CN103822993B - Method for detecting content of herba lagotis in preparation - Google Patents
Method for detecting content of herba lagotis in preparation Download PDFInfo
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- CN103822993B CN103822993B CN201410096039.8A CN201410096039A CN103822993B CN 103822993 B CN103822993 B CN 103822993B CN 201410096039 A CN201410096039 A CN 201410096039A CN 103822993 B CN103822993 B CN 103822993B
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- 238000002360 preparation method Methods 0.000 title claims abstract description 27
- 241000382298 Lagotis Species 0.000 title abstract description 5
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- 238000001514 detection method Methods 0.000 claims abstract description 32
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- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention relates to a method for detecting the content of herba lagotis in a preparation. A test solution is obtained through steps of degreasing treatment, extraction by water saturated n-butyl alcohol, impurity removing and concentration by a porous resin, and furthermore, methanol of different proportions and 0.1% phosphoric acid are used as a flow phase for gradient elution for performing separation detection on echinacoside in the preparation. The method is simple and convenient to operate, high in specificity, small in interference, high in separation degree and good in linear property within a certain concentration range, and can be applied to detection on the content of the herba lagotis in the preparation.
Description
Technical field
The present invention relates to a kind of detection method of content of preparation, particularly a kind of detection method of content of the preparation containing Lagotis glauca.
Background technology
Lagotis glauca is the dry herb of goatweed Lagotis brachystachya Maxim Lagotis brevituba Maxim. or Herba Lagotis integrae L.integraW.Smith..The Xia Qiuhua phase of containing gathers, and removing impurity, cleans, dry in the shade.This product is medicinal herbs most in use in Tibetan medicinal preparation, has effect that is clearing heat and detoxicating, promoting circulation of blood menstruation regulating.At present, Lagotis glauca has more than 20 as the prescribed preparation of raw medicinal material, as 25-component great Tang ball, and ershiwuwei datang powder, 25-component emblic ball, 25-component emblic falls apart, and 25-component Zhu Huang is loose, and 25-component Swertia patens falls apart, 25-component tuberculosis ball, 25-component swertia pill, ten taste myrobalan soup fall apart, eight taste main ingredients fall apart, eight taste swertia pill, and eight taste Swertia patens fall apart, the blue or green roc of nine tastes falls apart, and nine taste Zhu Huangs are loose, nine taste swertia pill, 30 five tastes agalloch eaglewood balls, the solar month brilliant ball, intelligence holder JIEBAI WAN etc.
Lagotis glauca act.std is Tibetan medicine ministerial standard, standard No.: WS3-BC-0063-95.This standard has only underdrawed the shape of Lagotis glauca and discriminating.Article " in Tibetan Herb Lagotis brevituba Maxim, the RP-HPLC of echinacoside and acteoside analyzes " describes one with methyl alcohol-1% glacial acetic acid solution (28:72, V:V) for mobile phase, the method for echinacoside and ergot steroid in Simultaneously test Lagotis brachystachya Maxim.Pharmacopeia version in 2010 describes flood and connects the detection method of content that (i.e. Lagotis glauca) is mobile phase with acetonitrile-methanol-1% acetic acid (10:15:75).But above method is only applicable to the content detection of medicinal material, when for content detection containing Lagotis glauca preparation, due to other flavour of a drug serious interference, simultaneously main to detect component content less, all can not reach the requirement of HPLC method content detection.Therefore a kind of detection method of content containing Lagotis glauca preparation with versatility of present stage urgent need.
Without the detection method of content be directly used in containing Lagotis glauca preparation in prior art, only have the detection method for Lagotis glauca medicinal material, and the method cannot be used for the detection of compound preparation.The steps such as detection method of the present invention is extracted by ungrease treatment, water-saturated n-butanol, macroreticular resin impurity elimination is concentrated obtain need testing solution, and adopt the methyl alcohol of different ratio and the phosphoric acid of percent by volume 0.1% to be that mobile phase carries out gradient elution further, detection is separated to echinacoside wherein, operate easier, specificity is strong, and disturb less, degree of separation is high, better in finite concentration scope internal linear, can be used for the content detection containing Lagotis glauca preparation.
Summary of the invention
For overcoming the deficiencies in the prior art, the invention provides a kind of detection method of content containing Lagotis glauca preparation.
Term illustrates:
Gradient elution mode of the present invention is change the ratio of A, B two kinds of eluents, to method components different on chromatographic column taken off at rational time wash-in according to time gradient.
Peace youngster peaceful particle, hide capsule for descend of blood fat, eight taste swertia pill, the blue or green rocs of nine tastes are loose, nine taste Zhus Huangs are loose, intelligence holder JIEBAI WAN is the nomenclature of drug that national standard for traditional Chinese medicines compilation (Chinese patent drug provincial standard rising national standard part) is recorded.Standard number is as follows: the peaceful particle of peace youngster: WS-10640 (ZD-0640)-2002; Hide capsule for descend of blood fat: WS-10864 (ZD-0864)-2002; Eight taste swertia pill: WS3-BC-0241-95; The blue or green roc of nine tastes falls apart: WS3-BC-0247-95; Nine taste Zhu Huangs are loose: WS3-BC-0248-95; Intelligence holder JIEBAI WAN: WS3-BC-0334-95.
