CN106526002B - Ginseng-astragalus blood-sugar lowering preparation content determining method and its application in global quality control - Google Patents
Ginseng-astragalus blood-sugar lowering preparation content determining method and its application in global quality control Download PDFInfo
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Abstract
The present invention provides a kind of ginseng-astragalus blood-sugar lowering preparation content determining method, what the method measured is the content of 10 active constituents in ginseng-astragalus blood-sugar lowering preparation: ginsenoside Rb1, Ginsenoside Rc, ginsenoside Rd, ginsenoside Rg1, ginsenoside Re, Astragaloside IV, schizandrin A, deoxyschizandrin, schizandrin, wuweizi alcohol B；The present invention can preferably react the quality level of entire compound preparation with the various active substance that compound hypoglycemic activity is in guiding detection compound；Meanwhile choosing Q-MS ion monitoring mode and carrying out assay, mass spectrum dosing accuracy is high, specificity is strong, high sensitivity, is better than tradition HPLC analysis method；The different batches ginseng-astragalus blood-sugar lowering preparation liquid phase figure obtained according to the method for the present invention constructs finger-print, carries out similarity evaluation, and the inherent quality of more scientific comprehensive evaluation ginseng-astragalus blood-sugar lowering formulation products provides foundation to establish higher levels of quality standard.
Description
(1) technical field
The invention belongs to Control of drug quality fields, and in particular to the mono- level four bars mass spectrometric hyphenated technique of high performance liquid chromatography-
(HPLC-Q-MS) to the method for ginseng-astragalus blood-sugar lowering preparation multicomponent assay, and to the ginseng-astragalus blood-sugar lowering preparation that thus method obtains
Liquid phase figure constructs finger-print, further relates to this method in the application in ginseng-astragalus blood-sugar lowering preparation global quality control.
(2) background technique
Diabetes (Diabetes Mellitus, DM) belong to Chinese medicine " diabete " scope: " drinking a lot of water yearningly, urine number,
It is all diabete without rouge like bran flakes sweet tea person ".International Diabetes Federation (IDF) points out, the current whole world have 4.15 hundred million diabetes at
Year patient, 3.18 hundred million people, which exist, suffers from diabetes risk, wherein and type-2 diabetes mellitus accounts for the 90% of all diabetes cases in the whole world, because
The hyperglycemia of diabetes accompanying easily leads to various tissues, especially eye, kidney, heart, blood vessel, the chronic lesion of nerve, function barrier
The complication such as hinder, greatly reduces the quality of life of diabetic.
Currently, clinically the treatment of diabetes mainly takes orally sulfonylureas and biguanide antidiabetic medicament in addition to insulin injection,
But there is also numerous safety issues and adverse reactions, such as serious hypoglycemia, cream while blood glucose is effectively reduced for Western medicine
Acid acid poisoning, gastrointestinal reaction and hepatic injury, permanent neurologic function damage, headache, dizziness or even death etc..It grinds in recent years
Study carefully discovery, the hypoglycemic effect of Chinese medicine is mildly lasting, and curative effect is stablized, and Small side effects compensate for the deficiency of chemical drug just, increasingly by
To the great attention of domestic and international endocrine circle.
SHENQI JIANGTANG KELI (Shenqi Jiangtang Granule, SJG), is made of 11 taste traditional Chinese medicines, it is pure in
Medicine compound preparation belongs to national four kind new medicines, and national second level Chinese medicine protects kind, is clinically primarily adapted for use in II type sugar
Sick syndrome of deficiency of both qi and yin patient is urinated, clinical data display effect is good.Ginseng is the non-defective unit of strengthening YANG and invigorating YIN in side, and Radix Astragali is tonifying Qi liter
The key medicine of sun, the two are monarch drug in a prescription altogether；Minister with Radix Rehmanniae, Radix Ophiopogonis, radices trichosanthis fluid dryness and quench the thirst；It helps with fructus lycii, Schisandra chinensis, cover
Basin sealing kidney closes；Make with Chinese yam, Poria cocos, three Jiao Bingli of rhizoma alismatis.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 ingredient of regulation ginseng stem and leave general saponin is ginsenoside Rd, ginsenoside Re, ginsenoside Rg1；Radix Astragali
Middle Astragaloside IV, formoononetin, calycosin have hypoglycemic activity, and 2015 editions Chinese Pharmacopoeias provide the quality of Milkvetch Root
Con trolling index ingredient is Astragaloside IV；Modern pharmacology research also proves that oligosaccharide, Catalpol, martynoside D in Radix Rehmanniae have drop
Sugared effect；Schizandrin is the index ingredient that schisandra chinensis medicinal material is detected specified in Chinese Pharmacopoeia；Radix Ophiopogonis, radices trichosanthis, Chinese holly
Matrimony vine, Chinese yam, Poria cocos play mainly polysaccharose substance or the Pleurotus Ostreatus compound of hypoglycemic effect.
