CN109115923A - A kind of quality determining method of Fructus Sophorae granule - Google Patents
A kind of quality determining method of Fructus Sophorae granule Download PDFInfo
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Abstract
The invention discloses a kind of detection methods of Fructus Sophorae granule, and the invention also discloses a kind of detection methods of Sophoricoside in huaijiao agent.The quality determining method of Fructus Sophorae granule of the present invention, it creates comprising Fructus Sophorae granule thin-layered chromatography, Fructus Sophorae granule content determination, detection method is simple, the present invention also provides the detection methods of Sophoricoside in huaijiao agent, the accuracy in detection of this method is high, has a good application prospect.
Description
Technical field
Present invention relates particularly to a kind of quality determining methods of Fructus Sophorae granule.
Background technique
The Fructus Sophorae is the dry mature fruit of leguminous plant Chinese scholartree Sophora japonica L..
Chinese scholartree (Chinese scholar tree) phytomorph: deciduous tree, 15-25 meters high, the diameter of a cross-section of a tree trunk 1.3 meters above the ground is up to 1 meter.Tree crown spheroidal is wealthy oval, tree
Skin is coarse, there is irregular lobe, dark gray, and endothelium foresythia has odor.Imparipinnate leaf alternate, petiole base expand,
Rachis hairiness, vanelets 7-17, petiolule hairiness；Stipule sickle shaped, vanelets ovum shape lanceolar or ovate oblong, apex tool
Thin prominent point, full edge, basal circular or wide wedge shape, above bottle green, smoothly, lower colourless green lies prostrate raw white hair, master pulse is significant, side
Arteries and veins is unobvious.Basidixed large size panicle, bennet and the equal hairiness of pedicel；Calyx is bell；Corolla butterfly, yellow-white；Stamen 10, from
Raw or base portion slightly joint, filigree Length discrepancy；Ovary is upper, tubular, there is elongated hair, style bending.Pod is cylindrical, hairless, green
Color, meat do not crack, sagging, and fruit apex has apicule beak object, and contracting of obviously hanging between seed has 1-6 seed in beads shape.Seed
Brownish black, kidney shape.Position with medical value is the fruit of its drying and ripening.The ripening fruits for harvesting Chinese scholartree in winter, removes
Impurity, it is dry.
The Fructus Sophorae contains the multiple compounds such as flavonoids and osajin, mainly there is genistein (Genistein), sophoricoside
(Sopho-ricoside), sophorabioside (Sophorabioside), Kaempferol glucosides-C (Kaempferol glycoside-
C), the chemical components such as Sophora flavonoid glycoside (Sophoraflavonoloside) and rutin (Rutin)." Chinese Pharmacopoeia " 2015
Detection method under year one item of version in Fructus Sophorae quality standard is identified only using Sophoricoside as reference substance by high liquid chromatography method
And assay, to judge Fructus Sophorae quality.Method is single, and accuracy is not high, is not applied for the detection of huaijiao agent class Chinese medicine.
Summary of the invention
To solve the above problems, the present invention provides the quality determining methods and huaijiao agent of a kind of Fructus Sophorae granule
The detection method of middle Sophoricoside.
A kind of detection method of huaijiao agent of the present invention, it includes following operating procedure:
A, the preparation of control medicinal material solution:
Fructus Sophorae control medicinal material is taken, extracting in water filters, and filtrate is evaporated, and residue adds methanol to dissolve, molten as control medicinal material
Liquid；
B, the preparation of test solution:
Test sample is taken, methanol is added to extract, is filtered, filtrate is evaporated, and residue adds methanol to dissolve, as test solution.
C, thin-layer chromatography measures:
Aforementioned control medicinal material solution and test solution are taken, puts on silica gel thin-layer plate, is with ethyl acetate-formic acid-water
Solvent, detection.
The test sample is Huaijiao granule.
The volume ratio of water described in step a and control medicinal material is 100mL:1g；It is extracted as boiling extraction described in step a；Step a
The additional amount of the methanol is 4 times of ml/g of Fructus Sophorae control medicinal material.