Technical scheme of the present invention is as follows:
A detection method of content containing Lagotis glauca preparation, comprises the steps:
A, chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Determined wavelength 330-340nm; Number of theoretical plate calculates should be not less than 4000 by echinacoside peak;
B, mobile phase: take methyl alcohol as mobile phase A, with the phosphoric acid of percent by volume 0.1% for Mobile phase B; Wash-out is carried out: 0min → 25min → 40min according to gradient elution mode, in the above-mentioned time period, mobile phase A: 20% → 35% → 60%; Mobile phase B: 80% → 65% → 40%;
C, reference substance solution: get echinacoside reference substance appropriate, accurately weighed, put in brown measuring bottle, methyl alcohol-0.1% phosphoric acid solution adding volume ratio 20:80 makes the solution containing 0.25mg in every 1ml, to obtain final product;
D, need testing solution: get be equivalent to Lagotis glauca 0.2-1g containing Lagotis glauca formulation samples powder, accurately weighed, filter paper is wrapped, sherwood oil soxhlet extraction process 2-3h is to remove liposoluble constituent, sample powder after process is transferred in brown measuring bottle, add water-saturated n-butanol 40-60ml, soak 20-40 minute, ultrasonic process 30-40 minute, let cool, filter, the water-soluble solution of 100ml is added after filtrate evaporate to dryness, inject the D101 macroporous resin column of column volume 100ml, rinse with the water of 200-300ml, discard washing fluid, rinse with percent by volume 5% methyl alcohol 80-100ml again, discard washing fluid, add percent by volume 80% methyl alcohol 100ml to rinse, gained washing fluid is at 30-40 DEG C of evaporate to dryness, methyl alcohol-0.1% phosphoric acid mixed solution that residue adds 10ml volume ratio 20:80 dissolves, filter, get subsequent filtrate, obtain,
E, determination method: get reference substance solution and each 10 μ l of need testing solution, inject high performance liquid chromatograph, and record peak area, calculates the content of echinacoside by external standard method.
The present invention is preferred, and a kind of detection method of content containing Lagotis glauca preparation, comprises the steps:
A, chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Determined wavelength 334nm; Number of theoretical plate calculates should be not less than 4000 by echinacoside peak;
B, mobile phase: take methyl alcohol as mobile phase A, with the phosphoric acid of percent by volume 0.1% for Mobile phase B; Wash-out is carried out: 0min → 25min → 40min according to gradient elution mode, in the above-mentioned time period, mobile phase A: 20% → 35% → 60%; Mobile phase B: 80% → 65% → 40%;
C, reference substance solution: get echinacoside reference substance appropriate, accurately weighed, put in brown measuring bottle, methyl alcohol-0.1% phosphoric acid solution adding volume ratio 20:80 makes the solution containing 0.25mg in every 1ml, to obtain final product;
D, need testing solution: get be equivalent to Lagotis glauca 0.5g containing Lagotis glauca formulation samples powder, accurately weighed, filter paper is wrapped, sherwood oil soxhlet extraction process 3h is to remove liposoluble constituent, sample powder after process is transferred in brown measuring bottle, add water-saturated n-butanol 50ml, soak 30 minutes, ultrasonic process 40 minutes, let cool, filter, the water-soluble solution of 100ml is added after filtrate evaporate to dryness, inject the D101 macroporous resin column of column volume 100ml, rinse with the water of 300ml, rinse with percent by volume 5% methyl alcohol 100ml again, discard washing fluid, add percent by volume 80% methyl alcohol 100ml to rinse, gained washing fluid is at 30-40 DEG C of evaporate to dryness, methyl alcohol-0.1% phosphoric acid mixed solution that residue adds 10ml volume ratio 20:80 dissolves, filter, get subsequent filtrate, obtain,
E, determination method: get reference substance solution and each 10 μ l of need testing solution, inject high performance liquid chromatograph, and record peak area, calculates the content of echinacoside by external standard method.
Lagotis glauca of the present invention is Lagotis brachystachya Maxim or Herba Lagotis integrae, preferred Lagotis brachystachya Maxim.
Preparation containing Lagotis glauca of the present invention refers to the bulk drug containing Lagotis glauca, technique routinely, add customary adjuvant and be prepared into clinical acceptable any one formulation, comprise capsule, pill, micropill, dripping pill, tablet, soft capsule, particle, oral liquid.
The preparation of the present invention preferably containing Lagotis glauca comprises the peaceful particle of peace youngster, Tibetan capsule for descend of blood fat, eight taste swertia pill, the blue or green rocs of nine tastes are loose, nine taste Zhu Huangs are loose or intelligence holder JIEBAI WAN.More preferably pacify the peaceful particle of youngster or hide capsule for descend of blood fat.
Beneficial effect
Compared with prior art, advantage of the present invention is as follows:
Accompanying drawing explanation
Fig. 1 is peace youngster peaceful particulate samples solution high-efficient liquid phase chromatogram;
Fig. 2 is echinacoside reference substance solution high-efficient liquid phase chromatogram;
Fig. 3 is echinacoside linear relationship chart;
Wherein, the horizontal ordinate of Fig. 1, Fig. 2 is the time, unit: minute (minute min); Ordinate is voltage, unit: volt (Volts).
Embodiment
Following experimental example and embodiment are used for further illustrating but are not limited to the present invention.
Experimental example 1: the content detection of Lagotis brachystachya Maxim in the peaceful particle of peace youngster
1. instrument, reagent and test sample
Instrument: Agilent1260Infinity type high performance liquid chromatograph; Shimadzu AUW220D electronic balance.
Reference substance: echinacoside reference substance (Nat'l Pharmaceutical & Biological Products Control Institute, lot number: 111670-200503).
Sample: the peaceful particle of peace youngster (scold Tibetan medicine medicine company incorporated company and provide, and lot number is respectively: 20121110,20121114,20121119) by Qinghai gold.
2. the selection of determined wavelength
Get echinacoside reference substance solution, scan in 190 ~ 500nm wavelength coverage, at 334nm wavelength, there is absorption maximum at place, therefore to select 334nm according to ultraviolet absorpting spectrum be determined wavelength.
3. mobile phase is selected
Research finds, take methyl alcohol as mobile phase A, with the phosphoric acid of percent by volume 0.1% for Mobile phase B, carry out wash-out by table 1 mode, echinacoside can reach good chromatographic resolution effect.
Table 1 type of elution 1
| Time (minute) | Mobile phase A (%) | Mobile phase B (%) |
| 0～25 | 20→35 | 80→65 |
| 25～40 | 35→60 | 65→40 |
The present invention has attempted other mobile phase ratio: as table 2 and table 3.
Table 2 type of elution 2
| Time (minute) | Mobile phase A (%) | Mobile phase B (%) |
| 0～20 | 20→35 | 80→65 |
| 20～40 | 35→60 | 65→40 |
Table 3 type of elution 3
| Time (minute) | Mobile phase A (%) | Mobile phase B (%) |
| 0～25 | 15→30 | 85→70 |
| 25～40 | 30→60 | 65→40 |
Result shows: the separating effect of type of elution 2 and 3 is all not as type of elution 1 of the present invention.
