CN110068628A

CN110068628A - Radix Angelicae Sinensis standard decoction finger-print, characteristic spectrum is established and content assaying method

Info

Publication number: CN110068628A
Application number: CN201910418740.XA
Authority: CN
Inventors: 相雨; 杜蔼媚; 卢国扬; 马存
Original assignee: Sinopharm Dezhong Foshan Pharmaceutical Co Ltd
Current assignee: Sinopharm Dezhong Foshan Pharmaceutical Co Ltd
Priority date: 2019-05-20
Filing date: 2019-05-20
Publication date: 2019-07-30

Abstract

The present invention discloses that a kind of Radix Angelicae Sinensis standard decoction finger-print, characteristic spectrum is established and content assaying method, the method for building up of finger-print, comprising the following steps: the 1) preparation of test solution；2) chromatographic condition and system suitability；3) test solution is injected into liquid chromatograph, measures, obtains finger-print.By the research of the ultra performance liquid chromatography finger-print to Radix Angelicae Sinensis standard decoction, a kind of preferable Radix Angelicae Sinensis standard decoction finger-print has been obtained, has provided new technical method for control said preparation quality.

Description

Radix Angelicae Sinensis standard decoction finger-print, characteristic spectrum is established and content assaying method

Technical field

The present invention relates to Pharmaceutical Analysis field, a kind of Radix Angelicae Sinensis standard decoction finger-print is related generally to, characteristic spectrum is established And content assaying method.

Background technique

Decoction is in China's Traditional Chinese Medicine clinic using one of earliest, widest dosage form, is that patient takes Chinese medicine most Main dosage form.The Quality of Sliced Herbal Medicine is irregular, under background of medicine materical crude slice diversification of forms, the quality of decoction quality is evaluation drink The quality of piece even evaluates the most direct mode of Chinese Herbs.It is guidance that prepared slices of Chinese crude drugs standard decoction, which is with theory of traditional Chinese medical science, is faced Based on bed application, with reference to modern extracting method, single medicine materical crude slice water decoction that normalized technique is prepared.Prepared slices of Chinese crude drugs mark Quasi- decoction can be used as measure other medicine materical crude slice forms it is whether consistent with clinical decoction.Prepared slices of Chinese crude drugs standard decoction represents traditional decoction Ordinary circumstance, preparation method standard, specification, be easy to carry out qualitative and quantitative analysis.

After traditional Chinese medicine fingerprint refers to that certain Chinese medicines or Chinese materia medica preparation are appropriately processed, using certain analysis means, What is obtained can indicate the chromatogram or spectrogram of its chemical feature.It can more fully reflect chemical component contained by medicinal material Type and quantity, effectively embody traditional Chinese medicine ingredients globality and comprehensive function, it is quick with its, accurate the features such as transported extensively For Analysis of Chinese Traditional Medicine identification and quality control etc..

When the dry root for being classified as umbelliferae angelica Angelica sinensis (Oliv.) Diels, property is sweet, pungent, Temperature returns liver, the heart, the spleen channel, has the effect of replenishing and activating blood, regulating menstruation and relieving pain, moistening intestines and relaxing bowels.Radix Angelicae Sinensis standard decoction is by angelica sinensis Processed to be prepared, nature and flavor, channel tropism, effect and traditional decoction are almost the same.But it is led in Radix Angelicae Sinensis medicinal material analytical technology Domain, also useful ultrahigh speed liquid chromatography (UPLC) does not carry out Quality Control Analysis to Radix Angelicae Sinensis standard decoction and reports that it refers to yet Line map.

Therefore, the existing technology needs to be improved and developed.

Summary of the invention

In view of above-mentioned deficiencies of the prior art, the purpose of the present invention is to provide a kind of Radix Angelicae Sinensis standard decoction finger-print, Characteristic spectrum is established and content assaying method, by the research of the ultra performance liquid chromatography finger-print to Radix Angelicae Sinensis standard decoction, A kind of preferable Radix Angelicae Sinensis standard decoction finger-print has been obtained, has provided new technical method for control said preparation quality, it is intended to It solves the problems, such as insufficient to the Quality Control Technology of Radix Angelicae Sinensis standard decoction at present.

Technical scheme is as follows:

A kind of method for building up of Radix Angelicae Sinensis standard decoction finger-print, wherein the following steps are included:

1) preparation of test solution: taking Radix Angelicae Sinensis standard decoction, adds water, carries out ultrasonic extraction, lets cool, supply less loss with water Weight, be centrifuged, filtering；

2) chromatographic condition and system suitability: being stream with acetonitrile using octadecylsilane chemically bonded silica as filler Dynamic phase A, using 0.1% formic acid solution as Mobile phase B；Column temperature is 30-40 DEG C；Flow velocity is 0.2-0.4ml/min, and Detection wavelength is 270nm；

Wherein, gradient elution program is as follows: 0~3min, mobile phase A: 0%, Mobile phase B: 100%；3~5min, flowing Phase A:0% → 4%, Mobile phase B: 100% → 96%；5~16min, mobile phase A: 4% → 30%, Mobile phase B: 96% → 70%；16~17min, mobile phase A: 30% → 100%, Mobile phase B: 70% → 0%；17~20min, mobile phase A: 100%, Mobile phase B: 0%；

3) test solution is injected into liquid chromatograph, measures, obtains finger-print.

The method for building up of the Radix Angelicae Sinensis standard decoction finger-print, wherein in step 1) preferably, take Radix Angelicae Sinensis standard soup Agent 0.2g, be added water 25ml, ultrasonic extraction 10 minutes；Power is 400W, frequency 40kHz during the ultrasonic extraction.

The method for building up of the Radix Angelicae Sinensis standard decoction finger-print, wherein step 2) preferably uses CORTECS UPLC T3 chromatographic column, packing material size are 1.6 μm, internal diameter 2.1, column length 100mm；Column temperature is 30 DEG C；Flow velocity is 0.3ml/min；Step It is rapid 3) in test solution dosage be 5 μ l.

The method for building up of the Radix Angelicae Sinensis standard decoction finger-print, wherein further comprising the steps of:

4) obtained finger-print is subjected to similarity calculation with map is compareed.It is commented by chromatographic fingerprints of Chinese materia medica similarity Valence system, can be used for the overall evaluation of test sample quality, test article fingerprint with compare map through similarity calculation, similarity It must not be advisable lower than 0.90.

The method for building up of the Radix Angelicae Sinensis standard decoction finger-print, wherein the control map is as shown in figure 15.

A kind of method for building up of Radix Angelicae Sinensis standard decoction characteristic spectrum, wherein the following steps are included:

1) preparation of reference solution: taking ferulic acid reference substance, adds methanol that the solution that every 1ml contains 12 μ g is made；

2) preparation of test solution: taking Radix Angelicae Sinensis standard decoction, adds water, carries out ultrasonic extraction, lets cool, supply less loss with water Weight, be centrifuged, filtering；

3) chromatographic condition and system suitability: being stream with acetonitrile using octadecylsilane chemically bonded silica as filler Dynamic phase A, using 0.1% formic acid solution as Mobile phase B；Column temperature is 30-40 DEG C；Flow velocity is 0.2-0.4ml/min, and Detection wavelength is 270nm；

4) reference solution, test solution are injected into liquid chromatograph, measurement obtains characteristic spectrum.

The method for building up of the Radix Angelicae Sinensis standard decoction characteristic spectrum, wherein in step 2) preferably, take Radix Angelicae Sinensis standard soup Agent 0.2g, be added water 25ml, ultrasonic extraction 10 minutes；Power is 400W, frequency 40kHz during the ultrasonic extraction.

The method for building up of the Radix Angelicae Sinensis standard decoction finger-print, wherein step 3) preferably uses CORTECS UPLC T3 chromatographic column, packing material size are 1.6 μm, internal diameter 2.1, column length 100mm；Column temperature is 30 DEG C；Flow velocity is 0.3ml/min；Step It is rapid 4) in, reference solution dosage be 1 μ l, test solution dosage be 5 μ l.

