CN106033075A - A method of measuring the content of astragaloside in heart-tonifying pulse-restoring granules - Google Patents
A method of measuring the content of astragaloside in heart-tonifying pulse-restoring granules Download PDFInfo
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Abstract
The invention relates to a method of measuring an effective component in a traditional Chinese medicine, and particularly relates to a method of measuring the content of astragaloside in heart-tonifying pulse-restoring granules. The method includes a step of preparing a reference substance solution, namely a step of measuring a proper amount of an astragaloside reference substance and adding water-containing methanol or a mobile phase to prepare into a solution containing 0.16-0.24 mg1/mL of astragaloside; a step of preparing a sample solution to be measured, namely a step of weighting the heart-tonifying pulse-restoring granules, extracting with an alkali water solution having a concentration of 2-5%, adding the extract liquid into a solid phase extraction column, and eluting with methanol, and a step of measuring, namely a step of separately injecting 5-20 muL of the reference substance solution and the sample solution to be measured into a high performance liquid chromatograph.
Description
Technical field
The present invention relates to the content assaying method of effective ingredient in a kind of Chinese medicine preparation, particularly toA kind of Determination of Astragaloside method in the heartsupplementing and pulse restoring granule。
Background technology
The heartsupplementing and pulse restoring granule is the medicine that Tianjin Tasly (Liaoning) Pharmaceutical Co., Ltd. produces, and its function cures mainly as supplementing QI and nourishing YIN, multiple arteries and veins of invigorating blood circulation；For deficiency of both QI and YIN, painstaking effort internal resistance, obstruction of qi in the chest and cardialgia, uncomfortable in chest do not relax, cardiopalmus irregularly intermittent and regularly intermittent pulse.Have that drug loading is high, rapid-action, free from extraneous odour, the characteristic such as easy to carry.The compound preparation that the heartsupplementing and pulse restoring granule is made up of Radix Ginseng, the Radix Astragali, Radix Ophiopogonis, Fructus Schisandrae Chinensis, Radix Salviae Miltiorrhizae, Rhizoma Chuanxiong 6 taste medical material, wherein
Radix Ginseng: there is strongly invigorating primordial QI, benefit blood, the effect of tranquilizing by nourishing the heart.Radix Ginseng can improve brain, physical work capacity and immunologic function, strengthen anti-stress, resisting fatigue, antitumor, defying age, radioprotective, the benefit heart, Fu Mai, calm the nerves promote the production of body fluid, tonifying the lung spleen invigorating.
Radix Ophiopogonis: have sweet cold clear and rich, the heat of the kind lung that clears away heart-fire and yin nourishing relieving restlessness, double can clear and rich gastrointestinal and quench the thirst and moisturize.On Antagonizing Experimental Arrhythmia, increase coronary flow.
Fructus Schisandrae Chinensis: be that one has five kinds of property of medicine pungent, sweet, sour, bitter, salty.Tradition certified products Chinese Magnoliavine is selected to be used as medicine, best in quality.Restrain astringent or styptic treatment for spontaneous sweating, supplementing QI for promoting the production of body fluid, kidney calming.For chronic cough dyspnea due to deficiency, emission, enuresis frequent micturition, incessant chronic diarrhea, spontaneous perspiration, night sweat, Tianjin wound is thirsty, deficient pulse of losing heart, and interior-heat is quenched one's thirst, palpitation and insomnia.
The Radix Astragali: have the power that tonifying Qi and lifting yang, hemopoietic row are stagnant, present pharmacological research finds, the Radix Astragali also can scavenging activated oxygen, alleviate body lipid peroxidation injury.
Radix Salviae Miltiorrhizae: have the merit of blood circulation promoting and blood stasis dispelling, is possible not only to suppress platelet aggregation, and inhibition thrombosis and contained danshensu may also suppress the synthesis of cholesterol, anti-cell oxidation.
Rhizoma Chuanxiong: blood-activating and qi-promoting, wind-expelling pain-stopping.CAMP content in platelet can be improved, reduce TXA2 activity；Reduce surface activity of blood platelet, suppression platelet aggregation, make to assemble platelet disaggregation.
The active constituents of medicine of the heartsupplementing and pulse restoring granule is a lot, specifically includes that astragaloside, ligustrazine, arasaponin, TANSHINONES etc..
Owing to active component is numerous, it is thus desirable to the active component of the heartsupplementing and pulse restoring granule is detected by sensitive detection method, but existing detection method only comprises character, coherent detection item under 2 colour developing discriminatings and check item, do not carry out the qualitative or quantitative detection of medical material, the quality condition of product can not be reflected comprehensively, the present invention is directed to the monarch drug Radix Astragali and establish the method for Determination of Astragaloside in product, fill up the blank that the heartsupplementing and pulse restoring particle quantitative controls, make this product can be carried out more effective quality analysis, more comprehensively reflect the quality condition of product, ensure the quality stability of this product, the method saves testing cost simultaneously.
Summary of the invention
The present invention relates to a kind of measures the method for Astragaloside content in the heartsupplementing and pulse restoring granule, and the method uses high performance liquid chromatography, comprises the steps:
Step 1, the preparation of reference substance solution: take astragaloside reference substance appropriate, adds aqueous methanol or the solution containing astragaloside 0.16～0.24mg1/ml is made in flowing mutually.
Step 2, the preparation of need testing solution: take the heartsupplementing and pulse restoring granule, with concentration be 2～5% aqueous alkali extract, extracting solution crosses solid-phase extraction column, by methanol-eluted fractions.
Step 3, algoscopy: each to above-mentioned reference substance solution and reference substance solution 5～20 μ l are injected in high performance liquid chromatograph.
Wherein, in step (1), described aqueous methanol is the methanol of 50～100%, preferably 60～80%, most preferably 70% methanol.Flowing is mutually: acetonitrile: water=31～36:69～64.