4. system suitability
Under above-mentioned chromatographic condition, accurate absorption reference substance solution, need testing solution, each 10 μ l of negative control solution respectively, injection liquid chromatography, record chromatogram.Result shows, the degree of separation that in test sample chromatogram, echinacoside is adjacent chromatographic peak is all greater than 1.5, and negative control is noiseless.
5. reference substance solution preparation
It is appropriate that precision takes echinacoside reference substance, puts in brown measuring bottle, and methyl alcohol-0.1% phosphoric acid adding volume ratio 20:80 makes the solution of every 1ml containing echinacoside reference substance 0.25mg, to obtain final product.
6. need testing solution preparation
6.1 need testing solution petroleum ether degreasing times were investigated
Get (4 parts, the peaceful particle powder sample of the peace youngster being equivalent to Lagotis glauca 0.5g, every part of about 8g), accurately weighed, filter paper is wrapped, sherwood oil soxhlet extraction process 0 hour respectively, 1 hour, 2 hours, after 3 hours, sample and filter paper are put in brown measuring bottle, add water-saturated n-butanol 50ml, soak after 30 minutes, ultrasonic process 40 minutes, let cool, filter, add the water-soluble solution of 100ml after filtrate evaporate to dryness and inject D101 macroporous resin column (column volume 100ml), rinse with the water of 300ml, rinse with 5% methyl alcohol 100ml again, discard washing fluid, add 80% methyl alcohol 100ml to rinse, washing fluid, low temperature (less than 40 DEG C) evaporate to dryness, methyl alcohol-0.1% phosphoric acid mixed solution that residue adds 10ml volume ratio 20:80 dissolves, filter, get subsequent filtrate, obtain.To investigate in different degreasing time sample chromatogram figure echinacoside content in echinacoside and adjacent chromatographic peak degree of separation and need testing solution respectively.Test findings: after petroleum ether degreasing, in chromatogram, chromatographic peak quantity reduces, and degreasing time longer chromatographic peak quantity is fewer, petroleum ether degreasing 2 hours and 3 hours chromatographic peak quantity and echinacoside content substantially identical, show that 2 hours petroleum ether degreasings are complete, for ensureing complete degreasing, therefore the petroleum ether degreasing time is selected to be 3 hours.
6.2 need testing solution lucifuges are investigated
Get (2 parts, the peaceful particle powder sample of the peace youngster being equivalent to Lagotis glauca 0.5g, every part of about 8g), accurately weighed, filter paper is wrapped, sherwood oil soxhlet extraction process 3h, removing liposoluble constituent, put brown measuring bottle (lucifuge) respectively, in colourless measuring bottle (not lucifuge), add water-saturated n-butanol 50ml, soak after 30 minutes, ultrasonic process 40 minutes, let cool, filter, add the water-soluble solution of 100ml after filtrate evaporate to dryness and inject D101 macroporous resin column (column volume 100ml), rinse with the water of 300ml, rinse with 5% methyl alcohol 100ml again, discard washing fluid, add 80% methyl alcohol 100ml to rinse, washing fluid, low temperature (less than 40 DEG C) evaporate to dryness, methyl alcohol-0.1% phosphoric acid mixed solution that residue adds 10ml volume ratio 20:80 dissolves, filter, get subsequent filtrate, obtain.Investigation lucifuge and not lucifuge need testing solution echinacoside content respectively.Test findings: in the test sample of lucifuge process, echinacoside content is higher than the test sample of not lucifuge process, therefore select to adopt brown measuring bottle, lucifuge process test sample.The results are shown in Table 4.
Table 4 need testing solution lucifuge investigates test findings
| Condition | Echinacoside content (mg/g) |
| Lucifuge | 0.307 |
| Not lucifuge | 0.286 |
6.3 need testing solution Extraction solvent are investigated
Get (3 parts, the peaceful particle powder sample of the peace youngster being equivalent to Lagotis glauca 0.5g, every part of about 8g), accurately weighed, filter paper is wrapped, sherwood oil soxhlet extraction process 3h, removing liposoluble constituent, sample after process is put in brown measuring bottle, add water-saturated n-butanol respectively, methyl alcohol, ethanol 50ml, soak after 30 minutes, ultrasonic process 40 minutes, let cool, filter, add the water-soluble solution of 100ml after filtrate evaporate to dryness and inject D101 macroporous resin column (column volume 100ml), rinse with the water of 300ml, rinse with 5% methyl alcohol 100ml again, discard washing fluid, add 80% methyl alcohol 100ml to rinse, washing fluid, low temperature (less than 40 DEG C) evaporate to dryness, methyl alcohol-0.1% phosphoric acid mixed solution that residue adds 10ml volume ratio 20:80 dissolves, filter, get subsequent filtrate, obtain.Investigate three kinds of different solvents need testing solution echinacoside content respectively.Test findings: Extraction solvent is that in the need testing solution of water-saturated n-butanol, echinacoside content is greater than the content that Extraction solvent is echinacoside in the need testing solution of methyl alcohol, ethanol, therefore select to adopt water-saturated n-butanol to be Extraction solvent.The results are shown in Table 5.
Table 5 need testing solution Extraction solvent investigates test findings
| Extraction solvent | Echinacoside content (mg/g) |
| Water-saturated n-butanol | 0.309 |
| Methyl alcohol | 0.291 |
| Ethanol | 0.293 |
6.4 need testing solution extracting method are investigated
Get (3 parts, the peaceful particle powder sample of the peace youngster being equivalent to Lagotis glauca 0.5g, every part of about 8g), accurately weighed, filter paper is wrapped, sherwood oil soxhlet extraction process 3h, removing liposoluble constituent, sample after process is put in brown measuring bottle, add water-saturated n-butanol 50ml, soak after 30 minutes, ultrasonic process 40 minutes respectively, refluxing extraction 40 minutes, jolting 40 minutes, let cool, filter, add the water-soluble solution of 100ml after filtrate evaporate to dryness and inject D101 macroporous resin column (column volume 100ml), rinse with the water of 300ml, rinse with 5% methyl alcohol 100ml again, discard washing fluid, add 80% methyl alcohol 100ml to rinse, washing fluid, low temperature (less than 40 DEG C) evaporate to dryness, methyl alcohol-0.1% phosphoric acid mixed solution that residue adds 10ml volume ratio 20:80 dissolves, filter, get subsequent filtrate, obtain.Investigate three kinds of Different Extraction Method need testing solution echinacoside content respectively.Test findings: in the need testing solution of ultrasonic extraction and refluxing extraction, echinacoside content is suitable, and be greater than the content of echinacoside in the need testing solution of jolting extraction, for experimental implementation is easy, therefore select to adopt ultrasonic extraction.The results are shown in Table 6.
