CN108445115A - A kind of method that high performance liquid chromatography detects neoline and/or songorine and/or Fuziline - Google Patents
A kind of method that high performance liquid chromatography detects neoline and/or songorine and/or Fuziline Download PDFInfo
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Abstract
The invention discloses the methods of a kind of high performance liquid chromatography detection neoline and/or songorine and/or Fuziline.Using neoline and/or songorine and/or Fuziline solution as reference substance, then using octadecylsilane chemically bonded silica as stationary phase and the mobile phase containing triethylamine aqueous solution is to be detected under chromatographic condition, mobile phase therein is that volume ratio is (10 80):The acetonitrile triethylamine aqueous solution of (90 20) is isocratic or gradient elution, evaporative light scattering detector detect, the triethylamine aqueous solution a concentration of 0.02~0.2%.This method separating effect is good, simple and efficient, reproducible, and the quality control for monkshood (tag) or monkshood and its extract, Chinese patent drug containing neoline and/or songorine and/or Fuziline provides efficient detection method.
Description
Technical field
The present invention relates to efficient liquid phase chromatographic analysis technologies, more particularly it relates to a kind of high performance liquid chromatography
The method for detecting neoline and/or songorine and/or Fuziline.
Background technology
Monkshood is the processed goods of the sub- roots of ranunculaceae plant rhizome of Chinese monkshood Aconitum carmichaelii Debx., has Hui Yang
Inverse, mends fire supporing yang, eliminating cold to stop pain and other effects are rescued, the processing medicine materical crude slice of monkshood has raw tag, steaming tag, stir-fry tag etc..Monkshood is hair
Dry female root of gelsemium section plant rhizome of Chinese monkshood Aconitum carmichaelii Debx., there is dispelling wind and eliminating dampness, antalgic to act on,
The processing medicine materical crude slice of monkshood has Radix Aconiti, aconiti preparata,radix.The main active of monkshood and monkshood is diterpene alkaloid, according to C4 and
C8 whether there is or not ester groups, can be divided into diester-type alkaloids (aconitine, mesaconine, Hypaconitine), monoester alkaloid (benzoyl
Aconine, benzoylmesaconine, benzoyl time aconine) and amine alcohol type alkaloid (neoline, songorine, monkshood
Spirit etc.).Current edition Chinese Pharmacopoeia uses high performance liquid chromatography (HPLC), UV detector (UV) detection to measure monoester type life
The quality control index of alkaloids and/or diester-type alkaloids content as monkshood, monkshood and its medicine materical crude slice.In recent years, research shows that
Amine alcohol type diterpene alkaloid has the various actives effects such as cardiac stimulant, as there is neoline cardiac stimulant, analgesic activity, Fuziline to have strong
The heart, improves the effects that myocardial ischemia at Hemorrhagic shock, and songorine has the effects that anti-inflammatory and antalgic, antianxiety.
As described above, generally measuring ester alkaloid content as monkshood, monkshood and its processed goods using HPLC-UV methods
Therefore the quality evaluation index of (medicine materical crude slice, Chinese patent drug, extract), the rarely seen content for measuring amine alcohol type diterpene alkaloid lack matter
Measure the globality and science of control.In terms of amine alcohol type alkaloid content determination, there is document report to use UPLC-MS/MS methods
The content of a variety of higenamine alcohol type alkaloid components is measured simultaneously and the content of songorine, but the former are measured using HPLC-UV methods
Appointed condition requires height, universality poor, and the latter's amine alcohol type alkaloid UV absorption is weak, and poor sensitivity, interference are big.
Invention content
The present invention overcomes the deficiencies in the prior art, a kind of high performance liquid chromatography detection neoline and/or Song fruit are provided
The method of spirit and/or Fuziline, it is expected that can solve existing measurement method can not accurately measure amine alcohol type alkaloid
The problem of and existing measurement method universality difference problem.
