CN107764924A - The detection method of active ingredient in asthma particle - Google Patents
The detection method of active ingredient in asthma particle Download PDFInfo
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- CN107764924A CN107764924A CN201610697728.3A CN201610697728A CN107764924A CN 107764924 A CN107764924 A CN 107764924A CN 201610697728 A CN201610697728 A CN 201610697728A CN 107764924 A CN107764924 A CN 107764924A
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The present invention relates to the detection method of active ingredient in Pharmaceutical Analysis technical field, more particularly to asthma particle., can be qualitative to wherein 10 kinds of active ingredients progress in method provided by the invention the invention provides the quantitative and qualitative checking method to active ingredient in asthma particle, wherein 7 kinds of active ingredients are quantified, separating degree is not less than 1.5 between each material.Wherein 7 kinds of active ingredients can be quantified using method provided by the invention, recovery of standard addition has good accuracy between 97.29%~103.78%.
Description
Technical field
The present invention relates to the detection method of active ingredient in Pharmaceutical Analysis technical field, more particularly to asthma particle.
Background technology
Asthma particle is by the Chinese medicine of the treatment allergic asthma of Kangyuan Pharmaceutical Co., Ltd., Jiangsu Prov's exploitation, the medicine
Patent application, Application No. CN201510885057.9 are proposed on December 6th, 2015.The medicine by windproof, perilla leaf, process fiber crops
Huang, the fruit of Chinese magnoliavine, gingko, semen armeniacae amarae, aster, dogwood fruit, the flower bud of lily magnolia are made.Specific preparation method is to steam perilla leaf, the flower bud of lily magnolia with water
Steam distillation method extracts volatile ingredient, and using standby after beta-cyclodextrin inclusion compound, the dregs of a decoction merge with windproof, ephedra, semen armeniacae amarae, divides
Do not add water to cook 2-3 times, each 1-3 hours, merge extract solution, it is 1.10~1.15 to be condensed into relative density under the conditions of 50 DEG C
Clear cream, alcohol are sink to alcohol content 60%-70%, take supernatant concentration be made relative density under the conditions of 50 DEG C be 1.25~1.30 it is clear
Cream is standby；The fruit of Chinese magnoliavine, gingko, aster, dogwood fruit are extracted 2-3 times with 60%-70% ethanol, each 1-3 hours, merge alcohol extracting
The clear cream that relative density under the conditions of 50 DEG C is 1.25~1.30 is made in liquid, concentration；Above-mentioned each clear cream is merged, dry, pulverize, add
Enter volatile oil clathrate compound, mix, produce (referring specifically to Application No. CN201510885057.9 patent application document embodiment
1).The effect of experiment shows, asthma particle has dispelling wind emergency profit key, and respectful lung is relievingd asthma.Be mainly used in asthma it " pathogenic wind impairing lung,
Respectful drop is not normal, and air flue contraction is anxious " burst or recurrent exerbation, or intolerance factors and cause to pant feel oppressed, shortness of breath, breath it is urgent, in larynx
Wheezing is sound to wait the symptoms such as bronchial asthma in acute attack.
In order to preferably control the quality of medicine, ensure the effect of clinical, medicine must be grasped in medicine production process
The active ingredient and its content of thing.But due to the complicated component of asthma particle, wherein active ingredient mainly includes glycoside, alkaloid
The compositions such as class, lignanoids, volatile oil, and wherein also include substantial amounts of adjunct ingredient beta-schardinger dextrin.Due to detection target into
Divide complexity, each active ingredient physics, chemical property differ greatly, substantial amounts of auxiliary material sense again be present and disturb.With current detection method
Method there is no to carry out effectively quantitative or qualitative detection to the active ingredient in asthma particle.
The content of the invention
In view of this, the technical problem to be solved in the present invention the active ingredient in asthma particle is provided detection method,
Method provided by the invention has good precision, linear relationship, stability, repeatability, and the rate of recovery is high, good tolerance；
Separating degree, the reappearance of asthma particle finger-print are preferable, and information is comprehensive, indicate 20 shared peaks, to wherein 10 into
Point carry out it is qualitative, each batch sample similarity is more than 0.90.The assay of 7 kinds of compositions in the asthma particle that the present invention establishes
And finger-print research method provides foundation for the quality control of asthma particle；This method is simple and feasible, quick and precisely, Ke Yizuo
To evaluate the effective ways of asthma granular mass.
