CN110118841A - A kind of construction method of the HPLC characteristic spectrum of oral liquid for clearing liver and gallbladder - Google Patents
A kind of construction method of the HPLC characteristic spectrum of oral liquid for clearing liver and gallbladder Download PDFInfo
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- CN110118841A CN110118841A CN201910496854.6A CN201910496854A CN110118841A CN 110118841 A CN110118841 A CN 110118841A CN 201910496854 A CN201910496854 A CN 201910496854A CN 110118841 A CN110118841 A CN 110118841A
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Abstract
The present invention relates to Quality of Chinese Traditional Proprietary Medicine control technology field, in particular to the construction method of the HPLC characteristic spectrum of a kind of oral liquid for clearing liver and gallbladder.The construction method includes the following steps: for test solution and reference solution to be measured using high performance liquid chromatography；The condition of high performance liquid chromatography are as follows: C18 chromatographic column；Using acetonitrile as mobile phase A, using 0.05%~0.15% phosphate aqueous solution of mass percentage as Mobile phase B, gradient elution is carried out.This method can integrally control product quality comprehensively, it is ensured that product safety is effective, overcomes the shortcomings of detection method in the prior art, the unicity and one-sidedness for avoiding quality from controlling, and has the characteristics that convenient, fast, stable, accurate, favorable reproducibility.
Description
Technical field
The present invention relates to Quality of Chinese Traditional Proprietary Medicine control technology field, in particular to the HPLC feature of a kind of oral liquid for clearing liver and gallbladder
The construction method of map.
Background technique
Oral liquid for clearing liver and gallbladder is by bear gall powder, cortex moutan, Sculellaria barbata, Radix Codonopsis, Radix Angelicae Sinensis, the fruit of glossy privet, agalloch eaglewood, 8 taste of malt
Medicinal material composition.With clearing heat secreting bile, the effect of recuperating qi-blood.For liver and gallbladder damp-heat, side of body rib secret anguish caused by qi and blood disorder, mouth
It is dry, bitter taste, the adjuvant treatment for the symptoms such as deficiency of food is out of strength.
Oral liquid for clearing liver and gallbladder examination criteria is recorded in the national drug standards, standard number: WS-5124 (B-0124)-
2014Z.It include bear gall powder, Sculellaria barbata, the thin layer identification item of Radix Angelicae Sinensis and the content determination item of Paeonol in standard.Its quality standard
The method for qualitative and quantitative detection for only controlling medicine simply lacks the method for the whole product quality of control comprehensively.
The content assaying method of Paeonol is also only disclosed in existing open source literature.If Yan Mingming is in delivering 2006
A kind of detection method of content of Paeonol in oral solution, tool are disclosed in the HPLC of Content of Paeonol in Gandan Shuangqing Oral " measure "
Body is as follows:
Chromatographic condition:
Shimadzu VP-ODS C18 chromatographic column (250mm × 4.6mm, 5 μm), with methanol~0.4% phosphate aqueous solution (60:40)
For mobile phase；Examine the long 274nm of side wave；25 DEG C of column temperature, number of theoretical plate is calculated by Paeonol, should be not less than 4000.Separating degree is
L.83, tailing factor 1.02.With this condition, the available good separation of other materials in Paeonol and sample, and
Negative fluid is noiseless.
Paeonol reference substance, sample and negative controls (take the other medicinal materials removed other than cortex moutan in prescription, with oral
Liquid and preparation method thereof is made without cortex moutan feminine gender oral solution, and negative fluid is made with test sample preparation method).
The preparation of reference substance solution:
Precision weighs Paeonol reference substance 2.16mg, adds methanol to dissolve and is settled in 10mL measuring bottle to get Paeonol pair
According to product solution.
The preparation of test solution:
Precision draws this product 10mL, sets in separatory funnel, with extracted by ether 4 times, each 10mL, merges ether solution, natural
It volatilizes.Residue, which adds methanol in right amount, to be made to dissolve and be quantitatively transferred in 100mL measuring bottle, with methanol dilution to scale, is shaken up, as confession
Test sample solution.
Therefore, it is necessary to develop a kind of detection method of content of a variety of active ingredients in oral liquid for clearing liver and gallbladder, so as to simultaneously
The component group character of the more batch samples of analysis detection judges the quality difference between product batch, overall monitor product quality；Also
The problem of can tracing to its source, finding technological operation, raw medicinal material.
