CN105911176A - Detection method of peimine and peiminine in thunberg fritillary bulb - Google Patents
Detection method of peimine and peiminine in thunberg fritillary bulb Download PDFInfo
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- CN105911176A CN105911176A CN201610234905.4A CN201610234905A CN105911176A CN 105911176 A CN105911176 A CN 105911176A CN 201610234905 A CN201610234905 A CN 201610234905A CN 105911176 A CN105911176 A CN 105911176A
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- peiminine
- peimine
- bulbus fritillariae
- fritillariae thunbergii
- extraction
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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- Chemical & Material Sciences (AREA)
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Abstract
The invention discloses a detection method of peimine and peiminine in thunberg fritillary bulb. The method comprises the following steps: Step 1, crushing thunberg fritillary bulb and extracting by an ASE extraction method to obtain an extracting solution; Step 2, carrying out centrifugal separation on the extracting solution to obtain a supernatant; and Step 3, analyzing the supernatant by liquid chromatography. The invention provides a detection method of peimine and peiminine in thunberg fritillary bulb. The method is simple to operate and has high detection accuracy.
Description
Technical field
The present invention relates to test in laboratory field, the assay method of peimine, peiminine in a kind of Bulbus Fritillariae Thunbergii.
Background technology
Pharmacopeia in 2010 have recorded peimine, the method for peiminine in extraction Bulbus Fritillariae Thunbergii: takes this product powder (crossing No. four sieves) about
2g, accurately weighed, put in flask, add strong ammonia solution 4ml and infiltrate 1 hour, mixing of accurate addition chloroform-methanol (4:1)
Close solution 40ml, weighed weight, mixing, put in 80 DEG C of water-baths and be heated to reflux 2 hours, let cool, more weighed weight, add above-mentioned
Mixed solution supplies the weight of less loss, filters.Precision measures subsequent filtrate 10ml, puts in evaporating dish and is evaporated, and residue adds methanol to be made molten
Solve and be transferred in 2ml measuring bottle, adding methanol to scale, shake up, to obtain final product.Algoscopy precision absorption reference substance solution 10 μ l respectively,
20 μ l, need testing solution 5～15 μ l, note people's chromatograph of liquid, measures, calculates peimisine respectively with external standard two-point method logarithmic equation
The content of first, peiminine, to obtain final product.
The method operating process of pharmacopeia is complicated, and the operant skill of operator is required height.
Summary of the invention
It is an object of the invention to provide the assay method of peimine, peiminine in a kind of Bulbus Fritillariae Thunbergii, the method is simple to operate,
Accuracy of detection is high.
The technical scheme that the present invention provides is: the assay method of peimine, peiminine in a kind of Bulbus Fritillariae Thunbergii, including following step
Rapid:
Step 1: use ASE extraction that extract is obtained by extraction after being pulverized by Bulbus Fritillariae Thunbergii；
Step 2: after extract centrifugation, obtain supernatant；
Step 3: use liquid chromatography to be analyzed supernatant；
Wherein, the analysis condition of liquid chromatography is:
A) instrument: double ternary liquid phase chromatographs
B) chromatographic column specification: carbon 18 chromatographic column, 3*100mm 3um
C) column temperature: 40 DEG C
D) flow velocity: 0.5mL/min
E) flowing phase: acetonitrile-water-diethylamine, volume ratio 70:30:0.03
F) detection wavelength: evaporative light scattering detector.
In above-mentioned Bulbus Fritillariae Thunbergii in the assay method of peimine, peiminine, described step 1 particularly as follows:
S11: after Bulbus Fritillariae Thunbergii is pulverized, sieve；
S12: weigh 1 weight portion Bulbus Fritillariae Thunbergii powder, mix homogeneously stand-by with 1 weight portion kieselguhr；
S13: in the abstraction pool putting filter membrane in advance well, adds appropriate kieselguhr to the Chi Kou reaching abstraction pool after mix homogeneously；
The lid covering abstraction pool carries out static extracting；Extraction is extracted liquid after terminating；
Extraction conditions is: extractant is percent by volume 75% ethanol；Extraction temperature 80 DEG C；Static extracting time 3min；Punching
Wash volume 60%；Quiet cycle number of times 2 times.
In above-mentioned Bulbus Fritillariae Thunbergii in the assay method of peimine, peiminine, the volume of described abstraction pool is 10ml.
In above-mentioned Bulbus Fritillariae Thunbergii in the assay method of peimine, peiminine, the pressure of described static extracting is 1500psi.
In above-mentioned Bulbus Fritillariae Thunbergii in the assay method of peimine, peiminine, the purge time of described static extracting is
100s。
In above-mentioned Bulbus Fritillariae Thunbergii in the assay method of peimine, peiminine, step 2 is particularly as follows: exist extract
Under conditions of 15000r/min, centrifugal 3min, takes supernatant.
In above-mentioned Bulbus Fritillariae Thunbergii in the assay method of peimine, peiminine, described instrument is Thermo U3000
UHPLC chromatograph of liquid.