The method for building up of the Radix Angelicae Sinensis standard decoction characteristic spectrum, wherein further comprising the steps of:

4) characteristic spectrum that will be obtained, peak corresponding with ferulic acid reference solution are the peak S, calculate characteristic peak and the peak S Relative retention time, relative retention time should be within ± the 5% of specified value.Specifically, should have 7 in test sample characteristic spectrum A characteristic peak, peak 1: uridine；Peak 2: adenosine；Peak 3: guanosine；Peak 5 (S): ferulic acid；Peak 6: senkyunolide I；Peak 7: foreign Rhizoma Chuanxiong Lactone H.

The method for building up of the Radix Angelicae Sinensis standard decoction characteristic spectrum, wherein the control map is as shown in figure 15, relatively Retention time specified value are as follows: peak 1,0.199；Peak 2,0.374；Peak 3,0.431；Peak 4,0.655；Peak 5,1；Peak 6,1.163；Peak 7, 1.207.The foundation of the liquid phase characteristic spectrum of the Radix Angelicae Sinensis standard decoction, can be used as the global quality control side of Radix Angelicae Sinensis standard decoction Method.

A kind of content assaying method of Radix Angelicae Sinensis standard decoction, wherein the following steps are included:

3) chromatographic condition and system suitability: using octadecylsilane chemically bonded silica as filler；With acetonitrile- 0.085% phosphoric acid solution is mobile phase, and the volume ratio of -0.085% phosphoric acid solution of acetonitrile is 17:83；25-40 DEG C of column temperature；Detection Wavelength is 316nm, flow velocity 0.2-0.8ml/min；Number of theoretical plate is calculated by forulic acid peak should be not less than 4000；

4) reference substance solution and each 1~5 μ l of test solution are drawn, liquid chromatograph is injected, measures test solution Ferulaic acid content.

The content assaying method of the Radix Angelicae Sinensis standard decoction, wherein in step 2) preferably, take Radix Angelicae Sinensis standard decoction 0.2g, be added water 25ml, ultrasonic extraction 30 minutes；During the ultrasonic extraction, power 400W, frequency 40kHz.

The content assaying method of the Radix Angelicae Sinensis standard decoction, wherein CORTECS UPLC is preferably used in step 3) C18 chromatographic column, packing material size are 2.7 μm, internal diameter 4.6, column length 50mm, and column temperature is 30 DEG C, flow velocity 0.5ml/min；Step 4) In preferably, reference substance solution dosage be 2 μ l, test solution dosage be 2 μ l.

Radix Angelicae Sinensis standard decoction is calculated by dry product, mean value 0.1361%, and SD value is 0.0418%.Assay mean value 70%~130% range is containing ferulic acid (C10H10O4) 0.0953%~0.177%, and assay mean value adds and subtracts 3 times of SD Range be containing ferulic acid (C10H10O4) 0.0108%~0.261%.The rate of transform mean value of ferulic acid in Radix Angelicae Sinensis standard decoction It is 7.08% for 67.71%, SD value.Every gram of Radix Angelicae Sinensis standard decoction is equivalent to 2 grams of angelica sinensis.The content of this Radix Angelicae Sinensis standard decoction Measuring method can be used for measuring Chinese medicinal granule and the whether consistent benchmark of clinical decoction, carry out with Chinese medicinal granule Comparative study.

The utility model has the advantages that establishing Radix Angelicae Sinensis standard decoction using similarity evaluation in the present invention UPLC finger-print, fingerprint similarity is good (>=0.90)；Using ferulic acid in UPLC method measurement Radix Angelicae Sinensis standard decoction Content, calculate the rate of transform of the Radix Angelicae Sinensis from medicine materical crude slice to standard decoction, the mean transferred rate of ferulic acid is in Radix Angelicae Sinensis standard decoction 67.71%, SD (standard deviation) are 7.08%.Therefore, the Radix Angelicae Sinensis standard decoction quality standard that the present invention establishes is reliable and stable, can Quality for Radix Angelicae Sinensis standard decoction controls.The finger-print and characteristic spectrum construction method of Radix Angelicae Sinensis standard decoction of the present invention with And content assaying method, have the advantages that following significant: (1) present invention is for the first time using UPLC technology, to Radix Angelicae Sinensis standard decoction into The research of row finger-print provides new analysis method for the control of Radix Angelicae Sinensis standard decoction quality.(2) optimize the building of finger-print Method, the finger-print baseline made is steady, separating degree is good, peak type is preferable, appearance amount is more.(3) test sample system of the present invention Make simply, chromatographic condition is easy to accomplish, and precision is high, and its stability and reproducibility are preferable, the detection wave that the present invention uses Long appearance is more, and the information of reflection is complete.(4) the on-line analysis time is short, saves material cost and time cost.

Detailed description of the invention

Fig. 1 is 190~400nm wave band 3D chromatogram in the embodiment of the present invention 1.

Fig. 2 is the chromatogram in the embodiment of the present invention 1 under 270nm wavelength.

Fig. 3 is the 3D chromatogram that mobile phase is under methanol-water (190~400nm) in the embodiment of the present invention 1.

It is mobile phase is 3D chromatography under acetonitrile-water (190~400nm) that Fig. 4, which is in the embodiment of the present invention 1,.

Fig. 5 is the 3D chromatogram that mobile phase is under acetonitrile-water (190~400nm) in the embodiment of the present invention 1.

Fig. 6 be the embodiment of the present invention 1 in 0.1% glacial acetic acid of mobile phase water phase (on) with 0.1% formic acid (under) to separate imitate The comparison diagram of fruit.

Fig. 7 be the embodiment of the present invention 1 in various concentration formic acid as mobile phase water phase to the comparison diagram of separating effect.

Fig. 8 is filler chromatographic columns different in the embodiment of the present invention 1 to the comparison diagram of separating effect.

Fig. 9 is column temperatures different in the embodiment of the present invention 1 to the comparison diagram of separating effect.

Figure 10 is the mobile phase comparison diagram different in flow rate to separating effect in the embodiment of the present invention 1.

Figure 11 is flow gradients different in the embodiment of the present invention 1 to the comparison diagram of separating effect.

Figure 12 is the comparison diagram of chromatogram under the conditions of different solvents in the embodiment of the present invention 1.

Figure 13 is the comparison diagram of different extracting mode chromatograms in the embodiment of the present invention 1.

Figure 14 is 19 batches of different Radix Angelicae Sinensis batch mark soup finger-prints in the embodiment of the present invention 1.

Figure 15 is that control map is generated in the embodiment of the present invention 1.

Figure 16 is the comparison diagram of finger-print specificity map test in the embodiment of the present invention 2.

Figure 17 is reference substance solution in the embodiment of the present invention 3, negative solution, test solution chromatogram.

Figure 18 is ferulic acid purity figure in the embodiment of the present invention 3.

Figure 19 is ferulic acid linear relationship chart and regression equation in the embodiment of the present invention 3.

Specific embodiment

The present invention provides that a kind of Radix Angelicae Sinensis standard decoction finger-print, characteristic spectrum is established and content assaying method, to make this The purpose of invention, technical solution and effect are clearer, define, and the present invention is described in more detail below.It should be appreciated that this Locate described specific embodiment to be only used to explain the present invention, be not intended to limit the present invention.

Below by way of specific embodiment, the present invention will be further described.