Wherein, in step (2), described aqueous alkali is ammonia, a kind of in sodium bicarbonate.Preferably ammonia.Solid phase extraction column stuffing described in step (2) is C18 or PLS (polystyrene/divinylbenzene copolymer), preferably C18.It is supersound extraction that aqueous alkali described in step (2) extracts, and extracting solution is centrifuged.
The preferably step of step (2) is as follows: take the heartsupplementing and pulse restoring granule, precision adds 37 times amount 3～5% ammonia, supersound process 20～make for 40 minutes fully to leach, centrifugal, precision pipettes supernatant, and the speed with 0.5～1.5ml/min passes through processed good C18 solid phase extraction column, water elution, eluent discards, and again with methanol is eluted in measuring bottle, to obtain final product.
High Performance Liquid Chromatography condition described in step (3):
Evaporative light scattering detector: include yield value: 50～200；Drift tube temperature: 40～60 DEG C；Aerochamber temperature is arranged: cooling cooling；Gas pressure: 20～30psi；Second sampling site number: 1～10；
Chromatographic column: C 18；
Flowing phase: acetonitrile: water=31～36:69～64；
Column temperature: 35～42 DEG C；
Flow velocity: 0.8～1.5ml/min.
Except step 1, outside 2, the present invention can also include the preparation process of negative sample solution, method is by the heartsupplementing and pulse restoring granule prescription proportioning, remove the ingredients of the Radix Astragali, by the heartsupplementing and pulse restoring particle recipe preparation negative sample without astragaloside, then by step (2) need testing solution preparation method, make negative sample solution, according to step 3 injecting chromatograph.
Preferred assay method of the present invention, comprises the steps:
(1) preparation of reference substance solution: take astragaloside reference substance appropriate, accurately weighed, add 70% methanol and make every 1ml 0.20mg Han astragaloside, to obtain final product；
(2) preparation of need testing solution: take the heartsupplementing and pulse restoring, accurately weighed, to 50ml conical flask, precision adds 5 times amount 4% ammonia, supersound process makes fully to leach for 25 minutes, and centrifugal, precision pipettes supernatant, processed good C18 solid phase extraction column is passed through with the speed of 1ml/min, washing with water, eluent discards, and again with methanol is slowly eluted in measuring bottle, add methanol dilution to shake up to scale, to obtain final product.
(3), the preparation of negative sample solution, by the heartsupplementing and pulse restoring granule prescription proportioning, remove the ingredients of the Radix Astragali, by the heartsupplementing and pulse restoring particle recipe preparation negative sample without the Radix Astragali, then by step (2) need testing solution preparation method, make negative sample solution.
(4), injecting chromatograph of liquid, its chromatographic condition is: chromatographic column C18,4.6 × 150mm, 3.5 μm or 4.6 × 250mm, 5 μm；Flowing phase: acetonitrile: water=35:65；Column temperature: 40 DEG C；Flow velocity: 1.0ml/min；Sample size: 10 μ l；Evaporative light scattering detector condition: yield value: 100；Drift tube temperature: 50 DEG C；Aerochamber temperature is arranged: cooling cooling；Gas pressure: 30psi；Second sampling site number: 2.
Test example
Currently preferred instrument used is as follows with material:
1, high performance liquid chromatograph: U.S. WatersHPLC 2,695 2424, Empower work station.
2, chromatographic column
1. reversed phase chromatographic column, is suitable for low pH value chromatographic column, fixing with octadecylsilane chemically bonded silica as filler, Agilent ZORBAX SB C18 (4.6 × 150mm, 3.5 μm)；
2. reversed phase chromatographic column, versatility width pH value chromatographic column, the fixing high-purity silica gel with high purity 99.999% as filler, OmniBond HPLC Column Orca C18 (4.6 × 250mm, 5 μm)；
3. reversed phase chromatographic column, is suitable for low pH value chromatographic column, fixing with octadecylsilane chemically bonded silica as filler, Agilent ZORBAX SB C18 (4.6 × 250mm, 5 μm)；
3, reagent: acetonitrile (chromatographically pure, Tianjin Concord Technology Co., Ltd.).
Methanol (analytical pure, Tianjin Concord Technology Co., Ltd.).
4, reference substance: astragaloside (assay is used for Nat'l Pharmaceutical & Biological Products Control Institute, lot number: 110,781 201314).
5, sample: the heartsupplementing and pulse restoring granule, Tianjin Tasly (Liaoning) Pharmaceutical Co., Ltd., numbers (or lot number): test 1, test 2, test 3,20130205,20130304.
Currently preferred experimental technique, chromatographic condition and solvent extraction process, obtain through screening, and screening process is as follows:
The prescription of the heartsupplementing and pulse restoring granule and preparation method
The heartsupplementing and pulse restoring granule prescription of the present invention is as follows: Radix Ginseng 75 225g, the Radix Astragali 75 225g, 225g Radix Ophiopogonis 75, Fructus Schisandrae Chinensis 50 150g, Radix Salviae Miltiorrhizae 100 300g, Rhizoma Chuanxiong 50 150g and appropriate amount of auxiliary materials are made.
Preparing of preferred the heartsupplementing and pulse restoring granule is as follows:
[prescription] Radix Ginseng 150g Radix Astragali 150g 150g Radix Ophiopogonis Fructus Schisandrae Chinensis 100g Radix Salviae Miltiorrhizae 200g Rhizoma Chuanxiong 100g
6 taste medical material more than [preparation method], boiling 2 times, add water 10 times amount for the first time, decocts 2 hours, add water 8 times amount for the second time, decocts 1 hour, merges decoction liquor, stands 12 hours, Aspirate supernatant, is concentrated into the clear paste of relative density 1.20-1.30 (80 DEG C of surveys), adds the stevioside of 1%, mix homogeneously, qinghuo reagent 1 part, adds 1 part of dextrin, dry, pulverize, cross 60 mesh sieves, add lactose 2 parts, mixing, make 600g, subpackage, to obtain final product.
