CN103267812B - Method for detecting quality of Bazhen particles - Google Patents
Method for detecting quality of Bazhen particles Download PDFInfo
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- CN103267812B CN103267812B CN201310159676.0A CN201310159676A CN103267812B CN 103267812 B CN103267812 B CN 103267812B CN 201310159676 A CN201310159676 A CN 201310159676A CN 103267812 B CN103267812 B CN 103267812B
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Abstract
The invention discloses a method for detecting quality of Bazhen particles, comprising the following steps of: using octadecylsilane chemically bonded silica as a filler; using acetonitrile-0.1% phosphoric acid solution as a mobile phase, wherein a detecting wavelength is 230nm and a flow velocity of the mobile phase is 0.8-1.2ml/min; precisely weighting a paeoniflorin reference substance, and adding 70% ethanol to prepare a paeoniflorin solution; precisely weighting the Bazhen particles, and placing the particles into an erlenmeyer flask with a plug, then precisely adding 50ml 70%ethanol, sealedly plugging the erlenmeyer flask, weighting the erlenmeyer flask, ultrasonically processing for 28-32min, cooling, then weighting, supplementing losing weight by the 70% ethanol, shaking up; taking 10mul each of the a reference substrate solution and a tested object solution, injecting it into a liquid chromatograph, and then detecting. The method of the invention has advantages of rapid and timely completion, and good stabilization and reappearance, can control product quality, can reduce body damage of working personnel, and can reduce unfavorable influence on working environment.
Description
Technical field:
The invention belongs to the Quality Control Technology field of Chinese medicine preparation, be specifically related to a kind of quality determining method of Bazhen granule.
Background technology:
Bazhen granule records in " Chinese Pharmacopoeia " version in 2010, be made up of Radix Codonopsis, rhizoma atractylodis macrocephalae, Poria cocos, honey-fried licorice root, Radix Angelicae Sinensis, stir-baked RADIX PAEONIAE ALBA, Ligusticum wallichii, prepared rhizome of rehmannia eight taste Chinese medicine, Bazhen granule is made up of Radix Codonopsis 60g, rhizoma atractylodis macrocephalae 60g, Poria cocos 60g, honey-fried licorice root 30g, Radix Angelicae Sinensis 90g, stir-baked RADIX PAEONIAE ALBA 60g, Ligusticum wallichii 45g, prepared rhizome of rehmannia 90g eight taste medicine.Eight taste medicinal materials of Bazhen granule composition, except the root of herbaceous peony and Radix Glycyrrhizae, not yet set up the content of main pharmacodynamics composition in medicinal material, or content of drug effect components lower being difficult in compound preparation is detected or can't detect; Although glycyrrhizic acid content is higher in Radix Glycyrrhizae, but after prescription, because Radix Glycyrrhizae plays coordinating the drug actions of a prescription effect, after compatibility closes and decocts, glycyrrhizic acid content declines to a great extent, content in Bazhen granule is unstable, so that when Bazhen granule rises to 2010 editions standards of pharmacopoeia from ministerial standard, the discriminating item of Radix Glycyrrhizae in former Bazhen granule is left out.Now, many content by detecting effective component Paeoniflorin in the root of herbaceous peony, thus detect the quality of Bazhen granule.
Bazhen granule has invigorating qi and benefiting blood function, is used for the treatment of qi-blood deficiency, sallow complexion, poor appetite, limbs fatigue, menorrhalgia etc.And how effectively to detect the quality of Bazhen granule, thus make this finished medicines realize above-mentioned effect, be the problem that this field is studied always.At present, Bazhen granule adopts methanol-water (38: 62), acetonitrile-0.1% phosphoric acid solution-triethylamine (13: 87: 0.04) to carry out the quality control of Bazhen granule for flow phase system respectively.At present, the above-mentioned method of quality control of prior art, wherein adopts methyl alcohol as mobile phase, because methyl alcohol is the liquid of highly volatile, the mobile phase be equipped with will join timely use in time, can not place for a long time, therefore, each detection all will reconfigure, process is loaded down with trivial details, also first must configure just passable if need to detect as early as possible, therefore, be unfavorable for completing detection fast, timely.And methyl alcohol toxicity is relatively large, be easy to again volatilization, Long Term Contact, is not easy to the healthy of staff.In addition, triethylamine has the colourless transparent liquid having strong ammonia smelly, is micro-ly in atmosphere fuming, and Long Term Contact, very easily damages human body, and makes a very bad impression to working environment.