The volume ratio of methanol described in step b and test sample is 20mL:1g；Ultrasonic extraction is extracted as described in step b, preferably
Ground, Ultrasound Instrument the power 300W, frequency 25kHz of the ultrasonic extraction；The extraction time is 30min；Alcohol described in step b adds
Enter 2 times of ml/g that amount is Fructus Sophorae granule.
The amount that methanol is added in residue described in step a and step b is 2ml.
The acetic ether-methanoic acid of solvent described in step c-water ratio is 8: 1: 0.1；Detecting step described in step c includes: pre-
Balance is unfolded, and takes out, dries, and sprays with 1% alchlor ethanol solution, sets and inspect under ultraviolet lamp；Preferably, described pre- flat
The weighing apparatus time is 30min, and the ultraviolet lamp wavelength is 254nm.
The present invention also provides a kind of detection methods of Sophoricoside in huaijiao agent, include the following steps:
1) preparation of reference substance solution
Sophoricoside reference substance is taken, methanol is added to dissolve, as reference substance solution；
2) preparation of test solution
Test sample is taken, ethyl alcohol extracts, and filtering draws filtrate methanol dilution, as test solution；
3) assay
Draw reference substance solution and test solution respectively, inject liquid chromatograph, measurement to get；
Chromatographic condition is as follows:
Detection wavelength: 260nm；
Chromatographic column: C18 chromatographic column；
Mobile phase: mobile phase is the mixed solution of mobile phase A, Mobile phase B and mobile phase C, their volume ratio is
10%:18%:72%, isocratic elution.
The test sample is Huaijiao granule.
The reference substance solution concentration that step 1) the Sophoricoside reference substance adds methanol to configure is 50 μ g/ml.
In step 2), the ethyl alcohol that the ethyl alcohol is 70% is described to be extracted as ultrasonic extraction；Step 2) the test sample and second
The mass volume ratio of alcohol is 1g:125mL；The Fructus Sophorae granule, Fructus Sophorae medicine materical crude slice test sample filtrate and methanol dilution ratio are
1:5。
The sample-adding amount of reference substance solution and test solution amount that liquid chromatograph is injected separately into described in step 3) is 10 μ
l；Step 3) the C18 chromatographic column be different brands C18 (250 × 4.6mm, 5 μm), preferably 1) iamonsilC18 (250 ×
4.6mm, 5 μm).
The column temperature of chromatographic condition described in step 3) is 25 DEG C, 30 DEG C, 35 DEG C, preferably 30 DEG C；Step 3) the step f
The flow velocity of the chromatographic condition is 0.80mlmin-1、1.00ml·min-1, preferably 1.00mlmin-1。
The quality determining method of Fructus Sophorae granule of the present invention is created comprising Fructus Sophorae granule thin-layered chromatography, Chinese scholartree
Angle granule content determination, detection method is simple, and the present invention also provides the detection methods of Sophoricoside in huaijiao agent, should
The accuracy in detection of method is high, has a good application prospect.
Obviously, above content according to the present invention is not being departed from according to the ordinary technical knowledge and customary means of this field
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made.
The specific embodiment of form by the following examples remakes further specifically above content of the invention
It is bright.But the range that this should not be interpreted as to the above-mentioned theme of the present invention is only limitted to example below.It is all to be based on above content of the present invention
The technology realized all belongs to the scope of the present invention.
Detailed description of the invention
Fig. 1: thin-layer chromatography spectrogram, (1411103) 3. Fructus Sophorae granule of 1. Fructus Sophorae control medicinal material, 2. Fructus Sophorae granule
(15003952) 6. Fructus Sophorae granule of (130501) 5. Fructus Sophorae granule of (130301) 4. Fructus Sophorae granule
8. Fructus Sophorae granule (1305001S) of (1411001S) 7. Fructus Sophorae granule (1401001W), 9. Fructus Sophorae granule
(5062871) 10. Fructus Sophorae granules (407304L).
Fig. 2: acetic ether-methanoic acid-water is the thin-layer chromatography spectrogram of solvent.1. 2~4. Fructus Sophorae of Fructus Sophorae control medicinal material is matched
Square particle.
Fig. 3: acetate-methanol-formic acid is the thin-layer chromatography spectrogram of solvent.1. 2~4. Fructus Sophorae of Fructus Sophorae control medicinal material
Granule.
