CN107764908A - A kind of method for determining alkaloid component content in blood-nourishing and brain-refreshing the water extracted immersing paste - Google Patents
A kind of method for determining alkaloid component content in blood-nourishing and brain-refreshing the water extracted immersing paste Download PDFInfo
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Abstract
The present invention relates to a kind of method for determining alkaloid component content in blood-nourishing and brain-refreshing the water extracted immersing paste, the method for the invention, comprise the following steps：The preparation of need testing solution, the preparation of reference substance solution and assay, the method of the invention can effectively remove interference component, alkaloid component can be effectively enriched with simultaneously, this method is efficiently quick, stability is good, high sensitivity, efficiently separating for four kinds of alkaloid components can be realized in 5 minutes, the method for quality control precision of the empirical tests present invention, reappearance, have good stability, can effectively determine alkaloid component in blood-nourishing and brain-refreshing the water extracted immersing paste.
Description
Technical field
The present invention relates to a kind of content assaying method of active ingredient in Chinese medical extract, more particularly to a kind of measure blood-nourishing
The method of alkaloid component content in cephalocathartic the water extracted immersing paste.
Background technology
Blood-nourishing and brain-refreshing granules and blood-nourishing and brain-refreshing ball be by Radix Angelicae Sinensis, Ligusticum wallichii, the root of herbaceous peony, prepared rhizome of rehmannia, yncaria stem with hooks, reticulate millettia, selfheal,
Cassia seed, mother-of-pearl, corydalis tuber, a herb of asarum ten and herbal mixture made of auxiliary material.Blood-nourishing and brain-refreshing the water extracted immersing paste is foster
The preparation intermediate of blood brain-refreshing granules and blood-nourishing and brain-refreshing ball.Preparation technology is：Prepared rhizome of rehmannia, yncaria stem with hooks, reticulate millettia, selfheal, pearl
Mother, asarum add water to cook, and filter, and concentration, add ethanol to stand, and filter, and reclaim ethanol and are concentrated into right amount, produce blood-nourishing and brain-refreshing water
Extracted extract.
《Chinese Pharmacopoeia》Version in 2015 discloses the content detection of two kinds of preparations of blood-nourishing and brain-refreshing, is all the inspection to Paeoniflorin
Survey, the preparation method of its test sample is：This product content is taken, it is finely ground, about 0.08g is taken, it is accurately weighed, put in 20ml beakers, add
0.2% sodium acid carbonate, it is ultrasonically treated 5 minutes, by D101 large pore resin absorption columns, is eluted with water, discards eluent, then use first
Alcohol elutes, and collects eluent, adds water to scale, shake up, and centrifuges 5 minutes, takes supernatant, filters, takes subsequent filtrate, produce；Chromatogram
Condition is：Using octadecylsilane chemically bonded silica as filler, with isopropanol-methanol citric acid soln (2：18：80) it is flowing
Phase, Detection wavelength 240nm, number of theoretical plate calculates according to Paeoniflorin peak should be not less than 2000.
In blood-nourishing and brain-refreshing preparation, yncaria stem with hooks plays an important role in formula, has a The flat liver of heat-clearing, dispelling wind and relieving convulsion effect,
Mutually agree with the effect of blood-nourishing and brain-refreshing preparation " blood-nourishing soothing the liver, promoting blood circulation and removing obstruction in channels ".And alkaloid be Uncaria chief active into
Point, the report not detected in pharmacopeia to the yncaria stem with hooks composition in blood-nourishing and brain-refreshing preparation.
The alkaloid component that inventor is directed in blood-nourishing and brain-refreshing the water extracted immersing paste early stage has made intensive studies, and utilizes SPE-
UPLC methods, the isolation and purification method of alkaloid component is established, realize efficiently separating for alkaloid component.Using series connection level Four
Bar flight time mass spectrum (Q-TOF/MS) LC-MS instrument has carried out structure to the alkaloid component in blood-nourishing and brain-refreshing the water extracted immersing paste
Identification, by being compared to one-level, secondary fragment analysis and reference substance, it is determined that mainly containing four kinds of alkaloid components, respectively hook
Rattan alkali, isorhynchophylline, corynoxeine and isocorynoxeine.
For blood-nourishing and brain-refreshing the water extracted immersing paste, a whole set of Process Quality Control system comprehensively has been had been built up at present,
But also without the mass analysis method for alkaloid component.
For further Improving The Quality of Products controlled level, product quality is grasped comprehensively, and inventor is directed to blood-nourishing and brain-refreshing water extraction
Alkaloid component in medicinal extract establishes content assaying method, and has carried out Method validation.
The content of the invention
The present invention relates to a kind of method for determining alkaloid component content in blood-nourishing and brain-refreshing the water extracted immersing paste, this method is using super
High performance liquid chromatography.
The method of the invention, comprise the following steps：The preparation of need testing solution, the preparation of reference substance solution and containing measurement
It is fixed.
Wherein, the preparation of the need testing solution comprises the following steps：Blood-nourishing and brain-refreshing the water extracted immersing paste about 0.2g is taken, precision claims
It is fixed, 35%-45% methanol 10mL ultrasonic dissolutions, up to processed good ProElut C18- U (500mg/6mL) post, uses 35%-
45% methanol 5mL is washed, and cleaning solution discards, and is eluted with methanol 4.5mL, is collected eluent, with methanol constant volume to 5mL, is shaken up, i.e.,
；
Wherein, the preparation of the reference substance solution comprises the following steps：
Using rhynchophyllin, isorhynchophylline, corynoxeine and isocorynoxeine as reference substance, every milli is configured to methanol
Rise containing rhynchophyllin, isorhynchophylline, corynoxeine and isocorynoxeine be 0.008-0.012mg, 0.01-0.02mg,
0.001-0.002mg and 0.004-0.006mg solution, both；
Wherein, the assay, comprises the following steps：Reference substance solution and each 1-3ul of need testing solution are taken respectively, are noted
Enter liquid chromatograph, record peak area, external standard method calculates rhynchophyllin in need testing solution, isorhynchophylline, corynoxeine and different
The content of corynoxeine；
Wherein, the liquid chromatograph chromatographic condition in the content detection is as follows：Chromatographic column ACQUITY UPLC CSH C18
(2.1 × 100mm), wherein gradient elution, mobile phase are：Mobile phase A is 0.02%-0.2% ammonium acetate solutions, Mobile phase B
For acetonitrile；Flow velocity：0.35-0.45ml/min；Detection wavelength：240-250nm.