Therefore, be badly in need of exploitation one and can complete detection fast, timely, and effectively reduce the quality determining method of the Bazhen granule of the actual bodily harm to staff and the adverse effect to working environment.
Summary of the invention:
The present invention is directed to the above-mentioned deficiency of prior art, there is provided one can complete detection detecting method fast, timely, and the Bazhen granule quality determining method that stability, favorable reproducibility, the carrying out that can reduce the actual bodily harm to staff and the adverse effect to working environment control.
In order to solve the problems of the technologies described above, the technical solution used in the present invention is: a kind of quality determining method of Bazhen granule, is characterized in that: comprise the steps:
(1) chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; With acetonitrile-0.1% phosphoric acid solution (mass percent) for mobile phase; Determined wavelength is 230nm; Flow rate of mobile phase is 0.8 ~ 1.2ml/min; Number of theoretical plate calculates by Paeoniflorin peak and is not less than 2000;
(2) preparation of reference substance solution: get Paeoniflorin reference substance appropriate, accurately weighed, add 70% ethanol and make the solution of every 1ml containing 28 ~ 32g Paeoniflorin, to obtain final product;
(3) preparation of need testing solution: get Bazhen granule content, porphyrize, sugar-containing type gets 2g or Sugarless type gets 1g, accurately weighed, inserts to have stopper and obtain in conical flask; Precise 70% ethanol 50ml adds in conical flask, by stopper jam-pack taper bottleneck, weighs overall weight; Then ultrasonic process 28 ~ 32 minutes, in conical flask, solution is put to room temperature, more weighed weight, calculates twice weight difference, supplies the weight of less loss, shakes up, to obtain final product with 70% ethanol;
(4) determination method: accurately draw reference substance solution and each 10 μ l of need testing solution respectively, injection liquid chromatography, measures.
Flow rate of mobile phase described in step of the present invention (1) is preferably 1.0ml/min.
The solution of every 1ml containing 30 μ g Paeoniflorins made by the preferred 70% described ethanol of step of the present invention (2).
The preferred ultrasonic process 30 points of step of the present invention (3).
The power of step of the present invention (3) ultrasonic process is 300W, and frequency is 50kHz.
The volume ratio of acetonitrile of the present invention and 0.1% phosphoric acid solution is 12 ﹕ 88.
The Bazhen granule quality determining method that the present invention is above-mentioned, wherein: this product every bag is (containing the root of herbaceous peony with Paeoniflorin (C
23h
28o
11) meter, must not be less than 4.3mg(and determine according to multiple batches of assay, see below in table 9 and carry out assay to Paeoniflorin in multiple batches of Bazhen granules, find that part is batch at 4.3mg, all the other content are all higher than 4.3mg).