Fig. 4: 1~4. Fructus Sophorae control medicinal material of thin-layer chromatography spectrogram (1 μ l, 2 μ l, 5 μ l, 10 μ l) under different point sample amounts, 5
~8. Fructus Sophorae granules (1 μ l, 2 μ l, 5 μ l, 10 μ l).
Fig. 5: 1. Fructus Sophorae control medicinal material of thin-layer chromatography spectrogram, 2~4. Fructus Sophorae granule of Qingdao Haiyang gel GF 254 plate.
Fig. 6: 1. Fructus Sophorae control medicinal material of thin-layer chromatography spectrogram, 2~4. Fructus Sophorae formula of Tianjin Si Lida gel GF 254 plate
Grain.
1. Fructus Sophorae control medicinal material of thin-layer chromatography spectrogram, 2~4. Fructus Sophorae granule under Fig. 7: 8 DEG C of environmental conditions.
1. Fructus Sophorae control medicinal material of thin-layer chromatography spectrogram, 2~4. Fructus Sophorae granule under Fig. 8: 25 DEG C of environmental conditions.
1. Fructus Sophorae control medicinal material of thin-layer chromatography spectrogram, 2~4. Fructus Sophorae granule under Fig. 9: 42% humidity atmosphere.
1. Fructus Sophorae control medicinal material of thin-layer chromatography spectrogram, 2~4. Fructus Sophorae formula under Figure 10: 75% humidity atmosphere
Grain.
Figure 11: Sophoricoside full wavelength scanner figure.
Figure 12: mobile phase methanol: acetonitrile: 0.07% phosphoric acid (12:20:68) sample chromatogram figure.
Figure 13: mobile phase methanol: acetonitrile: 0.07% phosphoric acid (10:18:72) sample chromatogram figure.
Figure 14: Sophoricoside reference substance chromatogram.
Figure 15: Fructus Sophorae granule sample chromatogram figure.
Figure 16: reference substance, sample and scarce Fructus Sophorae negative sample chromatogram.
Figure 17: Sophoricoside canonical plotting.
Specific embodiment
Embodiment 1
1) identification of Fructus Sophorae granule:
A, the preparation of control medicinal material solution:
Fructus Sophorae control medicinal material 0.5g is taken, water 50ml is added, is boiled 30 minutes, is filtered, filtrate is evaporated, and residue adds methanol 2ml, system
At control medicinal material solution；
B, the preparation of test solution:
9 batch Fructus Sophorae granules are taken respectively, it is finely ground, 1.0g is respectively taken, methanol 20ml is added, is ultrasonically treated 30 minutes (function
Rate 300W, frequency 25kHz), filtration, filtrate is evaporated, and residue adds methanol 2ml to make to dissolve, as test solution；
C, thin-layer chromatography measures:
Aforementioned control medicinal material solution and test solution are taken, is put respectively on same silica GF254 lamellae, with acetic acid second
Ester-formic acid-water (8: 1: 0.1) is solvent, is pre-equilibrated 30 minutes, is unfolded, and takes out, dries, and is sprayed molten with 1% alchlor ethyl alcohol
Liquid is set and is inspected under ultraviolet lamp (254nm), in sample chromatogram, on position corresponding with reference medicine chromatography, shows identical face
The fluorescence spot of color.Such as Fig. 1
2) Sophoricoside assay in Fructus Sophorae granule；
D, the preparation of reference substance solution
Sophoricoside reference substance is taken, it is accurately weighed, add methanol that the solution of every 50 μ g/ml is made, as reference substance solution；
E, the preparation of test solution
9 batch Fructus Sophorae granules are taken respectively, it is finely ground, 0.2g is respectively taken, it is accurately weighed, it sets in stuffed conical flask, it is accurate
70% ethyl alcohol 25ml, weighed weight is added, ultrasonic treatment (power 300W, frequency 25kHz) 45 minutes is let cool, then weighed heavy
Amount, the weight of less loss is supplied with 70% ethyl alcohol.Precision measures 2ml and sets in 10ml volumetric flask, and methanol dilution is added to shake up to scale,
Filtration, as test solution；
F, assay
It is accurate respectively to draw reference substance solution and each 10 μ l of test solution, inject liquid chromatograph, measurement；
Chromatographic condition is as follows:
Detection wavelength: 260nm；
Chromatographic column: DiamonsilC18 (250 × 4.6mm, 5 μm)；
Mobile phase: -0.07% phosphoric acid solution (C) of methanol (A)-acetonitrile (B), isocratic elution (10%A:18%B:72%C)；
Flow velocity: 1.0ml/min
Column temperature: 30 DEG C
Measure the content of Sophoricoside in different batches Fructus Sophorae granule.It the results are shown in Table 1
Sophoricoside assay in 1 sample of table
According to the test result of above-mentioned 9 batches of Fructus Sophorae granules, the content of Sophoricoside is put down between 22.2-79.9mg/g
Mean value is 47.89mg/g.Sophoricoside (C21H20O10) content of Fructus Sophorae granule is no less than 20.0mg/g.