The ammonium acetate of the mobile phase is preferably 0.02-01%.
Preferably, the method for the invention comprises the following steps：
Step 1：The preparation of reference substance solution
It is appropriate that precision weighs rhynchophyllin, isorhynchophylline, corynoxeine and isocorynoxeine reference substance, is respectively placed in appearance
In measuring bottle, methanol is added to dissolve, be made every milliliter respectively is containing rhynchophyllin, isorhynchophylline, corynoxeine and isocorynoxeine
0.01mg, 0.015mg, 0.0016mg and 0.005mg solution, both；
Step 2：The preparation of need testing solution
Blood-nourishing and brain-refreshing the water extracted immersing paste about 0.2g is taken, it is accurately weighed, 35%-45% methanol 10mL ultrasonic dissolutions, up to locate
The ProElut C managed18- U (500mg/6mL) post, is washed with 35%-45% methanol 5mL, and cleaning solution discards, with methanol 4.5mL
Elution, eluent is collected, with methanol constant volume to 5mL, shakes up, produces；
Step 3：Content determination
Reference substance solution and each 2ul of need testing solution are taken respectively, injects liquid chromatograph, record peak area, external standard method meter
Calculate content；
Described liquid chromatograph chromatographic condition is as follows：
Chromatographic column ACQUITY UPLC CSH C18(2.1 × 100mm), mobile phase：Mobile phase A is 0.02%-0.2% second
Sour aqueous ammonium, Mobile phase B are acetonitrile, and gradient elution program is shown in Table 1；Flow velocity：0.35-0.45ml/min, Detection wavelength：240-
250nm；
The eluent gradient program of table 1
Most preferably, the method for the invention comprises the following steps：
Step 1：The preparation of reference substance solution
It is appropriate that precision weighs rhynchophyllin, isorhynchophylline, corynoxeine and isocorynoxeine reference substance, is respectively placed in appearance
In measuring bottle, methanol is added to dissolve, be made every milliliter respectively is containing rhynchophyllin, isorhynchophylline, corynoxeine and isocorynoxeine
0.01mg, 0.015mg, 0.0016mg and 0.005mg solution, both；
Step 2：The preparation of need testing solution
Take blood-nourishing and brain-refreshing the water extracted immersing paste about 0.2g, accurately weighed, 40% methanol 10mL ultrasonic dissolutions are up to processed good
ProElut C18- U (500mg/6mL) post, is washed with 40% methanol 5mL, and cleaning solution discards, and is eluted with methanol 4.5mL, and collection is washed
De- liquid, with methanol constant volume to 5mL, shakes up, produces；
Step 3：Content determination
Reference substance solution and each 2ul of need testing solution are taken respectively, injects liquid chromatograph, record peak area, external standard method meter
Calculate content；
Described liquid chromatograph chromatographic condition is as follows：
Chromatographic column ACQUITY UPLC CSH C18(2.1 × 100mm), mobile phase：Mobile phase A is 0.05% ammonium acetate water
Solution, Mobile phase B are acetonitrile, and gradient elution program is shown in Table 1；Flow velocity：0.35-0.45ml/min, Detection wavelength：246nm.
In method provided by the invention：
Methanol concentration is not defined, should be concentration specified in pharmacopeia as 100% methanol, 40% methanol,
Percentage implication therein is percent by volume, as contained 40ml methanol in 100ml solution.
Currently preferred method, chromatographic condition, are obtained by screening, and screening process is as follows：
1st, the foundation of pre-treating method
The selection of 1.1 test sample solvents
Blood-nourishing and brain-refreshing the water extracted immersing paste 0.2g is taken, it is ultrasonic with 20% methanol, 30% methanol, 40% methanol, 50% methanol respectively,
Sample introduction after progress SPE, chromatogram are shown in Fig. 3 (UPLC collection of illustrative plates of the Fig. 3 obtained by different solvents sample dissolution, wherein A：20%
The UPLC collection of illustrative plates that methanol dissolving test sample obtains；B：The UPLC collection of illustrative plates that 30% methanol dissolving test sample obtains；C：40% methanol is molten
The UPLC collection of illustrative plates that solution test sample obtains；D：The UPLC collection of illustrative plates that 50% methanol dissolving test sample obtains).
It was found from chromatogram, there is leakage in the sample of 50% methanol dissolving, peak area is less than 40% methanol.20% He
The sample of 30% methanol dissolving can not dissolve alkaloids well, and in addition to sample liquid is muddy, the rate of recovery is also than relatively low.Synthesis is examined
Consider, it is more suitable with 40% methanol sample dissolution to select.
The selection of 1.2 SPE posts
Selection ProElut C are respectively adopted18-U、ProElut C18, ProElut PXW solid-phase extraction columns are to sample solution
Purified, the results showed that ProElut C18- U solid-phase extraction columns selectivity and the rate of recovery are optimal.ProElut C18- U posts are
Silica gel bonded C18The anti-phase C of termination process is not carried out afterwards18Extraction column.The silicone hydroxyl and polar compounds of silica matrix surface residual
There is polar interaction in thing, thus enhance the reservation to polar compound especially amine compound, suitable for polarity and
Non-polar compound extracts.So inventor has selected ProElut C18- U SPE posts.