Instrument involved in embodiment:

Ultrahigh speed liquid phase H-CLASS UPLC (WATERS (China) Co., Ltd), Buchi R-100 Rotary Evaporators are (auspicious Shi Buqi Co., Ltd), Tianrey vacuum freeze drier, electronic balance (Sai Duolisi scientific instrument (Beijing) limited public affairs Department), YRE-501 Rotary Evaporators (Yuhua Instrument Co., Ltd., Gongyi City), DLSB-5/20 cryogenic liquid water circulating pump (Zhengzhou Greatwall Scientific Industrial & Trading Co., Ltd.), SHZ-D Ш vacuum pump using circulatory water (Yuhua Instrument Co., Ltd., Gongyi City), 101- 3BS electric drying oven with forced convection (Tianjing Huabei Laboratory Apparatus Co., Ltd.), 2-16N desk centrifuge, DC400 type Ultrasound Instrument (Dongguan City great Lang Su Jie ultrasonic device manufactory), II electric-heated thermostatic water bath of DK-98- (the limited public affairs of Tianjin Stettlen instrument Department), one piece of Shenzhen king's 500W health ceramic pot.

Reagent and material involved in embodiment:

Acetonitrile (chromatographically pure, Merck KGaA company), methanol (chromatographically pure, Merck KGaA company；Analyze pure, Jiangsu prosperity function Can chemical limited liability company), formic acid (chromatographically pure, Tianjin Ke Miou chemical reagent Co., Ltd), phosphoric acid (chromatographically pure, Tianjin Ke Miou chemical reagent Co., Ltd), ferulic acid reference substance (lot number 110773-201313, content 99.6%, Chinese drug food Research institute is determined in product examine).19 batches of Radix Angelicae Sinensis medicinal materials are purchased from Gansu, Qinghai and other places.

The preparation of Radix Angelicae Sinensis standard decoction:

Angelica sinensis 150g is taken, secondary, first time decoction plus 9 times of amount water, immersion 40 minutes, 60 points of decoction are added water to cook Clock is filtered while hot with 400 mesh screens, and filtrate is cooling with cold water rapidly.Second of decoction plus 7 times of amount water decoct 40 minutes, use 400 mesh screens filter while hot, and the rapid cold water of filtrate is cooling, merge filtrate twice.Filtrate after merging is transferred to rotary evaporation It is concentrated under reduced pressure in instrument, is concentrated into right amount, is freeze-dried to get Radix Angelicae Sinensis standard decoction.

Embodiment 1: finger-print is established

One, the investigation of Radix Angelicae Sinensis standard decoction chromatographic condition

1, chromatographic condition and system suitability

It is accurate respectively to draw 5 μ l of test solution, inject liquid chromatograph, measurement, record chromatogram to get.Radix Angelicae Sinensis mark Quasi- decoction finger-print primary condition is as shown in table 1, and eluent gradient table is as shown in table 2.According to high performance liquid chromatography (" China Pharmacopeia " 2015 years four general rules 0512 of version) measurement.

1 Radix Angelicae Sinensis standard decoction finger-print primary condition of table

2 initial liquid phase gradient table of table

Time (minute)	Mobile phase A (%)	Mobile phase B (%)
			0	10	90
7	90	10
			10	100	0

(1) selection of Detection wavelength

On the basis of the experiment condition drafted above, 190~400nm wave is carried out to test solution using PDA detector Section scanning, and the chromatogram of test solution is extracted, as shown in Figure 1.

Under ultraviolet spectra 190 to 400nm, mobile wavelength finds at low wavelength (190-220nm), peak response is very strong, Peak type is more；Into high wavelength moving process, the response at peak integrally weakens, and the Whole Response at each peak is preferable near 270nm.Figure 2 be wavelength be chromatogram under 270nm.

The result shows that: comprehensively consider the type of chromatographic peak, the stationarity of response intensity and baseline, select wavelength 220nm with 270nm.In addition to weak retained fraction response difference is larger, 220nm and 270nm, difference is not very big on the whole, more stable to seek Baseline, while convenient for mass spectrometry analyze, select 270nm.

(2) selection of organic phase

It is tested using methanol-water, two kinds of acetonitrile-water combinations as organic phase, using PDA detector to test solution The scanning of 190~400nm wave band is carried out, and extracts the chromatogram of test solution, as in Figure 3-5, Fig. 3 is that mobile phase is first 3D chromatogram under alcohol-water (190~400nm), Fig. 4 are that mobile phase is 3D chromatogram under acetonitrile-water (190~400nm), figure 5 be mobile phase be 3D chromatogram under acetonitrile-water (190~400nm).

As in Figure 3-5, the organic phase of methanol and acetonitrile as chromatographic isolation is respectively adopted in same test solution, Under same water phase gradient condition, the appearance quantity of acetonitrile and methanol is not much different, but in the separating degree of baseline smoothness, certain peaks From the point of view of, acetonitrile is slightly excellent.

(3) selection of mobile phase water phase

Using the type and concentration for comparing different acid, investigating chromatography global behavior influences, and selection is best.

1) same sample and chromatographic condition (water phase is different), compare 0.1% glacial acetic acid and 0.1% formic acid to separating effect It influences.Conclusion: as shown in fig. 6, the comparison diagram of 0.1% glacial acetic acid of mobile phase water phase (1) and 0.1% formic acid (2) to separating effect, The chromatogram of 0.1% formic acid is slightly better than 0.1% glacial acetic acid, selects formic acid for water phase.

2) same sample and chromatographic condition (water phase is different), formic acid various concentration compares: as shown in fig. 7, conclusion: 0.1% formic acid is slightly better than other concentration.

(4) selection of chromatographic column

Same batch sample, identical solution manufacturing method are separated using different chromatographic columns.Retained according to chromatographic column The difference of ability adjusts eluent gradient, under the premise of main chromatographic peak retains and shows comparable, more different filler chromatographic columns Separating effect:

1) chromatographic column: Waters CORTECS T3 (1.6 μm, 2.1*100mm)；

2) chromatographic column: Waters BEH C18 (1.7 μm, 2.1*50mm)；

3) chromatographic column: Waters BEH RP18 (1.7 μm, 2.1*100mm)；

4) chromatographic column: Waters CORTECS C18 (2.7 μm, 3.0*100mm)；

5) chromatographic column: Waters HSS T3 (1.8 μm, 2.1*100mm).

Conclusion: as shown in figure 8,2), 3), 4), 5) chromatographic peak of number chromatographic column compared with the 1) chromatographic peak of number chromatographic column: peak Type, separating degree are poor, and appearance quantity is few.Select chromatographic column: Waters CORTECS T3 (1.6 μm, 2.1*100mm).

(5) screening of column temperature

On the basis of the experiment condition drafted, investigated when being respectively 30 DEG C, 35 DEG C, 40 DEG C to column temperature.Such as Fig. 9 institute Show, the results showed that, for the separating degree at individual peaks, 30 DEG C of column temperature is preferably.

(6) screening of flow velocity

On the basis of the experiment condition drafted, respectively to flow velocity be 0.2mL/min, 0.3mL/min, 0.4mL/min when into Investigation is gone.As shown in Figure 10, the results showed that, when flow velocity is 0.3ml/min, chromatogram peak shape is preferable, and separating degree is moderate.Therefore Flow velocity is determined as 0.3ml/min.

(7) optimization of gradient

On the basis of the experiment condition drafted, using the mobile phase tonsure after initial liquid phase gradient table (table 4) and optimization Table (table 3) is investigated.It as shown in figure 11, is 1) the separating effect figure of initial flow gradient, 2) it is mobile phase ladder after optimization Spend the separating effect figure of table, the results showed that, under gradient after optimization, standard decoction main component realizes preferable separation.