The invention provides the detection method of active ingredient in asthma particle, asthma particle after pre-treatment, using methanol as
Mobile phase A phase, using acetonitrile as Mobile phase B phase, using water as mobile phase C phases, detected with HPLC methods and obtain chromatogram, according to chromatogram
Obtain the species and content of active ingredient.
Pre-treatment is to carry out the necessary step on HPLC before machine testing, is to remove the impurity of disturbed specimen analysis, carry
The precision and separating effect of high analyte, and improve the compatibility of sample and mobile phase.And if pre-treatment is improper, then it can cause point
It is deteriorated from effect, detects a series of problems, such as baseline is higher.
In the present invention, pre-treatment is specially：Filtered after asthma particle is extracted with methanol aqueous solution；It is described to be extracted as ultrasound
Assisted extraction.
In pretreatment process, the mass volume ratio of asthma particle and methanol aqueous solution is 1g：15mL~1g：50mL.
In the embodiment of the present invention, the mass volume ratio of asthma particle and methanol aqueous solution is 1g：25mL.
During ultrasonic extraction, methanol can be volatilized, and after extraction, the volume of loss is supplied with methanol.
The volume ratio that methanol in the methanol aqueous solution of asthma particle is extracted in pretreatment process is 30%~80%.
In the embodiment of the present invention, the volume ratio that methanol in the methanol aqueous solution of asthma particle is extracted in pretreatment process is
50%.
In pretreatment process, the power of ultrasound assisted extraction is 200W, and frequency 40kHz, the time is 30min~60min.
In the embodiment of the present invention, in pretreatment process, the time of ultrasound assisted extraction is 40min.
In pretreatment process, the aperture of the filtering is 0.22 μm.
It is currently used detection method to carry out quantitative detection to material using high performance liquid chromatography (HPLC), in face of needing
When analyzing the species and content of active ingredient in asthma particle, using high performance liquid chromatography be it is a kind of fast and effectively
Mode.But due to active ingredient species complexity in asthma particle, and the interference of substantial amounts of pharmaceutic adjuvant, prior art be present
In not suitable for the HPLC detection methods of asthma particle, and establish a kind of new method need to carry out this method it is accurate
Many identifications such as degree, stability, repeatability, it is specially：
Precision：The pin of same test sample (asthma particle, lot number 1411001) solution continuous sample introduction 6 is taken, with 5-O- Wei Sia
Rice alcohol glycosides is with reference to peak, calculates shared peak relative retention time and mainly shared peak relative peak area, and as a result RSD is respectively less than
1.8%.The similarity of finger-print obtained by 5 sample introductions calculated using finger-print obtained by the 1st sample introduction as reference again after, as a result
Similarity result is not less than 0.99.Standard solution is taken, repeats sample introduction 6 times, measures peak area.Morroniside, amarogentin, horse
Money glycosides, macrotin glycosides, 5-O- visamminols glycosides, the RSD of magnelin and schizandrin peak area are respectively 0.56%,
0.10%, 0.15%, 0.20%, 0.11%, 0.38%, 0.09%.
Stability：Same test sample (asthma particle, lot number 141101) appropriate content is taken, takes about 0.2g, it is accurately weighed,
Prepare need testing solution by the preparation method of above-mentioned need testing solution, precision draws 2 μ L, respectively at 0h, 3h, 6h, 9h, 12h and
18h injects liquid chromatograph.Using 5-O- visamminol glycosides 5-O- visamminols glycosides as with reference to peak, the relative guarantor in shared peak is calculated
Time and main chromatographic peak relative peak area are stayed, as a result RSD is respectively less than 2%.Ginseng is used as using finger-print obtained by the 1st sample introduction again
According to the similarity of finger-print obtained by 5 sample introductions after calculating, as a result similarity result is not less than 0.99.Need testing solution room temperature
Mode 24 hours, the RSD values of active ingredient peak area are all no more than 2%.
Repeatability：Same test sample (asthma particle, lot number 141101) appropriate content is taken, takes about 0.2g, it is accurately weighed,
Need testing solution is prepared by the preparation method of above-mentioned need testing solution, it is parallel to prepare 6 parts, determine, calculate.With 5-O- Wei Sia meter
Alcohol glycosides is with reference to peak, calculates shared peak relative retention time and main chromatographic peak relative peak area, and as a result RSD is respectively less than 2%.Again
Similarity with reference to 5 sample introductions gained finger-prints after calculating is used as using finger-print obtained by the 1st sample introduction, as a result similarity knot
Fruit is not less than 0.99.The RSD of six measure peak areas is respectively to be no more than 1.5%.