In order to solve the above technical problems, the present invention uses following technical scheme：
Present inventor is by deep experiment and research, using octadecylsilane chemically bonded silica as stationary phase, with second
Nitrile-is mobile phase containing triethylamine aqueous solution, and evaporative light scattering detector detection can be containing neoline and/or songorine and/or attached
The quality control of the monkshood (tag) or monkshood and its extract, Chinese patent drug of sub- spirit provides simple and feasible detection method.I.e. originally
Invention high performance liquid chromatography detects neoline and/or the method for songorine and/or Fuziline is：Using neoline and/or Song
Fruit spirit and/or Fuziline reference substance prepare reference substance solution, using octadecylsilane chemically bonded silica as stationary phase, with second
Nitrile-triethylamine aqueous solution is under the chromatographic condition of mobile phase, to the tested product containing neoline and/or songorine and/or Fuziline
The content of neoline and/or songorine and/or Fuziline in solution is detected, and the mobile phase is that volume ratio is (10-
80):The acetonitrile-triethylamine aqueous solution of (90-20), isocratic elution or gradient elution, are examined using evaporative light scattering detector
It surveys, the volumetric concentration of triethylamine is 0.02-0.2% in the triethylamine aqueous solution.
Preferably, the high performance liquid chromatography detects in neoline and/or the method for songorine and/or Fuziline,
The volumetric concentration of triethylamine is 0.1% in triethylamine aqueous solution.
Experiment display, the high performance liquid chromatography detects neoline and/or the method for songorine and/or Fuziline can
To use the chromatographic column of different brands, grain size, length, when using isocratic elution, the mobile phase can be that volume ratio is 25:75
Or 35:65 or 45:55 acetonitrile-triethylamine aqueous solution, wherein the volumetric concentration of triethylamine is preferably in triethylamine aqueous solution
0.1%.
The high performance liquid chromatography detection neoline and/or the method for songorine and/or Fuziline may be used more
The different elution program of kind, the preferred elution program of two of which are：
The first is：Using acetonitrile as mobile phase A in mobile phase, using triethylamine aqueous solution as Mobile phase B, carries out gradient and wash
De-, gradient elution program is as follows：0~8min, 10%A → 20%A；8~15min, 20%A → 25%A；15~25min, 25%
A → 25%A；25~30min, 25%A → 28%A；30~31min, 28%A → 60%A；31~42min, 60%A → 65%
A。
It is for second：Using acetonitrile as mobile phase A in mobile phase, using triethylamine aqueous solution as Mobile phase B, carries out gradient and wash
De-, gradient elution program is as follows：0~5min, 20%A → 25%A；5~15min, 25%A → 25%A；15~20min, 25%
A → 28%A；20~21min, 28%A → 55%A；21~28min, 55%A → 55%A；28~40min, 55%A → 80%
A。
Wherein, the volumetric concentration of triethylamine is preferably 0.1% in triethylamine aqueous solution.
The high performance liquid chromatography detects in neoline and/or the method for songorine and/or Fuziline, preferably chromatography
The number of theoretical plate of column is not less than 5000 based on neoline or songorine or monkshood Ling Feng Peak, and sample size is 5-80 μ L.Better sample introduction
Amount is 10-20 μ L.
Under normal circumstances, high performance liquid chromatography detection neoline and/or songorine and/or Fuziline of the present invention
Method in, reference substance solution and tested product solution are obtained through following manner respectively：
The reference substance for taking neoline and/or songorine and/or Fuziline is 0- with methanol volume fraction after accurately weighed
100% methanol aqueous solution is configured to reference substance solution；Such as accurate neoline and/or songorine and/or the Fuziline of weighing
Reference substance, it is that mixing of every 1mL containing above-mentioned reference substance 0.05-0.5mg is made in 50% methanol aqueous solution to add methanol volume fraction
Reference substance solution；
The tested product containing neoline and/or songorine and/or Fuziline are taken, after accurately weighed, add HCl containing 0.05mol/L
And ethyl alcohol or methanol volume fraction are the ethyl alcohol or methanol aqueous solution ultrasonic extraction of 0-80%, take supernatant to filter, take subsequent filtrate,
As tested product solution；Or filtration after taking supernatant that 5-10 times of concentration is recovered under reduced pressure in 60 DEG C or less, subsequent filtrate is taken, as tested
Product solution；Such as accurately weighed monkshood or monkshood powder 2.0g, it is 70% to add HCl containing 0.05mol/L and methanol volume fraction
Methanol aqueous solution 50mL, ultrasonic extraction 30 minutes take supernatant 25mL, and 60 DEG C or less are recovered under reduced pressure concentration and are settled to 5mL, mistake
Filter, takes subsequent filtrate to get tested product solution.