Eluent gradient table after the optimization of table 3

Time (minute)	Mobile phase A (%)	Mobile phase B (%)
			0~3	0	100
3~5	0→4	100→96
			5~16	4→30	96→70
16~17	30→100	70→0
			17~20	100	0

In conclusion Radix Angelicae Sinensis standard decoction finger-print chromatographic condition determines are as follows: be with octadecylsilane chemically bonded silica Filler (chromatographic column: Waters CORTECS T3 (1.6 μm, 2.1*100mm))；Using acetonitrile as mobile phase A, with 0.1% formic acid Solution is Mobile phase B, carries out gradient elution by the regulation in table 5；Column temperature is 30 DEG C；Flow velocity is 0.3ml/min, and Detection wavelength is 270nm。

Two, the foundation of sample solution preparation method

1, extraction time is investigated

This product (lot number B01) about 0.2g is taken, precision is weighed, set in triangular flask, and water 25ml is added in precision, is shaken up, and is claimed Determine weight, ultrasonic extraction 5,10,15min, let cool, weigh again, supply weight with water, shake up respectively, filtering to get.

The results are shown in Table 4, the results showed that, at the extraction between be 5 minutes when, can sufficiently extract, for guarantee dissolved Entirely, the selective extraction time is determined as 10 minutes.

The different extraction times of table 4 investigate (peak area/sample weighting amount)

2, Extraction solvent selects

This product (lot number B01) about 0.15g is taken, precision is weighed, set in triangular flask, accurate respectively that methanol, 50% is added Methanol, water 25ml, shake up, weighed weight, and ultrasonic 10min lets cool, weighs again, supply weight with coordinative solvent, filtering, i.e., ?.

The result shows that as shown in figure 12, when solvent is water, chromatographic peak contains much information, and separating degree is good.And water is as extraction Solvent is easy, nontoxic, therefore test sample Extraction solvent is determined as water.

3, extracting mode is investigated

This product (lot number B01) about 0.2g is taken, precision is weighed, set in triangular flask, and water 25ml is added in precision, is shaken up, and is claimed Determine weight, respectively reflux, ultrasonic extraction 10min, let cool, weigh again, supply weight with phase water, shake up, filtering to get.

The result shows that as shown in figure 13, it is consistent with effect when refluxing extraction to carry out ultrasonic extraction respectively to test sample, in addition to The chromatographic peak of refluxing extraction has more a small peak, and other peak positions are the same.Because ultrasonic extraction operation is more easy, and temperature It is low, therefore test sample extracting method is determined as ultrasonic extraction.

In conclusion the preparation method of Radix Angelicae Sinensis standard decoction finger-print test solution determines are as follows: take this product powder about 0.2g, it is accurately weighed, it sets in triangular flask, precision addition water 25ml, close plug, weighed weight, ultrasonic treatment (power 400W, Frequency 40kHz) 10 minutes, let cool, again weighed weight, the weight of less loss supplied with water, is shaken up, be centrifuged, filtering to get.

Three, methodology validation

1, specificity is tested

The preparation of test solution: by the aforementioned experiment condition drafted, Radix Angelicae Sinensis standard decoction test solution is prepared.It is negative The preparation of contrast solution: blank solvent (ultrapure water).Record chromatogram.

The result shows that negative control solution is almost noiseless.

2, precision test

(1) instrument precision

Precision weighs 1 part of Radix Angelicae Sinensis freeze-dried powder (lot number B01), carries out preparation and continuous sample introduction 6 times, note by test method is drafted Finger-print is recorded, similarity is calculated.The result shows that the similarity of map is 1 or 0.999, the instrument precision is good.

(2) repeated

Precision weighs 6 parts of Radix Angelicae Sinensis freeze-dried powder (lot number B01), is prepared and is measured by test method is drafted, and fingerprint is recorded Map calculates similarity.The result shows that the similarity of map is 1 or 0.999, this method is reproducible.

(3) Intermediate precision

Experimenter A1 prepares 6 parts of test solution in date B1, every part of 1 needle of sample introduction on instrument C1, records feature Or finger-print；Experimenter A2 prepares 6 parts of test solution in date B1, every part of 1 needle of sample introduction on instrument C1, records color Spectrogram map.Calculate the similarity of 6*2 map.The result shows that the similarity of map is 1 or 0.999, this method intermediate precision Degree is good.

3, durability

(a) study on the stability

On the basis of the experiment condition drafted above, take 1 part of test solution, respectively at different time (0,2,4,6,8, 12,16,20,24) hour sample introduction, records finger-print, calculates similarity.

The result shows that the similarity of map is 1 and 0.998, this method has good stability.

(b) phase concentration is flowed

On the basis of the experiment condition drafted above, 1 part of test solution is taken, the sample introduction under different flowing phase concentrations, note Finger-print is recorded, similarity is calculated.

The result shows that the slight change of this method mobile phase acidity influences very little to experimental result.

(c) column temperature

On the basis of the experiment condition drafted above, 1 part of test solution is taken, sample introduction under the conditions of different column temperatures records Finger-print calculates similarity.

The result shows that the slight change of this method column temperature influences very little to experimental result.

Four, the foundation of map is compareed

The measurement that using the method drafted 19 batches of samples of this product are carried out with single needle finger-print, is recorded finger-print, such as schemed Shown in 14, " similarity evaluation " (2.0 editions) similarity software is imported, uses average value for control Map generating mode, time window width 0.1, Supplements generate control map, and find 7 shared fingerprint peaks, such as Figure 15 institute Show.Wherein, peak 1: uridine；Peak 2: adenosine；Peak 3: guanosine；Peak 5 (S): ferulic acid；Peak 6: senkyunolide I；Peak 7: in foreign Rhizoma Chuanxiong Ester H.

Four, it measures

Using similarity evaluation, UPLC figure is generated to 19 batches of Radix Angelicae Sinensis standard decoction samples Spectrum, is matched, matching result is as shown in table 5 with finger-print.The experimental results showed that 19 batches of Radix Angelicae Sinensis standard decoction (freeze-dryings Powder) similarity is above 0.9, each quality of lot stable homogeneous.

5 different batches mark soup finger-print of table and finger-print matching result

The foundation of 2 characteristic spectrum of embodiment

Chromatographic condition: by the final experiment condition drafted in embodiment 1.

The preparation of reference solution: taking ferulic acid reference substance appropriate, accurately weighed, sets in brown measuring bottle, methanol is added to be made Every 1ml containing 12 μ g solution to get.

Measuring method: it is accurate respectively to draw 1 μ l of reference solution and 5 μ l of test solution, liquid chromatograph is injected, is surveyed It is fixed, record chromatogram to get.

One, the foundation of sample solution preparation method

1, Extraction solvent selects

As a result Figure 12 of visible embodiment 1, the results showed that, when mobile phase is water, chromatographic peak contains much information, separating degree It is good.And water is easy, nontoxic as Extraction solvent, therefore test sample Extraction solvent is determined as water.

2, extracting mode is investigated

The different extracting modes of table 6 investigate (peak area/sample weighting amount)

As a result Figure 12 and table 6 of visible embodiment 1, the results showed that, carry out ultrasonic extraction and reflux respectively to test sample Each characteristic peak (peak area/sample weighting amount) when extraction, ultrasound is preferable than reflowing result, and in addition to the chromatographic peak of refluxing extraction has more One small peak, other peak positions are the same.Test sample extracting method is determined as ultrasonic extraction.

3, extraction time is investigated

The different extraction times of table 7 investigate (peak area/sample weighting amount)

As a result as shown in Figure 14 and table 7, the results showed that, at the extraction between each extraction time peak area/sample weighting amount difference Less, to guarantee dissolution completely, the selective extraction time is determined as 10 minutes.

4, filter Absorbability

1 part of test solution is prepared, sample introduction under different filter conditions, investigates characteristic peak peak area after being centrifuged 30min Consistency the results are shown in Table 8.