Bazhen granule of the present invention is by Radix Codonopsis 60g, rhizoma atractylodis macrocephalae 60g, Poria cocos 60g, honey-fried licorice root 30g, Radix Angelicae Sinensis 90g, stir-baked RADIX PAEONIAE ALBA 60g, Ligusticum wallichii 45g, prepared rhizome of rehmannia 90g, above eight tastes, Radix Angelicae Sinensis, Ligusticum wallichii and rhizoma atractylodis macrocephalae priority 95% ethanol, 50% ethanol difference heating and refluxing extraction 2 hours, filter, filtrate merges, reclaim ethanol, filter, filtrate is for subsequent use; The five tastes boiling secondaries such as the dregs of a decoction and all the other Radix Codonopsis, each 1.5 hours, filter, filtrate merges, add above-mentioned filtrate for subsequent use, be concentrated into appropriate, add sucrose and appropriate dextrin, mixing, make particle, dry, make 1000g, be sugar-containing type specification, the sugar-containing type described in the present invention is this specification; Or adding appropriate soluble starch, dextrin, one or more auxiliary materials of starch and flavouring, mixing, makes particle, and dry, make 300g, be Sugarless type specification, the Sugarless type described in the present invention is this specification.The every packed 8g of sugar-containing type specification, the every packed 3.5g of Sugarless type specification.
Advantage of the present invention and beneficial effect:
1. the present invention is using Paeoniflorin in Bazhen granule as assay index components, content assaying method is determined by the test such as preparation selection, system suitability, linear relationship, precision, sample stability, sample reappearance, the sample pipetting volume recovery, sample determination of chromatography condition, need testing solution, Bazhen granule quality is more easily controlled, and method is stable, favorable reproducibility, is applicable to Sugarless type and sugar-containing type.
2. the present invention adopts acetonitrile-0.1% phosphoric acid solution as mobile phase, thus do not use the methyl alcohol that toxicity is relatively large, be easy to volatilization, the triethylamine having and have colourless transparent liquid that strong ammonia is smelly is not used yet, therefore, detection detecting method can be completed fast, timely, and stable, favorable reproducibility, be conducive to product quality, the actual bodily harm to staff and the adverse effect to working environment can be reduced.
Accompanying drawing explanation
Fig. 1, Paeoniflorin reference substance, Bazhen granule test sample, the blank test sample HPLC figure of the scarce root of herbaceous peony.
Fig. 2, Paeoniflorin canonical plotting.
Embodiment
1, instrument and reagent
1.1 instruments: HP1100 liquid chromatograph, HP1100 detecting device; TU-1901 ultraviolet spectrophotometer.
1.2 reference substances: Paeoniflorin reference substance, are provided (lot number is 110736-200321) by Nat'l Pharmaceutical & Biological Products Control Institute.
1.3 reagent: acetonitrile is chromatographically pure, water is redistilled water, and all the other are pure for analyzing.
1.4 reagents: Li Hua pharmaceutcal corporation, Ltd provides by Ningbo.
2, the selection of wavelength is measured: get Paeoniflorin reference substance appropriate, add 70% ethanol and make reference substance solution, scan within the scope of 200 ~ 400nm, result Paeoniflorin has absorption maximum at the wavelength place of 231.5nm, and with reference to pharmacopeia, therefore determined wavelength is chosen as 230nm.
3, the preparation of reference substance solution
Paeoniflorin reference substance solution is 1.: precision takes gets Paeoniflorin reference substance 13.51mg, puts in 100ml measuring bottle, adds 70% ethanol and dissolve and be diluted to scale, shake up, and obtains the solution of every 1ml containing Paeoniflorin 0.1351mg;
Paeoniflorin reference substance solution is 2.: precision measures Paeoniflorin reference substance solution 1. 3ml, puts in 10ml measuring bottle, adds 70% ethanol and is diluted to scale, shake up, and obtains the solution of every 1ml containing Paeoniflorin 40.53 μ g.
Paeoniflorin reference substance solution is 3.: precision measures Paeoniflorin reference substance solution 1. 2ml, puts in 10ml measuring bottle, adds 70% ethanol and is diluted to scale, shake up, and obtains the solution of every 1ml containing Paeoniflorin 27.02 μ g.
4, chromatographic condition test
When selecting acid to make mobile phase, chromatogram peak shape is better, pretends further investigation, finds with acetonitrile-0.1% phosphoric acid solution (12:88) as mobility good separating effect.