1.2.1 the selection of SPE loadings speed
Weigh 0.2g blood-nourishing and brain-refreshing the water extracted immersing pastes, the methanol ultrasonic dissolutions of 10mL 40%, respectively with 0.5mL/min, 1.0mL/
The ProElut C that min, 1.5mL/min flow velocity activated in medical methanol, water18- U SPE posts loadings and methanol elution collection five
Milliliter.Its rate of recovery is determined respectively, and the rate of recovery of rhynchophyllin is 100.5%, 100.0%, 94.5%；The isorhynchophylline rate of recovery is
100.2%th, 100.8%, 96.7%.Select loading speed more excellent for 1.0mL/min.
1.2.2 the selection of SPE loadings wash volumes
0.2g blood-nourishing and brain-refreshing the water extracted immersing pastes are weighed, the methanol ultrasonic dissolutions of 10mL 40%, are being used with 1.0mL/min flow velocitys
The ProElut C that methanol, water activated18- U SPE post loadings, eluted with methanol collect 3mL, 5mL, 6mL, 10mL respectively.Respectively
Its rate of recovery is determined, the rate of recovery of rhynchophyllin is 96.5%, 99.3%, 99.5%, 99.4%；The isorhynchophylline rate of recovery is
95.2%th, 100.5%, 100.9%, 101.2%.Illustrate that 5mL is enough sample elution and can save elution time, therefore select
5mL elution volume.
2.2 chromatographic condition
2.2.1 chromatographic column selects
That be respectively adopted is ACQUITY UPLC CSH C18Sample survey is carried out with ACQUITY UPLC HSS T3 chromatographic columns
It is fixed.
Fig. 4 (Fig. 4 is to use the UPLC collection of illustrative plates of ACQUITY UPLC HSS T3 chromatographic columns), 5 (Fig. 5 is using ACQUITY
UPLC CSH C18The UPLC collection of illustrative plates of chromatographic column) display, ACQUITY UPLC CSH C18Post has a preferable separating degree, and peak type is good
It is good, so selection ACQUITY UPLC CSH C18Chromatographic column.
2.2.2 the selection of wavelength
Hybrid standard product are prepared according to the method in " 2.1.1 ", carry out UV scanning, collection of illustrative plates is obtained, as a result sees Fig. 5.
From Fig. 6 (Fig. 6 is rhynchophyllin, isorhynchophylline mixing reference substance UV scanning figure) result, have most at 246nm
Big UV absorption, so selecting 246nm in an experiment as Detection wavelength.
2.2.3 the selection of mobile phase ratio
Take blood-nourishing and brain-refreshing the water extracted immersing paste 0.2g, prepared by method below " 2.1.6 " item, respectively with mobile phase acetonitrile solution (A),
0.05% ammonium acetate solution (B) is with mobile phase initial proportion 25：75、35：65、35：The 55 μ L of chromatographic process sample introduction 2, chromatogram
Figure is shown in that (Fig. 7 is the UPLC collection of illustrative plates of blood-nourishing and brain-refreshing the water extracted immersing paste under three kinds of mobile phase initial proportions, wherein A to Fig. 7：Mobile phase is initial
Ratio is 45：The UPLC collection of illustrative plates of 55 times blood-nourishing and brain-refreshing the water extracted immersing pastes；B：Mobile phase initial proportion is 35：65 times blood-nourishing and brain-refreshing water extractions
The UPLC collection of illustrative plates of medicinal extract；C：Mobile phase initial proportion is 25：The UPLC collection of illustrative plates of 75 times blood-nourishing and brain-refreshing the water extracted immersing pastes).
Fig. 7 results are shown, when mobile phase ratio is 35：When 65, reach optimal separation degree and there is steady baseline.
The structural confirmation of 2.3 detection compositions
UPLC-Q-TOF/MS combined instrument measure is carried out to sample, Mass Spectrometry Conditions are with tof tube detection pattern V-type, using just
Ion (ESI+) scan pattern, capillary voltage 3.5/kV (+), taper hole voltage 4/30V (+), extract taper hole voltage 5.0V, ion
100 DEG C of source temperature, 500 DEG C of desolvation temperature, taper hole air-flow 1L/h, desolventizing gas 594L/h, resolution ratio use
Resolution patterns, collision cell entrance potential 25V, collision cell exit potential 60V, sweep time 11min, trace interval
0.3s, mass charge ratio range：M/z 50~1200；Data collection form Centroid；Sensitivity Normal；Lock mass number POS
556.2771. press and sample is prepared under " 2.1.6 " item, liquid-phase condition is the same as " 2.2 " item.Obtaining Information in Mass Spectra, (Fig. 8 is blood-nourishing and brain-refreshing water
Extracted extract total ion current figure).
Analyzed by the comparison with reference substance mass spectrometric data, determine to mainly contain four kinds of biologies in blood-nourishing and brain-refreshing the water extracted immersing paste
Alkali composition, respectively rhynchophyllin, isorhynchophylline, corynoxeine and isocorynoxeine.
2.4 Method validation
2.4.1 linear and scope
Precision weighs appropriate through rhynchophyllin, isorhynchophylline, corynoxeine and isocorynoxeine reference substance, determines its peak
Area, reference substance collection of illustrative plates are shown in that Fig. 2 (because there is interference between reference substance, there is two reference substance collection of illustrative plates, i.e. Fig. 2-1,2-2, figure
2-1 is rhynchophyllin, isorhynchophylline；Fig. 2-2 is corynoxeine and isocorynoxeine).Determining the method for peak area is：Add first
Alcohol is configured to every milliliter
0.1mg, 0.04g, 0.08g and 0.1mg mixing contrast solution, then respectively taken amount take 0.5,1,2,5,10ml mixing contrast solutions
It is placed in 50ml measuring bottles, adds methanol to mix to scale, serial solution is made, inject liquid chromatograph, measure, with concentration to surveying
The peak area obtained carries out linear regression, tries to achieve regression equation.It the results are shown in Table 2, Fig. 9 (Fig. 8 is rhynchophyllin linear relationship), table 3, figure
(Figure 12 is by 10 (Fig. 9 is isorhynchophylline linear relationship), table 4, Figure 11 (Figure 11 is corynoxeine linear relationship), table 5, Figure 12
Isocorynoxeine linear relationship).