Also, experiment shows, according to collection of illustrative plates obtained by 10 batches of asthma particle detections, 20 shared peaks are demarcated, using national medicine
What allusion quotation was promulgated《Similarity evaluation》Version is analyzed within 2012, and establishing control with average method refers to
Line collection of illustrative plates (such as Fig. 2).10 batches survey test sample chromatogram and reference fingerprint similarity be respectively 0.987,0.995,
0.989th, 0.997,0.991,0.991,0.989,0.980,0.987 and 0.988.
Recovery testu is proved, asthma particle is detected in method provided by the invention, and each active ingredient is returned
Yield has good accuracy between 97.29%~103.78%.
Liquid chromatogram be sample component between column packing and mobile phase mass exchange and reach the purpose separated, therefore will
Ask flowing relative sample that there is certain solvability and do not produce chemical reaction with sample, its viscosity is as far as possible small, so as to
To good separating effect；And the physico-chemical property of mobile phase will be adapted with the detector used.UV-detector is such as used, then should
Prepared using to the relatively low solvent of UV absorption.Its boiling point can not ether it is low, otherwise easily produce bubble, cause experiment not enter
OK.For asthma particle, more preferable Detection results can be ensured by carrying out gradient elution with mobile phase provided by the invention, excellent
In the testing result of other mobile phases.The present invention compares three kinds of acetonitrile-water, methanol-water, methanol-acetonitrile-water mobile phase systems
System, as a result separated preferably using methanol-acetonitrile-water system chromatographic peak, and optimize elution requirement, finally determine condition of gradient elution
It is lower to complete measure.
In the present invention, the elution program of HPLC methods is：
0min~2min mobile phase As phase volume fraction is 15%；
The volume fraction of Mobile phase B phase is 2%；
The volume fraction of mobile phase C phases is 83%；
2min~6min mobile phase A phase volume fractions are by 15% to 54%；
The volume fraction of Mobile phase B phase is 2%；
The volume fraction of mobile phase C phases is by 83% to 44%；
16min~30min mobile phase As phase volume fraction is by 54% to 72%；
The volume fraction of Mobile phase B phase is 2%；
The volume fraction of mobile phase C phases is by 44% to 26%；
30min~36min mobile phase As phase volume fraction is by 72% to 98%；
The volume fraction of Mobile phase B phase is 2%；
The volume fraction of mobile phase C phases is by 26% to 0.
For method provided by the invention, C18 chromatographic columns are selected according to the polarity of active ingredient in asthma particle.Chromatogram
The size of post can have an impact to separating resulting, and its internal diameter can have an impact to the flow velocity of mobile phase, the shorter chromatographic column of length
Run time is short, and post pressure is relatively low；The longer chromatographic column resolution ratio original text of length, but run time increases.The present invention compares
Agilent C18Post, Waters Xbrige C18Post and Kromasil 100-3.5C18 posts, as a result in the chromatostrip of this method
Under part, each composition to be measured can obtain preferable separation.In the present invention, HPLC chromatographic column is Kromasil 100-3.5C18.
The size for the chromatographic column that the present invention uses for：4.6 × 150mm, 3.5 μm.
Selection to column temperature need to consider the characteristic of material to be separated in itself, and column temperature influences dissolving of the mobile phase to test substance
Degree can also influence post pressure.Generally, column temperature is improved to be advantageous to improve separating degree, but temperature is too high that post can be caused to press through is low,
It is unfavorable for the detection of material.The flow velocity of mobile phase is too high to reduce the number of plates, and the reduction of the number of plates can cause sample separating degree
Reduction.The present invention has investigated 35 DEG C, 40 DEG C, 45 DEG C of different column temperatures and 0.8mLmin-1、1.0mL·min-1、1.2mL·
min-1Influence different in flow rate to each composition to be measured, as a result in 40 DEG C of column temperature, flow velocity 1.0mLmin-1When separate best results.
In method provided by the invention, in the present invention, the column temperature of HPLC methods detection is 40 DEG C；The flow velocity of mobile phase is
1.0mL·min-1。
In the present invention, the wavelength of HPLC methods detection is 210nm and 240nm.
High performance liquid chromatography uses DAD detectors, and Detection wavelength need to be set according to the absorption collection of illustrative plates of determinand.It is each to make
Individual composition to be measured is preferably separated, and obtains higher sensitivity, using DAD detectors, in the range of 200nm to 400nm
Scanning, because chromatographic peak is more under 240nm, therefore select 240nm to check wavelength for finger-print, select each composition to be measured most preferably to survey
Standing wave is grown, and the final Detection wavelength for determining that 210nm is amarogentin and magnelin, 240nm is morroniside, loganin, rattletop
The Detection wavelength of plain glycosides, 5-O- visamminols glycosides and schizandrin.
