CN106526002A - Method for measuring content of Shenqi blood sugar reducing preparation and application thereof in overall quality control - Google Patents
Method for measuring content of Shenqi blood sugar reducing preparation and application thereof in overall quality control Download PDFInfo
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Abstract
The invention provides a method for measuring the content of a Shenqi blood sugar reducing preparation. The method is used for measuring the content of 10 active ingredients in the Shenqi blood sugar reducing preparation: ginsenoside Rb1, ginsenoside Rc, ginsenoside Rd, ginsenoside Rg1, ginsenoside Re, astragaloside, deoxyschizandrin, schisandrol A, schisandrol B. In the invention, with the guidance of blood sugar reducing activity of a compound, multiple active substances in the compound are detected, and the quality level of the whole compound preparation can be reflected better; meanwhile, a Q-MS ion monitoring mode is selected for content measurement, and the mass spectrum quantification has high accuracy, specificity and sensitivity and is superior to traditional HPLC analysis method; and moreover, fingerprint is established according to the liquid phase diagrams of different batches of Shenqi blood sugar reducing preparations obtained by the method, the similarity is evaluated, the inherent quality of the Shenqi blood sugar reducing preparation product is evaluated more scientifically and comprehensively, and a basis is provided for establishing a quality standard of higher level.
Description
(1) technical field
The invention belongs to Control of drug quality field, and in particular to high performance liquid chromatography-mono- level Four bar mass spectrometric hyphenated technique
(HPLC-Q-MS) method to ginseng-astragalus blood-sugar lowering preparation multicomponent assay, and the ginseng-astragalus blood-sugar lowering preparation obtained by thus method
Liquid phase figure builds finger-print, further relates to this method in the application in ginseng-astragalus blood-sugar lowering preparation global quality control.
(2) background technology
Diabetes (Diabetes Mellitus, DM) belong to the traditional Chinese medical science " diabete " category：" thirsty and drinking a lot of water, urine number,
Without fat like the sweet person of bran flakes, all it is diabete ".IDF (IDF) points out, the current whole world have 4.15 hundred million diabetes into
Year patient, 3.18 hundred million people exist and suffer from diabetes risk, and wherein, type ii diabetes account for the 90% of global all diabetes cases, because
The hyperglycaemia of diabetes accompanying, is easily caused various tissues, particularly eye, kidney, heart, blood vessel, the chronic lesion of nerve, function barrier
The complication such as hinder, greatly reduce the quality of life of diabetic.
At present, the clinically treatment of diabetes main oral sulfonylureas and biguanide antidiabetic medicament in addition to insulin injection,
But Western medicine there is also numerous safety issues and bad reaction while blood sugar is effectively reduced, such as serious hypoglycemia, breast
Acid acid poisoning, gastrointestinal reaction and hepatic injury, permanent neurologic function damage, headache, dizzy or even death etc..Grind in recent years
Study carefully discovery, gently persistently, curative effect is stable, Small side effects, compensate for just the deficiency of chemical drug, increasingly receives for the hypoglycemic activity of Chinese medicine
To the great attention of domestic and international endocrine circle.
SHENQI JIANGTANG KELI (Shenqi Jiangtang Granule, SJG), is made up of 11 taste traditional Chinese medicines, it is pure in
Medicine compound preparation, belongs to national four kind new medicine, and two grades of Chinese medicines of country protect kind, are clinically primarily adapted for use in II types sugar
The sick syndrome of deficiency of both qi and yin patient of urine, clinical data display effect are good.Non-defective unit of the ginseng for strengthening YANG and invigorating YIN in side, the Radix Astragali are tonifying Qi liter
The key medicine of sun, both are monarch drug in a prescription altogether；Minister with the dried rhizome of rehmannia, the tuber of dwarf lilyturf, root of Chinese trichosanthes fluid dryness and quench the thirst；Help with the fruit of Chinese wolfberry, the fruit of Chinese magnoliavine, cover
Basin sealing kidney is closed；Make with Chinese yam, Poria cocos, rhizoma alismatis three is burnt and manages.According to the literature：Ginsenoside Rb1, ginsenoside in ginseng
Rb2, ginsenoside Rb3, ginsenoside Rd, ginsenoside Re, ginsenoside Rg1 have hypoglycemic activity, 2015 editions Chinese Pharmacopoeias
The quality control index composition of regulation ginseng stem and leave general saponin is ginsenoside Rd, ginsenoside Re, ginsenoside Rg1；The Radix Astragali
Middle Astragaloside IV, formoononetin, calycosin have hypoglycemic activity, and 2015 editions Chinese Pharmacopoeias specify the quality of Milkvetch Root
Con trolling index composition is Astragaloside IV；Modern pharmacology research also proves that compound sugar in the dried rhizome of rehmannia, Catalpol, martynoside D have drop
Sugared effect；Schizandrin is the index composition for detecting schisandra chinensis medicinal material specified in Chinese Pharmacopoeia；The tuber of dwarf lilyturf, root of Chinese trichosanthes, Chinese holly
Matrimony vine, Chinese yam, Poria cocos play mainly polysaccharose substance or the Pleurotus Ostreatus compound of hypoglycemic effect.