8 filter Absorbability experimental result (peak area) of table

Number	Peak 1	Peak 2	Peak 3	Peak 4	Peak 5 (S)	Peak 6	Peak 7
								Film is not crossed	141352	193913	183381	156969	333317	209907	43717
It crosses film and does not discard solution	139504	192885	182606	151076	317225	199918	44200
								It crosses film and discards 0.5ml solution	142366	194937	184248	164041	339820	205014	44474
It crosses film and discards 1ml solution	141897	194637	184139	163797	335530	204117	44391
								It crosses film and discards 1.5ml solution	142042	194617	184106	163889	326407	203558	44079
It crosses film and discards 2ml solution	142524	194897	184198	163988	325566	203750	44045
								Average value	141614	194314	183780	160627	329644	204377	44151
RSD (%)	0.785	0.407	0.358	3.390	2.476	1.578	0.615

The result shows that RSD≤5% of characteristic peak peak area, filter without absorption or suction-operated very little, does not influence sample The response of chromatographic peak.

In conclusion the preparation method of Radix Angelicae Sinensis standard decoction characteristic spectrum test solution determines are as follows: take this product powder about 0.2g, it is accurately weighed, it sets in triangular flask, precision addition water 25ml, close plug, weighed weight, ultrasonic treatment (power 400W, Frequency 40kHz) 10 minutes, let cool, again weighed weight, the weight of less loss supplied with water, is shaken up, be centrifuged, filtering to get.

Two, methodology validation

1, specificity is tested

The preparation of test solution: by the aforementioned final experiment condition drafted, Radix Angelicae Sinensis standard decoction test solution is prepared.

The preparation of negative control solution: blank solvent (ultrapure water).

Record chromatogram.The result shows that as shown in figure 16, negative control solution is noiseless to characteristic peak, reference solution It is consistent with one of characteristic peak appearance time.Peak corresponding with object of reference peak is the peak S (peak 5), confirms that No. 5 peaks are ferulic acid. The relative retention time of each characteristic peak Yu the peak S is calculated, it should be within the scope of ± the 5% of specified value.Relative retention time specified value Are as follows: 0.199 (peak 1), 0.374 (peak 2), 0.431 (peak 3), 0.655 (peak 4), 1 (peak 5), 1.163 (peaks 6), 1.207 (peaks 7).

2, precision

1) instrument precision

Precision weighs 1 part of Radix Angelicae Sinensis freeze-dried powder (lot number B01), carries out preparation and continuous sample introduction 6 times, note by test method is drafted Map is recorded, the consistency of characteristic peak relative retention time and relative peak area is investigated, the results are shown in Table 9 and table 10.

9 precision experiment result of table (relative retention time)

Number	Peak 1	Peak 2	Peak 3	Peak 4	Peak 5 (S)	Peak 6	Peak 7
								1	0.203	0.384	0.441	0.661	1	1.160	1.203
2	0.204	0.386	0.441	0.661	1	1.159	1.203
								3	0.204	0.386	0.441	0.660	1	1.159	1.203
4	0.203	0.382	0.438	0.658	1	1.160	1.204
								5	0.202	0.380	0.437	0.658	1	1.160	1.203
6	0.203	0.384	0.439	0.659	1	1.160	1.204
								Average value	0.203	0.384	0.440	0.659	1	1.160	1.203
RSD (%)	0.390	0.598	0.361	0.197	0	0.038	0.043

10 precision experiment result of table (relative peak area)

Number	Peak 1	Peak 2	Peak 3	Peak 4	Peak 5 (S)	Peak 6	Peak 7
								1	0.241	0.278	0.225	0.496	1	0.199	0.035
2	0.242	0.281	0.227	0.504	1	0.201	0.035
								3	0.249	0.287	0.232	0.522	1	0.201	0.036
4	0.241	0.278	0.225	0.499	1	0.198	0.035
								5	0.244	0.279	0.224	0.497	1	0.198	0.034
6	0.242	0.278	0.224	0.495	1	0.194	0.035
								Average value	0.243	0.280	0.226	0.502	1	0.199	0.035
RSD (%)	1.177	1.228	1.321	1.989	0	1.343	1.807

The result shows that RSD≤3% of characteristic peak relative retention time and relative peak area, the instrument precision is good.

2) repeated

Precision weighs 6 parts of Radix Angelicae Sinensis freeze-dried powder (lot number B01), is prepared and is measured by test method is drafted, and feature is investigated The consistency of peak relative retention time and relative peak area the results are shown in Table 11 and table 12.

11 repeated experiment result (relative retention time) of table

12 repeated experiment result (relative peak area) of table

Number	Peak 1	Peak 2	Peak 3	Peak 4	Peak 5 (S)	Peak 6	Peak 7
								1	0.267	0.358	0.333	0.464	1	0.293	0.050
2	0.264	0.355	0.330	0.498	1	0.292	0.050
								3	0.265	0.356	0.331	0.500	1	0.293	0.050
4	0.265	0.357	0.332	0.501	1	0.294	0.050
								5	0.266	0.358	0.330	0.500	1	0.293	0.050
6	0.266	0.357	0.331	0.201	1	0.293	0.050
								Average value	0.265	0.357	0.331	0.494	1	0.293	0.050
RSD (%)	0.400	0.330	0.347	2.981	0	0.230	0.292

The result shows that RSD≤3% of characteristic peak relative retention time and relative peak area, this method are reproducible.

3) Intermediate precision

Experimenter A1 prepares 6 parts of test solution in date B1, every part of 1 needle of sample introduction on instrument C1, records feature Or finger-print；Experimenter A2 prepares 6 parts of test solution in date B1, every part of 1 needle of sample introduction on instrument C1, investigates special The consistency for levying peak relative retention time and relative peak area, the results are shown in Table 13 and table 14.

13 Intermediate precision experimental result (relative retention time) of table

Number	Peak 1	Peak 2	Peak 3	Peak 4	Peak 5 (S)	Peak 6	Peak 7
								1	0.199	0.373	0.430	0.653	1	1.162	1.206
2	0.200	0.375	0.431	0.653	1	1.162	1.206
								3	0.200	0.373	0.432	0.654	1	1.162	1.206
4	0.200	0.377	0.433	0.654	1	1.162	1.206
								5	0.200	0.376	0.432	0.654	1	1.162	1.206
6	0.199	0.375	0.432	0.655	1	1.162	1.206
								7	0.200	0.377	0.433	0.653	1	1.163	1.207
8	0.200	0.377	0.433	0.654	1	1.163	1.207
								9	0.200	0.377	0.433	0.654	1	1.162	1.206
10	0.200	0.377	0.433	0.654	1	1.163	1.207
								11	0.200	0.376	0.433	0.654	1	1.162	1.206
12	0.200	0.377	0.433	0.654	1	1.162	1.206
								Average value	0.200	0.376	0.432	0.654	1	1.162	1.206
RSD (%)	0.229	0.333	0.208	0.081	0	0.018	0.018

14 Intermediate precision experimental result (relative peak area) of table

The result shows that RSD≤3% of characteristic peak relative retention time and relative peak area, this method Intermediate precision is good It is good.