Table 6 need testing solution extracting method investigates test findings
| Extracting method | Echinacoside content (mg/g) |
| Ultrasonic extraction | 0.309 |
| Refluxing extraction | 0.307 |
| Jolting is extracted | 0.277 |
6.5 need testing solution soak times are investigated
Get (4 parts, the peaceful particle powder sample of the peace youngster being equivalent to Lagotis glauca 0.5g, every part of about 8g), accurately weighed, filter paper is wrapped, sherwood oil soxhlet extraction process 3h, removing liposoluble constituent, sample after process is put in brown measuring bottle, add water-saturated n-butanol 50ml, portion does not soak, other three points are soaked 20 minutes respectively, 30 minutes, after 40 minutes, ultrasonic process 40 minutes, let cool, filter, add the water-soluble solution of 100ml after filtrate evaporate to dryness and inject D101 macroporous resin column (column volume 100ml), rinse with the water of 300ml, rinse with 5% methyl alcohol 100ml again, discard washing fluid, add 80% methyl alcohol 100ml to rinse, washing fluid, low temperature (less than 40 DEG C) evaporate to dryness, methyl alcohol-0.1% phosphoric acid mixed solution that residue adds 10ml volume ratio 20:80 dissolves, filter, get subsequent filtrate, obtain.Investigate respectively and do not soak and three kinds of different soak time need testing solution echinacoside content.Test findings: in unsoaked need testing solution, echinacoside content is starkly lower than the content of echinacoside in the need testing solution soaked, and it is suitable to soak 20 minutes, 30 minutes, 40 minutes echinacoside content, for ensureing to extract completely, therefore select to adopt immersion 30 points of clock method process test samples.The results are shown in Table 7.
Table 7 need testing solution soak time investigates test findings
| Soak time | Echinacoside content (mg/g) |
| Do not soak | 0.276 |
| 20 minutes | 0.308 |
| 30 minutes | 0.309 |
| 40 minutes | 0.306 |
6.6 need testing solution extraction times were investigated
Get (3 parts, the peaceful particle powder sample of the peace youngster being equivalent to Lagotis glauca 0.5g, every part of about 8g), accurately weighed, filter paper is wrapped, sherwood oil soxhlet extraction process 3h, removing liposoluble constituent, sample after process is put in brown measuring bottle, add water-saturated n-butanol 50ml, soak after 30 minutes, ultrasonic process 20 minutes respectively, 30 minutes, 40 minutes, let cool, filter, add the water-soluble solution of 100ml after filtrate evaporate to dryness and inject D101 macroporous resin column (column volume 100ml), rinse with the water of 300ml, rinse with 5% methyl alcohol 100ml again, discard washing fluid, add 80% methyl alcohol 100ml to rinse, washing fluid, low temperature (less than 40 DEG C) evaporate to dryness, residue adds 10ml volume ratio 20:80 methyl alcohol-0.1% phosphoric acid mixed solution and dissolves, filter, get subsequent filtrate, obtain.Investigation three kinds need testing solution echinacoside content of different extraction time respectively.Test findings: in the ultrasonic extraction need testing solution of 20 minutes echinacoside content be starkly lower than ultrasonic extraction 30 minutes and 40 minutes need testing solution in the content of echinacoside, and ultrasonic extraction 30 minutes, 40 minutes echinacoside content is suitable, for ensureing to extract completely, therefore select to adopt ultrasonic extraction 40 points of clock method process test samples.The results are shown in Table 8.
Table 8 need testing solution Extraction solvent investigates test findings
| Extraction solvent | Echinacoside content (mg/g) |
| 20 minutes | 0.288 |
| 30 minutes | 0..307 |
| 40 minutes | 0.305 |
6.7 need testing solution water flushing doses are investigated
Get (4 parts, the peaceful particle powder sample of the peace youngster being equivalent to Lagotis glauca 0.5g, every part of about 8g), accurately weighed, filter paper is wrapped, sherwood oil soxhlet extraction process 3h, removing liposoluble constituent, sample after process is put in brown measuring bottle, add water-saturated n-butanol respectively, methyl alcohol, ethanol 50ml, soak after 30 minutes, ultrasonic process 40 minutes, let cool, filter, add the water-soluble solution of 100ml after filtrate evaporate to dryness and inject D101 macroporous resin column (column volume 100ml), a copy of it need testing solution does not adopt water to rinse, another three parts of need testing solutions are respectively with 100ml, 200ml, the water of 300ml rinses, rinse with 5% methyl alcohol 100ml again, discard washing fluid, add 80% methyl alcohol 100ml to rinse, washing fluid, low temperature (less than 40 DEG C) evaporate to dryness, residue adds 10ml volume ratio 20:80 methyl alcohol-0.1% phosphoric acid mixed solution and dissolves, filter, get subsequent filtrate, obtain.Investigate respectively and not adopt in washing and different washing amount sample chromatogram figure echinacoside content in echinacoside and adjacent chromatographic peak degree of separation and need testing solution.Test findings: do not adopt echinacoside peak and adjacent chromatographic peak in the test sample chromatogram of washing not to reach baseline separation, after adopting washing, test sample chromatographic peak quantity reduces, and echinacoside chromatographic peak and adjacent chromatographic peak reach baseline separation after 200ml and 300ml washing, and echinacoside content is roughly the same in test sample, show that 200ml water rinses technical ability and rinses completely by impurity, for ensureing that washing fully, therefore washing amount is selected to be 300ml.