Test example 1 flows the selection of phase；
The investigation carried out according to the liquid phase chromatogram condition separating astragaloside of embodiment 1, on the premise of meeting separating degree requirement, according toTable 1Adjusting the ratio of flowing phase, flow phase: acetonitrile: water=31 36:69 64, chromatographFigure such as figure 1、Figure 2、Figure 3、Figure 4WithFigure 5Shown in.Preferably A%: B%=acetonitrile: under the conditions of water (35:65) flowing mutually, astragaloside is optimum with the separating degree at other impurities peak, a length of 20min during analysis.
Table 1The ratio of flowing phase
The selection of test example 2 chromatographic column
Take off the method mensuration stating chromatographic column according to embodiment respectively.
1. Agilent ZORBAX SB C18 (4.6 × 150mm, 3.5 μm)；
2. OmniBond HPLC Column Orca C18 (4.6 × 250mm, 5 μm)；
3.: Agilent ZORBAX SB C18 (4.6 × 250mm, 5 μm),
ChromatographFigure such as figure 6、Figure 7WithFigure 8Shown in.
AnalyzeFigure 6‐Figure 8, compare the separating degree with adjacent chromatographic peak and retention time, it is seen thatFigure 7Optimum, therefore select the chromatographic column that Agilent ZORBAX SB C18 (4.6 × 150mm, 3.5 μm) measures as method.
Embodiment 3 Extraction solvent and sideMethod is investigated
Method is 1.
Take the heartsupplementing and pulse restoring granule about 3.0g, accurately weighed, put in 50ml conical flask, accurate addition 15ml4% ammonia, sealing, supersound process (power 120W, frequency 40KHZ) makes fully to leach for 20 25 minutes, let cool, shake up, centrifugal (3000 turns, 10min), precision pipettes supernatant 10ml, passes through processed good C with the speed of 1ml/min18Solid phase extraction column (500mg, first with methanol 5ml eluting, then with water 5ml eluting) water 5ml eluting, eluent discards, and again with methanol 2ml is slowly eluted in 2ml volumetric flask, adds methanol dilution to scale, shakes up, to obtain final product.This test sample processing method records the content of astragaloside: 0.1947mg/g chromatographFigure such as figure 9Shown in.
Method is 2.
Precision weighs the heartsupplementing and pulse restoring granule 2.5g, puts in 50ml triangular flask respectively, addsTable 2Shown in each 25ml of methanol solution, seal, supersound extraction 30 minutes, let cool, filter, reclaim methanol, residue 2ml methanol constant volume, to obtain final product.Result is respectively chromatographFigure such as figure 11、Figure 12、Figure 13 HesFigure 1Shown in 4.
Table 2Methanol solubility
| Group | Methanol concentration % | The content of astragaloside |
| 100% | 0.0127mg/g | |
| 90% | 0.0152mg/g | |
| 80% | 0.0170mg/g | |
| 70% | 0.0201mg/g |
Method is 3.
Precision weighs the heartsupplementing and pulse restoring granule 2.5g, puts in conical flask, and precision adds methanol 50ml, ultrasonic 30min.Filtering, water bath method, the residue 20ml that adds water makes dissolving, water liquid water saturated n-butanol extraction 3 times, each 20ml, merge n-butyl alcohol liquid, wash 3 times with ammonia solution, each 20ml, discard ammonia solution, n-butyl alcohol liquid water bath method, residue adds methanol and dissolves, is quantitatively transferred in 2ml measuring bottle, add methanol dilution to scale, shake up, filter with microporous filter membrane, as need testing solution, obtaining final product, the method records the content of astragaloside and is: 0.0482mg/g.ChromatographFigure such as figure 1Shown in 5.
Method is 4.
Take the heartsupplementing and pulse restoring granule appropriate, finely ground, take 3g, accurately weighed, add methanol 50ml, ultrasonic 30 minutes, put and be evaporated in water-bath, the residue 40ml that adds water makes to leach, extract 2 times by ethyl acetate, 40ml every time, discard acetic acid ethyl fluid, water liquid saturated n-butanol extraction 4 times, 30ml merges n-butyl alcohol liquid every time, wash 2 times with ammonia solution, 50ml every time, it is washed with water and washs 2 times, 50ml every time, take n-butyl alcohol liquid to be evaporated, residue adds proper amount of methanol and dissolves, put in 2ml volumetric flask, add methanol dilution to scale, shake up, filter, take subsequent filtrate, obtain, the method records the content of astragaloside: 0.0627mg/g.ChromatographFigure such as figure 1Shown in 6.
Method is 5.
Precision weighs the heartsupplementing and pulse restoring granule 5g, and add water 40ml, ultrasonic dissolution, accurate draw 20ml, put in separatory funnel, with water saturated n-butanol extraction 4 times, 20ml every time, merge n-butyl alcohol liquid, wash 3 times with ammonia solution, each 20ml, n-butyl alcohol liquid is evaporated, residue adds methanol dissolving and puts in 5ml volumetric flask, and as need testing solution, the method measures the content of astragaloside and is: 0.1259mg/g.ChromatographFigure such as figure 1Shown in 7.
The above-mentioned 5 kinds of test sample processing method results of Integrated comparative are shown inTable 3, it has been found that the content of the astragaloside that 1. test sample preparation method records is the highest, therefore using this test sample processing method as the test sample processing method of the heartsupplementing and pulse restoring granule Determination of Astragaloside in research afterwards.
