CN109425671A - A kind of ginsenoside Rg1, double internal standard high-efficiency liquid chromatography method for detecting of Re, Rb1 - Google Patents
A kind of ginsenoside Rg1, double internal standard high-efficiency liquid chromatography method for detecting of Re, Rb1 Download PDFInfo
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Abstract
The invention belongs to Pharmaceutical Analysis fields, are related to a kind of double internal standard high-efficiency liquid chromatography method for detecting of ginsenoside, in particular to ginsenoside Rg1 in a kind of ginseng, double internal standard high-efficiency liquid chromatography method for detecting of Re, Rb1.In detection method of the invention, high performance liquid chromatography uses C18 chromatographic column, and phosphate aqueous solution, acetonitrile are eluent gradient elution, and the qualitative analysis of ginsenoside is carried out according to the retention time of sample, carries out quantitative analysis according to its peak area and internal standard peak area ratio.The method has many advantages, such as that quick, sensitive, favorable reproducibility, the rate of recovery are high.
Description
Technical field
The invention belongs to Pharmaceutical Analysis fields, are related to a kind of double internal standard high-efficiency liquid chromatography method for detecting, and in particular to
A kind of double internal standard high-efficiency liquid chromatography method for detecting of ginsenoside, in particular to ginsenoside Rg1 in a kind of ginseng, Re, Rb1
Double internal standard high-efficiency liquid chromatography method for detecting.
Background technique
Ginseng has had the history of more than one thousand years as the drug of tonic and treatment disease, has and increases quantity of leucocyte, mentions
High body immunity promotes the effects of metabolism, antifatigue, anti-aging.It is main in ginseng that research, which discloses ginsenoside,
Ingredient, and the main component of ginseng pharmacological activity is played, currently, more than 40 kinds of ginseng soaps are had found from panax species
Glycosides, wherein the ginsenoside of most bioactivity has ginsenoside Rg1, ginsenoside Re and ginsenoside Rb1, and
It is proved to it with pharmacological actions such as oncotherapy, Liver protection, protection angiocarpy.But research is also shown, different sources, different product
Kind ginseng its saponin content have differences larger, therefore, measure ginsenoside Rg1, ginsenoside Re and ginseng soap in ginseng
The content of glycosides Rb1 is particularly important in evaluation ginseng curative effect, guarantee drug safety.
Ginsenoside ultraviolet detection method based on efficient liquid phase it has been reported that however, since extraction process technique is cumbersome,
Constituent of ginseng is complicated, therefore so far, still rapidly and accurately ginsenoside in Ginseng extract can not be measured in the industry.Internal standard method
It is more more accurate than external standard method quantitative approach in chromatography, wherein in analyzing test sample when constituent content, be added
A kind of internal standard compound is to calibrate and eliminate fluctuation due to operating condition and on resulting influence is analyzed, to improve analysis result
Accuracy.Since ginsenoside Rg1 and ginsenoside Re, ginsenoside Rb1's content difference are larger in different ginseng kinds, this
The inventor of application is quasi- differ biggish ingredient with three kinds of concentration simultaneously using double internal standards and compares, and analyzes result with raising
Accuracy, specifically, proposed found a kind of double internal standard high-efficiency liquid chromatography method for detecting accurately to detect people in different ginseng kinds
Join saponin(e Rg1, the content of Re, Rb1 provide reliable analysis means for identification Ginseng Quality and effect.
Summary of the invention
The purpose of the present invention is to provide a kind of quick, accurate, specificities by force, ginsenoside in the ginseng of high sensitivity
Double internal standard high-efficiency liquid chromatography method for detecting of Rg1, Re, Rb1.