American Diabetes Association advocates, and the target for the treatment of diabetes is blood glucose to be down to normal level or close to normal water
It is flat, while reducing the generation of complication, SHENQI JIANGTANG KELI multicomponent, multiple target point action character be more suitable for controlling for diabetes
It treats.Domestic and international pertinent literature discovery is consulted, currently, the reported quality control to SHENQI JIANGTANG KELI is mainly: by thin
Layer authenticated ginsenoside and astragaloside, and with 5% vanillic aldehyde-glacial acetic acid method measurement general ginsenoside and astragalus root total saponin
Content；The content of ginsenoside Re and Rg1 in SHENQI JIANGTANG KELI are determined using HPLC method.Above method is mostly only to ginseng
One or both of stilbene antihypelipidemic preparation ingredient carries out qualitative and quantitative analysis, cannot reflect the quality level of entire compound preparation,
And respectively have deficiency, and such as: poor selectivity, low, the reagent contamination environment of sensitivity etc..HPLC-Q-MS combination has become complex system
The good platform of separation analysis and compound structure identification, but had no so far using this technology to more in ginseng-astragalus blood-sugar lowering preparation
The document report that active component is analyzed simultaneously, it is often more important that, HPLC-Q-MS's is highly sensitive, highly selective and high
Resolution ratio can make up for it the deficiency of traditional HPLC, and can the multiclass ingredient to complicated compound Chinese medicinal preparation carry out while surveying
It is fixed.
(3) summary of the invention
It is described the purpose of the present invention is ginseng-astragalus blood-sugar lowering preparation being established while being measured the method for the content of 10 active constituents
10 active constituents be respectively as follows: ginsenoside Rb1, Ginsenoside Rc, ginsenoside Rd, ginsenoside Rg1, ginsenoside
Re, Astragaloside IV, schizandrin A, deoxyschizandrin, schizandrin, wuweizi alcohol B.Also, the present invention utilizes
Chempattern software establishes finger-print to the ginseng-astragalus blood-sugar lowering preparation liquid phase figure that above-mentioned content assaying method obtains, and carries out phase
It is analyzed like degree, to the inherent quality of more scientific comprehensive evaluation ginseng-astragalus blood-sugar lowering formulation products, to establish higher levels of quality
Standard provides foundation.The method of the present invention can be applied to the global quality control of ginseng-astragalus blood-sugar lowering preparation.
To achieve the above object, the present invention adopts the following technical scheme:
A kind of ginseng-astragalus blood-sugar lowering preparation (usually tablet, capsule or granule) content assaying method, the method measurement
Be 10 active constituents in ginseng-astragalus blood-sugar lowering preparation content, 10 active constituents are respectively as follows: ginsenoside Rb1, people
Join saponin(e Rc, ginsenoside Rd, ginsenoside Rg1, ginsenoside Re, Astragaloside IV, schizandrin A, deoxyschizandrin, five
Taste alcohol first, wuweizi alcohol B, described method includes following steps:
(1) mixed reference substance solution is prepared
Reference substance is weighed respectively: ginsenoside Rb1, Ginsenoside Rc, ginsenoside Rd, ginsenoside Rg1, ginsenoside
Re, Astragaloside IV, schizandrin A, deoxyschizandrin, schizandrin, wuweizi alcohol B are configured to mix using methanol as solvent
Close reference substance solution mother liquor；
In step (1), recommend in prepared mixed reference substance solution mother liquor, ginsenoside Rb1's is final concentration of
99.3325 μ g/mL, final concentration of 109.2657 μ g/mL of Ginsenoside Rc, ginsenoside Rd final concentration of 141.0522 μ
G/mL, final concentration of 152.5747 μ g/mL of ginsenoside Rg1, ginsenoside Re final concentration of 224.4914 μ g/mL, Huang
The end of final concentration of 52.4476 μ g/mL of stilbene first glycosides, final concentration of 42.9116 μ g/mL of schizandrin A, deoxyschizandrin
Concentration be 55.6262 μ g/mL, final concentration of 55.6262 μ g/mL of schizandrin, wuweizi alcohol B it is final concentration of
127.1456μg/mL。
(2) internal standard compound standard solution is prepared
Internal standard compound tanshinone IIA is weighed, using methanol as solvent, is configured to internal standard compound standard solution；
In step (2), recommend in prepared internal standard compound standard solution, the concentration of tanshinone IIA is 80 μ g/mL.
