CN101813674B - Method for measuring content of kaempferol glucose rhamnoside contained in folium ginkgo or related preparation thereof - Google Patents
Method for measuring content of kaempferol glucose rhamnoside contained in folium ginkgo or related preparation thereof Download PDFInfo
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Abstract
The invention discloses a method for measuring the content of kaempferol glucose rhamnoside contained in folium ginkgo or a related preparation thereof, comprising the following steps of: measuring the kaempferol glucose rhamnoside contained in the folium ginkgo as a test article or the related preparation thereof by utilizing a high performance liquid chromatography and using the kaempferol glucose rhamnoside as a reference substance under the condition without negative disturbance; and computing to obtain the content of the kaempferol glucose rhamnoside contained in the folium ginkgo or the related preparation thereof by an external reference method. The method overcomes the shortages of inaccurate measuring result, time and labor waste in the hydrolyzing process, and the like of the traditional measuring method without hydrolysis when preparing a test article solution, can accurately measure the real content of the kaempferol glucose rhamnoside contained in the folium ginkgo or the preparation thereof and is beneficial to effectively controlling the quality of related products.
Description
Technical field
The invention belongs to drug quality control field, relate to a kind of method of measuring Kaempferol glucose rhamnoside content in ginkgo leaf or its related preparations.
Background technology
Ginkgo leaf has another name called folium ginkgo bilobae, derives from the dried leaves of Ginkgoaceae plant Ginkgo biloba (Ginkgo biloba L.), in China long medicinal history is arranged, and is mainly used in the treatment deficiency syndrome of the lung and coughs and breathe heavily and disease such as palpitation and severe palpitation.
Modern pharmacology research shows that ginkgo leaf has the oxygen radical of removing, reducing blood lipid, enhancing central nervous system function, regulates neurotransmitter and hormonal readiness, improves effects such as hemorheology state, anti-inflammatory, antiallergy.Thereby more and more with the preparation variety of ginkgo leaf development, clinical practice is more and more widely.Si Tailong, Ginkgo Leaf Agent, Tian Bao are peaceful for having of using clinically at present, Gin Kgo Plus, Shu Xuening, network are glad logical; The tablet of multiple brand such as ketone for curing coronary heart disease, granule, injection are mainly used in the various cardiovascular and cerebrovascular diseases of treatment, diabetes, the nervous system disease and respiratory disease etc.
Main effective constituent is flavonoids and lactone two constituents in ginkgo leaf and the preparation thereof, and up to the present, the method for quality control about ginkgo leaf and preparation thereof generally is a content of measuring this two constituents both at home and abroad.Method about flavonoids assay in ginkgo leaf and the preparation thereof; Though high performance liquid chromatography and thin layer chromatography scanning have been arranged; But its measuring principle all is on the basis of hydrolysis; With Quercetin, Kaempferide and three kinds of flavone aglycones of Isorhamnetin is contrast, measures through conversion, and the major defect of these two kinds of methods is: (1) needs hydrolysis.In hydrolytic process, owing to many-sided reasons such as operations, phenomenons such as the not enough or excessive hydrolysis of hydrolysis possibly take place, it is inaccurate to cause measuring the result; And hydrolytic process wastes time and energy, and has a strong impact on work efficiency.What (2) measure is the content of flavone aglycone.Because what measure is the content of the flavone aglycone after the hydrolysis, can not accurately reflect the real content of flavonoid glycoside composition in ginkgo leaf and the preparation thereof, can not reach the purpose of real control ginkgo leaf and quality of the pharmaceutical preparations homogeneity thereof.
Summary of the invention
The purpose of this invention is to provide a kind of method of measuring Kaempferol glucose rhamnoside content in ginkgo leaf or its related preparations.
The method of Kaempferol glucose rhamnoside content comprises the steps: in mensuration ginkgo leaf provided by the invention or its related preparations
Do not having under the negative situation about disturbing; With the Kaempferol glucose rhamnoside is reference substance; Utilize high performance liquid chromatography to measuring, calculate the content of Kaempferol glucose rhamnoside in said ginkgo leaf or its related preparations by external standard method as the said ginkgo leaf of test sample or the Kaempferol glucose rhamnoside in its related preparations.