4) study on the stability

On the basis of the experiment condition drafted above, take 1 part of test solution, respectively at different time (0,2,4,6,8, 12,16,20,24) hour sample introduction, investigates the consistency of characteristic peak relative retention time and relative peak area, it the results are shown in Table 15~ 17。

15 stability experiment result (relative retention time) of table

Time	Peak 1	Peak 2	Peak 3	Peak 4	Peak 5 (S)	Peak 6	Peak 7
								0	0.190	0.354	0.416	0.651	1	1.162	1.206
2	0.191	0.355	0.417	0.652	1	1.162	1.207
								4	0.192	0.359	0.420	0.651	1	1.162	1.206
6	0.195	0.367	0.424	0.651	1	1.163	1.207
								8	0.196	0.368	0.426	0.651	1	1.163	1.207
12	0.196	0.368	0.426	0.651	1	1.162	1.207
								16	0.197	0.370	0.427	0.650	1	1.163	1.208
20	0.197	0.370	0.428	0.651	1	1.163	1.207
								24	0.201	0.381	0.434	0.655	1	1.162	1.206
Average value	0.195	0.366	0.424	0.651	1	1.162	1.207
								RSD (%)	1.670	2.292	1.351	0.225	0	0.045	0.057

16 stability experiment result (relative peak area) of table

Time	Peak 1	Peak 2	Peak 3	Peak 4	Peak 5 (S)	Peak 6	Peak 7
								0	0.216	0.231	0.193	0.415	1	0.172	0.030
2	0.215	0.231	0.193	0.414	1	0.172	0.030
								4	0.209	0.235	0.193	0.440	1	0.173	0.029
6	0.205	0.225	0.193	0.441	1	0.172	0.030
								8	0.206	0.226	0.194	0.437	1	0.174	0.030
12	0.207	0.227	0.194	0.439	1	0.172	0.030
								16	0.211	0.228	0.195	0.436	1	0.172	0.031
20	0.209	0.229	0.196	0.441	1	0.173	0.031
								24	0.186	0.224	0.188	0.440	1	0.171	0.031
Average value	0.207	0.228	0.193	0.434	1	0.172	0.030
								RSD (%)	4.253	1.449	1.189	2.543	0	0.450	2.101

17 stability experiment result (relative peak area) of table

The result shows that 0~24 hour characteristic peak relative retention time RSD≤3%, and No. 1 characteristic peak relative peak area RSD > 3%, illustrate that this method is bad in 0~24 hour stability；0~20 hour characteristic peak relative retention time and relative peak area RSD≤3% illustrates that this method had good stability at 0~20 hour.

Three, the foundation of map is compareed

Referring to the finger-print that embodiment 1 generates, characteristic peak number is as shown in table 18 below with relative retention time.

18 characteristic peak of table number and relative retention time

Characteristic peak number	Relative retention time
		1	0.199
2	0.374
		3	0.431
4	0.655
		5	1
6	1.163
		7	1.207

19 batches of different batches mark soup Fingerprints peak relative retention times and relative peak area are tested, are surveyed Test result is as shown in table 19 and 20.The result shows that different batches mark soup characteristic spectrum characteristic peak relative retention time RSD (%) < 3%, and relative peak area RSD (%) value is very big, therefore, using relative retention time as the index of characteristic peak.

19 different batches Radix Angelicae Sinensis mark soup characteristic peak experimental result (relative retention time) of table

20 stability experiment result (relative peak area) of table

3 Radix Angelicae Sinensis standard decoction assay of embodiment

Using the content of ferulic acid as the quality evaluation index of its medicinal material in pharmacopeia.This method refers to " Chinese Pharmacopoeia " 2015 Content assaying method under year one Radix Angelicae Sinensis medicinal material item of version is worked out, and is verified to it.

One, chromatographic condition and system suitability

The preparation of reference substance solution: taking ferulic acid reference substance appropriate, accurately weighed, sets in brown measuring bottle, methanol is added to be made Every 1ml containing 12 μ g solution to get.

Radix Angelicae Sinensis standard decoction content assaying method primary condition is as shown in table 21.

21 Radix Angelicae Sinensis standard decoction content assaying method primary condition of table

1, the determination of Detection wavelength

The acquisition of 200~400nm wave spectrum is carried out to the forulic acid peak of test solution, by the analysis to spectrogram, And with reference to the detection wave in the measuring method of " ferulic acid " under one Radix Angelicae Sinensis [assay] item of " Chinese Pharmacopoeia " version in 2015 It is long, determine that the best inspection wavelength of the assay of ferulic acid in Radix Angelicae Sinensis standard decoction is 316nm.

2, mobile phase

On the basis of the experiment condition drafted above, 1 part of test solution is taken, investigates 1) acetonitrile: 0.085% phosphoric acid respectively =13:87,2) methanol: 2% acetic acid=18:82,3) acetonitrile: the chromatography under the conditions of 2% acetic acid=10:90, tri- kinds of flow phase systems Index.The relevant parameter of different mobile phase ratios is more as shown in table 22.

The comparison of the different mobile phase ratios of table 22

Mobile phase ratio	Retention time	Separating degree	Symmetrical factor	Number of theoretical plate
					Acetonitrile: 0.085% phosphoric acid=13:87	4.384	2.657	1.139	4173
Methanol: 2% acetic acid=18:82	10.762	4.903	1.017	5112
					Acetonitrile: 2% acetic acid=10:90	9.261	5.018	1.061	5738

The result shows that three kinds of mobile phases can be separated and be detected to ferulic acid, mobile phase acetonitrile: 0.085% phosphoric acid The retention time of=13:87 is most short, and separating degree, symmetrical factor, number of theoretical plate are ideal, therefore selects the mobile phase to elute item Part.

3, flow velocity is investigated

On the basis of the experiment condition drafted above, 1 part of test solution is taken, the chromatography under the conditions of investigation is different in flow rate refers to Number.Relevant parameter different in flow rate is more as shown in table 23.

The relevant parameter different in flow rate of table 23 compares

Flow velocity	Retention time	Separating degree	Symmetrical factor	Number of theoretical plate
					0.2ml/min	8.039	1.855	1.141	4847
0.3ml/min	5.347	1.970	1.105	5501
					0.4ml/min	4.006	2.019	1.245	5740
0.5ml/min	3.204	2.131	1.141	6686
					0.6ml/min	2.681	2.159	1.161	6827
0.8ml/min	2.009	2.169	1.173	6855

The result shows that above-mentioned flow velocity can be separated and be detected to ferulic acid, and as flow velocity increases, separating degree and theory Plate number gradually improves, and symmetrical factor reduces again from low to high, therefore selecting intermediate flow velocity 0.5ml/min is elution flow rate.

4, column temperature

On the basis of the experiment condition drafted above, 1 part of test solution is taken, the chromatography under the conditions of different column temperatures is investigated and refers to Number.The relevant parameter comparison result of different column temperatures is as shown in table 24.

The relevant parameter of the different column temperatures of table 24 compares

Column temperature	Retention time	Separating degree	Symmetrical factor	Number of theoretical plate
					25℃	4.785	2.556	1.102	4637
30℃	4.384	2.657	1.139	4173
					35℃	4.066	1.693	1.153	3749
40℃	3.804	1.310	1.124	3555

The result shows that the separating degree of ferulic acid chromatographic peak is significantly better than other column temperatures, therefore column temperature selects under 30 DEG C of column temperatures 30℃。

(5) different sample volumes

On the basis of the experiment condition drafted above, 1 part of test solution is taken, the chromatography under the conditions of different sample volumes is investigated Index.The comparison result of different sample volume relevant parameters is as shown in Table 25.

The comparison of the different sample volume relevant parameters of table 25

Sample volume	Retention time	Separating degree	Symmetrical factor	Number of theoretical plate
					1μl	4.102	2.180	1.233	6358
2μl	4.006	2.019	1.245	5740
					3μl	4.001	1.903	1.245	5093
4μl	3.997	1.754	1.232	4409
					5μl	3.985	1.531	1.198	3611

The result shows that separating degree and number of theoretical plate gradually decrease as sample volume increases, the accuracy of sample introduction is considered, therefore Sample volume selects 2 μ l.

(6) chromatographic column

Same test sample (Radix Angelicae Sinensis mark soup B01) is taken, test solution is prepared by the method for drafting, under the conditions of different chromatographic columns Sample introduction records chromatogram.

Chromatographic column 1:Waters CORTECS C18 (2.7 μm, 4.6*50mm)；

Chromatographic column 2:Thermo Acclaim^TMRSLC (2.2 μm,100*2.1mm)；

Chromatographic column 3:Waters BEH C18 (1.7 μm, 2.1*50mm).

The comparison of different chromatographic column relevant parameters is as shown in table 26.

The comparison of the different chromatographic column relevant parameters of table 26

Chromatographic column	Retention time	Separating degree	Symmetrical factor	Number of theoretical plate
					1	4.010	1.755	1.204	4403
2	6.091	3.538	1.295	6901
					3	1.153	2.347	1.163	6209

The result shows that this experimental method can be separated and be detected to ferulic acid in different chromatographic columns.