The rhynchophyllin linear relationship of table 2
| Concentration (mg/ml) | 0.001028 | 0.002056 | 0.004112 | 0.008224 | 0.02056 |
| Peak area | 9185 | 21377 | 43923 | 50960 | 100892 |
The isorhynchophylline linear relationship of table 3
| Concentration (mg/ml) | 0.00405 | 0.0081 | 0.01619 | 0.04048 | 0.08096 |
| Peak area | 4643 | 10937 | 21940 | 26222 | 51814 |
The corynoxeine linear relationship of table 4
| Concentration (mg/ml) | 0.04106 | 0.08212 | 0.16424 | 0.32848 | 0.8212 |
| Peak area | 10040 | 23143 | 46188 | 54043 | 107382 |
The isocorynoxeine linear relationship of table 5
| Concentration (mg/ml) | 0.00716 | 0.01431 | 0.03578 | 0.07155 | 0.1431 |
| Peak area | 13614 | 31705 | 62675 | 73378 | 145242 |
2.4.2 sample introduction precision test：
Take D20140204 batch samples, prepare need testing solution in accordance with the law, repeat sample introduction 6 times, record rhynchophyllin, isorhynchophylline,
The peak area of corynoxeine, isocorynoxeine, calculate relative standard deviation.It the results are shown in Table 6.
The rhynchophyllin of table 6, isorhynchophylline, corynoxeine and isocorynoxeine sample introduction precision
2.4.3 reappearance test：
D20140204 batches of samples are taken, prepare 6 parts of need testing solution in accordance with the law, determine rhynchophyllin, isorhynchophylline, dehydrogenation respectively
The content of rhynchophyllin, isocorynoxeine, calculate relative standard deviation.It the results are shown in Table 7.
The rhynchophyllin of table 7, isorhynchophylline, corynoxeine and isocorynoxeine reappearance
2.4.4 recovery test：
Precision weighs D20140204 batches of 6 parts of medicinal extract, every part of about 0.1g, puts in 50ml measuring bottles, each addition mixing accurate respectively
(every milliliter is respectively reference substance solution 2ml containing rhynchophyllin, isorhynchophylline, corynoxeine and isocorynoxeine
0.01384mg, 0.0936mg, 0.0631mg and 0.1618mg), rise, shine from " adding 70% methanol solution appropriate, ultrasonic dissolution "
Handled in accordance with the law under assay item, as need testing solution, take 2ul injecting chromatographs, respectively calculate rhynchophyllin, isorhynchophylline,
The rate of recovery of corynoxeine and isocorynoxeine.It the results are shown in Table 8, table 9, table 10 and table 11.
The yncaria stem with hooks alkali recovery (mg, n=6) of table 8
The isorhynchophylline rate of recovery (mg, n=6) of table 9
The corynoxeine rate of recovery (mg, n=6) of table 10
The isocorynoxeine rate of recovery (mg, n=6) of table 11
2.4.5 stability
Take D20140204 batch to prepare need testing solution, respectively at 0,2,4,8,12,24h sample introductions, record rhynchophyllin, different hook
The peak area of rattan alkali, corynoxeine and isocorynoxeine, calculate relative standard deviation.It the results are shown in Table 12.
The rhynchophyllin of table 12, isorhynchophylline, corynoxeine and isocorynoxeine reappearance
2.4.6 sample determines
Take sample, test sample collection of illustrative plates is measured according to method provided by the invention (see Fig. 1).
Verify conclusion：
For determining rhynchophyllin, isorhynchophylline, corynoxeine, isocorynoxeine simultaneously in blood-nourishing and brain-refreshing the water extracted immersing paste
Deng the method for four kinds of compositions, Method validation has been carried out, the results showed that, this assay method linear relationship and the rate of recovery are good, essence
Density, reappearance and sample stability meet checking and required, can be used for the survey of alkaloid component in blood-nourishing and brain-refreshing the water extracted immersing paste
It is fixed.
2.5 testing conditions scopes
Test sample solvent：35-45% methanol solutions；Mobile phase solvent A：0.02-0.2% ammonium acetate solutions；Solid phase extracts
Take post：Waters Oasis MAX, Cleanert S C can be used18Substituted etc. model.
Beneficial effects of the present invention
1st, the present invention is measured using ultra-performance liquid chromatography, and efficiently quick, stability is good, high sensitivity, can
Efficiently separating for four kinds of alkaloid components was realized in 5 minutes.
2nd, the present invention uses solid phase extraction techniques, can effectively remove interference component, while alkaloid component can be entered
Row effectively enrichment.
3rd, prior art：
It is clear that CN103630614A (Application No. 201210301559.9, lower abbreviation patent in 2012) discloses a kind of blood-nourishing
The high-efficiency liquid chromatography method for detecting of brain particle, in this method：The preparation of test sample is carried by the use of 25% methanol as solvent, ultrasound
Take；Mobile phase A is 0.01-0.05% phosphoric acid solutions in chromatographic condition, and B is acetonitrile.Compared with patent in 2012：The master of the document
It is different to distinguish the composition for being to determine, the composition that the document determines is：Gallic acid, protocatechuic acid, chlorogenic acid, caffeic acid, Ah
Wei's acid, Paeoniflorin, albiflorin, the present invention is directed rhynchophyllin, isorhynchophylline, corynoxeine and different dehydrogenation yncaria stem with hooks
Alkali.
In " ultra-performance liquid chromatography determines yncaria stem with hooks alkali content in different extraction process samples " text, (author Liu Ping,
Wang Yingfeng, Capital Normal University's journal, the phase of volume 32 the 2nd) in the method that uses, using ACQUITY UPLC BEH C18Chromatographic column,
Adjust pH=9.8 (60: 40) to carry out isocratic elution for mobile phase with methanol -5mmol/L ammonium acetate solutions ammoniacal liquor, detect ripple
A length of 240nm., measure composition are rhynchophyllin, and appearance time is 4.87 minutes.Compared with this method, this patent analyze speed is more
It hurry up, more preferably, analysis ingredient is more for separating degree, and flowing phase composition is simpler.