Table 3, the Astragaloside content value of distinct methods
| Method group | The content of astragaloside |
| ① | 0.1947mg/g |
| ② | 0.0127mg/g、0.0152mg/g、0.0170mg/g、0.0201mg/g |
| ③ | 0.0482mg/g |
| ④ | 0.0627mg/g |
| ⑤ | 0.1259mg/g |
The selection of test example 4 aqueous alkali
According to the method for embodiment 1, aqueous alkali condition according toTable 4Processing, result is as follows:
Table 4, variety classes and the Astragaloside content value of concentration aqueous alkali process
| Aqueous alkali condition | The content of astragaloside |
| 1% ammonia | 0.1422mg/g |
| 2% ammonia | 0.1876mg/g |
| 4% ammonia | 0.1947mg/g |
| 5% ammonia | 0.1863mg/g |
| 6% ammonia | 0.1788mg/g |
| 4% sodium bicarbonate | 0.1654mg/g |
| 5% sodium bicarbonate | 0.1819mg/g |
Result shows, ammonia, the 5% sodium bicarbonate extraction effect of 2 5% are preferable, preferably 4% ammonia.
The different solid-phase extraction column raw material impact on Astragaloside content value of test example 5
According to the method for embodiment 1, solid-phase extraction column according toTable 5Processing, result is as follows:
Table 5, the Astragaloside content value of different solid-phase extraction columns
| Solid-phase extraction column | The content of astragaloside |
| C18 solid phase extraction column | 0.1947mg/g |
| PLS extracts pillar | 0.1896mg/g |
| DIKMA proElut NH2 | Before and after astragaloside chromatographic peak, impurity is more, and separating degree is bad |
| DIKMA proElut SAX | Astragaloside attached collection amount is little, it is virtually impossible to this material detected |
| DIKMA proElut AL-B-alkali alumina | 0.1062mg/g. |
[0084] Result shows, the Astragaloside content value after C18 solid phase extraction column and LS extraction pillar process is high, preferably C18 solid phase extraction column.
The methodological study of Determination of Astragaloside in test example 6 the heartsupplementing and pulse restoring granule
1, the preparation of standard curve
Take astragaloside reference substance appropriate, accurately weighed, add 70% methanol be respectively prepared every 1ml containing astragaloside 0.007988,0.01997,0.03994,0.07988, the serial solution of 0.15976mg, the most accurate sample introduction 20 μ l, inject chromatograph of liquid, the chromatographic condition of step 4 is analyzed preferably, measure respective peak area, with the logarithm of reference substance concentration as abscissa, the logarithm of peak area value is vertical coordinate, try to achieve regression equation: astragaloside Y=1.4203X+6.907, r=0.9993.Result shows that astragaloside is linear good in the range of 0.07616～0.7616mg/ml, and result is shown inTable 6, linearlyFigureSpectrum is shown inFigure 18。
Table 6Astragaloside standard curve
2, sample introduction precision test
Take astragaloside reference substance, according to preferred version step 1 reference substance solution preparation manipulation, the chromatographic condition of step 4 is analyzed preferably, continuous sample introduction 6 times, measuring astragaloside peak area, record peak area meansigma methods and be respectively 797256, the RSD of peak area is respectively 1.6%, meeting the requirements, result is shown inTable 7,FigureSpectrum is shown inFigure 19。
Table 7Sample introduction precision test
3, replica test
Take with a collection of (numbering: test 3) sample, prepare the need testing solution of basic, normal, high 3 kinds of concentration, concentration range is 75%～125%, totally 9 parts, according to preferred version step 2 need testing solution preparation manipulation, the chromatographic condition of step 4 is analyzed preferably, measures the content of astragaloside in every part of sample.In results sample, astragaloside average content is that 0.1907mg/g, RSD are respectively 2.4%, and repeatability is good, and result is shown inTable 8,FigureSpectrum is shown inFigure 20。
Table 8Replica test
4, stability test
Take with a collection of (numbering: test 3) sample, take 1 part, accurately weighed 3.0g, according to preferred version step 2 need testing solution preparation manipulation, the chromatographic condition of step 4 is analyzed preferably, respectively at 0,2,4,6,10,14,18,20 and 24 hours, measure astragaloside peak area in sample, the RSD recording peak area value is respectively 1.8%, and it is stable that result shows that need testing solution measured in 24 hours, and result is shown inTable 9,FigureSpectrum is shown inFigure 21。
Table 9Stability test
5, recovery test
Take with a collection of (numbering: test 3) sample, take the 1/2 of test sample sample weighting amount, accurately weighed 9 parts, it is separately added into reference substance (solution) appropriate, according to the need testing solution containing corresponding reference substance of preferred version step 23 variable concentrations of need testing solution preparation manipulation (with the concentration of composition to be measured contained by need testing solution for 100%, 3 variable concentrations are 75%～125%), each concentration prepares 3 parts of need testing solutions, the chromatographic condition of step 4 is analyzed preferably, calculate the response rate, result astragaloside average recovery rate is 102.8%, RSD is respectively 4.0%, result is shown inTable 10,FigureSpectrum is shown inFigure 22, recovery test meets the requirements.
Table 1Astragaloside recovery test in 0 the heartsupplementing and pulse restoring granule
6, serviceability test
Take with a collection of (numbering: test 3) sample, according to preferred version step 2 need testing solution preparation manipulation, respectively with Agilent ZORBAX C 18 (4.6 × 250mm, 3.5 μm)；OmniBondhubble C 18 (5 μm 4.6 × 250mm) chromatographic column, the chromatographic condition of step 4 is analyzed preferably, measures Astragaloside content in sample, and result is shown inTable 11,FigureSpectrum is shown inFigure 23 HesFigure 24.Measurement result shows, different brands chromatographic column on the result of assay without impact.