To achieve the above object, technical solution of the present invention are as follows:
Ginsenoside Rg1 in a kind of ginseng, double internal standard high-efficiency liquid chromatography method for detecting of Re, Rb1 comprising, it uses
Chromatographic column is C18 filler, 5 μm of partial sizes, 4.6 × 250mm specification；Mobile phase is 0.1% phosphate aqueous solution (A), acetonitrile (B), ladder
Degree elution, flow velocity 1.3mL/min, 30 DEG C of Detection wavelength 203nm of column temperature；Chromatography condition of gradient elution is 0-26min 78%A+
22%B, 26.01-30min are 70%A+30%B by 78%A+22%B linear transformation, and 30.01-55min is by 70%A+30%B
Linear transformation is 68%A+32%B, and 55.01-60min is 55%A+35%B, 60- by 68%A+32%B linear transformation
60.01min is 78%A+32%B by 55%A+35%B linear transformation, and 60.01-70min 78%A+32%B detects ginseng soap
Glycosides Rg1, Re, Rb1.
In the present invention, this method separating degree with higher can be made using gradient elution, and reduce analysis time.
Further, the detecting instrument is UV detector.
Further, the method is double internal standard methods, and internal standard compound is notoginsenoside R and dexamethasone；
In the present invention, internal standard method can reduce operating error caused by sample pretreatment in the process, and avoid sample volume
Variation, chromatographic condition influence of the minor change to quantitative result, keep measurement result more accurate；
In the present invention, double internal standards can more accurately analyze the content of different saponin(es in ginseng, and dexamethasone and Radix Notoginseng
Saponin(e R1 and ginsenoside Rg1, Re, Rb1 structure is similar, has similar physicochemical property, it is ensured that the accuracy of internal standard method.
Further, the internal standard compound internal standard compound notoginsenoside R selection ginsenoside Rg1 quantifies, dexamethasone choosing
Ginsenoside Re is selected, ginsenoside Rb1 quantifies, the high specificity and high sensitivity of ensuring method.
Further, determinand is Ginseng extract.How much content of ginsenoside can intuitively reflect in Ginseng extract
The quality and drug effect of ginseng out provides reference for ginseng place of production screening varieties.
Further, the determinand pre-treating method is soxhlet extraction.Soxhlet extraction can effectively remove ginseng
Middle lipid material, and it is convenient, the effective components such as ginsenoside are fully extracted from Ginseng Root Powder.Above-mentioned technical proposal
It has the beneficial effect that
It is of the invention provide it is a kind of quickly, accurate, specificity is strong, ginsenoside Rg1 in high sensitivity ginseng, Re,
Double internal standard high-efficiency liquid chromatography method for detecting of Rb1；The high performance liquid chromatography has high specificity, highly sensitive spy
Point, core be that component to be measured post analysis first can be separated with other components, is particularly suitable for the more complicated ginseng of ingredient
Extracting solution, and then realize quickly and accurately to ginsenoside quantitative study in sample；In the present invention internal standard compound notoginsenoside R and
The application of dexamethasone can effectively reduce influence of the operating error to measurement result, further increase the accurate of detection method
Degree and precision, soxhlet extraction can effectively remove lipid material in ginseng in the present invention, and it is convenient, fully from ginseng
The effective components such as ginsenoside are extracted in powder.
Detailed description of the invention
Fig. 1 is samples of Ginseng extracting solution high-efficient liquid phase chromatogram.
Fig. 2 is ginsenoside Rg1, Re, Rb1 canonical plotting.
Specific embodiment
The present invention is described in further detail below in conjunction with drawings and examples.
Embodiment 1
Instrument and reagent:
1) analysis instrument
Shimadzu SPD-10A high performance liquid chromatograph, purchased from Japanese Shimadzu Corporation；Chromatographic column is Ultimate Polar RP,
5 μm, 4.6 × 250mm, it is purchased from Shanghai Welch company.
2) test drug
Ginsenoside Rb1, Re standard items are provided by Sinopharm Chemical Reagent Co., Ltd. (Wo Kai)；Ginsenoside Rg1
Standard items are provided by Aladdin chemical reagent Co., Ltd；Dexamethasone standard items, by Dalian U.S. logical sequence Technology Co., Ltd.