It is an advantage of the invention that：
1) blood-nourishing and brain-refreshing compound is directed to, compound preparation, complicated component, interfering material is more, adds measure difficulty.
2) to the detection of rhynchophyllin, isorhynchophylline, four kinds of corynoxeine, isocorynoxeine compositions, while four are determined
Kind alkaloid component, alkaloid component composition and content ratio has been grasped comprehensively, quick detection, separating difficulty is big, efficiency high.Carry
High control of product quality water product, data support is provided for the research of product effective component.
3) during prepared by test sample：Using methanol ultrasonic dissolution, using ProElut C18- U solid-phase extraction columns are purified, and are had
Effect eliminates interference impurity, and realizes the enrichment to determining object, improves chromatographic peak separating degree, helps to extend chromatogram
Column life.
4) in chromatographic condition mobile phase selection, screened by chromatographic column and mobile phase, by simply flowing phase composition,
Reach perfect peak shape and high separation, overcome the drawbacks of alkaloid easily trails in reversed phase chromatography separation.
5th, empirical tests method of quality control precision provided by the invention, reappearance, have good stability, can effectively determine
Alkaloid component in blood-nourishing and brain-refreshing the water extracted immersing paste.
Brief description of the drawings
Fig. 1 is test sample collection of illustrative plates；
Fig. 2-1,2-2 are reference substance collection of illustrative plates；
UPLC collection of illustrative plates of the Fig. 3 obtained by different solvents sample dissolution, wherein A：20% methanol dissolving test sample obtains
UPLC collection of illustrative plates；B：The UPLC collection of illustrative plates that 30% methanol dissolving test sample obtains；C：The UPLC figures that 40% methanol dissolving test sample obtains
Spectrum；D：The UPLC collection of illustrative plates that 50% methanol dissolving test sample obtains；
Fig. 4 is the UPLC collection of illustrative plates using ACQUITY UPLC HSS T3 chromatographic columns；
Fig. 5 is using ACQUITY UPLC CSH C18The UPLC collection of illustrative plates of chromatographic column；
Fig. 6 is rhynchophyllin, isorhynchophylline mixing reference substance UV scanning figure；
Fig. 7 is the UPLC collection of illustrative plates of blood-nourishing and brain-refreshing the water extracted immersing paste under three kinds of mobile phase initial proportions, wherein A：Mobile phase is initial
Ratio is 45：The UPLC collection of illustrative plates of 55 times blood-nourishing and brain-refreshing the water extracted immersing pastes；B：Mobile phase initial proportion is 35：65 times blood-nourishing and brain-refreshing water extractions
The UPLC collection of illustrative plates of medicinal extract；C：Mobile phase initial proportion is 25：The UPLC collection of illustrative plates of 75 times blood-nourishing and brain-refreshing the water extracted immersing pastes；
Fig. 8 is blood-nourishing and brain-refreshing the water extracted immersing paste total ion current figure；
Fig. 9 is rhynchophyllin linear relationship；
Figure 10 is isorhynchophylline linear relationship；
Figure 11 is corynoxeine linear relationship；
Figure 12 is isocorynoxeine linear relationship.
Embodiment
The present invention is further illustrated by the following examples.
Embodiment 1：A kind of method for determining alkaloid component content in blood-nourishing and brain-refreshing the water extracted immersing paste
1st, chromatographic condition and system suitability：Instrument is Waters ACQUITY UPLC, using ACQUITY UPLC
CSH C18(2.1 × 100mm) chromatographic column, with 0.05% ammonium acetate solution with mobile phase A, acetonitrile is Mobile phase B, and gradient is set
It is shown in Table 1；Detection wavelength is 246nm.
2nd, the preparation of reference substance solution：Precision weighs rhynchophyllin, isorhynchophylline, corynoxeine and isocorynoxeine pair
It is appropriate according to product, it is respectively placed in volumetric flask, adds methanol to dissolve, every milliliter is made respectively containing rhynchophyllin, isorhynchophylline, dehydrogenation yncaria stem with hooks
Alkali and the solution that isocorynoxeine is 0.01mg, 0.015mg, 0.0016mg and 0.005mg, both.
3rd, the preparation of need testing solution：Take blood-nourishing and brain-refreshing the water extracted immersing paste about 0.2g, accurately weighed, 40% methanol 10mL ultrasounds
Dissolving, up to processed good ProElut C18- U (500mg/6mL) post, is washed with 40% methanol 5mL, and cleaning solution discards, and is used
Methanol 4.5mL is eluted, and is collected eluent, with methanol constant volume to 5mL, is shaken up, produce.
4th, determination method：Reference substance solution and each 2ul of need testing solution are taken respectively, injects liquid chromatograph, record peak area,
External standard method calculates content.
5th, testing result：Take three batches of online blood-nourishing and brain-refreshing the water extracted immersing pastes to be measured, the results are shown in Table 13.
Table 13 uses the content of alkaloid component in the blood-nourishing and brain-refreshing the water extracted immersing paste that the method for embodiment 1 determines
| Number | Rhynchophyllin | Isorhynchophylline | Corynoxeine | Isocorynoxeine |
| 1 | 0.264 | 0.095 | 0.040 | 0.152 |
| 2 | 0.246 | 0.083 | 0.032 | 0.130 |
| 3 | 0.239 | 0.086 | 0.041 | 0.149 |
Embodiment 2：A kind of method for determining alkaloid component content in blood-nourishing and brain-refreshing the water extracted immersing paste
1st, chromatographic condition and system suitability：Instrument is Waters ACQUITY UPLC, using ACQUITY UPLC
CSH C18(2.1 × 100mm) chromatographic column, with 0.1% ammonium acetate solution with mobile phase A, acetonitrile is Mobile phase B, and gradient is set
It is shown in Table 1；Detection wavelength is 240nm.