According to the above-mentioned detection method of the present invention, a kind of preferred detection method carries out in the following manner：
1) chromatographic condition and system suitability：It is stream with acetonitrile using octadecylsilane chemically bonded silica as filler
Dynamic phase A carries out gradient elution using 0.1% triethylamine aqueous solution as Mobile phase B, and elution program is：0~5min, 20%A →
25%A；5~15min, 25%A → 25%A；15~20min, 25%A → 28%A；20~21min, 28%A → 55%A；21
~28min, 55%A → 55%A；28~40min, 55%A → 80%A；Evaporative light scattering detector detects, and number of theoretical plate presses Buddhist nun
Spirit difficult to understand or songorine or monkshood Ling Feng Peak meter are not less than 5000；
2) preparation of reference substance solution：The reference substance of neoline and/or songorine and/or Fuziline is taken, it is accurately weighed, it uses
It is molten that the methanol aqueous solution that methanol volume fraction is 50% is configured to mixing reference substances of every 1mL containing above-mentioned reference substance 0.05-0.5mg
Liquid；
3) it is detected the preparation of product solution：It took No. 3 monkshoods sieved or monkshood powder 2.0g accurately weighed, and set conical flask with cover
The methanol solution 50mL that middle accurate addition HCl containing 0.05mol/L and methanol volume fraction are 70%, through 300W and 40kHz ultrasounds
Processing 30 minutes, takes supernatant 25mL, and concentration, which is recovered under reduced pressure, in 60 DEG C or less is settled to 5mL, and filtering takes subsequent filtrate to get quilt
Inspection product solution；
4) it measures：It is accurate respectively to draw reference substance solution 10ul, 20ul and tested each 10-20ul of product solution, inject liquid phase
Chromatograph measures.
The present invention has carried out mobile phase chromatographic condition comparative studies extensively and profoundly, has investigated methanol-triethylamine gradient
(isocratic) elution system, methanol-diethylamine gradient (isocratic) elution system, methanol-acetic acid ammonium gradient (isocratic) elution system, with
And acetonitrile-triethylamine gradient (isocratic) elution system, acetonitrile-diethylamine gradient (isocratic) elution system, acetonitrile-ammonium acetate gradient
(isocratic) elution system etc., in addition, methanol (or acetonitrile)-formic acid gradient (isocratic) elution system has also been investigated, including to various
The investigation of ratio, concentration.The results show that in addition to acetonitrile-triethylamine gradient (isocratic) elution system, other mobile phase elution systems
Actual effect is dissatisfied, and it is poor or be difficult to reach baseline separation etc. shift of retention time, chromatography peak symmetry easily occur, and divides
It is longer to analyse the time.
Compared with prior art, the present invention at least has the advantages that：The present invention detects neoline and/or songorine
And/or the method for Fuziline content, it is simple and feasible, each sample can be made to reach baseline separation, and baseline is steady, it is quantitative to meet pharmacopeia
Analysis technology requirement, for containing neoline and/or songorine and/or Fuziline monkshood (tag), monkshood and its extract, in
The quality control of patent medicine provides efficient detection method.
Description of the drawings
Fig. 1 is the HPLC chromatogram of 1 reference substance of the embodiment of the present invention.
Fig. 2 is the HPLC chromatogram of 1 monkshood water-soluble biological alkali extract of the embodiment of the present invention.
Fig. 3 is the HPLC chromatogram that the embodiment of the present invention 1 steams tag.
Fig. 1 to Fig. 3 is the reference substance of the method for the present invention detection and the HPLC chromatogram of typical sample, and 1 peak in figure is attached
Sub- spirit, 2 peaks are neoline, and 3 peaks are songorine.
Specific implementation mode
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to the accompanying drawings and embodiments, right
The present invention is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, and
It is not used in the restriction present invention.
Embodiment 1
Monkshood extract and the assay for steaming neoline, songorine and Fuziline in tag：According to the efficient liquid of Chinese Pharmacopoeia
Phase chromatography measures.