Chromatographic column: octadecylsilane chemically bonded silica is filling agent (5 μm, Yi Lite C18 post, 250 × 4.6mm)
Flow velocity: 1.0ml/ minute
Determined wavelength: 230nm
The preparation of need testing solution:
The preparation of need testing solution: get Bazhen granule content, porphyrize, sugar-containing type gets 2g(Sugarless type 1g), accurately weighed, put in tool plug conical flask, precision adds 70% ethanol 50ml, close plug, weighed weight, ultrasonic process (power 300W, frequency 50kHz) 30 minutes, let cool, more weighed weight, the weight of less loss is supplied with 70% ethanol, shake up, obtain need testing solution.
Lack the preparation of root of herbaceous peony blank solution: the Radix Codonopsis that feeds intake, Poria cocos, honey-fried licorice root, Radix Angelicae Sinensis, stir-baked RADIX PAEONIAE ALBA, Ligusticum wallichii, prepared rhizome of rehmannia, namely do not add the root of herbaceous peony, according to Bazhen granule preparation method, lack the blank Bazhen granule of the root of herbaceous peony with legal system is standby.Get the blank Bazhen granule of the scarce root of herbaceous peony, with need testing solution preparation method, obtained scarce root of herbaceous peony blank solution.
With acetonitrile-0.1% phosphoric acid solution (12:88) for mobile phase, accurate absorption reference substance solution (C=27.02 μ g/ml) and need testing solution, the scarce each 10 μ l of root of herbaceous peony blank solution respectively, injection liquid chromatography, record chromatogram.
Result is as can be seen from test sample collection of illustrative plates, have between Paeoniflorin peak and other peak that good degree of separation, peak shape are good, post is imitated high, Paeoniflorin peak is better separated with impurity peak energy and scarce root of herbaceous peony blank is noiseless, therefore using the chromatographic condition of this chromatographic condition as this experiment.
5, the selection of extraction conditions:
The selection of 5.1 solvents:
Because Paeoniflorin is soluble in ethanol water, therefore select 30% ethanol, 70% ethanol, ethanol as Extraction solvent respectively, compared, the results are shown in Table 1.Result shows, take ethanol as Extraction solvent, Lower result, and basically identical by 30% ethanol, 70% ethanol content result, because this product contains sucrose, is the solubleness reducing sucrose, therefore selects 70% ethanol to be Extraction solvent.
The selection of table 1 Extraction solvent
The selection of 5.2 extracting method:
When determining solvent, comparing heating and refluxing extraction and ultrasonic extracting method, the results are shown in Table 2.Result is visible, and content is basically identical, therefore selects ultrasonic extraction 30 minutes.
The selection of table 2 extracting method
The comparison of 5.3 Extraction solvent amounts:
Under sampling amount same case, compare different Extraction solvent amounts, the results are shown in Table 3.Result is visible, and content is basically identical, therefore Extraction solvent amount elects 50ml as.
The selection of table 3 Extraction solvent amount
6 is linear:
Precision measures reference substance solution (40.53 μ g/ml) 0.5,1,2,5,10 μ l, reference substance solution (0.1351mg/ml) 5,10,15 μ l, injection liquid chromatography, measure peak area Y(in table 4), with sample size (ug) for horizontal ordinate, peak area is ordinate, makes typical curve (see figure 2), and calculate regression equation and obtain: Y=952.48X+36.033, R
2=0.9971, R=0.9985, result display sample size has good linear relationship within the scope of 0.023 ~ 2.027ug.
Table 4 Paeoniflorin linear relationship is investigated
7, instrument precision: get same test sample (lot number: criticize 2) solution, by above chromatographic condition, repeat sample introduction 6 times, measure peak area, result RSD is 0.76% (n=6), in table 5.
Table 5 Paeoniflorin precision test
8, durability (stability): get same test sample (lot number: criticize 2) solution, separated in time measures once, and result RSD=0.76% (n=6), the results are shown in Table 6.Result display need testing solution is basicly stable in 36 hours.