2nd, the preparation of reference substance solution：Precision weighs rhynchophyllin, isorhynchophylline, corynoxeine and isocorynoxeine pair
It is appropriate according to product, it is respectively placed in volumetric flask, adds methanol to dissolve, every milliliter is made respectively containing rhynchophyllin, isorhynchophylline, dehydrogenation yncaria stem with hooks
Alkali and the solution that isocorynoxeine is 0.01mg, 0.015mg, 0.0016mg and 0.005mg, both.
3rd, the preparation of need testing solution：Take blood-nourishing and brain-refreshing the water extracted immersing paste about 0.2g, accurately weighed, 40% methanol 10mL ultrasounds
Dissolving, up to processed good ProElut C18- U (500mg/6mL) post.Washed with 40% methanol 5mL, cleaning solution discards, and uses
Methanol 4.5mL is eluted, and is collected eluent, with methanol constant volume to 5mL, is shaken up, produce.
4th, determination method：Reference substance solution and each 2ul of need testing solution are taken respectively, injects liquid chromatograph, record peak area.
External standard method calculates content.
5th, testing result：Take three batches of online blood-nourishing and brain-refreshing the water extracted immersing pastes to be measured, the results are shown in Table 14：
The content of alkaloid component in the blood-nourishing and brain-refreshing the water extracted immersing paste of the use embodiment 2 of table 14
| Sequence number | Rhynchophyllin | Isorhynchophylline | Corynoxeine | Isocorynoxeine |
| 1 | 0.258 | 0.097 | 0.041 | 0.155 |
| 2 | 0.241 | 0.085 | 0.033 | 0.125 |
| 3 | 0.245 | 0.084 | 0.041 | 0.146 |
Embodiment 3：A kind of method for determining alkaloid component content in blood-nourishing and brain-refreshing the water extracted immersing paste
1st, chromatographic condition and system suitability：
Instrument is Waters ACQUITY UPLC, using ACQUITY UPLC CSH C18(2.1 × 100mm) chromatographic column,
With 0.02% ammonium acetate solution with mobile phase A, acetonitrile is Mobile phase B, and gradient sets and is shown in Table 1；Detection wavelength is 250nm.
2nd, the preparation of reference substance solution：Precision weighs rhynchophyllin, isorhynchophylline, corynoxeine and isocorynoxeine pair
It is appropriate according to product, it is respectively placed in volumetric flask, adds methanol to dissolve, every milliliter is made respectively containing rhynchophyllin, isorhynchophylline, dehydrogenation yncaria stem with hooks
Alkali and the solution that isocorynoxeine is 0.01mg, 0.015mg, 0.0016mg and 0.005mg, both.
3rd, the preparation of need testing solution：Take blood-nourishing and brain-refreshing the water extracted immersing paste about 0.2g, accurately weighed, 40% methanol 10mL ultrasounds
Dissolving, up to processed good ProElut C18- U (500mg/6mL) post.Washed with 40% methanol 5mL, cleaning solution discards, and uses
Methanol 4.5mL is eluted, and is collected eluent, with methanol constant volume to 5mL, is shaken up, produce.
4th, determination method：Reference substance solution and each 2ul of need testing solution are taken respectively, injects liquid chromatograph, record peak area.
External standard method calculates content.
5th, testing result：Take three batches of online blood-nourishing and brain-refreshing the water extracted immersing pastes to be measured, the results are shown in Table 15：
The content of alkaloid component in the blood-nourishing and brain-refreshing the water extracted immersing paste of the use embodiment 3 of table 15
| Sequence number | Rhynchophyllin | Isorhynchophylline | Corynoxeine | Isocorynoxeine |
| 1 | 0.260 | 0.096 | 0.040 | 0.156 |
| 2 | 0.247 | 0.084 | 0.033 | 0.127 |
| 3 | 0.240 | 0.081 | 0.040 | 0.143 |
Embodiment 4：A kind of method for determining alkaloid component content in blood-nourishing and brain-refreshing the water extracted immersing paste
1st, chromatographic condition and system suitability：
Instrument is Waters ACQUITY UPLC, using ACQUITY UPLC CSH C18(2.1 × 100mm) chromatographic column,
With 0.05% ammonium acetate solution with mobile phase A, acetonitrile is Mobile phase B, and gradient sets and is shown in Table 1；Detection wavelength is 246nm.
2nd, the preparation of reference substance solution：Precision weighs rhynchophyllin, isorhynchophylline, corynoxeine and isocorynoxeine pair
It is appropriate according to product, it is respectively placed in volumetric flask, adds methanol to dissolve, every milliliter is made respectively containing rhynchophyllin, isorhynchophylline, dehydrogenation yncaria stem with hooks
Alkali and the solution that isocorynoxeine is 0.01mg, 0.015mg, 0.0016mg and 0.005mg, both.
3rd, the preparation of need testing solution：Take blood-nourishing and brain-refreshing the water extracted immersing paste about 0.2g, accurately weighed, 45% methanol 10mL ultrasounds
Dissolving, up to processed good Cleanert S C18(500mg/6mL) post.Washed with 45% methanol 5mL, cleaning solution discards, and uses
Methanol 4.5mL is eluted, and is collected eluent, with methanol constant volume to 5mL, is shaken up, produce.
4th, determination method：Reference substance solution and each 2ul of need testing solution are taken respectively, injects liquid chromatograph, record peak area,
External standard method calculates content.
5th, testing result：Take three batches of online blood-nourishing and brain-refreshing the water extracted immersing pastes to be measured, the results are shown in Table 16：
The content of alkaloid component in the blood-nourishing and brain-refreshing the water extracted immersing paste of the use embodiment 4 of table 16
| Sequence number | Rhynchophyllin | Isorhynchophylline | Corynoxeine | Isocorynoxeine |
| 1 | 0.261 | 0.093 | 0.041 | 0.153 |
| 2 | 0.244 | 0.084 | 0.033 | 0.129 |
| 3 | 0.237 | 0.088 | 0.042 | 0.147 |
Embodiment 5：A kind of method for determining alkaloid component content in blood-nourishing and brain-refreshing the water extracted immersing paste
1st, chromatographic condition and system suitability：
Instrument is Waters ACQUITY UPLC, using ACQUITY UPLC CSH C18(2.1 × 100mm) chromatographic column,
With 0.05% ammonium acetate solution with mobile phase A, acetonitrile is Mobile phase B, and gradient sets and is shown in Table 1；Detection wavelength is 246nm.