Summary of the invention
In view of this, the present invention provides a kind of construction methods of the HPLC characteristic spectrum of oral liquid for clearing liver and gallbladder.The party
Method can integrally control product quality comprehensively, it is ensured that product safety is effective, overcomes the shortcomings of detection method in the prior art, avoids
The unicity and one-sidedness of quality control, and have the characteristics that convenient, fast, stable, accurate, favorable reproducibility.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides a kind of construction methods of the HPLC characteristic spectrum of oral liquid for clearing liver and gallbladder, include the following steps:
Test solution and reference solution are measured using high performance liquid chromatography；
The condition of high performance liquid chromatography are as follows: C18 chromatographic column；Using acetonitrile as mobile phase A, with mass percentage 0.05%~
0.15% phosphate aqueous solution is Mobile phase B, carries out gradient elution；The program of gradient elution are as follows:
Preferably, Mobile phase B is 0.1% phosphate aqueous solution of mass percentage.
Preferably, the column length of C18 chromatographic column is 25cm, internal diameter 4.6mm, partial size is 5 μm.
Preferably, C18 chromatographic column is Wondacract ODS C18 chromatographic column.
Preferably, the Detection wavelength of high performance liquid chromatography is 230~260nm.
Preferably, the Detection wavelength of high performance liquid chromatography is 245nm.
Preferably, the column temperature of high performance liquid chromatography is 30~40 DEG C.
Preferably, the column temperature of high performance liquid chromatography is 35 DEG C.
Preferably, the flow velocity of high performance liquid chromatography is 0.9~1.1mL/min.
Preferably, the flow velocity of high performance liquid chromatography is 0.9mL/min.
Preferably, the sample volume of reference solution or test solution is 5~15 μ L.
Preferably, the sample volume of reference solution or test solution is 10 μ L.
Preferably, reference solution include gallic acid solution, Paeoniflorin solution, root bark of tree peony phenol solution, asafoetide acid solution,
Four alcoholic solution of scutellarin solution, Specnuezhenide solution and agalloch eaglewood.
Preferably, the solvent of reference solution is methanol, the quality concentration expressed in percentage by volume of reference solution is 0.08~
0.12mg/mL。
Preferably, the quality concentration expressed in percentage by volume of reference solution is 0.1mg/mL.
In the present invention, test solution is the dilution of oral liquid for clearing liver and gallbladder stoste or oral liquid for clearing liver and gallbladder.
The dilution of oral liquid for clearing liver and gallbladder is that oral liquid for clearing liver and gallbladder adopts the solution after being diluted with water, clearing liver and gallbladder clothes
Volumn concentration of the liquid in the dilution of oral liquid for clearing liver and gallbladder is not less than 40%.
Preferably, volumn concentration of the oral liquid for clearing liver and gallbladder in the dilution of oral liquid for clearing liver and gallbladder is 80%.
The present invention provides a kind of construction methods of the HPLC characteristic spectrum of oral liquid for clearing liver and gallbladder.The construction method includes
Following steps: test solution and reference solution are measured using high performance liquid chromatography；The condition of high performance liquid chromatography
Are as follows: C18 chromatographic column；Using acetonitrile as mobile phase A, using 0.05%~0.15% phosphate aqueous solution of mass percentage as Mobile phase B,
Carry out gradient elution.The present invention has the following technical effect that
Using detection method can simultaneously the more batch samples of analysis detection component group character, judge product batch it
Between quality difference, overall monitor product quality.The problem of can also tracing to its source, finding technological operation, raw medicinal material.
This method can integrally control product quality comprehensively, it is ensured that product safety is effective, overcomes detection side in the prior art
The deficiency of method, the unicity and one-sidedness for avoiding quality from controlling, and there are the spies such as convenient, fast, stable, accurate, favorable reproducibility
Point.
15 characteristic peaks are presented in test sample characteristic spectrum of the present invention, wherein 7 peaks are protected with corresponding object of reference peak respectively
Stay the time identical.It is the peak S with Specnuezhenide object of reference peak respective peaks, other peak relative retention times should be ± the 5% of specified value
Within.Specified value are as follows: 0.16 (peak 1), 0.20 (peak 2), 0.21 (peak 3), 0.42 (peak 4), 0.58 (peak 5), 0.62 (peak 6),
0.72 (peak 7), 0.75 (peak 8), 0.81 (peak 9), 0.92 (peak 10), 0.96 (peak 11), 1.00 (peaks 12), 1.45 (peaks 13),
1.52 (peaks 14), 1.71 (peaks 15).