1) chromatographic condition and system suitability chromatographic column：Agilent Extend C18Chromatographic column (250mm ×
4.6mm, 5 μm)；Mobile phase：Acetonitrile (A) -0.1% (volume fraction) triethylamine aqueous solution (B), gradient elution (0~8min,
10%A → 20%A；8~15min, 20%A → 25%A；15~25min, 25%A → 25%A；25~30min, 25%A →
28%A；30~31min, 28%A → 60%A；31~42min, 60%A → 65%A)；Detector：Evaporative light scattering detector,
Nitrogen buffer gas, 50 DEG C of evaporating temperature, temperature of drifting about：40 DEG C, flow rate of carrier gas 1.5Lmin-1, gain 1；Number of theoretical plate is pressed
Neoline or songorine or monkshood Ling Feng Peak meter should be not less than 5000.
2) preparation of reference substance solution：Take each reference substance appropriate, it is accurately weighed, add 50% (volume) methanol solution to be made often
1mL 0.5mg containing neoline, Fuziline 0.3mg, the mixed reference substance solution of songorine 0.15mg.
3) preparation of extract test solution：Taking monkshood water-soluble biological alkali extract, (Luo Chunmei etc., monkshood are water-soluble
The extraction and purification process of alkaloid is studied, Chinese herbal medicine, 017,48 (12):2415) about 50mg, it is accurately weighed, set 10mL volumetric flasks
In, 50% (volume) methanol aqueous solution ultrasonic dissolution is added, is settled to scale, shakes up, 0.45 μm of filtering with microporous membrane takes continuous filter
Liquid to get.
4) preparation of tag test solution is steamed：It takes and steams tag powder 2.0g, it is accurately weighed, it sets in conical flask with cover, essence
Close addition 0.05mol/L hydrochloric acid solution 50mL, shake up, and are ultrasonically treated (power 250W, frequency 40kHz) 30min, let cool, shake up,
In 8000rmin-16min, accurate measurement supernatant 25mL are centrifuged, is recovered under reduced pressure to about 1-2mL at 60 DEG C, is transferred to 5mL amounts
Bottle in, add water washing to be settled to scale, shake up, 0.45 μm of filtering with microporous membrane, take subsequent filtrate to get.
5) measuring method：It is accurate respectively to draw reference substance solution 5ul, 10ul and tested each 10-20ul of product solution, inject liquid phase
Chromatograph measures.
Embodiment 2
Raw tag and the assay for frying neoline, songorine and Fuziline in tag：According to Chinese Pharmacopoeia high-efficient liquid phase color
Spectrometry measures.
1) chromatographic condition and system suitability chromatographic column：Supelco Discover-C18 (150mm × 4.6mm,
5.0μm)；Mobile phase：Acetonitrile (A) -0.1% (volume fraction) triethylamine aqueous solution (B), gradient elution (0~5min, 20%A →
25%A；5~15min, 25%A → 25%A；15~20min, 25%A → 28%A；20~21min, 28%A → 55%A；21
~28min, 55%A → 55%A；28~40min, 55%A → 80%A)；Detector：Evaporative light scattering detector is with nitrogen
Carrier gas, 50 DEG C of evaporating temperature, temperature of drifting about：35 DEG C, flow rate of carrier gas 1.2Lmin-1, gain 1；Number of theoretical plate presses neoline
Or songorine or monkshood Ling Feng Peak meter should be not less than 5000.
2) preparation of reference substance solution：Take each reference substance appropriate, it is accurately weighed, add 50% methanol aqueous solution that every 1mL is made
0.2mg containing neoline, Fuziline 0.1mg, the mixed reference substance solution of songorine 0.1mg.
3) preparation of test solution：Raw tag or stir-fry tag powder 2.0g are taken, it is accurately weighed, conical flask with cover is set respectively
In, the hydrochloric acid containing 0.05mol/L and the aqueous solution 50mL containing 70% (volume) methanol is added in precision, shakes up, and is ultrasonically treated (power
300W, frequency 40kHz) 30min, supernatant 25mL is taken, concentration, which is recovered under reduced pressure, in 60 DEG C or less is settled to 5mL, and filtering takes continuous filter
Liquid to get.
4) measuring method：It is accurate respectively to draw reference substance solution 10ul, 20ul and tested each 10-20ul of product solution, inject liquid
Chromatography measures.
Embodiment 3
The assay of neoline in monkshood and aconiti preparata,radix：According to Chinese Pharmacopoeia high effective liquid chromatography for measuring.