Beneficial effects of the present invention are illustrated below by way of test example:
1 thin-layer identification method of test example
1. instrument and reagent and experimental material
Instrument
Semi-automatic thin layer sample counter (CAMAG Lionmat-5 170605), thin layer imaging system CAMAG TLC
Visualizer 170513)；KQ5200DB type numerical control ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.)；Silica gel
GF254 lamellae (Haiyang Chemical Plant, Qingdao).
Reagent and reagent
The chemical reagent such as ethyl acetate, formic acid, methanol are that analysis is pure.
Experimental material
Fructus Sophorae granule (lot number: 1411103,130301,130501,15003952,1411001S, 1401001W,
1305001S,5062871,407304L)；Fructus Sophorae control medicinal material (Nat'l Pharmaceutical & Biological Products Control Institute provides, lot number:
121214-0101)。
2. prepared by solution
The preparation of test solution: taking this product, finely ground, takes 1.0g, adds methanol 20ml, is ultrasonically treated 30 minutes, filtration,
Filtrate is evaporated, and residue adds methanol 2ml to make to dissolve, as test solution.
The preparation of control medicinal material solution: taking Fructus Sophorae control medicinal material 0.5g, add water 50ml, boils 30 minutes, and filtration, filtrate is steamed
Dry, residue adds methanol 2ml, is made in the same way of control medicinal material solution.
3. the comparison of different solvents
Take control medicinal material solution and each 5 μ l of test solution respectively, put on same silica GF254 lamellae, respectively with
Acetic ether-methanoic acid-water (8: 1: 0.1), acetate-methanol-formic acid (4:1:
0.5) it is solvent, pre-equilibrates 30 minutes, be unfolded, take out, dry, sprays with 1% alchlor ethanol solution, set purple
It is inspected under outer smooth lamp (254nm), in sample chromatogram, on position corresponding with reference medicine chromatography, shows the glimmering of same color
Hot spot point.With acetic ether-methanoic acid-water (8: 1: 0.1) be solvent result see Fig. 2, with acetate-methanol-formic acid (4:1:
0.5) Fig. 3 is seen for solvent result.
With acetic ether-methanoic acid-water (8: 1: 0.1) be plate chromatography developing solvent ratio with acetate-methanol-formic acid (4:
1:0.5) more preferable for solvent, spot Rf value is moderate, and separating degree is good.Therefore using acetic ether-methanoic acid-water (8: 1:
It 0.1) is plate chromatography developing solvent as Fructus Sophorae granule quality Qualitive test.
4. the comparison of point sample amount
1 μ l of control medicinal material solution, 2 μ l, 5 μ l, 10 μ l, 1 μ l of test sample (lot number 1411103) solution, 2 μ l, 5 μ are taken respectively
L, 10 μ l points are unfolded on same silica GF254 lamellae, map such as Fig. 4.The result shows that 2 μ l of control medicinal material solution, examination
When product solution 2 μ l of point sample, optimum efficiency can reach.Therefore, 2 μ l of control medicinal material solution, 2 μ l of test sample solution point sample are selected, as
Best point sample amount.
5. the comparison of different lamellaes
Choose the prefabricated gel GF 254 plate of Haiyang Chemical Plant, Qingdao, Tianjin Si Lida Science and Technology Ltd. can cut out type thin layer
Chromatoplate (gel GF 254 plate) is tested by the test method drafted respectively, map such as Fig. 5, Fig. 6 the result shows that, two kinds of thin layers
Plate can reach identification and require.