In certain embodiments, the sample size of HPLC detections is 5 μ L.
The present invention establishes asthma particle finger-print and multicomponent assay method, has pointed out 10 shared peaks, and to it
In 7 compositions carried out quantitative analysis, be capable of the quality of more comprehensive Control of asthma particle.
It is qualitative to active ingredient according to the relative retention time of the chromatogram in the present invention.
The preparation method of standard curve is：Gradient dilution standard items, are ordinate with chromatographic peak area, and sample introduction concentration is
Abscissa, draw standard curve.
The invention provides the quantitative and qualitative checking method to active ingredient in asthma particle, with side provided by the invention
Method, can be qualitative to wherein 10 kinds of active ingredients progress, and 7 kinds of compositions are quantified, and separating degree is not less than 1.5 between each material.
Wherein 7 kinds of active ingredients can be quantified using method provided by the invention, recovery of standard addition is located at
Between 97.29%~103.78%, there is good accuracy.The asthma particle of 10 batches is entered in method provided by the invention
The quantitative analysis of row active ingredient.
Brief description of the drawings
Fig. 1 shows asthma particle reference fingerprint；
Fig. 2 shows 10 batches of asthma particle finger-prints；
Fig. 3 shows that peak is shared in asthma particle finger-print to be pointed out
Fig. 4 shows the HPLC chromatogram of 210nm Detection wavelengths；Wherein, Fig. 4-a show the control that the embodiment 2 under 210nm is prepared
Product chromatogram；Fig. 4-b show test sample (asthma particle, lot number 141101) chromatogram under 210nm；Fig. 4-c show the hardship under 210nm
The chromatogram of almond feminine gender (lacking semen armeniacae amarae in the bulk drug of asthma particle, lack amarogentin in chromatogram)；Fig. 4-d show
The chromatogram of the flower bud of lily magnolia negative (lacking the flower bud of lily magnolia in the bulk drug of asthma particle, lack magnelin in chromatogram) under 210nm；
Fig. 5 shows the HPLC chromatogram of 240nm Detection wavelengths；Wherein, Fig. 5-a show the control that the embodiment 2 under 240nm is prepared
The chromatogram of product；Fig. 5-b show test sample (asthma particle, lot number 141101) chromatogram under 240nm；Fig. 5-c show under 240nm
Fructus Corni feminine gender (lacking Fructus Corni in the bulk drug of asthma particle, the chemical composition such as loganin in Fructus Corni is lacked in chromatogram)
Chromatogram；What Fig. 5-d showed under 240nm windproof negative (lack windproof, rattletop is lacked in chromatogram in the bulk drug of asthma particle
The chemical compositions such as middle macrotin glycosides, 5-o- visamminol glycosides) chromatogram；Fig. 5-e show that the fruit of Chinese magnoliavine under 240nm is negative and (roared
Breathe heavily particle bulk drug in lack the fruit of Chinese magnoliavine, the first-class chemical composition of schisandrol is lacked in chromatogram) chromatogram.
Embodiment
The invention provides the detection method of active ingredient in asthma particle, those skilled in the art can be used for reference in this paper
Hold, be suitably modified technological parameter realization.In particular, all similar replacements and change are to those skilled in the art
For be it will be apparent that they are considered as being included in the present invention.The method of the present invention and application are by preferably implementing
Example is described, related personnel substantially can not depart from present invention, in spirit and scope to methods herein and application
It is modified or suitably changes with combining, realizes and using the technology of the present invention.
The instrument or reagent that the present invention uses are all common commercially available product, can all be bought in market.
The high performance liquid chromatographs of Agilent 1260, Agilent companies of the U.S.；MILLIPORE Milli-Q Century
Pure water meter, Millipore companies of the U.S.；BSA224S-CW type electronic analytical balances, German Sartorius companies；Mettler
Toledo X6 type electronic analytical balances, Mettler companies of Switzerland；KQ-250DB type ultrasonic washing instruments, city of Kunshan's ultrasonic instrument
Co., Ltd；Loganin reference substance (National Institute for Food and Drugs Control, lot number：111640-201005, content is with 99.2%
Meter)；Macrotin glycosides reference substance (National Institute for Food and Drugs Control, lot number：111522-201310, content is with 95.0%
Meter)；5-O- visamminol glycosides reference substance (National Institute for Food and Drugs Control, lot number：111523-201509, content with
95.8% meter)；Schizandrin reference substance (National Institute for Food and Drugs Control, lot number：110857-201412, content with
99.4% meter)；Deoxyschizandrin (National Institute for Food and Drugs Control, lot number：110765-200508, purity 100%)；Five
Taste alcohol second (Chengdu Puffy moral Bioisystech Co., Ltd, lot number：140330, purity >=98%)；(Chengdu is general for Schisantherin C
Fei De Bioisystech Co., Ltd, lot number：141021, purity purity >=98%)；Schizandrin A (Chengdu Puffy moral biology skill
Art Co., Ltd, lot number：141112, purity >=98%)；Methanol (the upper biochemical Co., Ltd of starfish, chromatographically pure)；Remaining reagent
It is that analysis is pure.