In this method, before said determination step, earlier said reference substance and said test sample are dissolved in the solvent; The solvent that dissolves said reference substance is selected from the WS of methyl alcohol, methanol in water, ethanol, ethanol and in the moving phase any one; The solvent that dissolves said test sample is selected from the WS of methyl alcohol, methanol in water, ethanol, ethanol and in the moving phase any one.
In the said determination step, the detection wavelength is 190nm～400nm, preferred 360nm.Moving phase is any one among following solvents a～solvent c: said solvent a is a methanol in water, and said solvent b is the WS of acetonitrile, and said solvent c is the mixed liquor that methyl alcohol, acetonitrile and water are formed.Said moving phase also can be the mixed liquor of at least a composition in any one and dressing agent, buffer solution, ionic inhibitor and the pH value correctives among the solvent a-solvent c; Wherein, said solvent a is a methanol in water, and said solvent b is the WS of acetonitrile, and said solvent c is the mixed liquor that methyl alcohol, acetonitrile and water are formed; Said dressing agent is selected from any one in tetrahydrofuran, isopropyl alcohol and the dioxane; Said buffer solution is selected from any one in PBS, acetate buffer solution and the citrate buffer solution; Said ionic inhibitor is selected from any one in sodium dodecylsulphonate, undecyl sodium sulfonate, sodium hexanesulfonate, sodium heptanesulfonate, perfluorooctane sulfonate and the nonane sodium sulfonate; Said pH value correctives is selected from any one in formic acid, acetate, phosphoric acid and the citric acid.The addition of said dressing agent is 0.5%～10.0%, preferred 3.0%～5.0% of said solvent a or solvent b or a solvent c volume; The addition of said buffer solution is 0.5%～10.0%, preferred 3.0%～5.0% of said solvent a or solvent b or a solvent c volume; The addition of said ionic inhibitor is 0.1%～3.0%, preferred 0.5%～1.0% of said solvent a or solvent b or a solvent c volume; The addition of said pH value correctives is 0.05%～5.0%, preferred 0.2%～1.0% of said solvent a or solvent b or a solvent c volume.Said related preparations is ginkgo leaf medicinal material, ginkgo biloba p.e, contain the solid pharmaceutical preparation of ginkgo leaf or contain the liquid preparation of ginkgo leaf; Wherein, Said solid pharmaceutical preparation is pill, granule, tablet, capsule, pill, powder or medicinal tea, and said liquid preparation is injection, oral liquid, tincture or soft extract.
The method of Kaempferol glucose rhamnoside content in mensuration ginkgo leaf provided by the invention or its related preparations can be the arbitrary method among the following method a-method c:
Said method a is:
1) solution of preparation reference substance: take by weighing Kaempferol glucose rhamnoside reference substance, add methyl alcohol and process the solution that every 1ml contains 0.04mg, obtain the solution of said reference substance;
2) preparation need testing solution:
(1) solution of preparation ginkgo leaf: get ginkgo leaf, pulverize, take by weighing 2g, the adding mass percentage concentration is 75% ethanol water 100ml, claims to decide weight; Placed the water-bath heating and refluxing extraction 40 minutes, and took out, put coldly, claim decide weight again, the use mass percentage concentration is that 75% ethanol water is supplied the weight that subtracts mistake; Shake up, filter, measure subsequent filtrate 25ml, evaporate to dryness, residue add water 2ml makes its dissolving; Place the polyamide column of having handled well, water 50ml wash-out discards water liquid, and using mass percentage concentration again is 75% ethanol water 100ml wash-out, collects eluent; Evaporate to dryness, residue add methyl alcohol makes its dissolving, quantitatively is transferred in the 5ml measuring bottle, is diluted to scale with methyl alcohol; Shake up,, get subsequent filtrate, obtain the solution of said ginkgo leaf with the miillpore filter filtration of 0.45 μ m; Wherein, Said polyamide column is handled according to following method: get polyamide granules and screen out fine powder with 60 mesh sieves, the water logging bubble that adds 2～3 times of weight makes it wetting, in the chromatographic column of packing into; The mass percentage concentration of using 2～5 times of column volumes is 2%～5% aqueous hydrochloric acid solution wash-out; Be eluted to neutrality with deionized water again, the mass percentage concentration of then using 2～5 times of column volumes is 2%～5% aqueous hydrochloric acid solution wash-out, is eluted to neutrality with deionized water again;