The present invention establishes 10 batch oral liquid for clearing liver and gallbladder HPLC characteristic spectrums, using Chinese Pharmacopoeia Commission's " Chinese medicine
Chromatographic fingerprinting similarity evaluation system " 2004A editions analyses, obtain the oral liquid for clearing liver and gallbladder being made of 15 characteristic peaks
HPLC standard feature map.No. 1 peak is gallic acid, and No. 5 peaks are Paeoniflorin, and No. 6 peaks are agalloch eaglewood tetrol, and No. 8 peaks are ferulic acid,
No. 11 peaks are scutellarin, and No. 12 peaks (S) are Specnuezhenide, No. 15 peaks are Paeonol.15 characteristic peaks are belonged to: ownership
There are 4 in the chromatographic peak of cortex moutan, respectively 1,4,5, No. 15 peak；The chromatographic peak for belonging to Sculellaria barbata has 3, respectively 9,
10, No. 11 peaks；The chromatographic peak for belonging to the fruit of glossy privet has 3, respectively 12,13, No. 14 peaks；The chromatographic peak for belonging to Radix Angelicae Sinensis is 1
It is a, it is No. 8 peaks；The chromatographic peak for belonging to agalloch eaglewood has 2, respectively 6, No. 7 peaks.
Detailed description of the invention
Fig. 1 shows 10 batches of oral liquid for clearing liver and gallbladder characteristic spectrum common pattern figures；
Fig. 2 shows oral liquid for clearing liver and gallbladder compare feature map.
Specific embodiment
The invention discloses a kind of construction method of the HPLC characteristic spectrum of oral liquid for clearing liver and gallbladder, those skilled in the art
Present disclosure can be used for reference, realization of process parameters is suitably modified.In particular, it should be pointed out that all similar substitutions and modifications pair
It is it will be apparent that they are considered as being included in the present invention for those skilled in the art.Method and application of the invention is
Through being described by preferred embodiment, related personnel obviously can not depart from the content of present invention, in spirit and scope to this
Methods and applications described in text are modified or appropriate changes and combinations, carry out implementation and application the technology of the present invention.
The construction method of oral liquid for clearing liver and gallbladder characteristic spectrum:
Chromatographic condition and system suitability: Wondacract ODS C18 chromatographic column (column length 25cm, internal diameter are used
For 4.6mm, partial size is 5 μm)；Using acetonitrile as mobile phase A, using 0.1% phosphoric acid solution as Mobile phase B, provide to carry out according to the form below
Gradient elution；Detection wavelength is 230~260nm；Column temperature is 30~40 DEG C；Flow velocity is 0.9~1.1mL/min；Theoretical cam curve
6000 should be not less than by calculating by Specnuezhenide peak.
The preparation of reference solution: it takes gallic acid, Paeoniflorin, Paeonol, ferulic acid, scutellarin, Specnuezhenide, sink
Fragrant tetrol reference substance is appropriate, accurately weighed, respectively with methanol be made solution of every 1mL containing 0.1mg to get.
The preparation of test solution: precision measures 10~20mL of this product, sets in 20~25mL measuring bottle, is diluted with water to quarter
Degree, shakes up, and filters, takes subsequent filtrate, take subsequent filtrate as test solution.
Measuring method: it is accurate respectively to draw reference solution and each 5~15 μ L of test solution, liquid chromatograph is injected, is surveyed
It is fixed, record chromatogram to get.
Agents useful for same or instrument are equal in the construction method of the HPLC characteristic spectrum of oral liquid for clearing liver and gallbladder provided by the invention
It is available on the market.
Below with reference to embodiment, the present invention is further explained:
The construction method of 1 oral liquid for clearing liver and gallbladder characteristic spectrum of embodiment
1 chromatographic condition and system suitability use Wondacract ODS C18 chromatographic column (column length 25cm, it is interior
Diameter is 4.6mm, and partial size is 5 μm)；Using acetonitrile as mobile phase A, using 0.1% phosphoric acid solution as Mobile phase B, provided according to the form below into
Row gradient elution；Detection wavelength is 245nm；Column temperature is 35 DEG C；Flow velocity is 0.9mL/min；Theoretical cam curve presses Specnuezhenide peak
6000 should be not less than by calculating.
The preparation of 2 reference solutions take gallic acid, Paeoniflorin, Paeonol, ferulic acid, scutellarin, Specnuezhenide,
Agalloch eaglewood tetrol reference substance is appropriate, accurately weighed, respectively with methanol be made solution of every 1mL containing 0.1mg to get.