(3) test solution is prepared
Ginseng-astragalus blood-sugar lowering preparation is crushed into (to 40 mesh), smashed ginseng-astragalus blood-sugar lowering powder formulation is weighed and mixes (material with methanol
Liquid mass ratio 3:50, g/mL), ultrasonic extraction (40KHz, 1h), extracting solution evaporating solvent under reduced pressure obtains medicinal extract；By gained medicinal extract
Be dissolved in water (feed liquid mass ratio 1:10, g/mL), then is saturated with water butanol solution extraction, collects extract liquor (n-butanol liquid layer)
Evaporating solvent under reduced pressure, freeze-drying, obtains ginseng-astragalus blood-sugar lowering preparation extract；Using methanol as solvent, gained ginseng-astragalus blood-sugar lowering system is prepared
Agent extract solution is centrifuged (13000rpm, 10min), takes supernatant, and crossing miillpore filter, (aperture is usually 0.45 μm or 0.22 μ
M), test solution is obtained；
In step (3), recommend in prepared ginseng-astragalus blood-sugar lowering preparation extract solution, ginseng-astragalus blood-sugar lowering preparation extract it is dense
Degree is 2mg/mL.
(4) test sample detects
Using HPLC-Q-MS instrument, the internal standard compound of step (2) preparation is added in the test solution of step (3) preparation
Standard solution (recommends the volumetric usage of the internal standard compound standard solution for 0.04 times of the test solution volume), sample introduction inspection
It surveys, obtains the mass spectrogram of test sample；
The running parameter condition of the HPLC-Q-MS instrument is as follows:
Liquid chromatogram separation condition are as follows: chromatographic column: using octadecylsilane chemically bonded silica as filler, partial size≤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, sampling volume 20 μ L, Detection wavelength 203nm；
Single level four bars Mass Spectrometry Conditions are as follows: use ESI electric spray ion source, voltage: 3000V is atomized 350 DEG C of room temperature, does
Pathogenic dryness flow velocity 12.0L/min, positive ion mode, SIM ion monitoring mode open ion monitoring channel 1, ion monitoring channel 2.
(5) standard curve is drawn
The mixed reference substance solution mother liquor for taking step (1) to prepare (recommends 7 at several pieces according to various concentration gradient dilution
Part) mixed reference substance solution sample, it is molten that internal standard compound standard prepared by step (2) is added in every part of mixed reference substance solution sample
Liquid (recommends the volumetric usage of the internal standard compound standard solution for 0.04 times of every part of mixed reference substance solution volume of sample), then
It using HPLC-Q-MS instrument, is detected according to the running parameter condition in step (4), obtains the mass spectrogram of mixing reference substance；
It is each single right in mass spectrogram for abscissa to mix each single respective sample introduction concentration of reference substance (μ g/ml) in reference substance
It is ordinate according to the ratio of the corresponding peak area of product and internal standard compound peak area, draws standard curve, and then linearly returned
Return equation, that is, respectively obtains reference substance ginsenoside Rb1, Ginsenoside Rc, ginsenoside Rd, ginsenoside Rg1, ginsenoside
Re, Astragaloside IV, schizandrin A, deoxyschizandrin, schizandrin, the standard curve of wuweizi alcohol B and linear regression side
Journey；
In step (5), recommend the mixed reference substance solution mother liquor by 1,2.5,5,10,25,50,100 times of gradient
It is diluted to 7 parts of mixed reference substance solution samples.