ADA advocates, and the target for the treatment of diabetes is that blood sugar is down to normal level or normal water is close to
Flat, while reducing the generation of complication, SHENQI JIANGTANG KELI multicomponent, the action character of Mutiple Targets are more suitable for controlling for diabetes
Treat.Consult domestic and international pertinent literature and find that at present, the quality control to SHENQI JIANGTANG KELI reported is mainly：By thin
Layer authenticated ginsenoside and astragaloside, and determine general ginsenoside and astragalus root total saponin with 5% vanillic aldehyde-glacial acetic acid method
Content；The content of ginsenoside Re and Rg1 in SHENQI JIANGTANG KELI is determined using HPLC methods.Above method is mostly simply to ginseng
One or two compositions in stilbene antihypelipidemic preparation carry out qualitative and quantitative analysis, it is impossible to reflect the quality level of whole compound preparation,
And respectively have deficiency, such as：Poor selectivity, low, the reagent contamination environment of sensitivity etc..HPLC-Q-MS combinations have become complex system
Separate analysis and compound structure identification good platform, but have no so far using this technology to ginseng-astragalus blood-sugar lowering preparation in it is many
The document report that active component is analyzed simultaneously, it is often more important that, the high sensitivity of HPLC-Q-MS, high selectivity and height
Resolution ratio can make up the deficiency of traditional HPLC, it is possible to which many constituents of complicated compound Chinese medicinal preparation are carried out while surveying
It is fixed.
(3) content of the invention
The purpose of the present invention is ginseng-astragalus blood-sugar lowering preparation to be set up while the method for determining the content of 10 active components, described
10 active components be respectively：Ginsenoside Rb1, Ginsenoside Rc, ginsenoside Rd, ginsenoside Rg1, ginsenoside
Re, Astragaloside IV, schizandrin A, deoxyschizandrin, schizandrin, wuweizi alcohol B.Also, the present invention is utilized
The ginseng-astragalus blood-sugar lowering preparation liquid phase figure that Chempattern softwares are obtained to above-mentioned content assaying method sets up finger-print, carries out phase
Like degree analysis, to the more scientific comprehensive inherent quality for evaluating ginseng-astragalus blood-sugar lowering formulation products, it is to set up higher levels of quality
Standard provides foundation.The inventive method can be applicable to the global quality control of ginseng-astragalus blood-sugar lowering preparation.
For achieving the above object, the present invention is adopted the following technical scheme that：
A kind of ginseng-astragalus blood-sugar lowering preparation (usually tablet, capsule or granule) content assaying method, methods described are determined
Be 10 active components in ginseng-astragalus blood-sugar lowering preparation content, 10 described active components are respectively：Ginsenoside Rb1, people
Ginseng saponin(e Rc, ginsenoside Rd, ginsenoside Rg1, ginsenoside Re, Astragaloside IV, schizandrin A, deoxyschizandrin, five
Taste alcohol first, wuweizi alcohol B, methods described comprise the steps：
(1) prepare mixed reference substance solution
Reference substance is weighed respectively：Ginsenoside Rb1, Ginsenoside Rc, ginsenoside Rd, ginsenoside Rg1, ginsenoside
Re, Astragaloside IV, schizandrin A, deoxyschizandrin, schizandrin, wuweizi alcohol B, with methyl alcohol as solvent, are configured to mix
Close reference substance solution mother liquor；
In step (1), in recommending prepared mixed reference substance solution mother liquor, ginsenoside Rb1's is final concentration of
99.3325 μ g/mL, the final concentration of 109.2657 μ g/mL of Ginsenoside Rc, final concentration of 141.0522 μ of ginsenoside Rd
G/mL, the final concentration of 152.5747 μ g/mL of ginsenoside Rg1, the final concentration of 224.4914 μ g/mL of ginsenoside Re, Huang
The final concentration of 52.4476 μ g/mL of stilbene first glycosides, the final concentration of 42.9116 μ g/mL of schizandrin A, the end of deoxyschizandrin
Concentration be 55.6262 μ g/mL, the final concentration of 55.6262 μ g/mL of schizandrin, wuweizi alcohol B it is final concentration of
127.1456μg/mL。
(2) prepare internal standard compound standard liquid
Internal standard compound tanshinone IIA is weighed, with methyl alcohol as solvent, internal standard compound standard liquid is configured to；
In step (2), in recommending prepared internal standard compound standard liquid, the concentration of tanshinone IIA is 80 μ g/mL.