In conclusion Radix Angelicae Sinensis standard decoction content detection chromatographic condition determines are as follows: be with octadecylsilane chemically bonded silica Filler；With -0.085% phosphoric acid solution of acetonitrile (17:83) for mobile phase；30 DEG C of column temperature；Detection wavelength is 316nm, and flow velocity is 0.5ml/min.Number of theoretical plate is calculated by forulic acid peak should be not less than 4000.

Two, the foundation of sample solution preparation method

1, the selection of solvent is extracted

Same batch sample (Radix Angelicae Sinensis mark soup B01) 8 parts of fine powder each about 0.2g are taken, it is accurately weighed, it sets in triangular flask, point Inaccurate that different solvents 25ml is added, close plug, weighed weight, ultrasonic 20min is let cool, then weighed weight, is supplied with coordinative solvent The weight of less loss, shakes up, stand, take supernatant, filter, take subsequent filtrate to get.Respectively take 2 μ l injecting chromatographs, analytical calculation its Respective content.Different solvents comparison result is as shown in table 27.

27 different solvents of table compare

The result shows that solvent is the test solution content phase of methanol, 70% methanol and 45% ethyl alcohol-glacial acetic acid (20:1) When, but sample is dissolved in the solvent of 70% methanol and 45% ethyl alcohol-glacial acetic acid (20:1) mixed liquor, the sad filter of solution, therefore select Selecting methanol is solvent.

2, extracting mode is investigated

Same batch sample (Radix Angelicae Sinensis mark soup B01) 4 parts of fine powder each about 0.2g are taken, it is accurately weighed, it sets in triangular flask, essence Close addition methanol 25ml, close plug, weighed weight, 2 parts of ultrasounds, 2 parts of reflux extract 30min, let cool, then weighed weight, use methanol The weight for supplying less loss, shakes up, stand, take supernatant, filter, take subsequent filtrate to get.Respectively take 2 μ l injecting chromatographs, analysis meter Calculate its respectively content.Shown in different extracting mode comparison result icons 28.

The different extracting modes of table 28 compare

The result shows that ultrasonic extracting method is easy, sample heating temperature is low, and content is higher, therefore ultrasound is selected to mention It takes.

3, ultrasonic time is investigated

Same batch sample (Radix Angelicae Sinensis mark soup B01) 6 parts of fine powder each about 0.2g are taken, it is accurately weighed, it sets in triangular flask, essence Close addition methanol 25ml, close plug, weighed weight, ultrasound 20min, 30min, 40min, let cool, then weighed weight, use methanol respectively The weight for supplying less loss, shakes up, stand, take supernatant, filter, take subsequent filtrate to get.Respectively take 2 μ l injecting chromatographs, analysis meter Calculate its respectively content.Different ultrasonic time comparison results are as shown in table 29.

The different ultrasonic times of table 29 compare

The result shows that ultrasonic 20min and ultrasound 30min result difference are smaller, to guarantee to extract completely, using ultrasonic extraction 30min mode.

In conclusion the preparation method of Radix Angelicae Sinensis standard decoction assay test solution determines are as follows: take this product powder about 0.2g, it is accurately weighed, it sets in triangular flask, methanol 25ml, close plug is added in precision, and weighed weight is ultrasonically treated (power 400W, frequency 40kHz) 30 minutes, it lets cool, again weighed weight, the weight of less loss is supplied with methanol, is shaken up, filter, take continuous filter Liquid to get.

Three, methodology validation

1, specificity

Test solution: it takes test sample (Radix Angelicae Sinensis mark soup B01) by the aforementioned experiment condition drafted, prepares Radix Angelicae Sinensis standard decoction Content test solution.

Reference substance solution: taking ferulic acid reference substance appropriate, accurately weighed, sets in brown measuring bottle, adds methanol that every 1ml is made and contains The solution of 12 μ g to get.

Negative control solution: blank solvent solution (methanol).

It is accurate respectively to draw test solution, reference substance solution and each 2 μ l of blank solvent solution, liquid chromatograph is injected, Measurement to get.Chromatogram is as shown in figure 17, and test solution has at retention time corresponding with ferulic acid reference substance solution Corresponding chromatographic peak；And negative solution at the corresponding retention time of ferulic acid without corresponding chromatographic peak, illustrate in test solution Ferulic acid composition measurement is noiseless, and method is exclusive.The purity testing result figure of ferulic acid is as shown in figure 18, the peak purity angle (0.456) < threshold angle (0.768), the peak purity are higher.

2, linear relationship is investigated

Ferulic acid reference substance (content 99.6%) is taken, phosphorus pentoxide is set and is dried under reduced pressure 12h or more, precision weighs in right amount, Add methanol to make to dissolve, the reference substance solution that concentration is 2.436,9.745,19.49,48.724,121.81 μ g/ml is made.Respectively Precision draws above-mentioned 5 concentrations control product solution, 2 μ l, and injecting chromatograph is analyzed to obtain respective peak area by the method for drafting, with right It is abscissa according to product solution concentration (X, μ g/ml), peak area (Y, microvolt * seconds) is that ordinate carries out linear regression.

Equation of linear regression: Y=2.5613.06X-5053.145, R=0.999988.

The result shows that ferulic acid is in the range of 2.436~121.81 μ g/ml of concentration, linear relationship is good, and see Table 3 for details 0 And Figure 19.

30 Radix Angelicae Sinensis standard curve of table analyzes result

3, accuracy

The test sample (Radix Angelicae Sinensis mark soup B01) of known content, takes 9 parts, every part of about 0.1g, points 3 groups, accurately weighed, is placed in tool It fills in conical flask.Respectively correspond accurate addition each 25ml of various concentration reference substance solution, high, medium and low concentrations control product additional amount With the ratio between ferulic acid amount control in taken test sample in 1.5:1,1:1,0.5:1 or so.It is molten that test sample is carried out by the method drafted The preparation and measurement of liquid calculate the rate of recovery, the results are shown in Table 31.Calculation formula is as shown in Equation 1:

Table 31 is loaded recovery test result

The result shows that the RSD value of rate of recovery result is 2.5%, this method accuracy is good.

4, precision

(1) instrument precision

1 part of test solution is prepared by the method for drafting, 6 needle of continuous sample introduction records target component peak area, and calculates RSD, As a result as shown in table 32.

32 precision of table investigates result

The result shows that the peak area RSD value of ferulic acid is 1.1% in precision investigation, the sample introduction precision of this method is good It is good.

(2) repeated

Same test sample (Radix Angelicae Sinensis mark soup B01) 0.2g is taken, accurately weighed 6 parts, is prepared by same operator by method of drafting Test solution calculates the content of 6 parts of test samples, the results are shown in Table 33.

33 repeatability of table investigates result

The result shows that the RSD value of content is 1.6% in repeatability investigation, the repeatability of this method is good.

(3) Intermediate precision

Same test sample (Radix Angelicae Sinensis mark soup B01) 0.2g is taken, it is accurately weighed, it is operated respectively by different personnel, different instrument, no With its content is measured under chromatography column condition, it the results are shown in Table 34.Wherein, the instrument model of scheme 1 is WATERS ARC, column model For Waters CORTECS C18 (2.7 μm, 4.6*50mm)；The instrument model of scheme 2 is WATERS H-CLASS, column model For Waters BEH C18,1.7 μm, 2.1*50mm Column；The instrument model of scheme 3 is THERMO UHPLC, column model For Thermo Acclaim^TMRSLC (2.2 μm,100*2.1mm)。

34 content Intermediate precision result of table

Personnel	Scheme 1	Scheme 2	Scheme 3
				A	0.2048%	0.2042%	0.2048%
B	0.2060%	0.2102%	0.2088%

Average value=0.2065%, RSD (%)=1.2%.The result shows that the RSD value of content is in this experiment investigation 1.2%, the Intermediate precision of this method is good.