6. the comparison of different temperatures
Lamellae after taking point sample is unfolded under 8 DEG C and 25 DEG C of room temperature of low temperature of temperature environment respectively, and map is as schemed
7, Fig. 8 the result shows that, this method is preferable to the adaptability of different temperatures.
7. the comparison of different humidity
Lamellae after taking point sample is unfolded under 42% and 75% humidity environment respectively, map such as Fig. 9, Figure 10.
The result shows that this method is preferable to the adaptability of different humidity.
The experiment results show that carrying out the qualitative detection of Huaijiao granule using the method for the present invention, accuracy in detection is high.
2 content determination of test example
1. instrument and reagent
Instrument
U.S.'s Waters2695 high performance liquid chromatograph；Waters2996 diode array detector, Empower chromatography
Work station；Electronic balance: BP211D (Sautoris, Germany) d=0.1mg/0.01mg, max=210g/80g；Ultrasonic wave is clear
Wash device: KQ5200DB type, Instrument Ltd. of city of Kunshan.
Reagent, reagent
Methanol (chromatographically pure), acetonitrile (chromatographically pure), water are redistilled water, and other reagents are that analysis is pure.
2. reference substance and sample source
Sophoricoside reference substance is purchased from Nat'l Pharmaceutical & Biological Products Control Institute (for assay, lot number 111695-
200501).Fructus Sophorae granule derives from granule manufacturer, (lot number: 1411103,130301,130501,
15003952、1411001S、1401001W、1305001S、5062871、 407304L)。
3, chromatographic condition and system suitability are tested
The selection of 3.1 Detection wavelengths
It takes Sophoricoside reference substance solution to be scanned in 200~400nm respectively, according to atlas analysis, it is found that Sophoricoside exists
There is absorption maximum at 259.6, with reference to the Detection wavelength under one Fructus Sophorae [assay] item of " Chinese Pharmacopoeia " version in 2015, determines
The Detection wavelength of Sophoricoside is 260nm, sees Figure 11.
The selection of 3.2 mobile phases
Consider the mobile phase of the assay under one Fructus Sophorae medicinal material item of " Chinese Pharmacopoeia " version in 2015, i.e. methanol: acetonitrile:
0.07% phosphoric acid solution (12:20:68), by sample drawing it is found that the symmetry at Sophoricoside peak is high, theoretical cam curve is high, separating degree
Preferably, but Sophoricoside peak retention time is shorter, such as Figure 12, table 2.
2 sample chromatogram result of table
The mobile phase under " Chinese Pharmacopoeia " 2015 editions Fructus Sophorae assays is optimized, i.e. methanol: acetonitrile: 0.07% phosphorus
Acid solution (10:18:72), the results showed that, the symmetry at Sophoricoside peak is high, and separating degree is good, and Sophoricoside retention time is moderate and manages
By number of plates height, therefore we determined that methanol: acetonitrile: 0.07% phosphoric acid solution (10:18:72) is Fructus Sophorae granule quality mark
The method of quasi- assay text.See Figure 13, table 3.
3 sample chromatogram result of table
The selection of 3.3 chromatographic columns
Investigate the chromatographic column separating effect of two kinds of different size chromatographic columns and three kinds of different brands.Different size: enlightening horse
Diamonsil-C18 (250 × 4.6mm, 5 μm), enlightening horse Diamonsil-C18 (150 × 4.6mm, 5 μm)；Different brands: enlightening
Horse Diamonsil-C18 (250 × 4.6mm, 5 μm), Agilent ZORBAX Eclipse XDB-C18 (Agilent, 250 ×
4.6mm, 5 μm), Féraud door C18 column (phenomenex company, 250 × 4.6mm, 5 μm)
Draw reference substance and each 10 μ l of test solution respectively, injection be respectively provided with two kinds of different sizes (250 ×
4.6mm, 5 μm), the liquid chromatograph of (150 × 4.6mm, 5 μm) chromatographic column.With chromatostrip determining under " 3.1 ", " 3.2 " item
Part, the separating effect of more each chromatographic column and each index, the former is better than the latter.This experiment use chromatographic column specification for (250 ×
4.6mm,5μm)。
For three kinds of different brands C18 (250 × 4.6mm, 5 μm) chromatographic column, it can achieve the effect that separation.Experiment card
Bright, which is suitable for the chromatographic column of different brands.