6.8 need testing solution 5% washed with methanol amounts are investigated
Get (4 parts, the peaceful particle powder sample of the peace youngster being equivalent to Lagotis glauca 0.5g, every part of about 8g), accurately weighed, filter paper is wrapped, sherwood oil soxhlet extraction process 3h, removing liposoluble constituent, sample after process is put in brown measuring bottle, add water-saturated n-butanol respectively, methyl alcohol, ethanol 50ml, soak after 30 minutes, ultrasonic process 40 minutes, let cool, filter, add the water-soluble solution of 100ml after filtrate evaporate to dryness and inject D101 macroporous resin column (column volume 100ml), after rinsing with the water of 300ml, a copy of it need testing solution does not adopt 5% washed with methanol, another three parts of need testing solutions are respectively with 50ml, 80ml, 5% washed with methanol of 100ml, discard washing fluid, add 80% methyl alcohol 100ml to rinse, washing fluid, low temperature (less than 40 DEG C) evaporate to dryness, residue adds 10ml volume ratio 20:80 methyl alcohol-0.1% phosphoric acid mixed solution and dissolves, filter, get subsequent filtrate, obtain.Investigate respectively and not adopt in 5% methanol wash column and different 5% methanol wash column amount sample chromatogram figure echinacoside content in echinacoside and adjacent chromatographic peak degree of separation and need testing solution.Test findings: do not adopt echinacoside peak and adjacent chromatographic peak in the test sample chromatogram of 5% methanol wash column not to reach baseline separation, after adopting 5% methanol wash column, test sample chromatographic peak quantity reduces, and echinacoside chromatographic peak and adjacent chromatographic peak reach baseline separation after 80ml and 100ml5% methanol wash column, and echinacoside content is roughly the same in test sample, show that impurity rinses completely by 80ml5% washed with methanol technical ability, for ensureing to rinse fully, therefore the 5% methanol wash column amount of selection is 100ml.
6.9 need testing solution 80% washed with methanol amounts are investigated
Get (3 parts, the peaceful particle powder sample of the peace youngster being equivalent to Lagotis glauca 0.5g, every part of about 8g), accurately weighed, filter paper is wrapped, sherwood oil soxhlet extraction process 3h, removing liposoluble constituent, sample after process is put in brown measuring bottle, add water-saturated n-butanol 50ml, soak after 30 minutes, ultrasonic process 20 minutes respectively, 30 minutes, 40 minutes, let cool, filter, add the water-soluble solution of 100ml after filtrate evaporate to dryness and inject D101 macroporous resin column (column volume 100ml), rinse with the water of 300ml, rinse with 5% methyl alcohol 100ml again, discard washing fluid, add 80% methyl alcohol 50ml respectively, 80ml, 100ml rinses, washing fluid, low temperature (less than 40 DEG C) evaporate to dryness, residue adds 10ml volume ratio 20:80 methyl alcohol-0.1% phosphoric acid mixed solution and dissolves, filter, get subsequent filtrate, obtain.Investigate echinacoside content in different 80% methanol wash column amount need testing solution respectively.Test findings: 50ml80% washed with methanol test sample in echinacoside content be starkly lower than and adopt echinacoside content in 80ml and 100ml80% washed with methanol gained test sample, and echinacoside content is roughly the same in 80ml and 100ml gained test sample, show that 80ml80% washed with methanol can be complete, for ensureing to rinse completely, therefore the 80% washed with methanol amount of selection is 100ml.The results are shown in Table 9.
Table 9 need testing solution Extraction solvent investigates test findings
| 80% quantity of methyl alcohol | Echinacoside content (mg/g) |
| 50ml | 0.283 |
| 80ml | 0.307 |
| 100ml | 0.305 |
The 6.10 need testing solution processing modes selected
Get the formulation samples powder being equivalent to Lagotis glauca 0.5g, accurately weighed, filter paper is wrapped, sherwood oil soxhlet extraction process 2-3h is to remove liposoluble constituent, sample powder after process is transferred in brown measuring bottle, add water-saturated n-butanol 40-60ml, soak 20-40 minute, ultrasonic process 30-40 minute, let cool, filter, the water-soluble solution of 100ml is added after filtrate evaporate to dryness, inject the D101 macroporous resin column of column volume 100ml, rinse with the water of 200-300ml, discard washing fluid, rinse with 5% methyl alcohol 80-100ml again, discard washing fluid, add 80% methyl alcohol 80-100ml to rinse, gained 80% washed with methanol liquid less than 40 DEG C evaporates to dryness, methyl alcohol-0.1% phosphoric acid mixed solution that residue adds 10ml volume ratio 20:80 dissolves, filter, get subsequent filtrate, obtain.
Wherein preferred, get the formulation samples powder being equivalent to Lagotis glauca 0.5g, accurately weighed, filter paper is wrapped, sherwood oil soxhlet extraction process 3h is to remove liposoluble constituent, sample powder after process is transferred in brown measuring bottle, add water-saturated n-butanol 40-60ml, soak 30 minutes, ultrasonic process 40 minutes, let cool, filter, the water-soluble solution of 100ml is added after filtrate evaporate to dryness, inject the D101 macroporous resin column of column volume 100ml, rinse with the water of 300ml, rinse with 5% methyl alcohol 100ml again, discard washing fluid, add 80% methyl alcohol 100ml to rinse, gained 80% washed with methanol liquid less than 40 DEG C evaporates to dryness, methyl alcohol-0.1% phosphoric acid mixed solution that residue adds 10ml volume ratio 20:80 dissolves, filter, get subsequent filtrate, obtain.
7. the preparation of typical curve and the investigation of linear relationship
Precision measures echinacoside reference substance stock solution solution (echinacoside content is 496.8 μ g/ml) 1ml, 3ml, 5ml, 8ml, 10ml, put in 10ml measuring bottle respectively, scale is diluted to volume ratio 20:80 methyl alcohol-0.1% phosphoric acid, shake up, each accurate sample introduction 10 μ l, with peak area (A), linear regression is carried out to reference substance concentration (C), obtain echinacoside regression equation: A=13.207C-97.852, related coefficient: R=0.9999.Result shows, echinacoside is within the scope of 49.68 μ g/ml ~ 496.80 μ g/ml, and the peak area (A) of echinacoside is good with reference substance concentration (C) linear relationship.The results are shown in Table 10.