(2) solution of preparation ginkgo biloba p.e: take by weighing the batch number of producing available from Zhejiang Conba Pharmaceutical Co., Ltd. and be 070301 ginkgo biloba p.e 0.035g; Add methyl alcohol 10ml; Sonicated 10 minutes; Using the aperture is the miillpore filter filtration of 0.45 μ m, gets subsequent filtrate, obtains the solution of said ginkgo biloba p.e;
(3) solution of preparation Shu Xuening injection: using the aperture is the miillpore filter filtration Shu Xuening injection of 0.45 μ m, gets subsequent filtrate, obtains the solution of said Shu Xuening injection;
3) content of Kaempferol glucose rhamnoside in the said need testing solution of mensuration:
Selecting model for use is that Agilent ZORBAX Eclipse Plus C18, specification are that 50mm * 4.6mm, filling agent particle diameter are the performance liquid chromatographic column of 1.8 μ m, and the filling agent in the said chromatographic column is an octadecylsilane chemically bonded silica, is that the mixed liquor that 0.4% phosphate aqueous solution is formed is a moving phase with acetonitrile and mass percentage concentration; Accurate respectively said reference substance solution and each 2 μ l of said need testing solution of drawing; Inject said high performance liquid chromatograph respectively, according to carrying out gradient elution shown in the table 1, the equilibration time of each said gradient elution is 10 minutes; The flow velocity of said moving phase is per minute 0.6ml; Said detection wavelength is 360nm, and the column temperature of said chromatographic column is 40 ℃, and number of theoretical plate calculates by Kaempferol glucose rhamnoside peak and is not less than 8000; In the said table 1; A represents acetonitrile, and B representation quality percentage concentration is 0.4% phosphate aqueous solution, and % represents percent by volume;
Table 1, condition of gradient elution
High-efficient liquid phase chromatogram according to said test sample of gained and reference substance calculates the peak area of said Kaempferol glucose rhamnoside and the peak area of said reference substance, calculates according to the external standard method shown in the following formula:
Obtain the concentration of Kaempferol glucose rhamnoside in the said need testing solution;
Said method b is:
1) solution of preparation reference substance: take by weighing Kaempferol glucose rhamnoside reference substance, add methyl alcohol and process the solution that every 1ml contains 0.04mg, obtain the solution of said reference substance;
2) preparation need testing solution:
(1) solution of preparation ginkgo leaf: get ginkgo leaf, pulverize, take by weighing 2g, the adding mass percentage concentration is 75% ethanol water 100ml, claims to decide weight; Placed the water-bath heating and refluxing extraction 40 minutes, and took out, put coldly, claim decide weight again, the use mass percentage concentration is that 75% ethanol water is supplied the weight that subtracts mistake; Shake up, filter, measure subsequent filtrate 25ml, evaporate to dryness, residue add water 2ml makes dissolving; Place the polyamide column of having handled well, water 50ml wash-out discards water liquid, and using mass percentage concentration again is 75% ethanol water 100ml wash-out, collects eluent; Evaporate to dryness, residue add methyl alcohol makes its dissolving, quantitatively is transferred in the 5ml measuring bottle, is diluted to scale with methyl alcohol; Shake up,, get subsequent filtrate, obtain the solution of said ginkgo leaf with the miillpore filter filtration of 0.45 μ m; Wherein, Said polyamide column is handled according to following method: get polyamide granules and screen out fine powder with 60 mesh sieves, the water logging bubble that adds 2～3 times of weight makes it wetting, in the chromatographic column of packing into; The mass percentage concentration of using 2～5 times of column volumes is 2%～5% aqueous hydrochloric acid solution wash-out; Be eluted to neutrality with deionized water again, the mass percentage concentration of then using 2～5 times of column volumes is 2%～5% aqueous hydrochloric acid solution wash-out, is eluted to neutrality with deionized water again;
(2) solution of preparation ginkgo biloba p.e: take by weighing the batch number of producing available from Zhejiang Conba Pharmaceutical Co., Ltd. and be 070301 ginkgo biloba p.e 0.035g; Add methyl alcohol 10ml; Sonicated 10 minutes; Using the aperture is the miillpore filter filtration of 0.45 μ m, gets subsequent filtrate, obtains the solution of said ginkgo biloba p.e;
(3) solution of preparation Shu Xuening injection: using the aperture is that 0.45 μ m miillpore filter filters Shu Xuening injection, gets subsequent filtrate, obtains the solution of said Shu Xuening injection.
