platelet function and antithrombus formation; stablize erythrocyte membrane; anti-hepatic fibrosis, reducing blood lipid and antiatherosclerosis, the reparation of promotion tissue and palingenesis etc.
Red sage root kind, the place of production and the difference of picking time can cause the difference of its end product quality.For ensureing the stability of product quality, to the assay of its water soluble ingredient and finger-print research more.A kind of detection method of efficient liquid-phase chromatograph finger print atlas of red rooted salvia water soluble ingredient is disclosed in " the efficient liquid-phase chromatograph finger print atlas research of red rooted salvia water soluble ingredient " (Zhou Xin etc., chromatogram, 03 phase in 2005,292 ~ 295).
In prior art, during in red rooted salvia, water soluble ingredient assay and finger-print detect, the general content only measuring wherein one to two kind of composition.In order to reduce separately repeatedly the error that the different water soluble ingredient content of mensuration red rooted salvia produces, if the content of energy single-time measurement Multiple components, not only can save time and cost, the test result under same system condition can also be made more stable, reliable.But not yet have Simultaneously test at present and detect the content of red sage root various ingredients and the relevant report of finger-print.
Summary of the invention
For solving the deficiencies in the prior art, the object of the present invention is to provide content assaying method and the application thereof of water soluble ingredient in a kind of red rooted salvia, can the content of multiple water soluble ingredient in Simultaneously test red rooted salvia, and detect its finger-print, described assay method, to the good separating effect of water soluble ingredient in red rooted salvia, has the feature of quick, accurate, high reappearance, high-recovery.
In order to realize above-mentioned target, the present invention adopts following technical scheme:
The content assaying method of water soluble ingredient in red rooted salvia, described method is with danshensu, protocatechualdehyde, Rosmarinic acid, tanshin polyphenolic acid B and salviandic acid A reference substance for contrast, with 0.026% phosphate aqueous solution-acetonitrile=85 ~ 10: 15 ~ 90 is the ultra-performance liquid chromatography of eluent gradient wash-out.
Concrete content assaying method is: according to ultra-performance liquid chromatography, measure according to following steps:
(1) chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; With 0.026% phosphate aqueous solution for mobile phase A, acetonitrile is Mobile phase B, A: B=85 ~ 10: 15 ~ 90, gradient elution: 0 ~ 5min:A: B=85 ~ 75: 15 ~ 25,5 ~ 8min:A: B=75 ~ 70: 25 ~ 30,8 ~ 12min:A: B=70 ~ 50: 30 ~ 50,12 ~ 12.01min:A: B=50 ~ 10: 50 ~ 90,12.01 ~ 17min:A: B=10: 90; Determined wavelength is 276 ~ 306nm; Flow velocity is 0.1 ~ 0.5mLmin-1; Column temperature is 25 DEG C ~ 35 DEG C; Number of theoretical plate calculates should be not less than 2000 by by tanshin polyphenolic acid B peak;
(2) preparation of reference substance solution: get danshensu, protocatechualdehyde, Rosmarinic acid, tanshin polyphenolic acid B and salviandic acid A reference substance appropriate, accurately weighed, add 5% methyl alcohol respectively to dissolve, make five kinds of reference substance storing solutions of 1mg/mL, draw each reference substance storing solution respectively, adding 5% methyl alcohol, to be mixed with danshensu reference substance concentration be 82.2 μ g/mL, protocatechualdehyde reference substance concentration is 15.5 μ g/mL, Rosmarinic acid reference substance concentration is 37.2 μ g/mL, tanshin polyphenolic acid B reference substance concentration is 522.5 μ g/mL, salviandic acid A reference substance concentration is the mixed solution of 32.2 μ g/mL, for subsequent use in 4 DEG C of refrigerations, product solution in contrast,
(3) preparation of need testing solution: get red rooted salvia powder 0.2g, accurately weighed, put in 100mL tool plug conical flask, add 40mL 5% methyl alcohol, ultrasonic extraction 30min, supplies weight with 5% methyl alcohol; Filter, get subsequent filtrate, to obtain final product;
(4) measure: accurate absorption reference substance solution and each 1 μ L of need testing solution respectively, inject Ultra Performance Liquid Chromatography instrument, measure, to obtain final product.