(6) test sample content detection result
Each active compound component respectively corresponding peak area and internal standard compound in the test sample mass spectrogram that step (4) is measured
The ratio of peak area substitutes into the equation of linear regression of each reference substance obtained in step (5), and people in test solution is calculated
Join saponin(e Rb1, Ginsenoside Rc, ginsenoside Rd, ginsenoside Rg1, ginsenoside Re, Astragaloside IV, schizandrin A, five
The content of taste B prime, schizandrin, wuweizi alcohol B, and then 10 kinds of active constituents are calculated in ginseng-astragalus blood-sugar lowering preparation
Content.
Further, in the step (4), the eluting solvent of the liquid chromatogram separation gradient elution presses following time sequencing
Carry out (meaning of the percentage sign in table is percentage by volume):
Further, in the step (4), under the list level four bars mass spectrum SIM ion monitoring mode, binary channels monitoring objective
The title of compound, molecular formula, monitoring opening time and monitoring ionic molecule amount are as follows:
The compound information that ion monitoring channel 1 monitors
The compound information that ion monitoring channel 2 monitors
The present invention is directed to the deficiency of existing ginseng-astragalus blood-sugar lowering quality of the pharmaceutical preparations control method, is put forward for the first time and is surveyed using HPLC-Q-MS
Determine the method for multi-target ingredient content in ginseng-astragalus blood-sugar lowering preparation, is to have hypoglycemic in guiding selection ginseng-astragalus blood-sugar lowering preparation with hypoglycemic activity
Activity and the higher ginsenoside Rb1 of content, ginsenoside Rd, ginsenoside Rg1, ginsenoside Re, Astragaloside IV, Schisandra chinensis
10 kinds of A prime, schizandrin, wuweizi alcohol B, Ginsenoside Rc, deoxyschizandrin compounds are as index ingredient.This hair
The bright quality evaluating method for making ginseng-astragalus blood-sugar lowering preparation more science, systematicness and specificity, while can also be people in bulk pharmaceutical chemicals
The identification of ginseng, Radix Astragali, Schisandra chinensis provides certain reference, for further increase the quality standard of ginseng-astragalus blood-sugar lowering preparation provide science according to
According to.
The beneficial effects of the present invention are:
(1) present invention is the various active substance being oriented in detection compound with compound hypoglycemic activity, avoids and only measures
One, two chemical components and determine the one-sidedness of ginseng-astragalus blood-sugar lowering preparation total quality, to preferably react entire compound preparation
Quality level；
(2) present invention chooses Q-MS ion monitoring mode (SIM) and carries out assay, and mass spectrum dosing accuracy is high, exclusive
Property strong, high sensitivity, be better than tradition HPLC analysis method；
(3) the different batches ginseng-astragalus blood-sugar lowering preparation liquid phase figure that the method for the present invention obtains is established using Chempattern software
Ginseng-astragalus blood-sugar lowering preparation finger carries out similarity evaluation, the inherent matter of more scientific comprehensive evaluation ginseng-astragalus blood-sugar lowering formulation products
Amount, provides foundation to establish higher levels of quality standard.
(4) Detailed description of the invention
Fig. 1: liquid chromatogram of the SHENQI JIANGTANG KELI test solution under elution program 1 in embodiment 1；
Fig. 2: liquid chromatogram of the SHENQI JIANGTANG KELI test solution under elution program 2 in embodiment 1；
Fig. 3: ion monitoring channel 1 mass spectrogram of the mixed reference substance solution under elution program 2 in embodiment 2；
Fig. 4: ion monitoring channel 2 mass spectrogram of the mixed reference substance solution under elution program 2 in embodiment 2；
Fig. 5: ion monitoring channel 1 mass spectrum of the SHENQI JIANGTANG KELI test solution under elution program 2 in embodiment 2
Figure；
Fig. 6: ion monitoring channel 2 mass spectrum of the SHENQI JIANGTANG KELI test 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 test solution in embodiment 2；
Fig. 9: fingerprint similarity evaluates schematic diagram between SHENQI JIANGTANG KELI batch in embodiment 2.