In above-mentioned Bulbus Fritillariae Thunbergii in the assay method of peimine, peiminine, described chromatographic column is Thermo
Syncronis C18。
In above-mentioned Bulbus Fritillariae Thunbergii in the assay method of peimine, peiminine, the instrument that described ASE extraction is used
For ASE350 Accelerate solvent extraction instrument.
The present invention is after using technique scheme, and it has the beneficial effect that
The detection method of the present invention is simple to operate, and precision is consistent with the precision of official method, reproducible.
Accompanying drawing explanation
Fig. 1 is the rectangular plots of the chromatography validation verification of peimine in the present invention；
Fig. 2 is the rectangular plots of the chromatography validation verification of peiminine in the present invention；
Fig. 3 is the peimine reference substance of the present invention, the chromatogram of peiminine reference substance；
Fig. 4 is the extraction of the present invention and method of testing obtains chromatogram.
Detailed description of the invention
Below in conjunction with detailed description of the invention, technical scheme is described in further detail, but does not constitute the present invention
Any restriction.
Embodiment 1:
Size-reduced for Bulbus Fritillariae Thunbergii machine is pulverized, excessively No. four sieves, about 1g, accurately weighed, mix homogeneously with 1g kieselguhr, stand-by,
It is moved into and put glass fiber filter in advance well10ml abstraction pool adds proper amount of silicon diatomaceous earth, gently shaking be allowed to
Chi Kou in the same horizontal line, tightens abstraction pool upper cover.After extraction terminates, extract is shifted in 25ml volumetric flask, uses first
Alcohol is diluted to scale, and under 15000r/min, centrifugal 3min, takes supernatant, enters LC and measures.
Extraction conditions (being shown in Table 1) is:
Table 1
Analysis method is LC liquid chromatography, liquid-phase chromatographic analysis condition:
A) the double ternary liquid phase (U-3000) of instrument: Thermo
B) chromatographic column: Thermo Syncronis C18 3*100mm 3um, i.e. carbon 18 chromatographic column, length 100mm,
Diameter 3mm, particle diameter 3 microns；
C) column temperature: 40 DEG C
D) flow velocity: 0.5mL/min
E) flowing phase: acetonitrile-water-diethylamine, volume ratio 70:30:0.03
F) detection wavelength: evaporative light scattering detector.
Chromatography validation verification:
Take peimine reference substance, peiminine reference substance in right amount, accurately weighed, add methanol and make every 1ml containing peimine
0.2mg, the mixed solution of peiminine 0.15mg, obtain peimine reference substance, peiminine reference substance.
Take peimine reference substance, peiminine reference substance in right amount, accurately weighed, add methanol and make every 1ml containing peimine
0.1791mg, the mixed solution of peiminine 0.1240mg, to obtain final product.Then respectively precision draw this solution 0.2 μ l, 0.5 μ l,
0.8 μ l, 1 μ l, 1.5 μ l, 2 μ l enter LC and measure, and measure according to said method, the results detailed in Table 2-1, table 2-2.
Accompanying drawing 1 is corresponding with table 2-1, and accompanying drawing 2 is corresponding with table 2-2, the abscissa indicated concentration of accompanying drawing 1 and accompanying drawing 2, vertical coordinate table
Show peak area.Accompanying drawing 3 is the chromatograph using above-mentioned LC liquid chromatography for measuring of peimine reference substance, peiminine reference substance
Figure.
Table 2-1 (peimine linear relationship)
Table 2-2 (peiminine linear relationship)
The repeatability checking of the extracting process of 1.1 embodiments 1:
Take sample (lot number: the 150827) 1.0g of identical lot number, totally 4 parts, accurately weighed, extract by ASE extracting method and supply
Test sample solution, sample size is 3 μ L, with above-mentioned chromatographic condition parallel test, records the content of peimine in sample and is shown in Table 3-1,
RSD is 2.54%；Recording the content of peiminine in sample and being shown in Table 3-2, RSD is 2.12%；Test shows ASE extraction side
Method repeatability is good, the results detailed in Table 3-1, table 3-2
Table 3-1
Table 3-2
The degree of accuracy test of the extracting process of 1.2 embodiments 1
Using the ASE extracting process of embodiment 1 and the extracting process of pharmacopeia to extract Bulbus Fritillariae Thunbergii, Bulbus Fritillariae Thunbergii is 4 batches, batch
Number be respectively 1510111,151102,150827,150618；
The ASE extracting process of embodiment 1 is to a batch of Bulbus Fritillariae Thunbergii parallel testing twice, respectively ASE1 and ASE2 table
Show.
Fig. 4 is that sample 150827 obtains according to ASE extracting process and LC liquid chromatography test (ASE2) of the present invention
Chromatogram.
Concrete test result see table 4；
Table 4
Above-described be only presently preferred embodiments of the present invention, all made in the range of the spirit and principles in the present invention any amendment,
Equivalent and improvement etc., should be included within the scope of the present invention.