In aforementioned content assaying method, in step (1), determined wavelength is 286nm, and flow velocity is 0.2mLmin-1, and column temperature is 30 DEG C.
The application of content assaying method in red rooted salvia finger-print detects of water soluble ingredient in red rooted salvia.
The application of aforementioned content assaying method, is process the chromatogram in obtain in content assaying method 17 minutes with finger-print software, obtains the finger-print of red rooted salvia water soluble ingredient.
In the application of aforementioned content assaying method, in the finger-print of described red rooted salvia water soluble ingredient, there are 17 total peaks.
Although danshensu, protocatechualdehyde, Rosmarinic acid, tanshin polyphenolic acid B and salviandic acid A all belong to water soluble ingredient in red rooted salvia, but it is respectively had any different, suitable assay condition is often not identical, want their content of Simultaneously test, must the terms and conditions parameter of UPLC be groped, be tested.In order to water soluble ingredient in red rooted salvia being effectively separated, to chromatographic condition especially mobile phase kind, mobile phase ratio and condition of gradient elution to grope, determine be very crucial.The present invention is on the basis of prior art, continue to explore, by groping the preparation method etc. of mobile phase kind, mobile phase ratio, condition of gradient elution, reference substance and test sample, screen, finally establish the content assaying method of water soluble ingredient in red rooted salvia.
One, key instrument and reagent
U.S. WATERS Ultra Performance Liquid Chromatography (UPLC) Acquity system, comprise automatic sampler, quaternary solvent manager, PDA detecting device and Empower 3 chromatographic work station, WATERS ACQUITY UPLC BEH C18 chromatographic column (2.1 × 100mm, 1.7 μm)
Acetonitrile is chromatographically pure, Fisher company of the U.S.; Other reagent is pure for analyzing, and buys in traditional Chinese medicines group.
Tanshin polyphenolic acid B, danshensu, protocatechualdehyde, Rosmarinic acid and salviandic acid A are HPLC level, purity >=98%.
Red rooted salvia totally 13 batches, wherein: 1 ~ 3 batch of medicinal material (S1, S2, S3 medicinal material) derives from Sichuan Feng Chun Pharmacy stock Co., Ltd (genunie medicinal materials), 4 ~ 13 batches of medicinal materials (S4 ~ S13 medicinal material) derive from Guizhou Xin Bang Chinese crude drug Development Co., Ltd.
Two, the method for assay and result
1, the selection of chromatographic condition
The selection of 1.1 column temperatures
For finding optimum column temperature, adopt different column temperature to test, observations, the results are shown in Table 1.
Table 1 column temperature experiment table
| Column temperature (DEG C) | 20 | 25 | 30 | 35 | 40 |
| Observations | Degree of separation is poor | Degree of separation is better | Degree of separation is good | Degree of separation is better | Solvent viscosity is large |
As shown in Table 1, good 25 ~ 35 DEG C of degree of separation, thus select 25 ~ 35 DEG C as column temperature.Wherein during column temperature 30 DEG C, separating effect is best.
The selection of 1.2 flow velocitys
For finding optimum flow rate, adopt different in flow rate to test, record chromatographic peak, the results are shown in Table 2.
Table 2 chromatographic peak record sheet
As shown in Table 3, arranging flow velocity is 0.1 ~ 0.5mL/min, and each peak-to-peak resolution is good, and chromatographic time is short, and peak area is moderate, and effect is best.
1.3 mobile phases are selected
For finding optimal flow phase, adopt different mobile phase to carry out UV detection, record chromatographic peak, the results are shown in Table 3.
The different mobile phase UV of table 3 detects table
As shown in Table 3, select with 0.026% phosphate aqueous solution as mobile phase A, acetonitrile is Mobile phase B, gradient elution: 0 ~ 5min:A: B=85 ~ 75: 15 ~ 25,5 ~ 8min:A: B=75 ~ 70: 25 ~ 30,8 ~ 12min:A: B=70 ~ 50: 30 ~ 50,12 ~ 12.01min:A: B=50 ~ 10: 50 ~ 90,12.01 ~ 17min:A: B=10: 90, its ghost peak produced is few, the peak shape obtained is good and degree of separation is high, is top condition.