(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, comprising the following steps:
Influence of the different gradients to ginseng-astragalus blood-sugar lowering preparation test solution chemical component separation situation is investigated.
(1) chromatographic column: Agilent ZORBAX SB-Aq C18 column (4.6 × 250mm i.d., 5 μm)；Mobile phase: with second
Nitrile is mobile phase A, and the aqueous formic acid for being 0.1% using percentage by volume is Mobile phase B；Type of elution are as follows: linear gradient elution,
Elution program 1 is shown in Table 1；Column temperature: 30 DEG C；Flow velocity: 1.0mL/min；Detection wavelength 203nm.Gained liquid chromatogram is shown in attached drawing 1,
As can be seen that the ingredient in SHENQI JIANGTANG KELI cannot be eluted chromatographic column completely by this elution program in figure.
1. elution program 1 of table
(2) chromatographic column: Agilent ZORBAX SB-Aq C18 column (4.6 × 250mm i.d., 5 μm)；Mobile phase: with second
Nitrile is mobile phase A, and the aqueous formic acid for being 0.1% using percentage by volume is Mobile phase B；Type of elution are as follows: linear gradient elution,
Elution program 2 is shown in Table 2；Column temperature: 30 DEG C；Flow velocity: 1.0mL/min；Detection wavelength 203nm.Gained liquid chromatogram is shown in attached drawing 2,
As can be seen that the separation of each chromatographic peak is good in figure, elution is complete.
2. elution program 2 of table
Embodiment 2
The building and application of ginseng-astragalus blood-sugar lowering preparation HPLC-Q-MS content assaying method, comprising the following steps:
(1) instrument and reagent:
1290 high performance liquid chromatograph of Agilent (Anjelen Sci. & Tech. Inc), the single level four bars matter of Agilent 6120
Spectrometer (Anjelen Sci. & Tech. Inc), KQ-250DE type numerical control ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.),
R210+V700 type rotates evaporimeter (Switzerland, BUCHI), AR224CN electronic balance (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 column (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: the production of Shandong Lunan Cortex Magnoliae Officinalis pharmaceutical factory, national drug standard Z10950075 are granule, every packet weight 3g.
(2) prepared by mixed reference substance solution:
Precision weighs ginsenoside Rb1, Ginsenoside Rc, ginsenoside Rd, ginsenoside Rg1, ginsenoside Re, Radix Astragali
First glycosides, schizandrin A, deoxyschizandrin, schizandrin, wuweizi alcohol B, are dissolved with methanol, are configured to mother liquor final concentration
Be respectively as follows: 99.3325 μ g/mL, 109.2657 μ g/mL, 141.0522 μ g/mL, 152.5747 μ g/mL, 224.4914 μ g/mL,
The mixing reference substance of 52.4476 μ g/mL, 42.9116 μ g/mL, 55.6262 μ g/mL, 55.6262 μ g/mL, 127.1456 μ g/mL
Solution is diluted to various concentration on demand.
(3) prepared by internal standard substance solution:
Precision weighs tanshinone IIA, and methanol dissolution is configured to the standard solution that concentration is 80 μ g/mL.