The selection of 3.4 column temperatures
Reference substance and each 10 μ l of test solution are drawn in this experiment respectively at a temperature of 25 DEG C, 30 DEG C, 35 DEG C, inject liquid
Chromatography.With chromatographic condition determining under " 3.1 ", " 3.2 " item, compare the separation feelings at index components Sophoricoside peak at each temperature
Condition.
The result shows that target peak can reach preferable separating effect, adaptation of this method to temperature at three temperature
Property is preferable.The column temperature that this experiment uses is 30 DEG C.
The selection of 3.5 flow velocitys
This experiment draws reference substance respectively under the flow velocity of 0.8ml/min, 1.0ml/min, 1.2ml/min and test sample is molten
Each 10 μ l of liquid injects liquid chromatograph.With chromatographic condition determining under " 3.1 ", " 3.2 " item, index components under more each flow velocity
The separation situation at Sophoricoside peak.
The result shows that target peak can reach preferable separating effect, this experiment under 0.8ml/min, 1.0ml/min flow velocity
The column flow rate 1.0ml/min of use.
4. the determination of chromatographic condition and system test
Chromatographic column: Di Ma company DiamonsilC18 (250 × 4.6mm, 5 μm)；Mobile phase: methanol: acetonitrile: 0.07% phosphorus
Sour (10:18:72) Detection wavelength is 360nm；Flow velocity is 1.0ml/min.
Under selected chromatographic condition, in Sophoricoside peak and sample other component chromatographic peaks can baseline separation, Sophoricoside peak
It is greater than 1.5 with the separating degree of adjacent chromatographic peak；It is calculated by Sophoricoside peak, theoretical cam curve (N) is greater than 3000, can reach detection
It is required that therefore this method can be as the quality determining method of Fructus Sophorae granule.See Figure 14, Figure 15.
5, the preparation of reference substance solution: taking Sophoricoside reference substance, accurately weighed, and methanol is added to be made containing 50 μ g/ml of Sophoricoside
Mixed solution to get.
6, the preparation of test sample
6.1 the investigation of Extraction solvent
Sample (lot number 1411103) 0.2g is weighed respectively, it is four parts, accurately weighed, it sets in stuffed conical flask, it is accurate respectively
Different solvents 25ml, weighed weight is added, ultrasonic treatment (power 300W, frequency 25kHz) 45 minutes is let cool, then weighed weight,
The weight of less loss is supplied with coordinative solvent.Precision measures 2ml and sets in 10ml volumetric flask, adds methanol dilution to scale, shakes up, filter
Cross to get.It is shown in Table 4.
The investigation of 4 different solvents of table
The above result shows that: the total content highest that 70% ethyl alcohol extracts, therefore select 70% ethyl alcohol as Extraction solvent.
6.2 extracting methods are investigated
Sample (lot number 1411103) 0.2g is weighed respectively, it is three parts, accurately weighed, it sets in stuffed conical flask, precision is added
70% ethyl alcohol 25ml, weighed weight, respectively ultrasound, flow back, stand overnight 3 kinds of processing methods, let cool, then weighed weight, use
70% ethyl alcohol supplies the weight of less loss.Precision measures 2ml and sets in 10ml volumetric flask, and methanol dilution is added to shake up to scale, filters,
To obtain the final product.It is shown in Table 5.
The comparison of 5 Different Extraction Method of table
The above result shows that: ultrasound is all higher than the content extraction for being heated to reflux and standing overnight, and ultrasound procedure is easy, nothing
Pollution, therefore select extracting method of the ultrasonic extracting method as this experiment.
6.3 extraction times were investigated
Sample (lot number 1411103) 0.2g is weighed respectively, it is four parts, accurately weighed, it sets in stuffed conical flask, precision is added
70% ethyl alcohol 25ml, weighed weight, respectively be ultrasonically treated (power 300W, frequency 25kHz) 15min, 30min, 45min,
60min different time, lets cool, then weighed weight, and the weight of less loss is supplied with 70% ethyl alcohol.Precision measures 2ml and sets 10ml capacity
In bottle, methanol dilution is added to shake up to scale, filtration to get.It is shown in Table 6.