1) chromatographic condition and system suitability chromatographic column：Phenomenex-C18(250mm × 4.6mm, 5.0 μm)；Stream
Dynamic is mutually -0.2% triethylamine aqueous solution (35 of acetonitrile:65, v/v)；Detector：Evaporative light scattering detector, nitrogen buffer gas,
50 DEG C, flow rate of carrier gas 1.5Lmin-1 of evaporating temperature, gain 1；Number of theoretical plate should be not less than 5000 based on neoline peak.
2) preparation of reference substance solution：Take neoline reference substance appropriate, it is accurately weighed, add methanol that every 1mL is made difficult to understand containing Buddhist nun
The reference substance solution of clever 0.2mg.
3) preparation of test solution：Monkshood and aconiti preparata,radix powder 2.0g are taken, it is accurately weighed, conical flask with cover is set respectively
In, hydrochloric acid containing 0.05mol/L and the aqueous solution 50mL containing 50% (volume) ethyl alcohol is added in precision, shakes up, and is ultrasonically treated (power
300W, frequency 40kHz) 30min, precision measures supernatant 25mL, and concentration, which is recovered under reduced pressure, at 60 DEG C is settled to 5ml, filters, takes continuous
Filtrate to get.
4) measuring method：It is accurate respectively to draw reference substance solution 10ul, 20ul and tested each 20-30ul of product solution, inject liquid
Chromatography measures.
Embodiment 4
The assay of neoline, songorine and Fuziline in unprocessed Radix Aconiti Lateralis and Radix Aconiti：According to Chinese Pharmacopoeia high-efficient liquid phase color
Spectrometry measures.
1) chromatographic condition and system suitability chromatographic column：Agilent Extend C18 (250mm × 4.6mm, 5.0 μ
m)；Mobile phase：Acetonitrile (A) -0.05% (volume fraction) triethylamine aqueous solution (B), gradient elution (0~5min, 20%A →
25%A；5~15min, 25%A → 25%A；15~20min, 25%A → 28%A；20~21min, 28%A → 55%A；21
~28min, 55%A → 55%A；28~40min, 55%A → 80%A)；Detector：Evaporative light scattering detector is with nitrogen
Carrier gas, 50 DEG C of evaporating temperature, temperature of drifting about：35 DEG C, flow rate of carrier gas 1.2Lmin-1, gain 1；Number of theoretical plate presses neoline
Or songorine or monkshood Ling Feng Peak meter should be not less than 5000.
2) preparation of reference substance solution：Take each reference substance appropriate, it is accurately weighed, add water that every 1mL is made and contains neoline
The mixing reference substance of 0.2mg, Fuziline 0.1mg, songorine 0.1mg.
3) preparation of test solution：Unprocessed Radix Aconiti Lateralis or Radix Aconiti powder 2.0g are taken, it is accurately weighed, conical flask with cover is set respectively
In, the aqueous solution 50mL of the hydrochloric acid containing 0.05mol/L is added in precision, shakes up, and is ultrasonically treated (power 250W, frequency 40kHz)
50min takes supernatant to filter, take subsequent filtrate to get.
4) measuring method：It is accurate respectively to draw reference substance solution 5ul, 10ul and tested each 50-80ul of product solution, inject liquid phase
Chromatograph measures.
Following tests result further demonstrates that the method for the present invention is simple and feasible, and the technology for meeting pharmacopeia quantitative analysis is wanted
It asks, can be used for the quality controls such as monkshood, monkshood and quality evaluation.
The content assaying method of the above embodiment of the present invention 1:
1. chromatographic condition chromatographic column：Agilent Eclipse C18(250mm × 4.6mm, 5 μm)；Mobile phase：Acetonitrile (A)-
0.1% (volume fraction) triethylamine aqueous solution (B), gradient elution (0~8min, 10%A → 20%A；8~15min, 20%A →
25%A；15~25min, 25%A → 25%A；25~30min, 25%A → 28%A；30~31min, 28%A → 60%A；31
~42min, 60%A → 65%A)；Flow velocity：1.0mL·min-1；Column temperature：35℃；Detector：Evaporative light scattering detector, with nitrogen
Gas is carrier gas, 50 DEG C of evaporating temperature, flow rate of carrier gas 1.5Lmin-1, gain 1；20~80 μ L of sample size.In above-mentioned chromatostrip
Under part, the retention time of neoline, songorine and Fuziline is consistent with reference substance in sample, the chromatographic peak separating degree of 3 kinds of ingredients
Well, other ingredients are noiseless to the measurement of ingredient to be measured in sample, meet quantitative analysis requirement.HPLC chromatogram is shown in Fig. 1 extremely
Fig. 3.