With reference to embodiment, the present invention is expanded on further：
The qualitative detection of embodiment 1
1 chromatographic condition
Chromatographic column is Kromasil 100-3.5C18 (4.6X150mm, 3.5 μm), and mobile phase is methanol (A)-acetonitrile-
(B)-water (C) elution program is：0~2min, 15%A；2~16min, 15%~54%A, 16~30min, 54%~72%A；
30~36min, 72%~98%A；0~36min, 2%B；Column temperature：40℃；Flow velocity 1.0mLmin-1；Detection wavelength：210nm
(amarogentin, magnelin), 240nm (morroniside, loganin, macrotin glycosides, 5-O- visamminols glycosides, schisandrol
First), the μ L of sample size 5.
The selection of 2 objects of reference
Select retention time moderate, separate preferable 5-O- visamminols glycosides as object of reference.
The preparation of 3 need testing solutions
Take the asthma particle (lot number under content uniformity item：141101), mix, take in right amount, it is finely ground, about 1.0g is taken, it is accurate
It is weighed, put in conical flask with cover, precision adds 50% methanol 25mL, weighed weight, is ultrasonically treated (power 200W, frequency
40KHz) 40 minutes, then weighed weight, the weight of less loss is supplied with methanol, filters (0.22 μm of miillpore filter), takes subsequent filtrate, is made
For need testing solution.
4 precision tests
Take the pin of same test sample (asthma particle, lot number 141101) solution continuous sample introduction 6, using 5-O- visamminols glycosides as
With reference to peak, shared peak relative retention time and mainly shared peak relative peak area are calculated, as a result RSD is respectively less than 1.8%.Again with the
Similarity of the finger-print as the finger-print with reference to obtained by 5 sample introductions after calculating obtained by 1 sample introduction, as a result similarity result is equal
Not less than 0.99.
5 stability tests
Same test sample (asthma particle, lot number 141101) appropriate content is taken, takes about 0.2g, it is accurately weighed, by above-mentioned
The preparation method of need testing solution prepares need testing solution, and precision draws 2 μ L, is injected respectively at 0h, 3h, 6h, 9h, 12h and 18h
Liquid chromatograph.Using 5-O- visamminols glycosides as with reference to peak, calculate shared peak relative retention time and main chromatographic peak is relative
Peak area, as a result RSD be respectively less than 2%.Referred to again using finger-print obtained by the 1st sample introduction as after with reference to calculating obtained by 5 sample introductions
The similarity of line collection of illustrative plates, as a result similarity result is not less than 0.99.
6 replica tests
Same test sample (asthma particle, lot number 141101) appropriate content is taken, takes about 0.2g, it is accurately weighed, by above-mentioned
The preparation method of need testing solution prepares need testing solution, parallel to prepare 6 parts, determines, and calculates.Using 5-O- visamminols glycosides as
With reference to peak, shared peak relative retention time and main chromatographic peak relative peak area are calculated, as a result RSD is respectively less than 2%.Again with the 1st
Similarity of the finger-print as the finger-print with reference to obtained by 5 sample introductions after calculating obtained by secondary sample introduction, as a result similarity result is equal
Not less than 0.99.
7 results
The foundation of 7.1 finger-prints and technical parameter
According to collection of illustrative plates obtained by 10 batches of asthma particle detections, 16 shared peaks are demarcated, are promulgated using NF《Chinese medicine color
Compose fingerprint similarity evaluation system》Version is analyzed within 2012, is established reference fingerprint with average method, is seen Fig. 1.10
Batch survey test sample chromatogram and reference fingerprint similarity be respectively 0.987,0.995,0.989,0.997,0.991,
0.991st, 0.989,0.980,0.987 and 0.988.See Fig. 2.