2nd, the preparation of reference substance solution：Precision weighs rhynchophyllin, isorhynchophylline, corynoxeine and isocorynoxeine pair
It is appropriate according to product, it is respectively placed in volumetric flask, adds methanol to dissolve, every milliliter is made respectively containing rhynchophyllin, isorhynchophylline, dehydrogenation yncaria stem with hooks
Alkali and the solution that isocorynoxeine is 0.01mg, 0.015mg, 0.0016mg and 0.005mg, both.
3rd, the preparation of need testing solution：Blood-nourishing and brain-refreshing the water extracted immersing paste about 0.25g is taken, accurately weighed, 35% methanol 10mL surpasses
Sound dissolves, up to processed good Waters Sep-pak C18(500mg/6mL) post.Washed with 35% methanol 5mL, cleaning solution
Discard, eluted with methanol 4.5mL, collect eluent, with methanol constant volume to 5mL, shake up, produce.
4th, determination method：Reference substance solution and each 2ul of need testing solution are taken respectively, injects liquid chromatograph, record peak area,
External standard method calculates content.
5th, testing result：Take three batches of online blood-nourishing and brain-refreshing the water extracted immersing pastes to be measured, the results are shown in Table 17：
The content of alkaloid component in the blood-nourishing and brain-refreshing the water extracted immersing paste of the use embodiment 5 of table 17
| Sequence number | Rhynchophyllin | Isorhynchophylline | Corynoxeine | Isocorynoxeine |
| 1 | 0.265 | 0.093 | 0.041 | 0.151 |
| 2 | 0.251 | 0.082 | 0.032 | 0.132 |
| 3 | 0.237 | 0.081 | 0.041 | 0.146 |
Embodiment 6
1st, chromatographic condition and system suitability：
Instrument is Waters ACQUITY UPLC, using ACQUITY UPLC CSH C18(2.1 × 100mm) chromatographic column,
With 0.05% ammonium acetate solution with mobile phase A, acetonitrile is Mobile phase B, and gradient sets and is shown in Table 1；Detection wavelength is 246nm.
2nd, the preparation of reference substance solution：Precision weighs rhynchophyllin, isorhynchophylline, corynoxeine and isocorynoxeine pair
It is appropriate according to product, it is respectively placed in volumetric flask, adds methanol to dissolve, every milliliter is made respectively containing rhynchophyllin, isorhynchophylline, dehydrogenation yncaria stem with hooks
Alkali and the solution that isocorynoxeine is 0.01mg, 0.015mg, 0.0016mg and 0.005mg, both.
3rd, the preparation of need testing solution：Blood-nourishing and brain-refreshing the water extracted immersing paste about 0.15g is taken, accurately weighed, 40% methanol 10mL surpasses
Sound dissolves, up to processed good Waters Sep-pak C18(500mg/6mL) post.Washed with 40% methanol 5mL, cleaning solution
Discard, eluted with methanol 4.5mL, collect eluent, with methanol constant volume to 5mL, shake up, produce.
4th, determination method：Reference substance solution and each 2ul of need testing solution are taken respectively, injects liquid chromatograph, record peak area.
External standard method calculates content.
5th, testing result：Take three batches of online blood-nourishing and brain-refreshing the water extracted immersing pastes to be measured, the results are shown in Table 18：
The content of alkaloid component in the blood-nourishing and brain-refreshing the water extracted immersing paste of the use embodiment 6 of table 18
| Sequence number | Rhynchophyllin | Isorhynchophylline | Corynoxeine | Isocorynoxeine |
| 1 | 0.255 | 0.094 | 0.038 | 0.154 |
| 2 | 0.242 | 0.085 | 0.032 | 0.131 |
| 3 | 0.232 | 0.088 | 0.040 | 0.146 |
Embodiment data all of the above are collected, obtain table 19-1,19-2,19-3：
Table 19-1：01 batch of detection data
| Sequence number | Rhynchophyllin | Isorhynchophylline | Corynoxeine | Isocorynoxeine |
| Embodiment 1 | 0.264 | 0.095 | 0.040 | 0.152 |
| Embodiment 2 | 0.258 | 0.097 | 0.041 | 0.155 |
| Embodiment 3 | 0.260 | 0.096 | 0.040 | 0.156 |
| Embodiment 4 | 0.261 | 0.093 | 0.041 | 0.153 |
| Embodiment 5 | 0.265 | 0.093 | 0.041 | 0.151 |
| Embodiment 6 | 0.255 | 0.094 | 0.038 | 0.154 |
| Average value | 0.261 | 0.095 | 0.040 | 0.154 |
| RSD% | 1.4 | 1.7 | 1.9 | 1.2 |
Table 19-2：02 batch of detection data
| Sequence number | Rhynchophyllin | Isorhynchophylline | Corynoxeine | Isocorynoxeine |
| Embodiment 1 | 0.246 | 0.083 | 0.032 | 0.130 |
| Embodiment 2 | 0.241 | 0.085 | 0.033 | 0.125 |
| Embodiment 3 | 0.247 | 0.084 | 0.033 | 0.127 |
| Embodiment 4 | 0.244 | 0.084 | 0.033 | 0.129 |
| Embodiment 5 | 0.251 | 0.082 | 0.032 | 0.132 |
| Embodiment 6 | 0.242 | 0.085 | 0.032 | 0.131 |
| Average value | 0.245 | 0.084 | 0.033 | 0.129 |
| RSD% | 1.5 | 1.4 | 1.7 | 2.0 |
Table 19-3：03 batch of detection data
| Sequence number | Rhynchophyllin | Isorhynchophylline | Corynoxeine | Isocorynoxeine |
| Embodiment 1 | 0.239 | 0.086 | 0.041 | 0.149 |
| Embodiment 2 | 0.245 | 0.084 | 0.041 | 0.146 |
| Embodiment 3 | 0.240 | 0.086 | 0.040 | 0.143 |
| Embodiment 4 | 0.237 | 0.088 | 0.042 | 0.147 |
| Embodiment 5 | 0.237 | 0.085 | 0.041 | 0.146 |
| Embodiment 6 | 0.232 | 0.088 | 0.040 | 0.146 |
| Average value | 0.238 | 0.086 | 0.041 | 0.146 |
| RSD% | 1.8 | 1.9 | 1.8 | 1.3 |
Above-mentioned table 19-1,19-2,19-3 content results show, are determined according to the embodiment in embodiment, three above
The testing result no significant difference of batch, can obtain correct testing result.