2. the preparation of solution
The preparation of 2.1 mixing reference substance storing solutions takes each reference substance appropriate, accurately weighed, and 50% methanol solution is added to be made often
The mixing reference substance stock solution of 1mL 0.5mg containing neoline, Fuziline 0.3mg, songorine 0.15mg.
The preparation of 2.2 extract test solutions takes monkshood water-soluble biological alkali extract about 50mg, accurately weighed, sets
In 10mL volumetric flasks, 50% methanol-water ultrasonic dissolution is added, is settled to scale, shakes up, 0.45 μm of filtering with microporous membrane takes continuous filter
Liquid to get.
The preparations of 2.3 steaming tag test solutions, which take, steams tag powder 2.0g, accurately weighed, sets in conical flask with cover, essence
Close addition 0.05mol/L aqueous hydrochloric acid solution 50mL, shake up, and are ultrasonically treated (power 250W, frequency 40kHz) 30min, let cool, shake
It is even, in 8000rmin-16min, accurate measurement supernatant 25mL are centrifuged, is recovered under reduced pressure to about 1-2mL at 60 DEG C, is transferred to 5ml
In measuring bottle, water washing is added to be settled to scale, shaken up, 0.45 μm of filtering with microporous membrane, take subsequent filtrate to get.
3. linear relationship investigates accurate draw respectively and mixes reference substance storing solution 1,2,4,6,8,10mL under " 2.1 " item, point
It does not set in 10mL measuring bottles, with 50% methanol dilution to scale, shakes up to get serial mixed reference substance solution.Draw above-mentioned mixing
20 μ L of reference substance solution measure chromatographic peak area by chromatographic condition under " 1 " item.It is right using the logarithm of peak area as ordinate (Y)
Logarithm according to product concentration is abscissa (X), carries out linear regression analysis, calculates regression equation and related coefficient, by mixing pair
Sample introduction measures after gradually being diluted according to product solution, is that 3: 1 and S/N is 10: 1 determination detection limits and determines with signal-to-noise ratio S/N in chromatogram
Amount limit, the results are shown in Table 1.
1 neoline of table, the linearity and range of songorine and Fuziline, detection limit and quantitative limit
As it can be seen from table 1 the linear relationship of Fuziline, neoline and songorine is good, meet quantitative analysis method
Technology requirement.
4. Precision Experiment precision draws the 20 μ L of mixed reference substance solution of same concentration, connect by chromatographic condition under " 1 " item
Continuous sample introduction 6 times, measures peak area, calculates the RSD of peak area.The results show that the RSD of Fuziline, neoline, songorine is respectively
1.85%, 2.25%, 1.17%, show that instrument precision is good.
5. stability experiment takes same test solution, place at room temperature, respectively at 0,2,4,6,8,10,20 μ of 12h sample introductions
L is analyzed by chromatographic condition under " 1 " item, Fuziline, neoline, songorine peak area RSD be respectively 1.72%, 0.93%,
1.88%, the results showed that test solution, which is placed at room temperature in 12h, to be stablized.
6. repetitive test takes same batch of sample, precision to weigh 6 parts, by legal system available test sample solution below " 2 " item, press
Under " 1 " item chromatographic condition analyze, measure Fuziline, neoline, songorine content average value (n=6) be respectively 20.92,
46.32 15.44mgg-1.It is respectively 1.69%, 0.79%, 1.35% to calculate its RSD, shows that this method repeatability is good.
7. rate of recovery experiment precision weighs 6 parts of the same batch of sample of known content under " repetitive test " item, every part about
25mg, precision addition mixed reference substance solution is appropriate, according to legal system available test sample solution below " 2 " item, according to the chromatography under " 1 " item
Condition carries out assay, calculates the rate of recovery.As a result Fuziline, neoline, songorine average recovery rate be respectively 98.5%,
97.2%, 97.7%；RSD is respectively 1.26%, 1.29%, 1.70%.