Table 1The 1 different chromatographic column impacts on assay
7, the determination of number of theoretical plate
Consider separation situation and the theoretical cam curve result of 2 kinds of different chromatographic columns of the heartsupplementing and pulse restoring granule, be shown in Table, the number of theoretical plate of astragaloside must not be defined as less than 5000.
Beneficial effects of the present invention: the method for Astragaloside content in the liquid chromatogram measuring the heartsupplementing and pulse restoring granule that the present invention sets up, meets the requirement of Method validation, and easy and simple to handle, highly sensitive, measuring accurately, the suitability is strong, it is possible to for the quality control to this product.Meanwhile, the blank that this weight per unit length controls has been filled up so that this product can be carried out more effective quality analysis, more comprehensively reflect the quality condition of product, it is ensured that the quality stability of this product.
Accompanying drawing explanation
Figure 1A%: B%=acetonitrile: water (31:69) chromatographFigure
Figure 2A%: B%=acetonitrile: water (33:67) chromatographFigure
Figure 3A%: B%=acetonitrile: water (34:66) chromatographFigure
Figure 4A%: B%=acetonitrile: water (35:65) chromatographFigure
Figure 5A%: B%=acetonitrile: water (36:64) chromatographFigure
Figure 6OmniBond HPLC Column Orca C18 gathers chromatographFigure
Figure 7Agilent ZORBAX SB C18 (4.6 × 150mm, 3.5 μm) gathers chromatographFigure
Figure 8Agilent ZORBAX SB C18 (4.6 × 250mm, 5 μm) gathers chromatographFigure
Figure 9Test sample processes 1. gained chromatographFigure
Figure 10 test sample processes 3. 100% methanol gained chromatographFigure
Figure 11 test sample processes 3. 90% methanol gained chromatographFigure
Figure 12 test samples process 3. 80% methanol gained chromatographFigure
Figure 13 test samples process 3. 70% methanol gained chromatographFigure
Figure 14 test samples process 4. chromatographFigure
Figure 15 test samples process 5. chromatographFigure
Figure 16 test samples process 6. chromatographFigure
Figure 17 is linearFigureSpectrum
Figure 18 sample introduction precisionFigureSpectrum
Figure 19 repeatabilityFigureSpectrum
Figure 20 stabilityFigureSpectrum
Figure 2The 1 astragaloside response rateFigureSpectrum
Figure 22Agilent ZORBAX C 18 chromatographic column is composedFigure
Figure 23OmniBond Hubble C 18 chromatographic column is composedFigure
Figure 24 test 3 sample chromatogramsFigure
Figure 25 test 1 sample chromatogramsFigure
Figure 26 test 2 sample chromatogramsFigure
Figure 27 lot number 20130205 sample chromatogramsFigure
Figure 28 lot number 20130304 sample chromatogramsFigure
Detailed description of the invention
Further illustrate the present invention by the following examples, but not as limitation of the present invention.
Embodiment 1 takes the heartsupplementing and pulse restoring granule of numbered test 3, measures the content of its astragaloside
Step 1, the preparation of reference substance solution
Take astragaloside reference substance appropriate, accurately weighed, add 70% methanol and make every 1ml and containing astragaloside 0.20mg and get final product.
Step 2, the preparation of need testing solution, method is as follows: take the heartsupplementing and pulse restoring granule about 3.0g, accurately weighed, to 50ml conical flask, precision adds 4% ammonia 15ml, and supersound process makes fully to leach for 25 minutes, centrifugal (3000 turns, 10min), precision pipettes supernatant 10ml, and with the speed of 1ml/min, by processed good C18 solid phase extraction column, (500mg, first with methanol 5ml eluting, again with water 5ml eluting), with water 5ml eluting, eluent discards, and again with methanol 2ml is slowly eluted in 2ml measuring bottle, add methanol dilution to shake up to scale, to obtain final product.
Step 3, the preparation of negative sample solution, method is as follows: by the heartsupplementing and pulse restoring granule prescription proportioning, remove the ingredients of the Radix Astragali, by the heartsupplementing and pulse restoring particle recipe preparation negative sample without astragaloside, then by step 2 need testing solution preparation method, make negative sample solution.
Step 4, injects chromatograph of liquid, calculates Astragaloside content, and method is as follows: using liquid chromatography, its chromatographic condition is: C 18 chromatographic column；Flowing phase: A%: B%=acetonitrile: water (35:65)；Column temperature: 40 DEG C；Flow velocity: 1.0ml/min；Sample size: 10 20 μ l.Detector condition: yield value: 100；Drift tube temperature: 50 DEG C；Aerochamber temperature is arranged: cooling；Gas pressure: 30psi；Second sampling site number: 2.
Recording the content of astragaloside in every g the heartsupplementing and pulse restoring granule is 0.1907mg, and result is shown in Figure 24.
Embodiment 2 takes the heartsupplementing and pulse restoring granule of numbered test 1, measures the content of its astragaloside
Step 1, the preparation of reference substance solution
Take astragaloside reference substance appropriate, accurately weighed, add 70% methanol and make every 1ml and containing astragaloside 0.16 0.24mg and get final product.
Step 2, the preparation of need testing solution, method is as follows: take the heartsupplementing and pulse restoring granule about 3.0g, accurately weighed, to 50ml conical flask, precision adds 4% ammonia 15ml, and supersound process makes fully to leach for 25 minutes, centrifugal (3000 turns, 10min), precision pipettes supernatant 10ml, and with the speed of 1ml/min, by processed good C18 solid phase extraction column, (500mg, first with methanol 5ml eluting, again with water 5ml eluting), with water 5ml eluting, eluent discards, and again with methanol 2ml is slowly eluted in 2ml measuring bottle, add methanol dilution to shake up to scale, to obtain final product.