2, the preparation of reference substance solution
Get danshensu respectively, protocatechualdehyde, Rosmarinic acid, tanshin polyphenolic acid B and salviandic acid A reference substance appropriate, accurately weighed, add 5% methyl alcohol respectively to dissolve, make five kinds of reference substance storing solutions of 1mg/mL, draw each reference substance storing solution respectively, adding 5% methyl alcohol, to be mixed with danshensu reference substance concentration be 82.2 μ g/mL, protocatechualdehyde reference substance concentration is 15.5 μ g/mL, Rosmarinic acid reference substance concentration is 37.2 μ g/mL, tanshin polyphenolic acid B reference substance concentration is 522.5 μ g/mL, salviandic acid A reference substance concentration is the mixed solution of 32.2 μ g/mL, for subsequent use in 4 DEG C of refrigerations, product solution in contrast.
3, the preparation of need testing solution
Got the red rooted salvia powder 0.2g of No. three sieves, accurately weighed, put in 100mL tool plug conical flask, add 40mL5% methyl alcohol, ultrasonic extraction 30min, supplies weight with 5% methyl alcohol; Filter, get subsequent filtrate, to obtain final product.
4, the selection of determined wavelength
Under 276nm ~ 306nm is to 2, reference substance solution carries out spectrum UV scanning, it has maximum absorption wavelength at 286nm place, in red rooted salvia, other water soluble ingredient also has good display at this wavelength, and therefore in the present invention, in red rooted salvia, the determined wavelength of water soluble ingredient is preferably 286nm.
5, the investigation of linear relationship
Precision measures 2 lower reference substance mixed solutions, and draw 5 μ L, 25 μ L respectively, 50 μ L, 100 μ L, 200 μ L, 300 μ L, 400 μ L, 500 μ L are settled to 500 μ L, sample introduction respectively, measures peak area.With each chromatogram integrating peak areas value (Y), linear regression is carried out to its concentration (X) respectively, the results are shown in Table 4.
Table 4
Result shows, danshensu is good linear relation within the scope of 1.644 ~ 82.2 μ g/mL, protocatechualdehyde is good linear relation within the scope of 0.31 ~ 15.5 μ g/mL, Rosmarinic acid is good linear relation within the scope of 0.31 ~ 37.2 μ g/mL, tanshin polyphenolic acid B is in good linear relation within the scope of 10.45 ~ 522.5 μ g/mL, and salviandic acid A is good linear relation within the scope of 0.32 ~ 32.2 μ g/mL.
6, precision test
Get same batch of red rooted salvia, prepare need testing solution according to the method under 3, continuous sample introduction 6 times, chromatographic resolution also calculates the RSD of each Component peak area.The results are shown in Table 5, show that the method precision is good.
7, stability experiment
Get same batch of red rooted salvia, prepare need testing solution according to the method under 3, carry out measuring respectively at 0h, 4h, 8h, 12h, 16h, 24h and calculate the RSD of each Component peak area.The results are shown in Table 5, at room temperature 24h is interior stable to show the solution after processing, and has good stability.
8, repeated experiment
Get same batch of red rooted salvia 6 parts, prepare need testing solution according to the method under 3, chromatographic resolution also calculates the RSD of each Component peak area.The results are shown in Table 5, show that repeatability is good.
Table 5
9, average recovery experiment
The accurately weighed red rooted salvia 0.1g totally 9 parts having predicted content, be divided into three groups, it is the reference substance solution that 0.1g has predicted 60%, 100% and 140% of each content of material in the red rooted salvia sample of content that each group adds reference substance content respectively, prepares need testing solution according to the method under 3.Calculate the recovery, the results are shown in Table 6.Calculate its average recovery rate and RSD, the recovery is high, shows that this method is feasible.
Table 6
10, sample size measures
Get 5 batches of red rooted salvias, accurately weighed, be prepared by method under the preparation of the preparation of reference substance solution, need testing solution respectively, under the investigation item of linearly relation, method carries out assay (need testing solution, reference substance solution be sample introduction 1 μ L respectively), calculate the content of each water soluble ingredient, the results are shown in Table 7.