Claims (9)
1. peimine, the assay method of peiminine in a Bulbus Fritillariae Thunbergii, it is characterised in that comprise the following steps:
Step 1: use ASE extraction that extract is obtained by extraction after being pulverized by Bulbus Fritillariae Thunbergii；
Step 2: after extract centrifugation, obtain supernatant；
Step 3: use liquid chromatography to be analyzed supernatant；
Wherein, the analysis condition of liquid chromatography is:
A) instrument: double ternary liquid phase chromatographs
B) chromatographic column specification: carbon 18 chromatographic column, 3*100mm 3um
C) column temperature: 40 DEG C
D) flow velocity: 0.5mL/min
E) flowing phase: acetonitrile-water-diethylamine, volume ratio 70:30:0.03
F) detection wavelength: evaporative light scattering detector.
The assay method of peimine, peiminine in Bulbus Fritillariae Thunbergii the most according to claim 1, it is characterised in that described
Step 1 particularly as follows:
S11: after Bulbus Fritillariae Thunbergii is pulverized, sieve；
S12: weigh 1 weight portion Bulbus Fritillariae Thunbergii powder, mix homogeneously stand-by with 1 weight portion kieselguhr；
S13: in the abstraction pool putting filter membrane in advance well, adds appropriate kieselguhr to the Chi Kou reaching abstraction pool after mix homogeneously；
The lid covering abstraction pool carries out static extracting；Extraction is extracted liquid after terminating；
Extraction conditions is: extractant is percent by volume 75% ethanol；Extraction temperature 80 DEG C；Static extracting time 3min；Punching
Wash volume 60%；Quiet cycle number of times 2 times.
The assay method of peimine, peiminine in Bulbus Fritillariae Thunbergii the most according to claim 2, it is characterised in that described
The volume of abstraction pool be 10ml.
The method of peimine, peiminine in Bulbus Fritillariae Thunbergii the most according to claim 2, it is characterised in that described is quiet
The pressure of state extraction is 1500psi.
The method of peimine, peiminine in Bulbus Fritillariae Thunbergii the most according to claim 2, it is characterised in that described is quiet
The purge time of state extraction is 100s.
The method of peimine, peiminine in Bulbus Fritillariae Thunbergii the most according to claim 1, it is characterised in that step 2 has
Body is: by extract centrifugal 3min under conditions of 15000r/min, take supernatant.
The method of peimine, peiminine in Bulbus Fritillariae Thunbergii the most according to claim 1, it is characterised in that described instrument
Device is Thermo U3000UHPLC chromatograph of liquid.
The method of peimine, peiminine in Bulbus Fritillariae Thunbergii the most according to claim 7, it is characterised in that described color
Spectrum post is Thermo Syncronis C18.
The method of peimine, peiminine in Bulbus Fritillariae Thunbergii the most according to claim 1, it is characterised in that described
The instrument that ASE extraction is used is ASE350 Accelerate solvent extraction instrument.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610234905.4A CN105911176A (en) | 2016-04-15 | 2016-04-15 | Detection method of peimine and peiminine in thunberg fritillary bulb |
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| CN201610234905.4A CN105911176A (en) | 2016-04-15 | 2016-04-15 | Detection method of peimine and peiminine in thunberg fritillary bulb |
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| CN201610234905.4A Pending CN105911176A (en) | 2016-04-15 | 2016-04-15 | Detection method of peimine and peiminine in thunberg fritillary bulb |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106885862A (en) * | 2017-04-13 | 2017-06-23 | 广西壮族自治区梧州食品药品检验所 | A kind of ASE HPLC methods determine peimine, the method for the content of Peiminine in fritillaria thunbergii |
| CN113189223A (en) * | 2021-04-24 | 2021-07-30 | 无锡济煜山禾药业股份有限公司 | Method for determining content of peimine and peiminine in Huangshi rattle tea |
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| WO2001049279A2 (en) * | 1999-12-30 | 2001-07-12 | Yeda Research And Development Co. Ltd. | Use of steroidal alkaloids to reverse multidrug resistance |
| CN101601805A (en) * | 2009-06-30 | 2009-12-16 | 长春工程学院 | The detection method of peimine and peiminine in the Chinese medicine Bulbus Fritillariae Cirrhosae extract |
| CN104407064A (en) * | 2014-10-22 | 2015-03-11 | 浙江康恩贝制药股份有限公司 | Fritillaria medicinal material identification and content determination method |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106885862A (en) * | 2017-04-13 | 2017-06-23 | 广西壮族自治区梧州食品药品检验所 | A kind of ASE HPLC methods determine peimine, the method for the content of Peiminine in fritillaria thunbergii |
| CN113189223A (en) * | 2021-04-24 | 2021-07-30 | 无锡济煜山禾药业股份有限公司 | Method for determining content of peimine and peiminine in Huangshi rattle tea |
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