8. sample measurement takes 3 batches of monkshood water-soluble biological alkali extracts and three batches of steaming tags respectively, according to
Legal system available test sample solution below " 2 " item, each sample is 2 parts parallel, and is carried out according to the chromatographic condition under " 1 " item
It measures, calculates the content of 3 kinds of alkaloids in each batch of sample, assay the results are shown in Table 2.
23 batches of extracts of table and 3 crowdes of content (mgg for steaming 3 kinds of alkaloids in tag-1, n=2)
| Sample | Lot number | Yield (%) | Fuziline | Neoline | Songorine |
| Steam tag 1 | 151002 | 0.37 | 0.72 | 0.28 | |
| Steam tag 2 | 160201 | 0.38 | 0.77 | 0.28 | |
| Steam tag 3 | 160901 | 0.39 | 0.75 | 0.29 | |
| Extract 1 | 20161208 | 1.63 | 20.81 | 46.41 | 15.59 |
| Extract 2 | 20161211 | 1.75 | 20.22 | 45.29 | 15.02 |
| Extract 3 | 20161220 | 1.68 | 22.08 | 47.80 | 15.92 |
From table 2 it can be seen that three batches of contents for steaming Fuziline, neoline and songorine in tag and three batches of extracts are opposite
Stablize, it is quality controllable.
Although reference be made herein to invention has been described for explanatory embodiment of the invention, however, it is to be understood that ability
Field technique personnel can be designed that a lot of other modification and implementations, these modifications and implementations will be fallen in the application public affairs
Within the scope and spirit opened.It more specifically, can be to the group of theme combination layout in range disclosed in the present application
A variety of variations and modifications are carried out at component and/or layout.In addition to variations and improvements to the component parts and or layout,
To those skilled in the art, other purposes also will be apparent.
Claims (10)
1. a kind of method of high performance liquid chromatography detection neoline and/or songorine and/or Fuziline, it is characterised in that use
The reference substance of neoline and/or songorine and/or Fuziline prepares reference substance solution, is being with octadecylsilane chemically bonded silica
Stationary phase, using acetonitrile-triethylamine aqueous solution as under the chromatographic condition of mobile phase, to containing neoline and/or songorine and/or monkshood
Neoline and/or the content of songorine and/or Fuziline in the tested product solution of spirit are detected, and the mobile phase is volume
Than for (10-80):The acetonitrile-triethylamine aqueous solution of (90-20), is detected using evaporative light scattering detector, three second
The volumetric concentration of triethylamine is 0.02-0.2% in amine aqueous solution.
2. the side of high performance liquid chromatography detection neoline according to claim 1 and/or songorine and/or Fuziline
Method, it is characterised in that the volumetric concentration of triethylamine is 0.1% in the triethylamine aqueous solution.
3. high performance liquid chromatography according to claim 1 or 2 detection neoline and/or songorine and/or Fuziline
Method, it is characterised in that it is that volume ratio is 25 that elution program, which uses isocratic elution, the mobile phase,:75 or 35:65 or 45:55
Acetonitrile-triethylamine aqueous solution.
4. high performance liquid chromatography according to claim 1 or 2 detection neoline and/or songorine and/or Fuziline
Method, it is characterised in that elution program uses gradient elution, water-soluble with triethylamine using acetonitrile as mobile phase A in the mobile phase
Liquid is Mobile phase B, and gradient elution program is as follows：0~8min, 10%A → 20%A；8~15min, 20%A → 25%A；15~
25min, 25%A → 25%A；25~30min, 25%A → 28%A；30~31min, 28%A → 60%A；31~42min,
60%A → 65%A.
5. high performance liquid chromatography according to claim 1 or 2 detection neoline and/or songorine and/or Fuziline
Method, it is characterised in that elution program uses gradient elution, water-soluble with triethylamine using acetonitrile as mobile phase A in the mobile phase
Liquid is Mobile phase B, and gradient elution program is as follows：0~5min, 20%A → 25%A；5~15min, 25%A → 25%A；15~
20min, 25%A → 28%A；20~21min, 28%A → 55%A；21~28min, 55%A → 55%A；28~40min,
55%A → 80%A.
6. the side of high performance liquid chromatography detection neoline according to claim 1 and/or songorine and/or Fuziline
Method, it is characterised in that the number of theoretical plate of chromatographic column is not less than 5000 based on neoline or songorine or monkshood Ling Feng Peak, and sample size is
5-80μL。
7. the side of high performance liquid chromatography detection neoline according to claim 6 and/or songorine and/or Fuziline
Method, it is characterised in that the sample size is 10-20 μ L.