Table 10 echinacoside linear relationship investigates result
| Sequence number | Reference substance concentration C (μ g/ml) | Peak area A (μ Vs) |
| 1 | 49.68 | 583.5 |
| 2 | 149.04 | 1874.2 |
| 3 | 248.40 | 3130.5 |
| 4 | 347.76 | 4497.6 |
| 5 | 496.80 | 6483.9 |
8. precision test
Accurate absorption echinacoside reference substance solution 10 μ l, injection liquid chromatography, each METHOD FOR CONTINUOUS DETERMINATION 6 times, record peak area also calculates relative standard deviation.Result shows, instrument precision is good.The results are shown in Table 11.
Table 11 echinacoside Precision test result
9. stability test
After prepared by need testing solution, accurate absorption 10 μ l, injection liquid chromatography, record peak area, measured once every 2 hours later, investigates 8 hours, and record peak area also calculates relative standard deviation.Result shows, in need testing solution, echinacoside measurement result in 8 hours is stablized.The results are shown in Table 12.
Table 12 echinacoside sample stability test findings
10. replica test
Get with the peaceful particulate samples of a collection of peace youngster that (product batch number: 20121110), prepares need testing solution by the method under the preparation of need testing solution by totally 6 parts, respectively accurately draws 10 μ l, injection liquid chromatography, echinacoside content in calculation sample.Result shows, this analytical approach repeatability is good.The results are shown in Table 13.
Table 13 echinacoside content replica test result
11. recovery tests
Precision takes with the peaceful particulate samples of a collection of peace youngster (product batch number: 20121110) 6 parts, precision adds echinacoside reference substance, measures its content, the calculating recovery.Result shows, this detection method measurement result is accurate.The results are shown in Table 14.
Table 14 echinacoside recovery test result
12. sample determinations
Get the peaceful particle of Tibetan medicinal composition peace youngster three batches, measure and calculate echinacoside content.The results are shown in Table 15.
Table 15 sample echinacoside assay result
| Lot number | Content (mg/g) |
| 20121110 | 0.309 |
| 20121114 | 0.314 |
| 20121119 | 0.312 |
Following embodiment all can realize the effect described in above-mentioned experimental example.In embodiment
Instrument: Agilent1260Infinity type high performance liquid chromatograph; Shimadzu AUW220D electronic balance.
Reference substance: echinacoside reference substance (Nat'l Pharmaceutical & Biological Products Control Institute, lot number: 111670-200503)
Sample: hide capsule for descend of blood fat, eight taste swertia pill, the peaceful particle of peace youngster, intelligence holder JIEBAI WAN, nine taste Zhu Huangs fall apart, and (scold Tibetan medicine medicine company incorporated company by Qinghai gold to provide, lot number is respectively: 20120809,20120415,20121119,20120508,20111221)
Embodiment 1: the content detection of hiding Lagotis brachystachya Maxim in capsule for descend of blood fat
According to high effective liquid chromatography for measuring:
A, chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Determined wavelength 334nm.Number of theoretical plate calculates should be not less than 4000. by echinacoside peak
B, mobile phase: take methyl alcohol as mobile phase A, with the phosphoric acid of percent by volume 0.1% for Mobile phase B; Carry out wash-out as follows:
| Time (minute) | Mobile phase A (%) | Mobile phase B (%) |
| 0～25 | 20→35 | 80→65 |
| 25～40 | 35→60 | 65→40 |
C, reference substance solution: get echinacoside reference substance appropriate, accurately weighed, put in brown measuring bottle, add volume ratio 20:80 methyl alcohol-0.1% phosphoric acid and make the solution containing 0.25mg in every 1ml, to obtain final product.
D, need testing solution: get 5.5g(and be equivalent to Lagotis brachystachya Maxim 0.5g) hide capsule for descend of blood fat content powder, accurately weighed, filter paper is wrapped, sherwood oil soxhlet extraction process 3h, removing liposoluble constituent, sample after process is put in brown measuring bottle, add water inclusion normal butyl alcohol 50ml, soak after 30 minutes, ultrasonic process 40 minutes, let cool, filter, add the water-soluble solution of 100ml after filtrate evaporate to dryness and inject D101 macroporous resin column (column volume 100ml), rinse with the water of 300ml, rinse with percent by volume 5% methyl alcohol 100ml again, discard washing fluid, add percent by volume 80% methyl alcohol 100ml to rinse, washing fluid, low temperature (less than 40 DEG C) evaporate to dryness, residue adds 10ml volume ratio 20:80 methyl alcohol-0.1% phosphoric acid mixed solution and dissolves, filter, get subsequent filtrate, obtain.
E, determination method: get reference substance solution and each 10ul of need testing solution, inject high performance liquid chromatograph, and record peak area, calculates the content of echinacoside by external standard method.
F, result: hide Lagotis brevituba content in capsule for descend of blood fat, count 122.1 μ g with echinacoside (C35H46O20) for every.
The content detection of Lagotis glauca in embodiment 2: eight taste swertia pill
According to high effective liquid chromatography for measuring:
A, chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Determined wavelength 330nm.Number of theoretical plate calculates should be not less than 4000. by echinacoside peak
B, mobile phase: take methyl alcohol as mobile phase A, with the phosphoric acid of percent by volume 0.1% for Mobile phase B; Wash-out is carried out by the following time:
| Time (minute) | Mobile phase A (%) | Mobile phase B (%) |
| 0～25 | 20→35 | 80→65 |
| 25～40 | 35→60 | 65→40 |
C, reference substance solution: get echinacoside reference substance appropriate, accurately weighed, put in brown measuring bottle, add methyl alcohol-0.1% phosphoric acid (20:80) and make the solution containing 0.25mg in every 1ml, to obtain final product.