The comparison of the different extraction times of table 6
The above result shows that: it can sufficiently extract within ultrasonic extraction 45 minutes, ultrasonic time extends Sophoricoside content and no longer increases
Add.Therefore choosing ultrasound is used as extraction time in 45 minutes.
7, methodological study
Negative sample each 0.5g of 7.1 specificity test sampling product (lot number 1411103) and the scarce Fructus Sophorae, it is accurately weighed, it sets
In stuffed conical flask, test solution and negative control solution are prepared by the preparation method of test article drafted.It draws above two
Solution and each 10 μ l of reference substance solution inject liquid chromatograph.It can be obtained in result negative control chromatogram by experiment without Sophoricoside
As a result peak proves that auxiliary material is noiseless.See Figure 16.
7.2 linear relationships are investigated
It is accurate respectively to draw Sophoricoside reference substance solution (Sophoricoside concentration is 0.0572mg/ml) 1 μ l, 2 μ l, 5 μ l, 10 μ
L, 20 μ l inject liquid chromatograph, measure peak area.With the sample volume (μ g) of Sophoricoside for abscissa, with the peak face of Sophoricoside
Product (A) is that ordinate carries out linear regression, obtains the standard curve of Sophoricoside.Sophoricoside calibration curve equation is Y=3E+06X-
34097, r2=1, for Sophoricoside then in 0.0572~1.144 μ g range, the peak area and sample volume of reference substance have good line
Sexual intercourse.Such as Figure 17, table 7.
7 Sophoricoside reference substance measurement result of table
7.3 precision tests: accurate respectively to draw Sophoricoside reference substance solution (0.0572mg/ml) 10 μ l, continuous sample introduction 6
It is secondary, Sophoricoside peak area is measured, RSD is calculated, the results are shown in Table 8.
8 Sophoricoside Precision test result of table
Result above Sophoricoside RSD% is 0.54%, less than 3.0%, shows that instrument precision is good.
7.4 repetitive tests: weigh same batch of sample (lot number: 1411103) 6 parts of Fructus Sophorae granule, it is accurately weighed, press
Sample test solution is made in the above sample solution preparation method, measures Sophoricoside peak area respectively, calculates content, calculates
RSD the results are shown in Table 9.
9 Sophoricoside repetitive test result of table
The above result shows that: Sophoricoside RSD is 0.21%, and less than 3.0%, this method repeatability is good.
The test of 7.5 Intermediate precisions: three experimenters respectively sample (lot number: 1411103) one in two times respectively
Part, test solution is prepared by sample solution preparation method.Sophoricoside peak area is measured respectively, calculates content, and calculate
RSD the results are shown in Table 10.
10 Sophoricoside Intermediate precision test result of table
The RSD of Sophoricoside known to the above table is 0.65%, less than 3.0%, shows that this method Intermediate precision is good.
7.6 stability tests: sample (lot number 1411103) 0.2g is taken, prepares sample test sample by said determination method
Solution, set respectively different time 0,1,2,8,12, for 24 hours, 10 μ l of sample introduction measures peak area, calculates RSD.It the results are shown in Table 11.
11 Sophoricoside stability test result of table
Indicated above: Sophoricoside RSD is 0.49%, and less than 3.0%, Sophoricoside index ingredient is at 24 in results sample
Stablize in hour.
7.7 recovery tests: take the sample of known content (lot number 1411103, Sophoricoside content are 60.3mg/g)
0.1g, it is totally 6 parts, accurately weighed, it sets in stuffed conical flask, is separately added into Sophoricoside reference substance solution (concentration 0.0572mg/
Ml) volume, by above-mentioned sample preparation methods, preparation sample-adding recycling test solution is contained by above-mentioned chromatographic condition measurement Sophoricoside
Amount calculates sample recovery rate according to following formula, the results are shown in Table 12.
12 Sophoricoside recovery test result of table
Find out above, between 95%-105%, the average recovery rate of Sophoricoside is the sample pipetting volume rate of recovery
Less than 3.0%, test result shows that sample recovery rate is good by 100.38%, RSD 1.30%, RSD.