Step 3, the preparation of negative sample solution, method is as follows: by the heartsupplementing and pulse restoring granule prescription proportioning, remove the ingredients of the Radix Astragali, by the heartsupplementing and pulse restoring particle recipe preparation negative sample without astragaloside, then by step 2 need testing solution preparation method, make negative sample solution.
Step 4, injects chromatograph of liquid, calculates Astragaloside content, and method is as follows: using liquid chromatography, its chromatographic condition is: C 18 chromatographic column；Flowing phase: A%: B%=acetonitrile: water (35:65)；Column temperature: 40 DEG C；Flow velocity: 1.0ml/min；Sample size: 10 20 μ l.Detector condition: yield value: 100；Drift tube temperature: 50 DEG C；Aerochamber temperature is arranged: cooling；Gas pressure: 30psi；Second sampling site number: 2.
Recording the content of astragaloside in every g the heartsupplementing and pulse restoring granule is 0.1654mg, and result is shown in Figure 25.
Embodiment 3 takes the heartsupplementing and pulse restoring granule of numbered test 2, measures the content of its astragaloside
Step 1, the preparation of reference substance solution
Take astragaloside reference substance appropriate, accurately weighed, add 70% methanol and make every 1ml and containing astragaloside 0.16 0.24mg and get final product.
Step 2, the preparation of need testing solution, method is as follows: take the heartsupplementing and pulse restoring granule about 3.0g, accurately weighed, to 50ml conical flask, precision adds 4% ammonia 15ml, and supersound process makes fully to leach for 25 minutes, centrifugal (3000 turns, 10min), precision pipettes supernatant 10ml, and with the speed of 1ml/min, by processed good C18 solid phase extraction column, (500mg, first with methanol 5ml eluting, again with water 5ml eluting), with water 5ml eluting, eluent discards, and again with methanol 2ml is slowly eluted in 2ml measuring bottle, add methanol dilution to shake up to scale, to obtain final product.
Step 3, the preparation of negative sample solution, method is as follows: by the heartsupplementing and pulse restoring granule prescription proportioning, remove the ingredients of the Radix Astragali, by the heartsupplementing and pulse restoring particle recipe preparation negative sample without astragaloside, then by step 2 need testing solution preparation method, make negative sample solution.
Step 4, injects chromatograph of liquid, calculates Astragaloside content, and method is as follows: using liquid chromatography, its chromatographic condition is: C 18 chromatographic column；Flowing phase: A%: B%=acetonitrile: water (35:65)；Column temperature: 40 DEG C；Flow velocity: 1.0ml/min；Sample size: 10 20 μ l.Detector condition: yield value: 100；Drift tube temperature: 50 DEG C；Aerochamber temperature is arranged: cooling；Gas pressure: 30psi；Second sampling site number: 2.
Recording the content of astragaloside in every g the heartsupplementing and pulse restoring granule is 0.1090mg, and result is shown in Figure 26.
Embodiment 4 takes the heartsupplementing and pulse restoring granule that lot number is 20130205, measures the content of its astragaloside.
Step 1, the preparation of reference substance solution
Take astragaloside reference substance appropriate, accurately weighed, add 70% methanol and make every 1ml and containing astragaloside 0.16 0.24mg and get final product.
Step 2, the preparation of need testing solution, method is as follows: take the heartsupplementing and pulse restoring granule about 3.0g, accurately weighed, to 50ml conical flask, precision adds 4% ammonia 15ml, and supersound process makes fully to leach for 25 minutes, centrifugal (3000 turns, 10min), precision pipettes supernatant 10ml, and with the speed of 1ml/min, by processed good C18 solid phase extraction column, (500mg, first with methanol 5ml eluting, again with water 5ml eluting), with water 5ml eluting, eluent discards, and again with methanol 2ml is slowly eluted in 2ml measuring bottle, add methanol dilution to shake up to scale, to obtain final product.
Step 3, the preparation of negative sample solution, method is as follows: by the heartsupplementing and pulse restoring granule prescription proportioning, remove the ingredients of the Radix Astragali, by the heartsupplementing and pulse restoring particle recipe preparation negative sample without astragaloside, then by step 2 need testing solution preparation method, make negative sample solution.
Step 4, injects chromatograph of liquid, calculates Astragaloside content, and method is as follows: using liquid chromatography, its chromatographic condition is: C 18 chromatographic column；Flowing phase: A%: B%=acetonitrile: water (35:65)；Column temperature: 40 DEG C；Flow velocity: 1.0ml/min；Sample size: 10 20 μ l.Detector condition: yield value: 100；Drift tube temperature: 50 DEG C；Aerochamber temperature is arranged: cooling；Gas pressure: 30psi；Second sampling site number: 2.
Recording the content of astragaloside in every g the heartsupplementing and pulse restoring granule is 0.1147mg, and result is shown in Figure 27.
Embodiment 5 takes the heartsupplementing and pulse restoring granule that lot number is 20130304, measures the content of its astragaloside
Step 1, the preparation of reference substance solution
Take astragaloside reference substance appropriate, accurately weighed, add 70% methanol and make every 1ml and containing astragaloside 0.16 0.24mg and get final product.
Step 2, the preparation of need testing solution, method is as follows: take the heartsupplementing and pulse restoring granule about 3.0g, accurately weighed, to 50ml conical flask, precision adds 4% ammonia 15ml, and supersound process makes fully to leach for 25 minutes, centrifugal (3000 turns, 10min), precision pipettes supernatant 10ml, and with the speed of 1ml/min, by processed good C18 solid phase extraction column, (500mg, first with methanol 5ml eluting, again with water 5ml eluting), with water 5ml eluting, eluent discards, and again with methanol 2ml is slowly eluted in 2ml measuring bottle, add methanol dilution to shake up to scale, to obtain final product.