Pointing out for peak is shared in 7.2 finger-prints
Take morroniside, loganin, macrotin glycosides, 5-O- visamminols glycosides, magnelin, schizandrin, the fruit of Chinese magnoliavine
Alcohol second, Schisantherin C, schizandrin A and deoxyschizandrin reference substance are appropriate, add 50% methanol that mixed reference substance solution is made
(reference substance containing the above is 20,6,15,25,20,50,10,8,8,10 μ g/ml respectively).Analyzed by chromatographic condition sample introduction, pass through guarantor
Stay time and DAD scanning analysis, No. 3 peaks are morroniside in 10 shared peaks of demarcation in finger-print (Fig. 3), and No. 5 peaks are horse
Money glycosides, No. 6 peaks are macrotin glycosides, and No. 9 peaks are 5-O- visamminol glycosides, and No. 11 peaks are magnelin, and No. 15 peaks are the fruit of Chinese magnoliavine
Alcohol first, No. 16 peaks are wuweizi alcohol B, and No. 18 peaks are Schisantherin C, and No. 19 peaks are schizandrin A, and No. 20 peaks are the fruit of Chinese magnoliavine
B prime.The relative retention time of each active ingredient and relative peak area such as table 1：
The retention time and relative peak area of 1 each active ingredient of table
Embodiment 2 quantitatively detects
1 chromatographic condition
Chromatographic column is Kromasil 100-3.5C18 (4.6 × 150mm, 3.5 μm), and mobile phase is methanol (A)-acetonitrile-
(B)-water (C) elution program is：0~2min, 15%A；2~16min, 15%~54%A, 16~28min, 54%~72%A；
0~28min, 2%B；Column temperature：40℃；Flow velocity 1.0mLmin-1；Detection wavelength：210nm (amarogentin, magnelin),
240nm (morroniside, loganin, macrotin glycosides, 5-O- visamminols glycosides, schizandrin), the μ L of sample size 5.
2. prepared by reference substance stock solution
Precision weighs morroniside, amarogentin, loganin, macrotin glycosides, 5-O- visamminols glycosides, magnelin and five
Taste alcohol first reference substance is appropriate, and adding 50% methanol that hybrid standard stock solution is made, (morroniside content, amarogentin, loganin contain
Measure 640.93 μ gmL-1, the μ gmL of macrotin glycosides content 171.78-1, the μ gmL of 5-O- visamminol glycosides content 283.62-1,
Magnelin, schizandrin content are 547.99 μ gmL-1)
It is prepared by 3 need testing solutions
Take the asthma particle (lot number under content uniformity item：141101), mix, take in right amount, it is finely ground, about 1.0g is taken, it is accurate
It is weighed, put in conical flask with cover, precision adds 50% methanol 25mL, weighed weight, is ultrasonically treated (power 200W, frequency
40KHz) 40 minutes, then weighed weight, the weight of less loss is supplied with methanol, filters (0.22 μm of miillpore filter), takes subsequent filtrate, is made
For need testing solution.
4 quantitative limits
By above-mentioned chromatographic condition, tested with mixing reference substance stock solution dilution, as a result morroniside, amarogentin,
Loganin, macrotin glycosides, 5-O- visamminols glycosides, the quantitative limit of magnelin and schizandrin are respectively 1.010ng (S/
N=10.2), 0.339ng (S/N=9.8), 0.941ng (S/N=10.3), 0.563ng (S/N=10.5), 0.632ng (S/N
=10.5), 0.754ng (S/N=10.5), 0.441ng (S/N=10.5).
5 linear relationships are investigated
Precision measures above-mentioned mixing reference substance stock solution 0.1,0.2,0.5,1.0,1.5,2.0mL respectively at 5mL measuring bottles
It is interior, add methanol dilution to be settled to scale, shake up the mixed reference substance solution that 6 concentration are made, successively containing morroniside 5.02,
10.03,25.08,50.15,75.23 100.30 μ gmL-1,
Containing the μ gmL of amarogentin 46.42,92.83,232.08,464.16,696.23,928.31-1,
Containing the μ gmL of loganin 12.82,25.64,64.09,128.19,192.28,256.37-1,
The μ gmL of glycosides containing macrotin 3.44,6.87,17.18,34.36,51.35,68.71-1,
The μ gmL of the glycosides of visamminol containing 5-O- 5.67,11.34,28.36,56.72,85.08,113.45-1,
Containing the μ gmL of magnelin 4.22,8.44,21.20,42.20,63.30,84.40-1,
Containing the μ gmL of schizandrin 10.96,21.92,54.80,109.60,164.40,219.20-1,
Determined by condition under above-mentioned chromatographic condition item, record chromatogram, be ordinate with chromatographic peak area, sample introduction concentration
For abscissa, standard curve is drawn.As a result showing, each composition is in good linear relation in the range of respective concentration, the range of linearity,
Regression equation, coefficient correlation are shown in Table 2.