It provides；Notoginsenoside R standard items, Chinese biological drug inspection office provide.Ginseng, purchased from Chinese Taobao.
Methanol, the chromatography pure reagent that acetonitrile is the production of Xinlan's scape chemical industry Co., Ltd, during chloroform, n-butanol be
The analytical reagents of medicine (group) Solution on Chemical Reagents in Shanghai company, state production, phosphoric acid are raw for Sinopharm Chemical Reagent Co., Ltd.
The analytical reagents of production；Pure water is Millipore deionized water.
Test method and result:
1) prepared by Ginseng extract
Samples of Ginseng is taken, is crushed, No. four sieves is crossed, accurately weighs 200mg, it is accurately weighed, it sets in Soxhlet extractor, adds trichlorine
Heat methane flows back 3 hours, discards chloroform liquid, and the dregs of a decoction volatilize solvent, moves into 100mL conical flask together with filtration paper cylinder, essence
Close plus water-saturated n-butanol 50mL, close plug are stood overnight, and ultrasonic treatment (power 250W, frequency 50kHz) 30 minutes filters, and are abandoned
Primary filtrate is gone, precision measures subsequent filtrate 25mL, sets in evaporating dish and be evaporated, residue adds methanol to dissolve and is transferred in 5mL measuring bottle, adds
Methanol dilution shakes up to scale, filters, take subsequent filtrate to get；
2) chromatographic separation condition
Chromatographic column: WelchTM (moon rising sun) Ultimate Polar RP, 5 μm, 4.6*250mm；Mobile phase: mobile phase is
0.1% phosphate aqueous solution (A), acetonitrile (B), gradient elution, flow velocity 1.3mL/min, 30 DEG C of column temperature, Detection wavelength 203nm；Color
Spectrum condition of gradient elution is 0-26min 78%A+22%B, and 26.01-30min is 70%A+ by 78%A+22%B linear transformation
30%B, 30.01-55min are 68%A+32%B by 70%A+30%B linear transformation, and 55.01-60min is by 68%A+32%B
Linear transformation is 55%A+35%B, and 60-60.01min is 78%A+32%B, 60.01- by 55%A+35%B linear transformation
70min 78%A+32%B；
3) standard curve is established
Precision weighs ginsenoside Rg1 6.00mg, is placed in 10.00mL volumetric flask, adds methanol to dissolve and is diluted to scale,
Ginsenoside Rg1's Standard Reserving Solution to get 0.6mg/mL, accurate ginsenoside Re are shaken up, ginsenoside Rb1 40mg is placed in
To get the ginsenoside Re of 4mg/mL, Rb1 Standard Reserving Solution in 10mL volumetric flask；
Accurate draw is equivalent to 1200 μ g, 600 μ g, 120 μ g, 60 μ g from ginsenoside Rg1's Standard Reserving Solution, 30 μ g's
Sample is placed in 5 10.0mL measuring bottles, and 220 μ g notoginsenoside Rs and 500ug dexamethasone is added, with methanol constant volume, with ginseng
The ratio between the peak area and internal standard notoginsenoside R peak area of saponin(e Rg1 standard reference material (ARg1/AR1) to corresponding concentration (C, μ g/
ML linear regression) is carried out, obtains ginsenoside Rg1's calibration curve equation are as follows: y=0.0511x+0.124, R2=0.9997, linearly
Range 3-120 μ g/mL (ginsenoside Rg1's standard curve is as shown in Figure 2)；This method measures the minimum quantitative of ginsenoside Rg1
It is limited to 2.5 μ g/mL；
Accurate draw is equivalent to 8000 μ g, 4000 μ g, 800 μ g, 400 μ g, 200 μ from ginsenoside Re's Standard Reserving Solution
G, the sample of 40 μ g are placed in 6 10.0mL measuring bottles, and 220 μ g notoginsenoside Rs and 500ug dexamethasone is added, fixed with methanol
Hold, with the ratio between the peak area of ginsenoside Re's standard reference material and internal standard dexamethasone peak area (ARe/AGround) to corresponding concentration
(C, μ g/mL) carries out linear regression, obtains ginsenoside Re's calibration curve equation are as follows: y=0.0052x+0.0204, R2=
0.9996, the range of linearity 4-800 μ g/mL (ginsenoside Re's standard curve is as shown in Figure 2)；This law measures ginsenoside Re most
Low basis weight is limited to 2.5 μ g/mL；