Step 3, the preparation of negative sample solution, method is as follows: by the heartsupplementing and pulse restoring granule prescription proportioning, remove the ingredients of the Radix Astragali, by the heartsupplementing and pulse restoring particle recipe preparation negative sample without astragaloside, then by step 2 need testing solution preparation method, make negative sample solution.
Step 4, injects chromatograph of liquid, calculates Astragaloside content, and method is as follows: using liquid chromatography, its chromatographic condition is: C 18 chromatographic column；Flowing phase: A%: B%=acetonitrile: water (35:65)；Column temperature: 40 DEG C；Flow velocity: 1.0ml/min；Sample size: 10 20 μ l.Detector condition: yield value: 100；Drift tube temperature: 50 DEG C；Aerochamber temperature is arranged: cooling；Gas pressure: 30psi；Second sampling site number: 2.
Recording the content of astragaloside in every g the heartsupplementing and pulse restoring granule is 0.1754mg, and result is shown in Figure 28.
Embodiment 6 takes the heartsupplementing and pulse restoring granule of numbered test 3, measures the content of its astragaloside
Step 1, the preparation of reference substance solution
Take astragaloside reference substance appropriate, accurately weighed, add 80% methanol and make every 1ml and containing astragaloside 0.16mg and get final product.
Step 2, the preparation of need testing solution, method is as follows: take the heartsupplementing and pulse restoring granule about 3.0g, accurately weighed, to 50ml conical flask, precision adds 2% ammonia 9ml, and supersound process makes fully to leach for 20 minutes, centrifugal (3000 turns, 10min), precision pipettes supernatant 10ml, and with the speed of 0.5ml/min, by processed good C18 solid phase extraction column, (500mg, first with methanol 5ml eluting, again with water 5ml eluting), with water 5ml eluting, eluent discards, and again with methanol 2ml is slowly eluted in 2ml measuring bottle, add methanol dilution to shake up to scale, to obtain final product.
Step 3, the preparation of negative sample solution, method is as follows: by the heartsupplementing and pulse restoring granule prescription proportioning, remove the ingredients of the Radix Astragali, by the heartsupplementing and pulse restoring particle recipe preparation negative sample without astragaloside, then by step 2 need testing solution preparation method, make negative sample solution.
Step 4, injects chromatograph of liquid, calculates Astragaloside content, and method is as follows: using liquid chromatography, its chromatographic condition is: C 18 chromatographic column；Flowing phase: A%: B%=acetonitrile: water (35:65)；Column temperature: 35 DEG C；Flow velocity: 0.8ml/min；Sample size: 10 μ l.Detector condition: yield value: 50；Drift tube temperature: 40 DEG C；Aerochamber temperature is arranged: cooling；Gas pressure: 20psi；Second sampling site number: 1.
Recording the content of astragaloside in every g the heartsupplementing and pulse restoring granule is 0.1807mg.
Embodiment 7 takes the heartsupplementing and pulse restoring granule of numbered test 3, measures the content of its astragaloside
Step 1, the preparation of reference substance solution
Take astragaloside reference substance appropriate, accurately weighed, add 60% methanol and make every 1ml and containing astragaloside 0.24mg and get final product.
Step 2, the preparation of need testing solution, method is as follows: take the heartsupplementing and pulse restoring granule about 3.0g, accurately weighed, to 50ml conical flask, precision adds 5% ammonia 21ml, and supersound process makes fully to leach for 40 minutes, centrifugal (3000 turns, 10min), precision pipettes supernatant 10ml, and with the speed of 1.5ml/min, by processed good C18 solid phase extraction column, (500mg, first with methanol 5ml eluting, again with water 5ml eluting), with water 5ml eluting, eluent discards, and again with methanol 2ml is slowly eluted in 2ml measuring bottle, add methanol dilution to shake up to scale, to obtain final product.
Step 3, the preparation of negative sample solution, method is as follows: by the heartsupplementing and pulse restoring granule prescription proportioning, remove the ingredients of the Radix Astragali, by the heartsupplementing and pulse restoring particle recipe preparation negative sample without astragaloside, then by step 2 need testing solution preparation method, make negative sample solution.
Step 4, injects chromatograph of liquid, calculates Astragaloside content, and method is as follows: using liquid chromatography, its chromatographic condition is: C 18 chromatographic column；Flowing phase: A%: B%=acetonitrile: water (35:65)；Column temperature: 40 DEG C；Flow velocity: 1.5ml/min；Sample size: 20 μ l.Detector condition: yield value: 200；Drift tube temperature: 60 DEG C；Aerochamber temperature is arranged: cooling；Gas pressure: 20psi；Second sampling site number: 10.
Recording the content of astragaloside in every g the heartsupplementing and pulse restoring granule is 0.1925mg.
Embodiment 8 takes the heartsupplementing and pulse restoring granule of numbered test 3, measures the content of its astragaloside
Step 1, the preparation of reference substance solution
Take astragaloside reference substance appropriate, accurately weighed, add 70% methanol and make every 1ml and containing astragaloside 0.20mg and get final product.
Step 2, the preparation of need testing solution, method is as follows: take the heartsupplementing and pulse restoring granule about 3.0g, accurately weighed, to 50ml conical flask, precision adds 4% ammonia 15ml, and supersound process makes fully to leach for 25 minutes, centrifugal (3000 turns, 10min), precision pipettes supernatant 10ml, and with the speed of 1ml/min, by processed good PLS extraction pillar, (500mg, first with methanol 5ml eluting, again with water 5ml eluting), with water 5ml eluting, eluent discards, and again with methanol 2ml is slowly eluted in 2ml measuring bottle, add methanol dilution to shake up to scale, to obtain final product.