The range of linearity of table 2, regression equation and coefficient correlation
| Chemical composition | The range of linearity | Regression equation | r |
| Morroniside | 5.02~100.3 | Y=12.444X+2.8238 | R=0.999 9 |
| Amarogentin | 46.42~928.31 | Y=4.3097X+1.0088 | R=1 |
| Loganin | 12.82~256.37 | Y=7.5711X-1.1344 | R=1 |
| Macrotin glycosides | 3.44~68.71 | Y=10.601X-0.9102 | R=1 |
| 5-O- visamminol glycosides | 5.67~113.45 | Y=11.872X-0.2371 | R=1 |
| Magnelin | 4.22~84.4 | Y=36.032X+4.6157 | R=0.999 9 |
| Schizandrin | 10.96~219.2 | Y=11.658X-34.001 | R=0.999 9 |
6 precision tests
Line taking intermediate point (the μ gmL containing morroniside 25.08-1, the μ gmL of amarogentin 232.08-1, the μ of loganin 64.09
g·mL-1, the μ gmL of macrotin glycosides 17.18-1, the μ gmL of 5-O- visamminols glycosides 28.36-1, the μ g of magnelin 21.10
mL-1, the μ gmL of schizandrin 54.80-1Mixed reference substance solution), determined by condition under above-mentioned chromatographic condition item, repeat
Sample 6 times, measures peak area.Morroniside, amarogentin, loganin, macrotin glycosides, 5-O- visamminols glycosides, magnelin and
The RSD of schizandrin peak area is respectively 0.56%, 0.10%, 0.15%, 0.20%, 0.11%, 0.38%, 0.09%.
7 repeatability
Asthma particle (lot number 141101) is taken to prepare six parts of need testing solution by method under need testing solution preparation is parallel,
Being determined by condition under 2.1 chromatographic condition items, the RSD of six measure is respectively 1.22%, 0.39%, 1.18%, 1.27%,
0.55%, 1.01%, 0.34%.As a result this product contains morroniside, amarogentin, loganin, macrotin glycosides, 5-O- Wei Sia per 1g
The amount of rice alcohol glycosides, magnelin and schizandrin is respectively 0.65,5.43,1.28,0.40,0.54,0.45,1.03mg.
8 stability
Asthma particle need testing solution (lot number) is taken, presses 2.1 respectively at 0,4,8,12,16,20,24h at ambient temperature
Condition determines under chromatographic condition item, morroniside, amarogentin, loganin, macrotin glycosides, 5-O- visamminols glycosides, lily magnolia fat
The peak area RSD of element and schizandrin is respectively 1.5%, 0.8%, 1.8%, 1.3%, 1.1%, 1.7% and 0.5%.Knot
Fruit shows that need testing solution is stable in 24 hours.
9 recovery tests
Asthma particle (lot number 141101) is taken, this is tieed up containing morroniside, amarogentin, loganin, macrotin glycosides, 5-O- per 1g
The amount of ammiol glycosides, magnelin and schizandrin is respectively 0.65,5.43,1.28,0.40,0.54,0.45,1.03mg)
It is finely ground, weigh 6 parts, every part of about 0.5g respectively, it is accurately weighed, put in conical flask with cover, every part to be separately added into mixing reference substance molten
Liquid 1.0mL, after reference substance solution to be mixed and test sample are sufficiently mixed, every part accurate again to add 50% methanol 25mL, close plug, claims
Determine weight, be ultrasonically treated (power 200W, frequency 40KHz) 40 minutes, let cool to room temperature, the weight of less loss is supplied with 50% methanol
Amount, shakes up, and filters (0.22 μm of miillpore filter), takes subsequent filtrate, as need testing solution, produce.
The average recovery (n=6) of 37 tested compositions of table
3 samples determine
Asthma particle (lot number 141101) is taken by legal system available test sample solution below need testing solution preparation, by 2.1 colors
Condition determines under spectral condition item, records chromatogram, containing morroniside, amarogentin, loganin, macrotin glycosides, 5- in calculating per 1g
The content of O- visamminols glycosides, magnelin and schizandrin, the results are shown in Table 4.