Accurate draw is equivalent to 8000 μ g, 4000 μ g, 800 μ g, 400 μ g, 200 μ from ginsenoside Rb1's Standard Reserving Solution
G, the sample of 40 μ g are placed in 6 10.0mL measuring bottles, and 220 μ g notoginsenoside Rs and 500ug dexamethasone is added, fixed with methanol
Hold, with the ratio between the peak area of ginsenoside Rb1's standard reference material and internal standard dexamethasone peak area (ARb1/AGround) to corresponding dense
It spends (C, μ g/mL) and carries out linear regression, obtain ginsenoside Re's calibration curve equation are as follows: y=0.0048x+0.0173, R2=
0.9997, the range of linearity 4-800 μ g/mL (ginsenoside Rb1's standard curve is as shown in Figure 2)；This law measures ginsenoside Rb1's
It is minimum to be quantitatively limited to 2.5 μ g/mL；
4) preci-sion and accuracy is investigated
By above-mentioned same operation, the people of 5.9 μ g/mL, 59 μ g/mL, the basic, normal, high three kinds of various concentrations of 88.5 μ g/mL is prepared
Join saponin(e Rg1 standard items, internal standard concentration is 22 μ g/mL notoginsenoside Rs.Every kind concentration standard liquid sample introduction 3 times, substitute into standard
Curve calculates and acquires the rate of recovery by the ratio of measured amount and actual content, as a result basic, normal, high concentration samples measured amount difference
For 5.82 ± 0.11 μ g/mL, 58.54 ± 0.65 μ g/mL, 85.53 ± 0.29 μ g/mL, the rate of recovery is 98.69% ± 0.28%,
99.23% ± 1.11%, 96.64% ± 0.32%, RSD value is 1.90%, 1.12%, 0.34%.The rate of recovery and RSD value exist
In prescribed limit, illustrate that the detection method is accurate, reliable；
Prepare the ginsenoside of the basic, normal, high three kinds of various concentrations of 39.75 μ g/mL, 397.50 μ g/mL, 596.25 μ g/mL
Re standard items, internal standard concentration are 50 μ g/mL dexamethasone.Every kind concentration standard liquid sample introduction 3 times, substitute into standard curve, calculate
Acquire the rate of recovery by the ratio of measured amount and actual content, as a result basic, normal, high concentration samples measured amount be respectively 38.75 ±
0.40 μ g/mL, 413.13 ± 0.36 μ g/mL, 597.21 ± 0.92 μ g/mL, the rate of recovery be 97.49% ± 1.03%,
103.93% ± 0.09%, 100.16% ± 0.16%, RSD value is 1.03%, 0.09%, 0.15%.The rate of recovery and RSD value are equal
Within the specified scope, illustrate that the detection method is accurate, reliable；
Prepare the ginsenoside of the basic, normal, high three kinds of various concentrations of 39.00 μ g/mL, 390.00 μ g/mL, 595.00 μ g/mL
Rb1 standard items, internal standard concentration are 50 μ g/mL dexamethasone.Every kind concentration standard liquid sample introduction 3 times, substitute into standard curve, calculate
Acquire the rate of recovery by the ratio of measured amount and actual content, as a result basic, normal, high concentration samples measured amount be respectively 37.83 ±
0.31 μ g/mL, 399.41 ± 1.57 μ g/mL, 578.38 ± 1.54 μ g/mL, the rate of recovery be 97.00% ± 0.53%,
102.41% ± 2.66%, 98.87% ± 1.74%, RSD value is 0.82%, 0.39%, 0.27%.The rate of recovery and RSD value are equal
Within the specified scope, illustrate that the detection method is accurate, reliable；
5) sample recovery rate is investigated
Precision weighs tri- parts of Ginseng Root Powder 200mg, is separately added into containing 286 μ g of ginsenoside Rg1,270 μ of ginsenoside Re
G, 322 μ g of ginsenoside Rb1 mix standard solution, and the deposit containing 220 μ g and dexamethasone 500ug of notoginsenoside R
Liquid volatilizes at room temperature, extracts by aforementioned Ginseng Root Powder extraction method, and sample recovery rate is calculated in sample introduction.As a result ginsenoside
Rg1 sample recovery rate is 107.20% ± 11.07%, and ginsenoside Re's sample recovery rate is 91.65% ± 8.08%, ginseng soap
Glycosides Rb1 sample recovery rate is 87.37% ± 6.12%.Sample recovery rate within the specified scope, illustrate the detection method it is accurate,
Reliably.