(3) prepare need testing solution
Ginseng-astragalus blood-sugar lowering preparation is crushed (to 40 mesh), the ginseng-astragalus blood-sugar lowering powder formulation after crushing is weighed and is mixed (material with methyl alcohol
Liquid mass ratio 3：50, g/mL), ultrasonic extraction (40KHz, 1h), extract removes solvent under reduced pressure, obtains medicinal extract；By gained medicinal extract
Be dissolved in water (feed liquid mass ratio 1：10, g/mL), then with water-saturated n-butanol solution extract, collect extract (n-butanol liquid layer)
Remove solvent under reduced pressure, freeze-drying obtains ginseng-astragalus blood-sugar lowering preparation extract；With methyl alcohol as solvent, gained ginseng-astragalus blood-sugar lowering system is prepared
Agent extract solution, is centrifuged (13000rpm, 10min), takes supernatant, and (aperture is usually 0.45 μm or 0.22 μ to cross miillpore filter
M), obtain need testing solution；
In step (3), in recommending prepared ginseng-astragalus blood-sugar lowering preparation extract solution, ginseng-astragalus blood-sugar lowering preparation extract it is dense
Spend for 2mg/mL.
(4) test sample detection
Using HPLC-Q-MS instruments, in need testing solution prepared by step (3), add step (2) to prepare internal standard compound
Standard liquid (volumetric usage for recommending the internal standard compound standard liquid is 0.04 times of the need testing solution volume), sample introduction inspection
Survey, obtain the mass spectrogram of test sample；
The running parameter condition of the HPLC-Q-MS instruments is as follows：
Liquid chromatogram separation condition is：Chromatographic column：With octadecylsilane chemically bonded silica as filler, particle diameter≤5 μm are (preferably
5 μm, 3.5 μm or 1.8 μm), mobile phase A is acetonitrile, and Mobile phase B is the aqueous formic acid of volume fraction 0.1%, is washed using gradient
It is de-, 30 DEG C of column temperature, flow velocity 1.0mL/min, 20 μ L of sampling volume, Detection wavelength is 203nm；
Single level Four bar Mass Spectrometry Conditions are：Using ESI electric spray ion sources, voltage：3000V, is atomized 350 DEG C of room temperature, does
Pathogenic dryness flow velocity 12.0L/min, positive ion mode, SIM ion monitoring modes open ion monitoring passage 1, ion monitoring passage 2.
(5) draw calibration curve
The mixed reference substance solution mother liquor that step (1) is prepared is taken, (recommends 7 according to variable concentrations gradient dilution into several pieces
Part) mixed reference substance solution sample, in every part of mixed reference substance solution sample add internal standard compound standard prepared by step (2) molten
Liquid (volumetric usage for recommending the internal standard compound standard liquid is 0.04 times of every part of mixed reference substance solution volume of sample), then
Using HPLC-Q-MS instruments, detected according to the running parameter condition in step (4), obtain mixing the mass spectrogram of reference substance；
To mix each single reference substance respective sample introduction concentration (μ g/ml) in reference substance, as abscissa, in mass spectrogram, each is single right
It is ordinate according to the ratio of each self-corresponding peak area of product and internal standard compound peak area, draws calibration curve, and then linearly returned
Return equation, that is, respectively obtain reference substance ginsenoside Rb1, Ginsenoside Rc, ginsenoside Rd, ginsenoside Rg1, ginsenoside
Re, Astragaloside IV, schizandrin A, deoxyschizandrin, schizandrin, the calibration curve of wuweizi alcohol B and linear regression side
Journey；
In step (5), the gradient for recommending described mixed reference substance solution mother liquor to press 1,2.5,5,10,25,50,100 times
It is diluted to 7 parts of mixed reference substance solution samples.
(6) test sample content detection result
Each active compound component each corresponding peak area and internal standard compound in the test sample mass spectrogram that step (4) is measured
The equation of linear regression of each reference substance that the ratio of peak area is obtained in substituting into step (5), is calculated people in need testing solution
Ginseng saponin(e Rb1, Ginsenoside Rc, ginsenoside Rd, ginsenoside Rg1, ginsenoside Re, Astragaloside IV, schizandrin A, five
Taste B prime, schizandrin, the content of wuweizi alcohol B, and then it is calculated 10 kinds of active components in ginseng-astragalus blood-sugar lowering preparation
Content.