The preparation precision of 3 test solutions measures this product 20mL, sets in 25mL measuring bottle, is diluted with water to scale, shakes up,
Filtration, takes subsequent filtrate, takes subsequent filtrate as test solution.
4 measuring methods difference is accurate to draw reference solution and each 10 μ L of test solution, injects liquid chromatograph, measures,
Record chromatogram to get.
1 methodological study of test example embodiment
1, precision test
Same test solution is taken, is measured according to the above method, continuous sample introduction 6 times, chromatogram is recorded, calculates each characteristic peak
Relative retention time RSD value, RSD value show that instrument precision is good less than 1.0%.It the results are shown in Table 3.
1 Precision test result of table
2, repetitive test
It takes with a collection of test sample, prepares 6 parts of test solutions in accordance with the law, be measured, each characteristic peak in 6 measurement chromatograms
Relative retention time RSD value less than 1.0%, show method repeatability preferably.It the results are shown in Table 2.
2 repetitive test result of table
3, stability test
Same test solution is taken, was detected in accordance with the law at 0,2,4,8,12,18,24 hour respectively, the phase of each characteristic peak
To retention time RSD value less than 1.0%, show that test solution is stablized within 24 hours.It the results are shown in Table 3.
3 solution stability testing result of table
4, characteristic spectrum measures
The oral liquid for clearing liver and gallbladder of 10 batches is measured using the above method, records characteristic spectrum, when relatively retaining relatively
Between, as a result for 10 batches of oral liquid for clearing liver and gallbladder relative retention time RSD values less than 3.0%, detailed results are as follows:
Table 4
Using 2004A editions analyses of Chinese Pharmacopoeia Commission's " similarity evaluation ", 10 are obtained
The characteristic spectrum and common pattern of the oral liquid for clearing liver and gallbladder of a batch are as shown in Figure 1.To 10 batches of oral liquid for clearing liver and gallbladder features
Map carries out similarity calculation, and 10 batches of oral liquid for clearing liver and gallbladder characteristic spectrums and standard feature map similarity are all larger than 0.90.
Detailed results are as follows:
Table 5
10 batch oral liquid for clearing liver and gallbladder HPLC characteristic spectrums are established, using Chinese Pharmacopoeia Commission's " Chinese medicine chromatographic fingerprint
Map similarity evaluation system " 2004A editions analyses, obtain the oral liquid for clearing liver and gallbladder HPLC standard spy being made of 15 characteristic peaks
Levy map.No. 1 peak is gallic acid, and No. 5 peaks are Paeoniflorin, and No. 6 peaks are agalloch eaglewood tetrol, and No. 8 peaks are ferulic acid, and No. 11 peaks are open country
Scutelloside, No. 12 peaks (S) are Specnuezhenide, No. 15 peaks are Paeonol.15 characteristic peaks are belonged to: belonging to cortex moutan
Chromatographic peak has 4, respectively 1,4,5, No. 15 peak；The chromatographic peak for belonging to Sculellaria barbata has 3, respectively 9,10, No. 11 peaks；Return
The chromatographic peak for belonging to the fruit of glossy privet has 3, respectively 12,13, No. 14 peaks；The chromatographic peak for belonging to Radix Angelicae Sinensis is 1, is No. 8 peaks；Return
The chromatographic peak for belonging to agalloch eaglewood has 2, respectively 6, No. 7 peaks.
Comparative example 1:
Chromatographic condition and system suitability Shimadzu VP-ODS C18 chromatographic column (250mm × 4.6mm, 5 μm)；With first
Alcohol~0.4% phosphate aqueous solution (60:40) is mobile phase；Examine the long 274nm of side wave；25 DEG C of column temperature；
The preparation of reference solution takes gallic acid, Paeoniflorin, Paeonol, ferulic acid, scutellarin, Specnuezhenide, sinks
Fragrant tetrol reference substance is appropriate, accurately weighed, respectively with methanol be made solution of every 1mL containing 0.1mg to get.
The preparation precision of test solution measures this product 20mL, sets in 25mL measuring bottle, is diluted with water to scale, shakes up, filter
It crosses, takes subsequent filtrate, take subsequent filtrate as test solution.
Measuring method difference is accurate to draw reference solution and each 10 μ L of test solution, injects liquid chromatograph, measures,
Record chromatogram to get.
As a result, chromatographic peak quantity is few in chromatogram, separating degree is poor between each chromatographic peak, each reference substance chromatography of reference solution
Only Paeonol reference substance has corresponding chromatographic peak at peak, the non-appearance of remaining reference substance.