(4) prepared by test solution:
SHENQI JIANGTANG KELI 3.0g is weighed into measuring bottle, methanol 50mL, weighed weight is added in precision.(400KW) ultrasound mentions
60min is taken, rear stand lets cool, and makes up less loss weight with methanol.Natural filtration obtains SHENQI JIANGTANG KELI crude extract.Vacuum distillation,
SHENQI JIANGTANG KELI extract medicinal extract, 20mL deionized water dissolving, 20mL water-saturated n-butanol solution shaking out 3 times.It collects
Butanol extraction liquid is evaporated under reduced pressure to medicinal extract shape, is transferred to freeze dryer freeze-drying, is obtained ginseng-astragalus blood-sugar lowering extract powder.Methanol
Dissolution, 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 column (4.6x250mm i.d., 5 μm)；Mobile phase: it is with acetonitrile
Mobile phase A, the aqueous formic acid for being 0.1% using percentage by volume is Mobile phase B；Type of elution are as follows: linear gradient elution, elution
Program is shown in Table 4；Column temperature: 30 DEG C；Flow velocity: 1.0mL/min；
4. gradient elution program of table
(6) Mass Spectrometer Method condition:
Single level four bars mass spectrograph, ion source: ESI electric spray ion source；Voltage: 3000V is atomized 350 DEG C of room temperature, drying
Device flow velocity 12.0L/min, positive ion mode, SIM ion monitoring mode open ion monitoring channel 1, ion monitoring channel 2；
Ion monitoring channel 1 to respectively monitored under 8 compounds and 1 internal standard positive ion mode SIM ion and monitoring open
Time is as shown in table 5 below:
The compound information of 5. ion monitoring channel 1 of table monitoring
Ion monitoring channel 2 to respectively monitored under 2 compounds and 1 internal standard positive ion mode SIM ion and monitoring open
Time is as shown in table 6 below:
The compound information of 6. ion monitoring channel 2 of table monitoring
(7) drafting of standard curve:
Mixed reference substance solution dilutes 1,2.5,5,10,25,50,100 times on the basis of mother liquor, is configured to various concentration
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 20 μ l is taken to inject liquid chromatograph respectively, and 1 mass spectrogram of ion monitoring channel is shown in attached drawing 3,2 mass spectrum of ion monitoring channel
Figure is shown in attached drawing 4.With the sample introduction concentration (μ g/ml) of respective standard items for abscissa, with standard items each in mass spectrogram and internal standard compound peak
The ratio of area is ordinate, draws standard curve, carries out linear regression, obtaining regression equation 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 chromatography and Mass Spectrometry Conditions, precision is drawn continuous in mixed reference substance solution one day
Sample introduction 6 times, calculate 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) investigates withinday precision；
B, day to day precision: according to above-mentioned chromatography and Mass Spectrometry Conditions, 3 days sample introductions of mixed reference substance solution point is drawn, are calculated
The same withinday precision of method；
Day to day precision and withinday precision data display instrument precision are good；
8. instrument precision of table and method repeatability investigate result
(9) repeated experiment
5 parts of SHENQI JIANGTANG KELI sample of same (Shandong Lunan Cortex Magnoliae Officinalis pharmaceutical factory, batch 00113295) is taken, according to above-mentioned confession
Test sample solution preparation method prepares 5 parts with a batch of test solution, according to above-mentioned chromatography and Mass Spectrometry Conditions, accurate sample introduction.
RSD value is calculated, the repeatability of result presentation method is good.It is shown in Table 8.
(10) stability experiment
Same (Shandong Lunan Cortex Magnoliae Officinalis pharmaceutical factory, batch 00113295) SHENQI JIANGTANG KELI sample is taken, according to above-mentioned for examination
Product solution manufacturing method prepares test solution, according to above-mentioned chromatography and Mass Spectrometry Conditions, measures when 0,4,8,12 and 24.
The result shows that analyte is very stable during the experiment, it is shown in Table 9.
(11) sample recovery rate is tested
The Standard Addition Method for Determination rate of recovery investigates the accuracy of method, and addition is similar to analyte concentration in sample
0.5 times of each standard items, according to above-mentioned chromatography and Mass Spectrometry Conditions, accurate sample introduction 3 times surveys content, calculates sample recovery rate, as a result
It see the table below 9.
9. sample stability of table and sample recovery rate result
(12) ginseng-astragalus blood-sugar lowering formulation content measurement application
According to the content assaying method drafted to the ginseng stilbene of the lot number 00113295 from Shandong Lunan Cortex Magnoliae Officinalis pharmaceutical factory
Hypoglycemic granule carries out assay, and assay result is as shown in the following table 10, and 1 mass spectrogram of ion monitoring channel is shown in attached drawing 5, ion
Monitoring 2 mass spectrogram of channel is shown in attached drawing 6.
The SHENQI JIANGTANG KELI assay result (μ g/g) of 10. Shandong Lunan Cortex Magnoliae Officinalis 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 Cortex Magnoliae Officinalis pharmaceutical factory production
Measurement, 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: "/" indicates that peak area has been lower than quantitative limit.