Further, in step (4), the liquid chromatogram separates the eluting solvent of gradient elution and presses following time sequencing
Carry out (implication of the percentage sign in table is percentage by volume)：
Further, in step (4), under single level Four bar mass spectrum SIM ion monitoring modes, binary channels monitoring objective
The title of compound, molecular formula, monitoring opening time and monitoring ionic molecule amount are as follows：
The compound information of the monitoring of ion monitoring passage 1
The compound information of the monitoring of ion monitoring passage 2
Deficiency of the present invention for existing ginseng-astragalus blood-sugar lowering quality of the pharmaceutical preparations control method, proposes to survey using HPLC-Q-MS first
The method for determining multi-target ingredient content in ginseng-astragalus blood-sugar lowering preparation, has hypoglycemic with hypoglycemic activity as being oriented to choose in ginseng-astragalus blood-sugar lowering preparation
Activity and the higher ginsenoside Rb1 of content, ginsenoside Rd, ginsenoside Rg1, ginsenoside Re, Astragaloside IV, the fruit of Chinese magnoliavine
A prime, schizandrin, wuweizi alcohol B, Ginsenoside Rc, 10 kinds of compounds of deoxyschizandrin are used as index composition.This
The bright quality evaluating method for making ginseng-astragalus blood-sugar lowering preparation is more scientific, systematicness and specificity, while people alternatively in bulk drug
Ginseng, the Radix Astragali, the discriminating of the fruit of Chinese magnoliavine provide it is certain refer to, the quality standard for further improving ginseng-astragalus blood-sugar lowering preparation provide science according to
According to.
The invention has the beneficial effects as follows：
(1) present invention is with compound hypoglycemic activity as the various active material that is oriented in detection compound, it is to avoid only determine
First, two chemical compositions and judge the one-sidedness of ginseng-astragalus blood-sugar lowering preparation total quality, so as to preferably react whole compound preparation
Quality level；
(2) present invention chooses Q-MS ion monitoring modes (SIM) and carries out assay, and mass spectrum dosing accuracy is high, exclusive
The strong, sensitivity of property is high, better than traditional HPLC analysis methods；
(3) the different batches ginseng-astragalus blood-sugar lowering preparation liquid phase figure that the inventive method is obtained, is set up using Chempattern softwares
Ginseng-astragalus blood-sugar lowering preparation finger, carries out similarity evaluation, the more scientific comprehensive inherent matter for evaluating ginseng-astragalus blood-sugar lowering formulation products
Amount, provides foundation for setting up higher levels of quality standard.
(4) illustrate
Fig. 1：Liquid chromatogram of the SHENQI JIANGTANG KELI need testing solution under elution program 1 in embodiment 1；
Fig. 2：Liquid chromatogram of the SHENQI JIANGTANG KELI need testing solution under elution program 2 in embodiment 1；
Fig. 3：Ion monitoring passage 1 mass spectrogram of the mixed reference substance solution under elution program 2 in embodiment 2；
Fig. 4：Ion monitoring passage 2 mass spectrogram of the mixed reference substance solution under elution program 2 in embodiment 2；
Fig. 5：Ion monitoring passage 1 mass spectrum of the SHENQI JIANGTANG KELI need testing solution under elution program 2 in embodiment 2
Figure；
Fig. 6：Ion monitoring passage 2 mass spectrum of the SHENQI JIANGTANG KELI need testing solution under elution program 2 in embodiment 2
Figure；
Fig. 7：Liquid chromatogram of the SHENQI JIANGTANG KELI batch 001135 under elution program 2 in embodiment 2；
Fig. 8：The liquid-phase chromatograph finger print atlas of different batches SHENQI JIANGTANG KELI need testing solution in embodiment 2；
Fig. 9：In embodiment 2, between SHENQI JIANGTANG KELI batch, fingerprint similarity evaluates schematic diagram.
(5) specific embodiment
Below by specific embodiment, the present invention is further illustrated, but protection scope of the present invention is not limited in
This.
Embodiment 1
The optimization of ginseng-astragalus blood-sugar lowering preparation chromatographic separation condition, comprises the following steps：
The impact that different gradients separate situation to ginseng-astragalus blood-sugar lowering preparation need testing solution chemical composition is investigated.
(1) chromatographic column：Agilent ZORBAX SB-Aq C18 posts (4.6 × 250mm i.d., 5 μm)；Mobile phase：With second
Nitrile is mobile phase A, with aqueous formic acid that percentage by volume is 0.1% as Mobile phase B；Type of elution is：Linear gradient elution,
Elution program 1 is shown in Table 1；Column temperature：30℃；Flow velocity：1.0mL/min；Detection wavelength 203nm.Gained liquid chromatogram is shown in accompanying drawing 1,
As can be seen that the composition in SHENQI JIANGTANG KELI can not be eluted chromatographic column by this elution program completely in figure.