Table 7
Three, finger-print detect method and result
1, the selection of chromatographic condition: with reference in above-mentioned two 1.1 ~ 1.3 determine chromatographic condition.
2, the detection of red rooted salvia water soluble ingredient finger-print
The preparation of 2.1 reference substance solution: with reference to 2 preparations in above-mentioned two.
The preparation of 2.3 need testing solutions: with reference to 3 preparations in above-mentioned two.
The selection of 2.4 determined wavelength: with reference in above-mentioned two 4 determine.
The investigation of 2.5 linear relationships: with reference in above-mentioned two 5 test.
2.6 methodological study
2.6.1 Precision Experiment: with reference to 6 mensuration in above-mentioned two.
2.6.2 repeated experiment: with reference to 8 mensuration in above-mentioned two
2.6.3 stability experiment: with reference to 7 mensuration in above-mentioned two.
2.7 sample determinations: with reference to 10 mensuration in above-mentioned two, record the chromatogram in 17 minutes.
3, data, interpretation of result
3.1UPLC finger-print
Measuring 13 batches of red rooted salvia samples, indicating its contribution to total peak by comparing chromatographic peak retention time, the peak that retention time RSD value is less than 1% is the ownership peak of water soluble ingredient on medicinal material collection of illustrative plates in red rooted salvia.By chromatogram data importing " similarity evaluation " (2004A version), through establishing with reference to spectrum, Supplements, Auto-matching, generation contrast collection of illustrative plates and matched data, obtain Similarity value and determine 17 total peaks, Fig. 1 and Fig. 2 be shown in by its finger-print.
3.2 Similarity Measure and analysis
(" " similarity evaluation " (the 2004A version) " of National Drug Administration's recommendation carries out similarity evaluation to the UPLC chromatogram of red rooted salvia to adopt similarity evaluation system.Select the red rooted salvia finger-print in contrast that Sichuan Feng Chun Pharmacy stock Co., Ltd provides.Similarity result is in table 8.
The similarity of table 8 13 batch sample
| S1 | S2 | S3 | S4 | S5 | S6 | S7 | S8 | S9 | S10 | S11 | S12 | S13 | |
| S1 | 1 | ||||||||||||
| S2 | 0.998 | 1 | |||||||||||
| S3 | 0.999 | 0.999 | 1 | ||||||||||
| S4 | 0.983 | 0.99 | 0.986 | 1 | |||||||||
| S5 | 0.969 | 0.98 | 0.972 | 0.997 | 1 | ||||||||
| S6 | 0.84 | 0.867 | 0.847 | 0.921 | 0.946 | 1 | |||||||
| S7 | 0.91 | 0.928 | 0.916 | 0.967 | 0.981 | 0.985 | 1 | ||||||
| S8 | 0.441 | 0.487 | 0.452 | 0.581 | 0.636 | 0.841 | 0.746 | 1 | |||||
| S9 | 0.995 | 0.997 | 0.996 | 0.996 | 0.987 | 0.884 | 0.943 | 0.508 | 1 | ||||
| S10 | 0.878 | 0.903 | 0.886 | 0.948 | 0.969 | 0.994 | 0.99 | 0.797 | 0.916 | 1 | |||
| S11 | 0.572 | 0.615 | 0.583 | 0.701 | 0.75 | 0.915 | 0.84 | 0.982 | 0.636 | 0.884 | 1 | ||
| S12 | 0.795 | 0.826 | 0.804 | 0.887 | 0.918 | 0.994 | 0.969 | 0.88 | 0.843 | 0.985 | 0.944 | 1 | |
| S13 | 0.91 | 0.929 | 0.916 | 0.967 | 0.982 | 0.986 | 0.997 | 0.754 | 0.942 | 0.994 | 0.848 | 0.972 | 1 |
3.3 cluster analysis
Adopt IBM SPSS Statistics 19 analysis software to carry out cluster analysis, according to squared Euclidean distance, use Ward ' s method, result is as Fig. 3.From HCA (hierarchical cluster analysis) result, if medicinal material is divided into 3 classes, then S9 can be divided into a class with S1, S2, S3; S8, S11, S6, S13 can be divided into a class; What all the other were remaining is a class.