Above as the result is shown to the methodological study of the assay of ginseng-astragalus blood-sugar lowering preparation (particle): this method precision,
Repeatability, stability, rate of recovery result are good, can accurately and quickly measure ginsenoside Rb1 in sample, ginsenoside Rd,
Ginsenoside Rg1, ginsenoside Re, Astragaloside IV, schizandrin A, schizandrin, wuweizi alcohol B, Ginsenoside Rc,
The content of 10 kinds of ingredients 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 established
By the efficient liquid-phase chromatography method of above-mentioned assay, above-mentioned 20 μ l of SHENQI JIANGTANG KELI test solution is taken to inject
Liquid chromatograph obtains SHENQI JIANGTANG KELI liquid phase spectrogram, sees attached drawing 7.
10 ingredients of above-mentioned assay are demarcated on liquid phase figure, wherein ginsenoside Rb1 and Astragaloside IV
It is mixed with impurity in liquid phase peak, therefore using remaining 8 ingredients as characteristic peak, establishes ginseng-astragalus blood-sugar lowering using Chempattern software
Particle liquid-phase chromatograph finger print atlas carries out similarity evaluation.SHENQI JIANGTANG KELI liquid-phase chromatograph finger print atlas is shown in attached drawing 8, wherein
The peak 1-8 is respectively as follows: ginsenoside Re, ginsenoside Rg1, Ginsenoside Rc, ginsenoside Rd, schizandrin, Schisandra chinensis
Alcohol second, schizandrin A, deoxyschizandrin.Similarity evaluation schematic diagram is shown in attached drawing 9, and wherein ordinate represents similarity.
The results show that 10 batch SHENQI JIANGTANG KELI liquid phase spectrogram similarities are all larger than 0.985, similarity is good.
Claims (8)
1. a kind of ginseng-astragalus blood-sugar lowering preparation content determining method, which is characterized in that the method measurement is in ginseng-astragalus blood-sugar lowering preparation
The content of 10 active constituents, 10 active constituents are respectively as follows: ginsenoside Rb1, Ginsenoside Rc, ginsenoside
Rd, ginsenoside Rg1, ginsenoside Re, Astragaloside IV, schizandrin A, deoxyschizandrin, schizandrin, schisandrol
Second, described method includes following steps:
(1) mixed reference substance solution is prepared
Weigh reference substance respectively: ginsenoside Rb1, Ginsenoside Rc, ginsenoside Rd, ginsenoside Rg1, ginsenoside Re,
Astragaloside IV, schizandrin A, deoxyschizandrin, schizandrin, wuweizi alcohol B are configured to mix using methanol as solvent
Reference substance solution mother liquor；
(2) internal standard compound standard solution is prepared
Internal standard compound tanshinone IIA is weighed, using methanol as solvent, is configured to internal standard compound standard solution；
(3) test solution is prepared
Ginseng-astragalus blood-sugar lowering preparation is crushed, smashed ginseng-astragalus blood-sugar lowering powder formulation is weighed and is mixed with methanol, ultrasonic extraction, extracting solution
Evaporating solvent under reduced pressure obtains medicinal extract；Gained medicinal extract is dissolved in water, then is saturated with water butanol solution extraction, collects extract liquor
Evaporating solvent under reduced pressure, freeze-drying, obtains ginseng-astragalus blood-sugar lowering preparation extract；Using methanol as solvent, gained ginseng-astragalus blood-sugar lowering system is prepared
Agent extract solution, centrifugation take supernatant, cross miillpore filter, obtain test solution；
(4) test sample detects
Using HPLC-Q-MS instrument, the internal standard compound standard of step (2) preparation is added in the test solution of step (3) preparation
Solution, sample detection obtain the mass spectrogram of test sample；
The running parameter condition of the HPLC-Q-MS instrument is as follows:
Liquid chromatogram separation condition are as follows: chromatographic column: using octadecylsilane chemically bonded silica as filler, partial size≤5 μm, mobile phase A
For acetonitrile, Mobile phase B is the aqueous formic acid of volume fraction 0.1%, using gradient elution, 30 DEG C of column temperature, and flow velocity 1.0mL/
Min, sampling volume 20 μ L, Detection wavelength 203nm；
Single level four bars Mass Spectrometry Conditions are as follows: use ESI electric spray ion source, voltage: 3000V is atomized 350 DEG C of room temperature, dry gas
Flow velocity 12.0L/min, positive ion mode, SIM ion monitoring mode open ion monitoring channel 1, ion monitoring channel 2；