1. elution program 1 of table
(2) chromatographic column：Agilent ZORBAX SB-Aq C18 posts (4.6 × 250mm i.d., 5 μm)；Mobile phase：With second
Nitrile is mobile phase A, with aqueous formic acid that percentage by volume is 0.1% as Mobile phase B；Type of elution is：Linear gradient elution,
Elution program 2 is shown in Table 2；Column temperature：30℃；Flow velocity：1.0mL/min；Detection wavelength 203nm.Gained liquid chromatogram is shown in accompanying drawing 2,
As can be seen that each chromatographic peak is separated well in figure, wash-out is complete.
2. elution program 2 of table
Embodiment 2
The structure of ginseng-astragalus blood-sugar lowering preparation HPLC-Q-MS content assaying methods and application, comprise the following steps：
(1) instrument and reagent：
6120 single level Four bar matter of 1290 high performance liquid chromatographs of Agilent (Anjelen Sci. ＆ Tech. Inc), Agilent
Spectrometer (Anjelen Sci. ＆ Tech. Inc), KQ-250DE type numerical control ultrasonic cleaners (Kunshan Ultrasonic Instruments Co., Ltd.),
R210+V700 types revolving evaporimeter (Switzerland, BUCHI), AR224CN electronic balances (Ao Haosi Instrument Ltd.), liquid-transfering gun
A set of (big dragon Xing Chuan laboratory apparatus Co., Ltd), chromatographic column：Agilent ZORBAX SB-Aq C18 post (4.6 × 250mm
I.d., 5 μm)；
Reagent：Reference substance
The compound information of 3. 10 reference substances of table and internal standard compound tanshinone IIA
SHENQI JIANGTANG KELI：Shandong Lunan bark of official magnolia pharmaceutical factory produces, and Chinese medicines quasi-word Z10950075 is granule, per Bao Chong
3g。
(2) prepared by mixed reference substance solution：
Precision weighs ginsenoside Rb1, Ginsenoside Rc, ginsenoside Rd, ginsenoside Rg1, ginsenoside Re, the Radix Astragali
First glycosides, schizandrin A, deoxyschizandrin, schizandrin, wuweizi alcohol B, are dissolved with methyl alcohol, are configured to mother liquor final concentration
Respectively：99.3325μg/mL、109.2657μg/mL、141.0522μg/mL、152.5747μg/mL、224.4914μg/mL、
52.4476 μ g/mL, 42.9116 μ g/mL, 55.6262 μ g/mL, 55.6262 μ g/mL, the mixing reference substance of 127.1456 μ g/mL
Solution, is diluted to variable concentrations on demand.
(3) prepared by internal standard substance solution：
Precision weighs tanshinone IIA, and methyl alcohol dissolving is configured to the standard liquid that concentration is 80 μ g/mL.
(4) prepared by need testing solution：
SHENQI JIANGTANG KELI 3.0g is weighed into measuring bottle, precision adds methyl alcohol 50mL, weighed weight.(400KW) ultrasound is carried
60min is taken, is stood afterwards and is let cool, less loss weight is made up with methyl alcohol.Natural filtration, obtains SHENQI JIANGTANG KELI crude extract.Vacuum distillation,
Obtain SHENQI JIANGTANG KELI and extract medicinal extract, 20mL deionized water dissolvings, 20mL water-saturated n-butanol solution shaking out 3 times.Collect
Butanol extraction liquid vacuum distillation is transferred to freeze dryer freeze-drying to medicinal extract shape, obtains ginseng-astragalus blood-sugar lowering extract powder.Methyl alcohol
Dissolving, is configured to the solution of the SHENQI JIANGTANG KELI extract of 2mg/mL, and high speed centrifugation 10min (13000rpm) takes supernatant,
0.22 μm of miillpore filter is crossed, test sample solution is obtained；
(5) high performance liquid chromatography separation condition：
Chromatographic column：Agilent ZORBAX SB-Aq C18 posts (4.6x250mm i.d., 5 μm)；Mobile phase：With acetonitrile it is
Mobile phase A, with aqueous formic acid that percentage by volume is 0.1% as Mobile phase B；Type of elution is：Linear gradient elution, wash-out
Program is shown in Table 4；Column temperature：30℃；Flow velocity：1.0mL/min；
4. gradient elution program of table
(6) Mass Spectrometer Method condition：
Single level Four bar mass spectrograph, ion gun：ESI electric spray ion sources；Voltage：3000V, is atomized 350 DEG C of room temperature, is dried
Device flow velocity 12.0L/min, positive ion mode, SIM ion monitoring modes open ion monitoring passage 1, ion monitoring passage 2；
Under 1 pair of 8 compound of ion monitoring passage and 1 internal standard positive ion mode, each monitoring SIM ions are opened with monitoring
Time is as shown in table 5 below：
The compound information of the monitoring of 5. ion monitoring passage of table 1
Under 2 pairs of 2 compounds of ion monitoring passage and 1 internal standard positive ion mode, each monitoring SIM ions are opened with monitoring