Usefulness of the present invention is: the content assaying method of water soluble ingredient in red rooted salvia provided by the invention, can the content of multiple water soluble ingredient in Simultaneously test red rooted salvia, and can be applicable in the detection of red rooted salvia finger-print.This assay method, to the good separating effect of water soluble ingredient in red rooted salvia, has the feature of quick, accurate, high reappearance, high-recovery, and its specificity is strong, precision is high, reproducible, the recovery is high, stability is high, measurement result is accurate.Measured by application the method, the peak of finger-print is many, and the information of reflection is more complete; Peak shape is good, is easy to differentiate, similarity is high, accurately and reliably.
Accompanying drawing explanation
Fig. 1 is the finger-print stacking diagram of 13 batches of red rooted salvia samples;
Fig. 2 is the common pattern collection of illustrative plates of the red rooted salvia sample that the present invention records;
Fig. 3 is 13 batches of red rooted salvia cluster analysis result figure;
The implication of Reference numeral in figure: 1-danshensu, 2-protocatechualdehyde, 3-Rosmarinic acid, 4-tanshin polyphenolic acid B, 5-salviandic acid A.
Embodiment
Below in conjunction with specific embodiment, the present invention is further introduced.
Embodiment 1: in red rooted salvia, water soluble ingredient content assaying method is: according to ultra-performance liquid chromatography, measure according to following steps:
(1) chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; With 0.026% phosphate aqueous solution for mobile phase A, acetonitrile is Mobile phase B, A: B=85 ~ 10: 15 ~ 90, gradient elution: 0 ~ 5min:A: B=85 ~ 75: 15 ~ 25,5 ~ 8min:A: B=75 ~ 70: 25 ~ 30,8 ~ 12min:A: B=70 ~ 50: 30 ~ 50,12 ~ 12.01min:A: B=50 ~ 10: 50 ~ 90,12.01 ~ 17min:A: B=10: 90; Determined wavelength is 276nm; Flow velocity is 0.1mLmin-1; Column temperature is 25 DEG C; Number of theoretical plate calculates should be not less than 2000 by tanshin polyphenolic acid B peak;
(2) preparation of reference substance solution: get danshensu respectively, protocatechualdehyde, Rosmarinic acid, tanshin polyphenolic acid B and salviandic acid A reference substance appropriate, accurately weighed, add 5% methyl alcohol respectively to dissolve, make five kinds of reference substance storing solutions of 1mg/mL, draw each reference substance storing solution respectively, adding 5% methyl alcohol, to be mixed with danshensu reference substance concentration be 82.2 μ g/mL, protocatechualdehyde reference substance concentration is 15.5 μ g/mL, Rosmarinic acid reference substance concentration is 37.2 μ g/mL, tanshin polyphenolic acid B reference substance concentration is 522.5 μ g/mL, salviandic acid A reference substance concentration is the mixed solution of 32.2 μ g/mL, for subsequent use in 4 DEG C of refrigerations, product solution in contrast,
(3) preparation of need testing solution: get red rooted salvia powder 0.2g, accurately weighed, put in 100mL tool plug conical flask, add 40mL5% methyl alcohol, ultrasonic extraction 30min, supplies weight with 5% methyl alcohol; Filter, get subsequent filtrate, to obtain final product;
(4) measure: accurate absorption reference substance solution and each 1 μ L of need testing solution respectively, inject Ultra Performance Liquid Chromatography instrument, measure, to obtain final product.