Comparative example 2:
Chromatographic condition and system suitability: Wondacract ODS C18 chromatographic column (column length 25cm, internal diameter are used
For 4.6mm, partial size is 5 μm)；Mobile phase is A: acetonitrile and B:0.1% phosphate aqueous solution, eluent gradient elution requirement are as follows:
0min is 0% acetonitrile, and 40min is 50% acetonitrile, and 42min is 0% acetonitrile, and 50min is 0% acetonitrile；Flow velocity 1.0mL/min；Inspection
Survey wavelength 230nm；30 DEG C of column temperature.
The preparation of reference solution takes gallic acid, Paeoniflorin, Paeonol, ferulic acid, scutellarin, Specnuezhenide, sinks
Fragrant tetrol reference substance is appropriate, accurately weighed, respectively with methanol be made solution of every 1mL containing 0.1mg to get.
The preparation precision of test solution measures this product 20mL, sets in 25mL measuring bottle, is diluted with water to scale, shakes up, filter
It crosses, takes subsequent filtrate, take subsequent filtrate as test solution.
Measuring method difference is accurate to draw reference solution and each 10 μ L of test solution, injects liquid chromatograph, measures,
Record chromatogram to get.
As a result, separating degree and degree of asymmetry are low between each chromatographic peak in chromatogram, inferior separating effect.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of construction method of the HPLC characteristic spectrum of oral liquid for clearing liver and gallbladder, which comprises the steps of:
Test solution and reference solution are measured using high performance liquid chromatography；
The condition of the high performance liquid chromatography are as follows: C18 chromatographic column；Using acetonitrile as mobile phase A, with mass percentage 0.05%~
0.15% phosphate aqueous solution is Mobile phase B, carries out gradient elution；The program of the gradient elution are as follows:
2. construction method according to claim 1, which is characterized in that the Mobile phase B is 0.1% phosphorus of mass percentage
Aqueous acid.
3. construction method according to claim 1, which is characterized in that the column length of the C18 chromatographic column is 25cm, and internal diameter is
4.6mm, partial size are 5 μm.
4. construction method according to claim 1, which is characterized in that the C18 chromatographic column is Wondacract ODS
C18 chromatographic column.
5. construction method according to claim 1, which is characterized in that the Detection wavelength of the high performance liquid chromatography is 230
~260nm.
6. construction method according to claim 1, which is characterized in that the column temperature of the high performance liquid chromatography is 30~40
℃。
7. construction method according to claim 1, which is characterized in that the flow velocity of the high performance liquid chromatography be 0.9~
1.1mL/min；The sample volume of the reference solution or the test solution is 5~15 μ L.
8. construction method according to claim 1, which is characterized in that the reference solution include gallic acid solution,
Paeoniflorin solution, root bark of tree peony phenol solution, asafoetide acid solution, four alcoholic solution of scutellarin solution, Specnuezhenide solution and agalloch eaglewood.
9. construction method according to claim 8, which is characterized in that the solvent of the reference solution is methanol, reference
The quality concentration expressed in percentage by volume of object solution is 0.08~0.12mg/mL.
10. construction method according to any one of claim 1 to 9, which is characterized in that the test solution is liver and gallbladder
The dilution of double clear oral solution stostes or oral liquid for clearing liver and gallbladder.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112858495A (en) * | 2020-12-02 | 2021-05-28 | 吉林修正药业新药开发有限公司 | Construction method of HPLC (high Performance liquid chromatography) characteristic spectrum of bear gall Keming tablets |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1709320A (en) * | 2005-06-14 | 2005-12-21 | 吉林长白山药业集团股份有限公司 | Oral liquid for clearing liver and gallbladder |
-
2019
- 2019-06-10 CN CN201910496854.6A patent/CN110118841B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1709320A (en) * | 2005-06-14 | 2005-12-21 | 吉林长白山药业集团股份有限公司 | Oral liquid for clearing liver and gallbladder |
Non-Patent Citations (2)
| Title |
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| LING WANG ET AL.: "A bio-activity guided in vitro pharmacokinetic method to improve the quality control of Chinese medicines, application to SiWu Tang", 《INTERNATIONAL JOURNAL OF PHARMACEUTICS》 * |
| 严铭铭 等: "肝胆双清口服液中丹皮酚的HPLC测定", 《中国药师》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112858495A (en) * | 2020-12-02 | 2021-05-28 | 吉林修正药业新药开发有限公司 | Construction method of HPLC (high Performance liquid chromatography) characteristic spectrum of bear gall Keming tablets |
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