The eluting solvent that liquid chromatogram separates gradient elution is carried out by following time sequencing, and the meaning of the percentage sign in table is volume
Percentage:
；
(5) standard curve is drawn
The mixed reference substance solution mother liquor for taking step (1) to prepare mixes reference substance at several pieces according to various concentration gradient dilution
The internal standard compound standard solution of step (2) preparation is added in every part of mixed reference substance solution sample, then uses for liquor sample
HPLC-Q-MS instrument is detected according to the running parameter condition in step (4), obtains the mass spectrogram of mixing reference substance；With mixed
Closing each single respective sample introduction concentration of reference substance in reference substance is abscissa, and each single reference substance respectively corresponds in mass spectrogram
Peak area and the ratio of internal standard compound peak area be ordinate, draw standard 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, wuweizi alcohol B standard curve and equation of linear regression；
(6) test sample content detection result
Each active compound component respectively corresponding peak area and internal standard compound peak face in the test sample mass spectrogram that step (4) is measured
Long-pending ratio substitutes into the equation of linear regression of each reference substance obtained in step (5), and ginseng soap in test solution is calculated
Glycosides Rb1, Ginsenoside Rc, ginsenoside Rd, ginsenoside Rg1, ginsenoside Re, Astragaloside IV, schizandrin A, Schisandra chinensis
The content of B prime, schizandrin, wuweizi alcohol B, and then containing for 10 kinds of active constituents is calculated in ginseng-astragalus blood-sugar lowering preparation
Amount.
2. ginseng-astragalus blood-sugar lowering preparation content determining method as described in claim 1, which is characterized in that the ginseng-astragalus blood-sugar lowering preparation
For tablet, capsule or granule.
3. ginseng-astragalus blood-sugar lowering preparation content determining method as described in claim 1, which is characterized in that prepared in step (1)
In mixed reference substance solution mother liquor, final concentration of 99.3325 μ g/mL of ginsenoside Rb1, Ginsenoside Rc it is final concentration of
109.2657 μ g/mL, final concentration of 141.0522 μ g/mL of ginsenoside Rd, ginsenoside Rg1 final concentration of 152.5747
μ g/mL, final concentration of 224.4914 μ g/mL of ginsenoside Re, final concentration of 52.4476 μ g/mL of Astragaloside IV, Schisandra chinensis
Final concentration of 42.9116 μ g/mL of A prime, final concentration of 55.6262 μ g/mL of deoxyschizandrin, the end of schizandrin are dense
Degree is the final concentration of 127.1456 μ g/mL of 55.6262 μ g/mL, wuweizi alcohol B.
4. ginseng-astragalus blood-sugar lowering preparation content determining method as described in claim 1, which is characterized in that prepared in step (2)
In internal standard compound standard solution, the concentration of tanshinone IIA is 80 μ g/mL.
5. ginseng-astragalus blood-sugar lowering preparation content determining method as described in claim 1, which is characterized in that prepared in step (3)
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 described in claim 1, which is characterized in that in step (4), the Dan Si
Under grade bar mass spectrum SIM ion monitoring mode, title, molecular formula, monitoring opening time and the prison of binary channels monitoring objective compound
Measured ion molecular weight is as follows:
The compound information that ion monitoring channel 1 monitors
The compound information that ion monitoring channel 2 monitors
。
7. ginseng-astragalus blood-sugar lowering preparation content determining method as described in claim 1, which is characterized in that in step (5), described is 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.
8. application of the content assaying method as described in claim 1 in the global quality control of ginseng-astragalus blood-sugar lowering preparation.
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| CN109425671A (en) * | 2017-09-04 | 2019-03-05 | 复旦大学 | A kind of ginsenoside Rg1, double internal standard high-efficiency liquid chromatography method for detecting of Re, Rb1 |
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