Time is as shown in table 6 below：
The compound information of the monitoring of 6. ion monitoring passage of table 2
(7) drafting of calibration curve：
Mixed reference substance solution dilutes 1,2.5,5,10,25,50,100 times on the basis of mother liquor, is configured to variable concentrations
The mixed reference substance solution of gradient, the tanshinone IIA internal standard compound standard for being separately added into 0.04 times of volume of mixed reference substance solution are molten
Liquid, mixes, and takes 20 μ l injection liquid chromatographs respectively, and 1 mass spectrogram of ion monitoring passage is shown in accompanying drawing 3,2 mass spectrum of ion monitoring passage
Figure is shown in accompanying drawing 4.Sample introduction concentration (μ g/ml) with respective standard items as abscissa, with each standard items in mass spectrogram and internal standard compound peak
The ratio of area is ordinate, draws calibration curve, carries out linear regression, obtain regression equation and see the table below 7.
Table 7. SHENQI JIANGTANG KELI, 10 kinds of ingredient standard curves
(8) Precision Experiment
A, withinday precision：According to above-mentioned chromatogram and Mass Spectrometry Conditions, it is accurate draw it is continuous in mixed reference substance solution one day
Sample introduction 6 times, calculates the retention time (t of each chromatographic peakR) and peak area ratio (Pa, analyte peak area/internal standard compound peak area)
Relative standard deviation (RSD) is investigating withinday precision；
B, day to day precision：According to above-mentioned chromatogram and Mass Spectrometry Conditions, 3 days sample introductions of mixed reference substance solution point are drawn, is calculated
The same withinday precision of method；
Day to day precision is good with withinday precision data display instrument precision；
8. instrument precision of table investigates result with method repeatability
(9) repeated experiment
Take that same (Shandong Lunan bark of official magnolia pharmaceutical factory, batch 00113295) 5 parts of SHENQI JIANGTANG KELI sample, according to above-mentioned confession
Test sample solution preparation method prepares 5 parts with a batch of need testing solution, according to above-mentioned chromatogram and Mass Spectrometry Conditions, accurate sample introduction.
RSD values are calculated, the repeatability of result presentation method is good.It is shown in Table 8.
(10) stability experiment
Take that same (Shandong Lunan bark of official magnolia pharmaceutical factory, batch 00113295) SHENQI JIANGTANG KELI sample, according to above-mentioned for trying
Product solution manufacturing method prepares need testing solution, according to above-mentioned chromatogram and Mass Spectrometry Conditions, determines when 0,4,8,12 and 24.
As a result show that analyte is very stable in experimentation, be shown in Table 9.
(11) average recovery experiment
Investigating the accuracy of method, addition is similar to analyte concentration in sample to the Standard Addition Method for Determination rate of recovery
0.5 times of each standard items, according to above-mentioned chromatogram and Mass Spectrometry Conditions, accurate sample introduction 3 times, survey content, calculate average recovery, as a result
See the table below 9.
9. sample stability of table and average recovery result
(12) ginseng-astragalus blood-sugar lowering formulation content determines application
According to ginseng stilbene of the content assaying method drafted to the lot number 00113295 from Shandong Lunan bark of official magnolia pharmaceutical factory
Hypoglycemic granule carries out assay, and assay result is as shown in table 10 below, and 1 mass spectrogram of ion monitoring passage is shown in accompanying drawing 5, ion
Monitoring 2 mass spectrogram of passage is shown in accompanying drawing 6.
The SHENQI JIANGTANG KELI assay result (μ g/g) of 10. Shandong Lunan bark of official magnolia pharmaceutical factory lot number 00113295 of table
According to same method, content is carried out to the sample of other 9 different batches of Shandong Lunan bark of official magnolia pharmaceutical factory production
Determine, assay result see the table below 11 (batch 00113295 is equally listed in the table below).
The SHENQI JIANGTANG KELI assay result (μ g/g) of 11. 10 batches of table
Note："/" represents that peak area has been less than quantitative limit.