Embodiment 2: in red rooted salvia, water soluble ingredient content assaying method is: according to ultra-performance liquid chromatography, measure according to following steps:
(1) chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; With 0.026% phosphate aqueous solution for mobile phase A, acetonitrile is Mobile phase B, A: B=85 ~ 10: 15 ~ 90, gradient elution: 0 ~ 5min:A: B=85 ~ 75: 15 ~ 25,5 ~ 8min:A: B=75 ~ 70: 25 ~ 30,8 ~ 12min:A: B=70 ~ 50: 30 ~ 50,12 ~ 12.01min:A: B=50 ~ 10: 50 ~ 90,12.01 ~ 17min:A: B=10: 90; Determined wavelength is 306nm; Flow velocity is 0.5mLmin-1; Column temperature is 35 DEG C; Number of theoretical plate calculates should be not less than 2000 by tanshin polyphenolic acid B peak;
(2) preparation of reference substance solution: get danshensu respectively, protocatechualdehyde, Rosmarinic acid, tanshin polyphenolic acid B and salviandic acid A reference substance appropriate, accurately weighed, add 5% methyl alcohol respectively to dissolve, make five kinds of reference substance storing solutions of 1mg/mL, draw each reference substance storing solution respectively, adding 5% methyl alcohol, to be mixed with danshensu reference substance concentration be 82.2 μ g/mL, protocatechualdehyde reference substance concentration is 15.5 μ g/mL, Rosmarinic acid reference substance concentration is 37.2 μ g/mL, tanshin polyphenolic acid B reference substance concentration is 522.5 μ g/mL, salviandic acid A reference substance concentration is the mixed solution of 32.2 μ g/mL, for subsequent use in 4 DEG C of refrigerations, product solution in contrast,
(3) preparation of need testing solution: the red rooted salvia powder 0.2g getting No. three sieves, accurately weighed, put in 100mL tool plug conical flask, add 40mL5% methyl alcohol, ultrasonic extraction 30min, supplies weight with 5% methyl alcohol; Filter, get subsequent filtrate, to obtain final product;
(4) measure: accurate absorption reference substance solution and each 1 μ L of need testing solution respectively, inject Ultra Performance Liquid Chromatography instrument, measure, to obtain final product.
Embodiment 3: in red rooted salvia, water soluble ingredient content assaying method is: according to ultra-performance liquid chromatography, measure according to following steps:
(1) chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; With 0.026% phosphate aqueous solution for mobile phase A, acetonitrile is Mobile phase B, A: B=85 ~ 10: 15 ~ 90, gradient elution: 0 ~ 5min:A: B=85 ~ 75: 15 ~ 25,5 ~ 8min:A: B=75 ~ 70: 25 ~ 30,8 ~ 12min:A: B=70 ~ 50: 30 ~ 50,12 ~ 12.01min:A: B=50 ~ 10: 50 ~ 90,12.01 ~ 17min:A: B=10: 90; Determined wavelength is 286nm; Flow velocity is 0.2mLmin-1; Column temperature is 30 DEG C; Number of theoretical plate calculates should be not less than 2000 by tanshin polyphenolic acid B peak;
(2) preparation of reference substance solution: get danshensu respectively, protocatechualdehyde, Rosmarinic acid, tanshin polyphenolic acid B and salviandic acid A reference substance appropriate, accurately weighed, add 5% methyl alcohol respectively to dissolve, make five kinds of reference substance storing solutions of 1mg/mL, draw each reference substance storing solution respectively, adding 5% methyl alcohol, to be mixed with danshensu reference substance concentration be 82.2 μ g/mL, protocatechualdehyde reference substance concentration is 15.5 μ g/mL, Rosmarinic acid reference substance concentration is 37.2 μ g/mL, tanshin polyphenolic acid B reference substance concentration is 522.5 μ g/mL, salviandic acid A reference substance concentration is the mixed solution of 32.2 μ g/mL, for subsequent use in 4 DEG C of refrigerations, product solution in contrast,
(3) preparation of need testing solution: the red rooted salvia powder 0.2g getting No. three sieves, accurately weighed, put in 100mL tool plug conical flask, add 40mL5% methyl alcohol, ultrasonic extraction 30min, supplies weight with 5% methyl alcohol; Filter, get subsequent filtrate, to obtain final product;
(4) measure: accurate absorption reference substance solution and each 1 μ L of need testing solution respectively, inject Ultra Performance Liquid Chromatography instrument, measure, to obtain final product.