The experiment results show that the method for the present invention can detect the Sophoricoside in huaijiao agent with accurate and effective, application prospect is excellent.
Claims (10)
1. a kind of detection method of huaijiao agent, it is characterised in that: it includes following operating procedure:
A, the preparation of control medicinal material solution:
Fructus Sophorae control medicinal material is taken, extracting in water filters, and filtrate is evaporated, and residue adds methanol to dissolve, as control medicinal material solution；
B, the preparation of test solution:
Test sample is taken, methanol is added to extract, is filtered, filtrate is evaporated, and residue adds methanol to dissolve, as test solution.
C, thin-layer chromatography measures:
Take aforementioned control medicinal material solution and test solution, point is expansion with acetic ether-methanoic acid-water on silica gel thin-layer plate
Agent, detection.
2. quality determining method according to claim 1, it is characterised in that: the volume of water described in step a and control medicinal material
Than for 100mL:1g；It is extracted as boiling extraction described in step a；The additional amount of methanol described in step a is 4 times of Fructus Sophorae control medicinal material
ml/g。
3. quality determining method according to claim 1, it is characterised in that: the volume of methanol described in step b and test sample
Than for 20mL:1g；Ultrasonic extraction is extracted as described in step b, it is preferable that the Ultrasound Instrument power 300W of the ultrasonic extraction, frequency
25kHz；The extraction time is 30min；The additional amount of alcohol described in step b is 2 times of ml/g of Fructus Sophorae granule.
4. quality determining method according to claim 1, it is characterised in that: first is added in residue described in step a and step b
The amount of alcohol is 2ml.
5. quality determining method according to claim 1, it is characterised in that: the ethyl acetate of solvent described in step c-first
Acid-water ratio is 8: 1: 0.1；Detecting step described in step c includes: to pre-equilibrate, and is unfolded, and takes out, dries, and is sprayed with 1% tri-chlorination
Aluminium ethanol solution is set and is inspected under ultraviolet lamp；Preferably, the pre-equilibration time is 30min, and the ultraviolet lamp wavelength is
254nm。
6. the detection method of Sophoricoside in a kind of huaijiao agent, characterized by the following steps:
1) preparation of reference substance solution
Sophoricoside reference substance is taken, methanol is added to dissolve, as reference substance solution；
2) preparation of test solution
Test sample is taken, ethyl alcohol extracts, and filtering draws filtrate methanol dilution, as test solution；
3) assay
Draw reference substance solution and test solution respectively, inject liquid chromatograph, measurement to get；
Chromatographic condition is as follows:
Detection wavelength: 260nm；
Chromatographic column: C18 chromatographic column；
Mobile phase: mobile phase is the mixed solution of mobile phase A, Mobile phase B and mobile phase C, their volume ratio is 10%:
18%:72%, isocratic elution.
7. detection method according to claim 6, it is characterised in that: step 1) the Sophoricoside reference substance adds methanol to configure
Reference substance solution concentration be 50 μ g/ml.
8. detection method according to claim 6, it is characterised in that: in step 2), the ethyl alcohol that the ethyl alcohol is 70%, institute
It states and is extracted as ultrasonic extraction；The mass volume ratio of the step 2) test sample and ethyl alcohol is 1g:125mL；The Fructus Sophorae formula
Grain, Fructus Sophorae medicine materical crude slice test sample filtrate and methanol dilution ratio are 1:5.
9. detection method according to claim 6, it is characterised in that: be injected separately into liquid chromatograph described in step 3)
The sample-adding amount of reference substance solution and test solution amount is 10 μ l；Step 3) the C18 chromatographic column is the C18 (250 of different brands
× 4.6mm, 5 μm), preferably 1) iamonsilC18 (250 × 4.6mm, 5 μm).
10. detection method according to claim 6, it is characterised in that: the column temperature of the step 3) chromatographic condition
For 25 DEG C, 30 DEG C, 35 DEG C, preferably 30 DEG C；The flow velocity of chromatographic condition described in step 3) the step f is 0.80mlmin-1、
1.00ml·min-1, preferably 1.00mlmin-1。
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