D, need testing solution: get 2g eight taste swertia pill powder (being equivalent to Lagotis glauca 0.2g), accurately weighed, filter paper is wrapped, sherwood oil soxhlet extraction process 3h, removing liposoluble constituent, sample after process is put in brown measuring bottle, add water inclusion normal butyl alcohol 40ml, soak after 20 minutes, ultrasonic process 40 minutes, let cool, filter, add the water-soluble solution of 100ml after filtrate evaporate to dryness and inject D101 macroporous resin column (column volume 100ml), rinse with the water of 300ml, rinse with 5% methyl alcohol 80ml again, discard washing fluid, add 80% methyl alcohol 80ml to rinse, washing fluid, low temperature (less than 40 DEG C) evaporate to dryness, methyl alcohol-0.1% phosphoric acid mixed solution that residue adds 10ml volume ratio 20:80 dissolves, filter, get subsequent filtrate, obtain.
E, determination method: get reference substance solution and each 10 μ l of need testing solution, inject high performance liquid chromatograph, and record peak area, calculates the content of echinacoside by external standard method.
F, result: rabbit grass content in every 10 ball eight taste swertia pill, count 187.2 μ g with echinacoside (C35H46O20).
Embodiment 3: the content detection of Lagotis brachystachya Maxim in the peaceful particle of peace youngster
According to high effective liquid chromatography for measuring:
A, chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Determined wavelength 334nm.Number of theoretical plate calculates should be not less than 4000. by echinacoside peak
B, mobile phase: take methyl alcohol as mobile phase A, with the phosphoric acid of percent by volume 0.1% for Mobile phase B; Wash-out is carried out by the following time:
| Time (minute) | Mobile phase A (%) | Mobile phase B (%) |
| 0～25 | 20→35 | 80→65 |
| 25～40 | 35→60 | 65→40 |
C, reference substance solution: get echinacoside reference substance appropriate, accurately weighed, put in brown measuring bottle, add volume ratio 20:80 methyl alcohol-0.1% phosphoric acid and make the solution containing 0.25mg in every 1ml, to obtain final product.
D, need testing solution: get 8g and pacify the peaceful particle powder of youngster (being equivalent to Lagotis brachystachya Maxim 0.5g), accurately weighed, filter paper is wrapped, sherwood oil soxhlet extraction process 3h, removing liposoluble constituent, sample after process is put in brown measuring bottle, add water inclusion normal butyl alcohol 50ml, soak after 30 minutes, ultrasonic process 40 minutes, let cool, filter, add the water-soluble solution of 100ml after filtrate evaporate to dryness and inject D101 macroporous resin column (column volume 100ml), rinse with the water of 300ml, rinse with 5% methyl alcohol 100ml again, discard washing fluid, add 80% methyl alcohol 100ml to rinse, washing fluid, low temperature (less than 40 DEG C) evaporate to dryness, methyl alcohol-0.1% phosphoric acid mixed solution that residue adds 10ml volume ratio 20:80 dissolves, filter, get subsequent filtrate, obtain.
E, determination method: get reference substance solution and each 10 μ l of need testing solution, inject high performance liquid chromatograph, and record peak area, calculates the content of echinacoside by external standard method.
F, result: Lagotis brevituba content in the peaceful particle of peace youngster, count 0.313mg/g with echinacoside (C35H46O20).Embodiment 4: the content detection of Lagotis glauca in intelligence holder JIEBAI WAN
According to high effective liquid chromatography for measuring:
A, chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Determined wavelength 334nm.Number of theoretical plate calculates should be not less than 4000. by echinacoside peak
B, mobile phase: take methyl alcohol as mobile phase A, with the phosphoric acid of percent by volume 0.1% for Mobile phase B; Wash-out is carried out by the following time:
| Time (minute) | Mobile phase A (%) | Mobile phase B (%) |
| 0～25 | 20→35 | 80→65 |
| 25～40 | 35→60 | 65→40 |
C, reference substance solution: get echinacoside reference substance appropriate, accurately weighed, put in brown measuring bottle, add volume ratio 20:80 methyl alcohol-0.1% phosphoric acid and make the solution containing 0.25mg in every 1ml, to obtain final product.
D, need testing solution: get 8g intelligence holder JIEBAI WAN powder (being equivalent to Lagotis glauca 1g), accurately weighed, filter paper is wrapped, sherwood oil soxhlet extraction process 2h, removing liposoluble constituent, sample after process is put in brown measuring bottle, add water inclusion normal butyl alcohol 60ml, soak after 40 minutes, ultrasonic process 30 minutes, let cool, filter, add the water-soluble solution of 100ml after filtrate evaporate to dryness and inject D101 macroporous resin column (column volume 100ml), rinse with the water of 200ml, rinse with 5% methyl alcohol 100ml again, discard washing fluid, add 80% methyl alcohol 100ml to rinse, washing fluid, low temperature (less than 40 DEG C) evaporate to dryness, methyl alcohol-0.1% phosphoric acid mixed solution that residue adds 10ml volume ratio 20:80 dissolves, filter, get subsequent filtrate, obtain.
E, determination method: get reference substance solution and each 10 μ l of need testing solution, inject high performance liquid chromatograph, and record peak area, calculates the content of echinacoside by external standard method.
In F, every 10 pills, rabbit grass content in intelligence holder JIEBAI WAN, counts 191.12ug with echinacoside (C35H46O20).The content detection of Lagotis glauca during embodiment 5: nine taste Zhu Huang is loose
According to high effective liquid chromatography for measuring:
A, chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Determined wavelength 334nm.Number of theoretical plate calculates should be not less than 4000. by echinacoside peak
B, mobile phase: take methyl alcohol as mobile phase A, with the phosphoric acid of percent by volume 0.1% for Mobile phase B; Wash-out is carried out by the following time:
| Time (minute) | Mobile phase A (%) | Mobile phase B (%) |
| 0～25 | 20→35 | 80→65 |
| 25～40 | 35→60 | 65→40 |
C, reference substance solution: get echinacoside reference substance appropriate, accurately weighed, put in brown measuring bottle, add volume ratio 20:80 methyl alcohol-0.1% phosphoric acid and make the solution containing 0.25mg in every 1ml, to obtain final product.