Above the methodological study result of the assay of ginseng-astragalus blood-sugar lowering preparation (particle) is shown：The method precision,
Repeatability, stability, rate of recovery result are good, can accurately and quickly ginsenoside Rb1 in determination sample, ginsenoside Rd,
Ginsenoside Rg1, ginsenoside Re, Astragaloside IV, schizandrin A, schizandrin, wuweizi alcohol B, Ginsenoside Rc,
The content of 10 kinds of compositions of deoxyschizandrin, can be used in the quality evaluation research of ginseng-astragalus blood-sugar lowering preparation.
(13) ginseng-astragalus blood-sugar lowering preparation finger is set up
By the efficient liquid-phase chromatography method of above-mentioned assay, 20 μ l of above-mentioned SHENQI JIANGTANG KELI need testing solution injections are taken
Liquid chromatograph, obtains SHENQI JIANGTANG KELI liquid phase spectrogram, sees accompanying drawing 7.
10 compositions of above-mentioned assay are demarcated on liquid phase figure, wherein ginsenoside Rb1 and Astragaloside IV
Impurity is mixed with liquid phase peak, therefore using remaining 8 compositions as characteristic peak, sets up ginseng-astragalus blood-sugar lowering using Chempattern softwares
Particle liquid-phase chromatograph finger print atlas, carry out similarity evaluation.SHENQI JIANGTANG KELI liquid-phase chromatograph finger print atlas are shown in accompanying drawing 8, wherein
1-8 peaks are respectively：Ginsenoside Re, ginsenoside Rg1, Ginsenoside Rc, ginsenoside Rd, schizandrin, the fruit of Chinese magnoliavine
Alcohol second, schizandrin A, deoxyschizandrin.Similarity evaluation schematic diagram is shown in accompanying drawing 9, and wherein ordinate represents similarity.
As a result show, 10 batch SHENQI JIANGTANG KELI liquid phase spectrogram similarities are all higher than 0.985, and similarity is good.
Claims (10)
1. a kind of ginseng-astragalus blood-sugar lowering preparation content determining method, it is characterised in that during what methods described was determined is ginseng-astragalus blood-sugar lowering preparation
The content of 10 active components, 10 described active components are respectively：Ginsenoside Rb1, Ginsenoside Rc, ginsenoside
Rd, ginsenoside Rg1, ginsenoside Re, Astragaloside IV, schizandrin A, deoxyschizandrin, schizandrin, schisandrol
Second, methods described comprise the steps：
(1) prepare mixed reference substance solution
Reference substance is weighed respectively：Ginsenoside Rb1, Ginsenoside Rc, ginsenoside Rd, ginsenoside Rg1, ginsenoside Re,
Astragaloside IV, schizandrin A, deoxyschizandrin, schizandrin, wuweizi alcohol B, with methyl alcohol as solvent, are configured to mixing
Reference substance solution mother liquor；
(2) prepare internal standard compound standard liquid
Internal standard compound tanshinone IIA is weighed, with methyl alcohol as solvent, internal standard compound standard liquid is configured to；
(3) prepare need testing solution
Ginseng-astragalus blood-sugar lowering preparation is crushed, the ginseng-astragalus blood-sugar lowering powder formulation after crushing is weighed and is mixed with methyl alcohol, ultrasonic extraction, extract
Remove solvent under reduced pressure, obtain medicinal extract；Gained medicinal extract is dissolved in water, then is extracted with water-saturated n-butanol solution, collect extract
Remove solvent under reduced pressure, freeze-drying obtains ginseng-astragalus blood-sugar lowering preparation extract；With methyl alcohol as solvent, gained ginseng-astragalus blood-sugar lowering system is prepared
Agent extract solution, centrifugation take supernatant, cross miillpore filter, obtain need testing solution；
(4) test sample detection
Using HPLC-Q-MS instruments, in need testing solution prepared by step (3), add step (2) to prepare internal standard compound standard
Solution, sample detection obtain the mass spectrogram of test sample；
The running parameter condition of the HPLC-Q-MS instruments is as follows：
Liquid chromatogram separation condition is：Chromatographic column：With octadecylsilane chemically bonded silica as filler, particle diameter≤5 μm, mobile phase A
For acetonitrile, aqueous formic acid of the Mobile phase B for volume fraction 0.1%, using gradient elution, 30 DEG C of column temperature, flow velocity 1.0mL/
Min, 20 μ L of sampling volume, Detection wavelength is 203nm；
Single level Four bar Mass Spectrometry Conditions are：Using ESI electric spray ion sources, voltage：3000V, is atomized 350 DEG C of room temperature, is dried gas
Flow velocity 12.0L/min, positive ion mode, SIM ion monitoring modes open ion monitoring passage 1, ion monitoring passage 2；
(5) draw calibration curve
The mixed reference substance solution mother liquor that step (1) is prepared is taken, mixes reference substance according to variable concentrations gradient dilution into several pieces