Above embodiments are only the explanation of technical idea of the invention, and this does not limit the scope of protection of the present invention, all
It is any changes made on the basis of the technical scheme according to the technical idea provided by the invention, each falls within present invention protection model
Within enclosing.
Claims (6)
1. a kind of ginsenoside Rg1, double internal standard high-efficiency liquid chromatography method for detecting of Re, Rb1, characterized in that it comprises: make
It is C18 filler, 5 μm of partial sizes, 4.6 × 250mm specification with chromatographic column；Mobile phase be 0.1% phosphate aqueous solution (A), acetonitrile (B),
Gradient elution, flow velocity 1.3mL/min, 30 DEG C of column temperature；Detection wavelength 203nm；Chromatography condition of gradient elution is 0-26min
78%A+22%B, 26.01-30min are 70%A+30%B by 78%A+22%B linear transformation, and 30.01-55min is by 70%A+
30%B linear transformation is 68%A+32%B, and 55.01-60min is 55%A+35%B, 60- by 68%A+32%B linear transformation
60.01min is 78%A+32%B by 55%A+35%B linear transformation, and 60.01-70min 78%A+32%B detects ginseng soap
Glycosides Rg1, Re, Rb1.
2. ginsenoside Rg1 according to claim 1, double internal standard high-efficiency liquid chromatography method for detecting of Re, Rb1 are special
Sign is that the detecting instrument used in the method is UV detector.
3. ginsenoside Rg1 according to claim 1, double internal standard high-efficiency liquid chromatography method for detecting of Re, Rb1 are special
Sign is that the method is double internal standard methods, and internal standard compound is notoginsenoside R and dexamethasone.
4. according to the method described in claim 3, it is characterized in that, the ginsenoside Rg1 selects internal standard compound notoginsenoside R
It is quantified, ginsenoside Re, ginsenoside Rb1 selects dexamethasone to quantify.
5. the method according to claim 1, wherein the determinand in the method is Ginseng extract.
6. according to the method described in claim 5, it is characterized in that, it is soxhlet extraction that the determinand, which obtains pre-treating method,.
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| CN113960218A (en) * | 2021-11-23 | 2022-01-21 | 天津师范大学 | Method for rapidly and quantitatively detecting notoginsenoside |
| CN114152686A (en) * | 2021-11-10 | 2022-03-08 | 湖南易能生物医药有限公司 | Fingerprint construction method and application of traditional Chinese medicine compound containing cinnamon |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114152686A (en) * | 2021-11-10 | 2022-03-08 | 湖南易能生物医药有限公司 | Fingerprint construction method and application of traditional Chinese medicine compound containing cinnamon |
| CN114152686B (en) * | 2021-11-10 | 2023-11-03 | 湖南易能生物医药有限公司 | Fingerprint construction method of traditional Chinese medicine compound containing cinnamon and application of fingerprint construction method |
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