Embodiment 4: in red rooted salvia, water soluble ingredient content assaying method is: according to ultra-performance liquid chromatography, measure according to following steps:
(1) chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; With 0.026% phosphate aqueous solution for mobile phase A, acetonitrile is Mobile phase B, A: B=85 ~ 10: 15 ~ 90, gradient elution: 0 ~ 5min:A: B=85 ~ 75: 15 ~ 25,5 ~ 8min:A: B=75 ~ 70: 25 ~ 30,8 ~ 12min:A: B=70 ~ 50: 30 ~ 50,12 ~ 12.01min:A: B=50 ~ 10: 50 ~ 90,12.01 ~ 17min:A: B=10: 90; Determined wavelength is 280nm; Flow velocity is 0.3mLmin
-1; Column temperature is 32 DEG C; Number of theoretical plate calculates should be not less than 2000 by tanshin polyphenolic acid B peak;
(2) preparation of reference substance solution: get danshensu respectively, protocatechualdehyde, Rosmarinic acid, tanshin polyphenolic acid B and salviandic acid A reference substance appropriate, accurately weighed, add 5% methyl alcohol respectively to dissolve, make five kinds of reference substance storing solutions of 1mg/mL, draw each reference substance storing solution respectively, adding 5% methyl alcohol, to be mixed with danshensu reference substance concentration be 82.2 μ g/mL, protocatechualdehyde reference substance concentration is 15.5 μ g/mL, Rosmarinic acid reference substance concentration is 37.2 μ g/mL, tanshin polyphenolic acid B reference substance concentration is 522.5 μ g/mL, salviandic acid A reference substance concentration is the mixed solution of 32.2 μ g/mL, for subsequent use in 4 DEG C of refrigerations, product solution in contrast,
(3) preparation of need testing solution: get red rooted salvia powder 0.2g, accurately weighed, put in 100mL tool plug conical flask, add 40mL5% methyl alcohol, ultrasonic extraction 30min, supplies weight with 5% methyl alcohol; Filter, get subsequent filtrate, to obtain final product;
(4) measure: accurate absorption reference substance solution and each 1 μ L of need testing solution respectively, inject Ultra Performance Liquid Chromatography instrument, measure, to obtain final product.
Embodiment 5: the application of water soluble ingredient content assaying method in finger-print detects in red rooted salvia, namely red rooted salvia water soluble ingredient fingerprint atlas detection method is: according to ultra-performance liquid chromatography, comprise the following steps:
(1) chromatographic condition is: take octadecylsilane chemically bonded silica as filling agent; With 0.026% phosphate aqueous solution for mobile phase A, acetonitrile is Mobile phase B, A: B=85 ~ 10: 15 ~ 90, gradient elution, in gradient elution process, the ratio of mobile phase A, B is changed to: 0 ~ 5min:A: B=85 ~ 75: 15 ~ 25,5 ~ 8min:A: B=75 ~ 70: 25 ~ 30,8 ~ 12min:A: B=70 ~ 50: 30 ~ 50,12 ~ 12.01min:A: B=50 ~ 10: 50 ~ 90,12.01 ~ 17min:A: B=10: 90; Determined wavelength is 286nm; Flow velocity is 0.2mLmin
-1; Column temperature is 30 DEG C; Number of theoretical plate calculates should be not less than 2000 by tanshin polyphenolic acid B peak;
(2) preparation of reference substance solution: get danshensu respectively, protocatechualdehyde, Rosmarinic acid, tanshin polyphenolic acid B and salviandic acid A reference substance appropriate, accurately weighed, add 5% methyl alcohol respectively to dissolve, make five kinds of reference substance storing solutions of 1mg/mL, draw each reference substance storing solution respectively, adding 5% methyl alcohol, to be mixed with danshensu reference substance concentration be 82.2 μ g/mL, protocatechualdehyde reference substance concentration is 15.5 μ g/mL, Rosmarinic acid reference substance concentration is 37.2 μ g/mL, tanshin polyphenolic acid B reference substance concentration is 522.5 μ g/mL, salviandic acid A reference substance concentration is the mixed solution of 32.2 μ g/mL, for subsequent use in 4 DEG C of refrigerations, product solution in contrast,
(3) preparation of need testing solution: the red rooted salvia powder 0.2g getting No. three sieves, accurately weighed, put in 100mL tool plug conical flask, add 40mL5% methyl alcohol, ultrasonic extraction 30min, supplies weight with 5% methyl alcohol; Filter, get subsequent filtrate, to obtain final product;
(4) measure: accurate absorption reference substance solution and each 1 μ L of need testing solution respectively, inject Ultra Performance Liquid Chromatography instrument to measure, record the chromatogram in 17 minutes, by chromatogram data importing " similarity evaluation " (2004A version), through establishing with reference to spectrum, Supplements, Auto-matching, generation contrast collection of illustrative plates and matched data, obtain the finger-print of red rooted salvia water soluble ingredient.