Liquor sample, in every part of mixed reference substance solution sample, add step (2) to prepare internal standard compound standard liquid, then adopts
HPLC-Q-MS instruments, are detected according to the running parameter condition in step (4), obtain mixing the mass spectrogram of reference substance；With mixed
In closing reference substance, the respective sample introduction concentration of each single reference substance is abscissa, and in mass spectrogram, each single reference substance is each corresponded to
The ratio of peak area and internal standard compound peak area be ordinate, draw calibration curve, and then obtain equation of linear regression, that is, distinguish
Obtain reference substance ginsenoside Rb1, Ginsenoside Rc, ginsenoside Rd, ginsenoside Rg1, ginsenoside Re, Astragaloside IV,
Schizandrin A, deoxyschizandrin, schizandrin, the calibration curve of wuweizi alcohol B and equation of linear regression；
(6) test sample content detection result
Each active compound component each corresponding peak area and internal standard compound peak face in the test sample mass spectrogram that step (4) is measured
The equation of linear regression of each reference substance that long-pending ratio is obtained in substituting into step (5), is calculated ginseng soap in need testing solution
Glycosides Rb1, Ginsenoside Rc, ginsenoside Rd, ginsenoside Rg1, ginsenoside Re, Astragaloside IV, schizandrin A, the fruit of Chinese magnoliavine
B prime, schizandrin, the content of wuweizi alcohol B, and then it is calculated containing for 10 kinds of active components in ginseng-astragalus blood-sugar lowering preparation
Amount.
2. ginseng-astragalus blood-sugar lowering preparation content determining method as claimed in claim 1, it is characterised in that described ginseng-astragalus blood-sugar lowering preparation
For tablet, capsule or granule.
3. ginseng-astragalus blood-sugar lowering preparation content determining method as claimed in claim 1, it is characterised in that in step (1), prepared
In mixed reference substance solution mother liquor, the final concentration of 99.3325 μ g/mL of ginsenoside Rb1, Ginsenoside Rc it is final concentration of
109.2657 μ g/mL, the final concentration of 141.0522 μ g/mL of ginsenoside Rd, final concentration of the 152.5747 of ginsenoside Rg1
μ g/mL, the final concentration of 224.4914 μ g/mL of ginsenoside Re, the final concentration of 52.4476 μ g/mL of Astragaloside IV, the fruit of Chinese magnoliavine
The final concentration of 42.9116 μ g/mL of A prime, the final concentration of 55.6262 μ g/mL of deoxyschizandrin, schizandrin end it is dense
Spend the final concentration of 127.1456 μ g/mL for 55.6262 μ g/mL, wuweizi alcohol B.
4. ginseng-astragalus blood-sugar lowering preparation content determining method as claimed in claim 1, it is characterised in that in step (2), prepared
In internal standard compound standard liquid, the concentration of tanshinone IIA is 80 μ g/mL.
5. ginseng-astragalus blood-sugar lowering preparation content determining method as claimed in claim 1, it is characterised in that in step (3), prepared
In ginseng-astragalus blood-sugar lowering preparation extract solution, the concentration of ginseng-astragalus blood-sugar lowering preparation extract is 2mg/mL.
6. ginseng-astragalus blood-sugar lowering preparation content determining method as claimed in claim 1, it is characterised in that in step (4), the liquid phase
The eluting solvent of chromatographic isolation gradient elution is carried out by following time sequencing, and the implication of the percentage sign in table is percentage by volume：
7. ginseng-astragalus blood-sugar lowering preparation content determining method as claimed in claim 1, it is characterised in that in step (4), the Dan Si
Under level bar mass spectrum SIM ion monitoring modes, the title of binary channels monitoring objective compound, molecular formula, monitoring opening time and prison
Measured ion molecular weight is as follows：
The compound information of the monitoring of ion monitoring passage 1
The compound information of the monitoring of ion monitoring passage 2
8. ginseng-astragalus blood-sugar lowering preparation content determining method as claimed in claim 1, it is characterised in that in step (5), described are mixed
Reference substance solution mother liquor is closed by 1,2.5,5,10,25,50,100 times of gradient dilution into 7 parts of mixed reference substance solution samples.
9. the finger-print that the ginseng-astragalus blood-sugar lowering preparation liquid phase figure that content assaying method according to claim 1 is obtained builds.
10. application of the content assaying method as claimed in claim 1 in the global quality control of ginseng-astragalus blood-sugar lowering preparation.
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