D, need testing solution: get the yellow divided powder (being equivalent to Lagotis glauca 0.5g) of 5.5g nine taste Zhu, accurately weighed, filter paper is wrapped, sherwood oil soxhlet extraction process 2h, removing liposoluble constituent, sample after process is put in brown measuring bottle, add water inclusion normal butyl alcohol 60ml, soak after 40 minutes, ultrasonic process 30 minutes, let cool, filter, add the water-soluble solution of 100ml after filtrate evaporate to dryness and inject D101 macroporous resin column (column volume 100ml), rinse with the water of 200ml, rinse with 5% methyl alcohol 100ml again, discard washing fluid, add 80% methyl alcohol 100ml to rinse, washing fluid, low temperature (less than 40 DEG C) evaporate to dryness, methyl alcohol-0.1% phosphoric acid mixed solution that residue adds 10ml volume ratio 20:80 dissolves, filter, get subsequent filtrate, obtain.
E, determination method: get reference substance solution and each 10 μ l of need testing solution, inject high performance liquid chromatograph, and record peak area, calculates the content of echinacoside by external standard method.
F, result: rabbit grass content during nine taste Zhu Huangs are loose, count 88.2ug/g with echinacoside (C35H46O20).
Claims (6)
1. the detection method of content containing Lagotis glauca preparation, is characterised in that, comprises the steps:
A, chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Determined wavelength 334nm; Number of theoretical plate calculates should be not less than 4000 by echinacoside peak;
B, mobile phase: take methyl alcohol as mobile phase A, with the phosphoric acid of percent by volume 0.1% for Mobile phase B; Wash-out is carried out: 0min → 25min → 40min according to gradient elution mode, in the above-mentioned time period, mobile phase A: 20% → 35% → 60%; Mobile phase B: 80% → 65% → 40%;
C, reference substance solution: get echinacoside reference substance appropriate, accurately weighed, put in brown measuring bottle, methyl alcohol-0.1% phosphoric acid solution adding volume ratio 20:80 makes the solution containing 0.25mg in every 1ml, to obtain final product;
D, need testing solution: get the formulation samples powder containing Lagotis glauca being equivalent to Lagotis glauca 0.5g, accurately weighed, filter paper is wrapped, sherwood oil soxhlet extraction process 3h is to remove liposoluble constituent, sample powder after process is transferred in brown measuring bottle, add water-saturated n-butanol 40-60ml, soak 30 minutes, ultrasonic process 40 minutes, let cool, filter, the water-soluble solution of 100ml is added after filtrate evaporate to dryness, inject the D101 macroporous resin column of column volume 100ml, rinse with the water of 300ml, rinse with percent by volume 5% methyl alcohol 100ml again, discard washing fluid, add percent by volume 80% methyl alcohol 100ml to rinse, gained washing fluid is at 30-40 DEG C of evaporate to dryness, methyl alcohol-0.1% phosphoric acid mixed solution that residue adds 10ml volume ratio 20:80 dissolves, filter, get subsequent filtrate, obtain,
E, determination method: get reference substance solution and each 10 μ l of need testing solution, inject high performance liquid chromatograph, and record peak area, calculates the content of echinacoside by external standard method.
2. detection method of content according to claim 1, be characterised in that, the described preparation containing Lagotis glauca refers to the bulk drug containing Lagotis glauca, technique routinely, add customary adjuvant and be prepared into clinical acceptable any one formulation, comprise capsule, pill, micropill, dripping pill, tablet, soft capsule, particle, oral liquid.
3. detection method of content according to claim 2, is characterised in that, the described preparation containing Lagotis glauca is the peaceful particle of peace youngster, hide capsule for descend of blood fat, eight taste swertia pill, the blue or green rocs of nine tastes are loose, nine taste Zhus Huangs are loose or intelligence holder JIEBAI WAN.
4. detection method of content according to claim 1, is characterised in that, described Lagotis glauca is Lagotis brachystachya Maxim.
5. detection method of content according to claim 3, is characterised in that, the described preparation containing Lagotis glauca is for the peace peaceful particle of youngster or hide capsule for descend of blood fat.
6. detection method of content according to claim 1, is characterised in that, comprises the steps:
A, chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Determined wavelength 334nm; Number of theoretical plate calculates should be not less than 4000 by echinacoside peak;
B, mobile phase: take methyl alcohol as mobile phase A, with the phosphoric acid of percent by volume 0.1% for Mobile phase B; Wash-out is carried out: 0min → 25min → 40min according to gradient elution mode, in the above-mentioned time period, mobile phase A: 20% → 35% → 60%; Mobile phase B: 80% → 65% → 40%;
C, reference substance solution: get echinacoside reference substance appropriate, accurately weighed, put in brown measuring bottle, methyl alcohol-0.1% phosphoric acid solution adding volume ratio 20:80 makes the solution containing 0.25mg in every 1ml, to obtain final product;
D, need testing solution: get containing the peaceful particle powder 8g of the peace youngster being equivalent to Lagotis brachystachya Maxim 0.5g, accurately weighed, filter paper is wrapped, sherwood oil soxhlet extraction process 3h is to remove liposoluble constituent, sample powder after process is transferred in brown measuring bottle, add water-saturated n-butanol 50ml, soak 30 minutes, ultrasonic process 40 minutes, let cool, filter, the water-soluble solution of 100ml is added after filtrate evaporate to dryness, inject the D101 macroporous resin column of column volume 100ml, rinse with the water of 300ml, discard washing fluid, rinse with percent by volume 5% methyl alcohol 100ml again, discard washing fluid, add percent by volume 80% methyl alcohol 100ml to rinse, gained washing fluid is at 30-40 DEG C of evaporate to dryness, methyl alcohol-0.1% phosphoric acid mixed solution that residue adds 10ml volume ratio 20:80 dissolves, filter, get subsequent filtrate, obtain,
E, determination method: get reference substance solution and each 10 μ l of need testing solution, inject high performance liquid chromatograph, and record peak area, calculates the content of echinacoside by external standard method.
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| RP-HPLC 法测定短筒兔耳草中松果菊苷;宋杰 等;《药物评价研究》;20130430;第36卷(第2期);第132-134页 * |
| 藏药短管兔耳草中松果菊苷和麦角甾苷的含量测定;星玉秀 等;《药物分析杂志》;20120731;第32卷(第7期);全文 * |
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