(4) durability

1) stability of solution

Same test sample (Radix Angelicae Sinensis mark soup B01) is taken, prepares test solution by the method for drafting, is surveyed respectively in different time sections Its fixed content, the results are shown in Table 35.

35 stable content result of table

The result shows that under this experiment condition, the RSD value of test sample content is 0.9%, and test solution is steady in 48h It is qualitative good.

2) mobile phase acidity

Same test sample (Radix Angelicae Sinensis mark soup B01) is taken, test solution is prepared by the method for drafting, respectively in different mobile phase water Its content is measured under phase acidity condition, and calculates RSD, the results are shown in Table 36.

The different mobile phase acidity results of table 36

The result shows that under this experiment condition, the RSD value of test sample content is 0.3%, and mobile phase water phase phosphoric acid concentration is light It is slightly variable dynamic, test solution content is influenced little.

3) mobile phase

Same test sample (Radix Angelicae Sinensis mark soup B01) is taken, prepares test solution by the method for drafting, respectively in different mobile phase ratios Its content is measured under the conditions of example, and calculates RSD, the results are shown in Table 37.

The different mobile phase ratio content results of table 37

The result shows that under this experiment condition, the RSD value of test sample content is 1.3%, mobile phase ratio slight variations, Test solution content is influenced little.

4) column temperature

Same test sample (Radix Angelicae Sinensis mark soup B01) is taken, test solution is prepared by the method for drafting, respectively in different column temperature conditions Lower its content of measurement, and RSD is calculated, it the results are shown in Table 38.

The different column temperature content results of table 38

The result shows that this method temperature durability is good.

Four, it measures

Ferulaic acid content measurement is carried out to 19 batches of Radix Angelicae Sinensis standard decoctions using final content assaying method, as a result such as table 39 It is shown.

39 Radix Angelicae Sinensis standard decoction ferulaic acid content of table, rate of transform result

Using the content of ferulic acid in UPLC method measurement Radix Angelicae Sinensis standard decoction, calculates Radix Angelicae Sinensis and turn from medicine materical crude slice to standard decoction Shifting rate, it is 7.08% that the mean transferred rate of ferulic acid, which is 67.71%, SD (standard deviation), in Radix Angelicae Sinensis standard decoction.

It should be understood that the application of the present invention is not limited to the above for those of ordinary skills can With improvement or transformation based on the above description, all these modifications and variations all should belong to the guarantor of appended claims of the present invention Protect range.

Claims

1. a kind of method for building up of Radix Angelicae Sinensis standard decoction finger-print, which comprises the following steps:

1) preparation of test solution: taking Radix Angelicae Sinensis standard decoction, adds water, carries out ultrasonic extraction, is centrifuged, filtering；

2) chromatographic condition and system suitability: using octadecylsilane chemically bonded silica as filler, using acetonitrile as mobile phase A, using 0.1% formic acid solution as Mobile phase B；Column temperature is 30-40 DEG C；Flow velocity is 0.2-0.4ml/min, Detection wavelength 270nm；

Wherein, gradient elution program is as follows: 0~3min, mobile phase A: 0%, Mobile phase B: 100%；3~5min, mobile phase A: 0% → 4%, Mobile phase B: 100% → 96%；5~16min, mobile phase A: 4% → 30%, Mobile phase B: 96% → 70%；16 ~17min, mobile phase A: 30% → 100%, Mobile phase B: 70% → 0%；17~20min, mobile phase A: 100%, mobile phase B:0%；

3) test solution by step 1) preparation injects liquid chromatograph, is measured with the condition of step 2), obtains finger-print.

2. the method for building up of Radix Angelicae Sinensis standard decoction finger-print according to claim 1, which is characterized in that in step 1), It takes Radix Angelicae Sinensis standard decoction 0.2g, is added water 25ml, ultrasonic extraction 10 minutes；Power is 400W, frequency during the ultrasonic extraction Rate is 40kHz.

3. the method for building up of Radix Angelicae Sinensis standard decoction finger-print according to claim 1, which is characterized in that be in step 2) Using CORTECS UPLC T3 chromatographic column, packing material size is 1.6 μm, internal diameter 2.1, column length 100mm；Column temperature is 30 DEG C；Flow velocity is 0.3ml/min；The dosage of test solution is 5 μ l in step 3).

4. the method for building up of Radix Angelicae Sinensis standard decoction finger-print according to claim 1, which is characterized in that further include following Step:

4) obtained finger-print is subjected to similarity calculation with map is compareed；The control map is as shown in figure 15.

5. a kind of method for building up of Radix Angelicae Sinensis standard decoction characteristic spectrum, which comprises the following steps:

2) preparation of test solution: taking Radix Angelicae Sinensis standard decoction, adds water, carries out ultrasonic extraction, lets cool, the weight of less loss is supplied with water Amount is centrifuged, filtering；

3) chromatographic condition and system suitability: using octadecylsilane chemically bonded silica as filler, using acetonitrile as mobile phase A, using 0.1% formic acid solution as Mobile phase B；Column temperature is 30-40 DEG C；Flow velocity is 0.2-0.4ml/min, Detection wavelength 270nm；

6. the method for building up of Radix Angelicae Sinensis standard decoction characteristic spectrum according to claim 5, which is characterized in that in step 2), It takes Radix Angelicae Sinensis standard decoction 0.2g, is added water 25ml, ultrasonic extraction 10 minutes；Power is 400W, frequency during the ultrasonic extraction Rate is 40kHz.

7. the method for building up of Radix Angelicae Sinensis standard decoction characteristic spectrum according to claim 5, which is characterized in that step 3) is to adopt With CORTECS UPLC T3 chromatographic column, packing material size is 1.6 μm, internal diameter 2.1, column length 100mm；Column temperature is 30 DEG C；Flow velocity is 0.3ml/min；In step 4), reference solution dosage is 1 μ l, and test solution dosage is 5 μ l.

8. the method for building up of Radix Angelicae Sinensis standard decoction characteristic spectrum according to claim 5, which is characterized in that further include following Step:

4) characteristic spectrum that will be obtained should have 7 characteristic peaks in test sample characteristic spectrum, wherein with ferulic acid reference solution phase The peak answered be the peak S, calculate characteristic peak and the peak S relative retention time, relative retention time should specified value ± 5% it It is interior；Relative retention time specified value are as follows: peak 1,0.199；Peak 2,0.374；Peak 3,0.431；Peak 4,0.655；Peak 5,1；Peak 6, 1.163；Peak 7,1.207.

9. a kind of content assaying method of Radix Angelicae Sinensis standard decoction, which comprises the following steps:

3) chromatographic condition and system suitability: using octadecylsilane chemically bonded silica as filler；With acetonitrile -0.085% Phosphoric acid solution is mobile phase, and the volume ratio of -0.085% phosphoric acid solution of acetonitrile is 17:83；25-40 DEG C of column temperature；Detection wavelength is 316nm, flow velocity 0.2-0.8ml/min；Number of theoretical plate is calculated by forulic acid peak should be not less than 4000；

4) reference substance solution and each 1~5 μ l of test solution are drawn, liquid chromatograph is injected, measures the asafoetide of test solution Acid content.

10. the content assaying method of Radix Angelicae Sinensis standard decoction according to claim 9, which is characterized in that in step 2), take and work as Return standard decoction 0.2g, is added water 25ml, ultrasonic extraction 30 minutes；During the ultrasonic extraction, power 400W, frequency is 40kHz；

In step 3), using CORTECS UPLC C18 chromatographic column, packing material size is 2.7 μm, internal diameter 4.6, column length 50mm, column temperature It is 30 DEG C, flow velocity 0.5ml/min；

In step 4), reference substance solution dosage is 2 μ l, and test solution dosage is 2 μ l.