Step 3, the preparation of negative sample solution, method is as follows: by the heartsupplementing and pulse restoring granule prescription proportioning, remove the ingredients of the Radix Astragali, by the heartsupplementing and pulse restoring particle recipe preparation negative sample without astragaloside, then by step 2 need testing solution preparation method, make negative sample solution.
Step 4, injects chromatograph of liquid, calculates Astragaloside content, and method is as follows: using liquid chromatography, its chromatographic condition is: C 18 chromatographic column；Flowing phase: A%: B%=acetonitrile: water (35:65)；Column temperature: 40 DEG C；Flow velocity: 1.0ml/min；Sample size: 10 20 μ l.Detector condition: yield value: 100；Drift tube temperature: 50 DEG C；Aerochamber temperature is arranged: cooling；Gas pressure: 30psi；Second sampling site number: 2.
Recording the content of astragaloside in every g the heartsupplementing and pulse restoring granule is 0.1896mg.
Claims (10)
1. Determination of Astragaloside method in a heartsupplementing and pulse restoring granule, it is characterised in that comprise the steps:
(1) preparation of reference substance solution: take astragaloside reference substance appropriate, adds aqueous methanol or flowing phase
Make the solution containing astragaloside 0.16～0.24mg1/ml；
(2) preparation of need testing solution: take the heartsupplementing and pulse restoring granule, with concentration be 2～5% aqueous alkali extract,
Extracting solution crosses solid-phase extraction column, by methanol-eluted fractions；
(3) algoscopy: each to above-mentioned reference substance solution and reference substance solution 5～20 μ l are injected high-efficient liquid phase color
In spectrometer.
2. assay method as claimed in claim 1, it is characterised in that in step (2), described aqueous alkali is molten
Liquid is ammonia, a kind of in sodium bicarbonate.
3. assay method as claimed in claim 2, it is characterised in that described aqueous alkali is ammonia.
4. assay method as claimed in claim 1, it is characterised in that the Solid-Phase Extraction described in step (2)
Column packing is C18 or polystyrene/divinylbenzene copolymer.
5. assay method as claimed in claim 4, it is characterised in that the Solid-Phase Extraction described in step (2)
Column packing is C18.
6. assay method as claimed in claim 1, it is characterised in that: the aqueous alkali described in step (2)
Extracting supersound extraction, extracting solution is centrifuged.
7. assay method as claimed in claim 1, it is characterised in that: step (2) is as follows: take the heartsupplementing and pulse restoring
Grain, precision adds 3～the ammonia of 7 times amount 4%, supersound process 20～make for 40 minutes fully to leach, centrifugal,
Precision pipettes supernatant, and the speed with 0.5～1.5ml/min is little by processed good C18 Solid-Phase Extraction
Post, water elution, eluent discards, and again with methanol is eluted in measuring bottle, to obtain final product.
8. assay method as claimed in claim 1, it is characterised in that the high-efficient liquid phase color described in step (3)
Spectrometer chromatographic condition:
Evaporative light scattering detector: include yield value: 50～200；Drift tube temperature: 40～60 DEG C；Aerochamber
Temperature is arranged: cooling cooling；Gas pressure: 20～30psi；Second sampling site number: 1～10；
Chromatographic column: C 18；
Flowing phase: acetonitrile: water=31～36:69～64；
Column temperature: 35～42 DEG C；
Flow velocity: 0.8～1.5ml/min.
9. assay method as claimed in claim 1, it is characterised in that also include the preparation step of negative sample solution
Suddenly, method is by the heartsupplementing and pulse restoring granule prescription proportioning, removes the ingredients of the Radix Astragali, by the heartsupplementing and pulse restoring
The grain preparation method preparation negative sample without astragaloside, then by step (2) need testing solution preparation method,
Make negative sample solution.
10. assay method as claimed in claim 1, it is characterised in that comprise the steps:
(1) preparation of reference substance solution: take astragaloside reference substance appropriate, accurately weighed, add 70% methanol
Make every 1ml 0.20mg Han astragaloside, to obtain final product；
(2) preparation of need testing solution: take the heartsupplementing and pulse restoring, accurately weighed, to 50ml conical flask, accurate
Adding 5 times amount 4% ammonia, supersound process makes fully to leach for 25 minutes, and centrifugal, precision pipettes supernatant, with
The speed of 1ml/min passes through processed good C18 solid phase extraction column, washes with water, and eluent discards, then
Slowly it is eluted in measuring bottle with methanol, adds methanol dilution and shake up to scale, to obtain final product.
(3), the preparation of negative sample solution, by the heartsupplementing and pulse restoring granule prescription proportioning, remove each taste medicine of the Radix Astragali
Material, by the heartsupplementing and pulse restoring particle recipe preparation negative sample without the Radix Astragali, then by step (2) need testing solution
Preparation method, makes negative sample solution.
(4), injecting chromatograph of liquid, its chromatographic condition is: chromatographic column C18,4.6 × 150mm, 3.5 μm
Or 4.6 × 250mm, 5 μm；Flowing phase: acetonitrile: water=35:65；Column temperature: 40 DEG C；Flow velocity: 1.0ml/min；
Sample size: 10 μ l；Evaporative light scattering detector condition, including yield value: 100；Drift tube temperature: 50 DEG C；
Aerochamber temperature is arranged: cooling cooling；Gas pressure: 30psi；Second sampling site number: 2.
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| CN110274963A (en) * | 2018-03-13 | 2019-09-24 | 天士力医药集团股份有限公司 | One kind is quenched one's thirst clear fingerprint atlas detection method |
| CN108956811A (en) * | 2018-06-12 | 2018-12-07 | 江苏颐海药业有限责任公司 | One kind being used for the method for quality control of " invigorating heart dredging collateral oral solution " preparation |
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