The stability test of table 6 Paeoniflorin
9, repeatability: precision takes same sample lots (lot number: criticize 2) 6 parts, press text method to extract, measure peak area and calculate paeoniflorin content (mg/g), result: the average content of this product Paeoniflorin is 0.497mg/g, RSD=2.2%(n=6), in table 7.
Table 7 Paeoniflorin replica test
10, accuracy: precision takes 9 parts, known content sample (lot number: criticize 2), it is appropriate that precision adds Paeoniflorin reference substance, extracts, record peak area and calculate content by text method, result shows: this product paeoniflorin content average recovery rate is 99.9%, RSD=1.2%.(see table 8).
Table 8 paeoniflorin content recovery test
11, sample determination and limits: the sample measuring six batches by text method, the results are shown in Table 9, according to these six batch datas, therefore orders limit temporarily as every bag contains the root of herbaceous peony with Paeoniflorin (C
23h
28o
11) meter, must not 4.3mg be less than.
Table 9 paeoniflorin content measurement result
Concrete quality determining method, comprises the steps:
1, chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; With acetonitrile-0.1% phosphoric acid solution (12:88) for mobile phase; Determined wavelength is 230nm; Flow velocity 1.0ml/min.Number of theoretical plate is pressed Paeoniflorin peak and is calculated, and should be not less than 2000.
2, the preparation of reference substance solution: get Paeoniflorin reference substance appropriate, accurately weighed, add 70% ethanol and make the solution of every 1ml containing 30 μ g, to obtain final product.
3, the preparation of need testing solution: get Bazhen granule content, porphyrize, sugar-containing type gets 2g(Sugarless type 1g), accurately weighed, put in tool plug conical flask, precision adds 70% ethanol 50ml, close plug, weighed weight, ultrasonic process (power 300W, frequency 50kHz) 30 minutes, let cool, more weighed weight, the weight of less loss is supplied with 70% ethanol, shake up, to obtain final product.
4, determination method: accurate absorption reference substance solution and each 10 μ l of need testing solution respectively, injection liquid chromatography, measures, to obtain final product.This product every bag contains the root of herbaceous peony with Paeoniflorin (C
23h
28o
11) meter, must not 4.3mg be less than.
Claims (1)
1. a quality determining method for Bazhen granule, is characterized in that: comprise the steps:
(1) chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; With acetonitrile-0.1% phosphoric acid solution (mass percent) for mobile phase; Determined wavelength is 230nm; Flow rate of mobile phase is 1.0ml/min; Number of theoretical plate calculates by Paeoniflorin peak and is not less than 2000;
(2) preparation of reference substance solution: get Paeoniflorin reference substance, accurately weighed, add 70% ethanol and make the solution of every 1ml containing 30 μ g Paeoniflorins, to obtain final product;
(3) preparation of need testing solution: get Bazhen granule content, porphyrize, sugar-containing type gets 2g or Sugarless type gets 1g, accurately weighed, inserts to have stopper and obtain in conical flask; Precise 70% ethanol 50ml adds in conical flask, by stopper jam-pack taper bottleneck, weighs overall weight; Then ultrasonic process 30 minutes, in conical flask, solution is put to room temperature, more weighed weight, calculates twice weight difference, supplies the weight of less loss, shakes up, to obtain final product with 70% ethanol;
(4) determination method: accurately draw reference substance solution and each 10 μ l of need testing solution respectively, injection liquid chromatography, measures; The volume ratio of described acetonitrile and 0.1% phosphoric acid solution is 12 ﹕ 88.
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Address after: 315174 Xinlian village, Gaoqiao Town, Haishu District, Ningbo City, Zhejiang Province Patentee after: NINGBO LIWAH PHARMACEUTICAL Co.,Ltd. Address before: 315174 New Union Village, Gaoqiao Town, Yinzhou District, Ningbo, Zhejiang Patentee before: NINGBO LIWAH PHARMACEUTICAL Co.,Ltd. |
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