Content (the mgg of 7 kinds of compositions in the asthma particle of table 4-1)
Embodiment 3 lacks side's detection
Prepare respectively and lack semen armeniacae amarae, the flower bud of lily magnolia, Fructus Corni, windproof, the fruit of Chinese magnoliavine five kinds of controls medicine in formula.To implement
The reference substance solution and test sample (asthma particle, lot number 141101 that example 2 is prepared.) to compare, chromatogram detection is carried out, observes chromatogram
The difference of figure.
1 chromatographic condition
Chromatographic column is Kromasil 100-3.5C18 (4.6 × 150mm, 3.5 μm), and mobile phase is methanol (A)-acetonitrile-
(B)-water (C) elution program is：0~2min, 15%A；2~16min, 15%~54%A, 16~28min, 54%~72%A；
0~28min, 2%B；Column temperature：40℃；Flow velocity 1.0mLmin-1；Detection wavelength：210nm (amarogentin, magnelin),
240nm (morroniside, loganin, macrotin glycosides, 5-O- visamminols glycosides, schizandrin), the μ L of sample size 5.Testing result
Such as Fig. 4~5.
It can be seen that according to method provided by the invention, scarce prescription thing can be effectively distinguished, can interpolate that the prescription of product to be tested is
It is no complete.
It the above is only the preferred embodiment of the present invention, it is noted that come for those skilled in the art
Say, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should be regarded as
Protection scope of the present invention.
Claims (10)
1. the detection method of active ingredient in asthma particle, it is characterised in that asthma particle is after pre-treatment, using methanol as flowing
Phase A phases, using acetonitrile as Mobile phase B phase, using water as mobile phase C phases, detected with HPLC methods and obtain chromatogram, obtained according to chromatogram
The species and content of active ingredient.
2. detection method according to claim 1, it is characterised in that the pre-treatment is specially：By asthma particle with first
Filtered after alcohol solution extraction；It is described to be extracted as ultrasound assisted extraction.
3. detection method according to claim 2, it is characterised in that the mass body of the asthma particle and methanol aqueous solution
Product ratio is 1g：15mL~1g：50mL.
4. detection method according to claim 2, it is characterised in that the volume ratio of methanol is in the methanol aqueous solution
30%~80%.
5. detection method according to claim 2, it is characterised in that the power of the ultrasound assisted extraction is 200W, frequency
Rate is 40kHz, and the time is 30~60min.
6. detection method according to claim 1, it is characterised in that the elution program of the HPLC methods is：
0min~2min mobile phase As phase volume fraction is 15%；
The volume fraction of Mobile phase B phase is 2%；
The volume fraction of mobile phase C phases is 83%；
2min~16min mobile phase A phase volume fractions are by 15% to 54%；
The volume fraction of Mobile phase B phase is 2%；
The volume fraction of mobile phase C phases is by 83% to 44%；
16min~30min mobile phase As phase volume fraction is by 54% to 72%；
The volume fraction of Mobile phase B phase is 2%；
The volume fraction of mobile phase C phases is by 44% to 26%；
30min~36min mobile phase As phase volume fraction is by 72% to 98%；
The volume fraction of Mobile phase B phase is 2%；
The volume fraction of mobile phase C phases is by 26% to 0.
7. detection method according to claim 1, it is characterised in that the chromatographic column of the HPLC is Kromasil 100-
3.5C18。
8. detection method according to claim 1, it is characterised in that the column temperature of the HPLC methods detection is 38 DEG C~42
℃；The flow velocity of the mobile phase is 0.8~1.2mLmin-1。
9. detection method according to claim 1, it is characterised in that the wavelength of HPLC methods detection for 210nm and
240nm。
10. according to the detection method described in any one of claim 1~9, it is characterised in that according to the relative guarantor of the chromatogram
Stay the time qualitative to active ingredient；Wherein,
The active ingredient that relative retention time is 0.358 ± 20% is morroniside；
The active ingredient that relative retention time is 0.569 ± 20% is loganin；
The active ingredient that relative retention time is 0.717 ± 20% is macrotin glycosides；
The active ingredient that relative retention time is 1 is 5-O- visamminol glycosides；
The active ingredient that relative retention time is 1.416 ± 20% is magnelin；
The active ingredient that relative retention time is 1.511 ± 20% is schizandrin；
The active ingredient that relative retention time is 1.641 ± 20% is wuweizi alcohol B；
The active ingredient that relative retention time is 1.808 ± 20% is Schisantherin C；
The active ingredient that relative retention time is 1.95 ± 20% is schizandrin A；
The active ingredient that relative retention time is 2.113 ± 20% is deoxyschizandrin.
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