8. the side of high performance liquid chromatography detection neoline according to claim 1 and/or songorine and/or Fuziline
Method, it is characterised in that the reference substance solution and tested product solution are obtained through following manner respectively：
The reference substance for taking neoline and/or songorine and/or Fuziline is 0-100% with methanol volume fraction after accurately weighed
Methanol aqueous solution be configured to reference substance solution；
The tested product containing neoline and/or songorine and/or Fuziline are taken, after accurately weighed, add HCl containing 0.05mol/L and second
Alcohol or ethyl alcohol or methanol aqueous solution ultrasonic extraction that methanol volume fraction is 0-80%, take supernatant to filter, take subsequent filtrate, as
Tested product solution；Or filtration after taking supernatant that 5-10 times of concentration is recovered under reduced pressure in 60 DEG C or less, subsequent filtrate is taken, it is molten as tested product
Liquid.
9. the side of high performance liquid chromatography detection neoline according to claim 8 and/or songorine and/or Fuziline
Method, it is characterised in that the preparation method of the reference substance solution is：Precision weighs neoline and/or songorine and/or Fuziline
Reference substance, add methanol volume fraction to be that every 1mL is made in 50% methanol aqueous solution mixed containing above-mentioned reference substance 0.05-0.5mg
Close reference substance solution；The preparation method of the tested product solution is：Accurately weighed monkshood or monkshood powder 2.0g, add and contain
The methanol aqueous solution 50mL that 0.05mol/L HCl and methanol volume fraction are 70%, ultrasonic extraction 30 minutes take supernatant
25mL, 60 DEG C or less are recovered under reduced pressure concentration and are settled to 5mL, and filtering takes subsequent filtrate to get tested product solution.
10. the side of high performance liquid chromatography detection neoline according to claim 1 and/or songorine and/or Fuziline
Method, it is characterised in that carry out in the following manner：
1) chromatographic condition and system suitability：Using octadecylsilane chemically bonded silica as filler, using acetonitrile as mobile phase
A carries out gradient elution using 0.1% triethylamine aqueous solution as Mobile phase B, and elution program is：0~5min, 20%A → 25%A；5
~15min, 25%A → 25%A；15~20min, 25%A → 28%A；20~21min, 28%A → 55%A；21~28min,
55%A → 55%A；28~40min, 55%A → 80%A；Evaporative light scattering detector detects, and number of theoretical plate presses neoline or Song
Fruit spirit or monkshood Ling Feng Peak meter are not less than 5000；
2) preparation of reference substance solution：The reference substance of neoline and/or songorine and/or Fuziline is taken, it is accurately weighed, use methanol
The methanol aqueous solution that volume fraction is 50% is configured to mixed reference substance solutions of every 1mL containing above-mentioned reference substance 0.05-0.5mg；
3) it is detected the preparation of product solution：It took No. 3 monkshoods sieved or monkshood powder 2.0g accurately weighed, set smart in conical flask with cover
The methanol solution 50mL that close addition HCl containing 0.05mol/L and methanol volume fraction are 70%, is ultrasonically treated through 300W and 40kHz
30 minutes, supernatant 25mL is taken, concentration, which is recovered under reduced pressure, in 60 DEG C or less is settled to 5mL, and filtering takes subsequent filtrate to get tested product
Solution；
4) it measures：It is accurate respectively to draw reference substance solution 10ul, 20ul and tested each 10-20ul of product solution, inject liquid chromatogram
Instrument measures.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110907545A (en) * | 2019-11-14 | 2020-03-24 | 健民药业集团股份有限公司 | Method for simultaneously determining contents of six metabolites in small gold preparation |
| CN114965735A (en) * | 2022-04-13 | 2022-08-30 | 神威药业集团有限公司 | Multivariate statistical analysis method of monkshood |
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| CN110907545A (en) * | 2019-11-14 | 2020-03-24 | 健民药业集团股份有限公司 | Method for simultaneously determining contents of six metabolites in small gold preparation |
| CN114965735A (en) * | 2022-04-13 | 2022-08-30 | 神威药业集团有限公司 | Multivariate statistical analysis method of monkshood |
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