Claims (8)
1. a kind of method for determining alkaloid component content in blood-nourishing and brain-refreshing the water extracted immersing paste, methods described, comprises the following steps：For
The preparation of test sample solution, the preparation of reference substance solution and assay.
2. according to the method for claim 1, it is characterised in that wherein, the preparation of the need testing solution includes following step
Suddenly：Take blood-nourishing and brain-refreshing the water extracted immersing paste about 0.2g, accurately weighed, 35%-45% methanol 10mL ultrasonic dissolutions are up to processed good
ProElut C18- U (500mg/6mL) post, is washed with 35%-45% methanol 5mL, and cleaning solution discards, and is eluted with methanol 4.5mL,
Eluent is collected, with methanol constant volume to 5mL, shakes up, produces.
3. according to the method for claim 1, it is characterised in that wherein, the preparation of the reference substance solution includes following step
Suddenly：
Using rhynchophyllin, isorhynchophylline, corynoxeine and isocorynoxeine as reference substance, it is configured to every milliliter with methanol and contains
Rhynchophyllin, isorhynchophylline, corynoxeine and isocorynoxeine are 0.008-0.012mg, 0.01-0.02mg, 0.001-
0.002mg and 0.004-0.006mg solution, both.
4. according to the method for claim 1, it is characterised in that wherein, the assay, comprise the following steps：Respectively
Reference substance solution and each 1-3ul of need testing solution are taken, injects liquid chromatograph, records peak area, it is molten that external standard method calculates test sample
Rhynchophyllin in liquid, isorhynchophylline, the content of corynoxeine and isocorynoxeine.
5. the method according to claim 1 or 4, it is characterised in that wherein, liquid chromatograph chromatogram during the content detection
Condition is as follows：Chromatographic column ACQUITY UPLC CSH C18(2.1 × 100mm), wherein gradient elution, mobile phase are：Mobile phase A
For 0.02%-0.2% ammonium acetate solutions, Mobile phase B is acetonitrile；Flow velocity：0.35-0.45ml/min；Detection wavelength：240-
250nm。
6. according to the method for claim 5, it is characterised in that the ammonium acetate of the mobile phase is preferably 0.02-01%.
7. according to the method described in claim any one of 1-6, it is characterised in that the described method comprises the following steps：
Step 1：The preparation of reference substance solution
It is appropriate that precision weighs rhynchophyllin, isorhynchophylline, corynoxeine and isocorynoxeine reference substance, is respectively placed in volumetric flask
In, add methanol to dissolve, be made every milliliter respectively is containing rhynchophyllin, isorhynchophylline, corynoxeine and isocorynoxeine
0.01mg, 0.015mg, 0.0016mg and 0.005mg solution, both；
Step 2：The preparation of need testing solution
Take blood-nourishing and brain-refreshing the water extracted immersing paste about 0.2g, accurately weighed, 35%-45% methanol 10mL ultrasonic dissolutions are up to processed good
ProElut C18- U (500mg/6mL) post, is washed with 35%-45% methanol 5mL, and cleaning solution discards, and is washed with methanol 4.5mL
It is de-, eluent is collected, with methanol constant volume to 5mL, shakes up, produces；
Step 3：Content determination
Reference substance solution and each 2ul of need testing solution are taken respectively, injects liquid chromatograph, records peak area, and external standard method, which calculates, to be contained
Amount；
Described liquid chromatograph chromatographic condition is as follows：
Chromatographic column ACQUITY UPLC CSH C18(2.1 × 100mm), mobile phase：Mobile phase A is 0.02%-0.2% ammonium acetates
The aqueous solution, Mobile phase B are acetonitrile, and gradient elution program see the table below；Flow velocity：0.35-0.45ml/min, Detection wavelength：240-
250nm；
8. according to the method for claim 7, it is characterised in that the described method comprises the following steps：
Step 1：The preparation of reference substance solution
It is appropriate that precision weighs rhynchophyllin, isorhynchophylline, corynoxeine and isocorynoxeine reference substance, is respectively placed in volumetric flask
In, add methanol to dissolve, be made every milliliter respectively is containing rhynchophyllin, isorhynchophylline, corynoxeine and isocorynoxeine
0.01mg, 0.015mg, 0.0016mg and 0.005mg solution, both；
Step 2：The preparation of need testing solution
Take blood-nourishing and brain-refreshing the water extracted immersing paste about 0.2g, accurately weighed, 40% methanol 10mL ultrasonic dissolutions are up to processed good
ProElut C18- U (500mg/6mL) post, is washed with 40% methanol 5mL, and cleaning solution discards, and is eluted with methanol 4.5mL, and collection is washed
De- liquid, with methanol constant volume to 5mL, shakes up, produces；
Step 3：Content determination
Reference substance solution and each 2ul of need testing solution are taken respectively, injects liquid chromatograph, records peak area, and external standard method, which calculates, to be contained
Amount；
Described liquid chromatograph chromatographic condition is as follows：
Chromatographic column ACQUITY UPLC CSH C18(2.1 × 100mm), mobile phase：Mobile phase A is 0.05% ammonium acetate solution,
Mobile phase B is acetonitrile, and gradient elution program see the table below；Flow velocity：0.35-0.45ml/min, Detection wavelength：246nm；
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