17 total peaks are had in measured red rooted salvia finger-print.
Claims (6)
1. the content assaying method of water soluble ingredient in red rooted salvia, it is characterized in that: described method is with danshensu, protocatechualdehyde, Rosmarinic acid, tanshin polyphenolic acid B and salviandic acid A reference substance for contrast, with 0.026% phosphate aqueous solution-acetonitrile=85 ~ 10: 15 ~ 90 is the ultra-performance liquid chromatography of eluent gradient wash-out.
2. the content assaying method of water soluble ingredient in red rooted salvia according to claim 1, is characterized in that: measure according to following steps:
(1) chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; With 0.026% phosphate aqueous solution for mobile phase A, acetonitrile is Mobile phase B, A: B=85 ~ 10: 15 ~ 90, gradient elution: 0 ~ 5min:A: B=85 ~ 75: 15 ~ 25,5 ~ 8min:A: B=75 ~ 70: 25 ~ 30,8 ~ 12min:A: B=70 ~ 50: 30 ~ 50,12 ~ 12.01min:A: B=50 ~ 10: 50 ~ 90,12.01 ~ 17min:A: B=10: 90; Determined wavelength is 276 ~ 306nm; Flow velocity is 0.1 ~ 0.5mLmin-1; Column temperature is 25 DEG C ~ 35 DEG C; Number of theoretical plate calculates should be not less than 2000 by tanshin polyphenolic acid B peak;
(2) preparation of reference substance solution: get danshensu, protocatechualdehyde, Rosmarinic acid, tanshin polyphenolic acid B and salviandic acid A reference substance appropriate, accurately weighed, add 5% methyl alcohol respectively to dissolve, make five kinds of reference substance storing solutions of 1mg/mL, draw each reference substance storing solution respectively, adding 5% methyl alcohol, to be mixed with danshensu reference substance concentration be 82.2 μ g/mL, protocatechualdehyde reference substance concentration is 15.5 μ g/mL, Rosmarinic acid reference substance concentration is 37.2 μ g/mL, tanshin polyphenolic acid B reference substance concentration is 522.5 μ g/mL, salviandic acid A reference substance concentration is the mixed solution of 32.2 μ g/mL, for subsequent use in 4 DEG C of refrigerations, product solution in contrast,
(3) preparation of need testing solution: get red rooted salvia powder 0.2g, accurately weighed, put in 100mL tool plug conical flask, add 40mL 5% methyl alcohol, ultrasonic extraction 30min, supplies weight with 5% methyl alcohol; Filter, get subsequent filtrate, to obtain final product;
(4) measure: accurate absorption reference substance solution and each 1 μ L of need testing solution respectively, inject Ultra Performance Liquid Chromatography instrument, measure, to obtain final product.
3. the content assaying method of water soluble ingredient in red rooted salvia according to claim 2, is characterized in that: in described step (1), determined wavelength is 286nm, and flow velocity is 0.2mLmin
-1, column temperature is 30 DEG C.
4. in red rooted salvia as described in any one of claims 1 to 3 water soluble ingredient content assaying method red rooted salvia finger-print detect in application.
5. the application of the content assaying method of water soluble ingredient in red rooted salvia according to claim 4, it is characterized in that: with finger-print software, the chromatogram in obtain in content assaying method 17 minutes is processed, obtain the finger-print of red rooted salvia water soluble ingredient.
6. the application of the content assaying method of water soluble ingredient in red rooted salvia according to claim 5, is characterized in that: have 17 total peaks in the finger-print of described red rooted salvia water soluble ingredient.
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