3) content of Kaempferol glucose rhamnoside in the said need testing solution of mensuration:
Selecting model for use is that HP 1100 type Inertsil
ODS-3 C18, specification are that 250mm * 4.6mm, filling agent particle diameter are the performance liquid chromatographic column of 5 μ m; Filling agent in the said chromatographic column is an octadecylsilane chemically bonded silica; The mixed liquor of forming with mobile phase A and Mobile phase B is a moving phase; Draw each 10 μ l of said reference substance solution and said need testing solution respectively; Inject said high performance liquid chromatograph respectively, according to carrying out gradient elution shown in the table 2, the equilibration time of each wash-out is 10 minutes; The flow velocity of moving phase is per minute 1.0ml; The wavelength of said detection is 360nm, and the column temperature of said chromatographic column is 30 ℃, and number of theoretical plate calculates by Kaempferol glucose rhamnoside peak and is not less than 8000; Said mobile phase A be by water, acetonitrile, isopropyl alcohol and citric acid with volume ratio 1000: 470: 50: 6.08 mix the mixed liquor that get, and said Mobile phase B is water, acetonitrile, isopropyl alcohol and citric acid with volume ratio 1400: 200: 30: the mixed liquor that 6.88 mixing get; In the said table 2, A represents said mobile phase A, and B represents said Mobile phase B, and % represents percent by volume;
Table 2, condition of gradient elution
High-efficient liquid phase chromatogram according to said test sample of gained and reference substance calculates the peak area of said Kaempferol glucose rhamnoside and the peak area of said reference substance, calculates according to the external standard method shown in the following formula:
Obtain the concentration of Kaempferol glucose rhamnoside in the said need testing solution;
Said method c is:
1) solution of preparation reference substance: it is an amount of to take by weighing Kaempferol glucose rhamnoside reference substance, adds methyl alcohol and processes the solution that every 1ml contains 0.04mg, obtains the solution of said reference substance;
2) preparation need testing solution:
(1) solution of preparation ginkgo biloba p.e: take by weighing the batch number of producing available from Zhejiang Conba Pharmaceutical Co., Ltd. and be 070301 ginkgo biloba p.e 0.035g; Add methyl alcohol 10ml; Sonicated 10 minutes; Using the aperture is the miillpore filter filtration of 0.45 μ m, gets subsequent filtrate, obtains the solution of said ginkgo biloba p.e;
(2) solution of preparation Shu Xuening injection: using the aperture is that 0.45 μ m miillpore filter filters Shu Xuening injection, gets subsequent filtrate, obtains the solution of said Shu Xuening injection.
3) content of Kaempferol glucose rhamnoside in the said need testing solution of mensuration:
Selecting model for use is that Inertsil ODS-3 C18, specification are that 250mm * 4.6mm, filling agent particle diameter are the performance liquid chromatographic column of 5 μ m; Filling agent in the said chromatographic column is an octadecylsilane chemically bonded silica; With acetonitrile and mass percentage concentration is that the mixed liquor that 0.4% phosphate aqueous solution is formed is a moving phase, draws respectively 10 μ l of said reference substance solution and said need testing solution respectively, injects said high performance liquid chromatograph respectively; According to carrying out gradient elution shown in the table 3; The equilibration time of each wash-out is 10 minutes, and the flow velocity of moving phase is per minute 1.0ml, and the wavelength of said detection is 360nm; The column temperature of said chromatographic column is 30 ℃, and number of theoretical plate calculates by Kaempferol glucose rhamnoside peak and is not less than 8000; In the said table 3, A represents acetonitrile, and B representation quality percentage concentration is 0.4% phosphate aqueous solution, and % represents percent by volume;
Table 3, condition of gradient elution
High-efficient liquid phase chromatogram according to said test sample of gained and reference substance calculates the peak area of said Kaempferol glucose rhamnoside and the peak area of said reference substance, calculates according to the external standard method shown in the following formula:
Obtain the concentration of Kaempferol glucose rhamnoside in the said need testing solution.
In the said determination method; The said situation of not having negative interference is meant in the compound preparation that contains ginkgo leaf in the prescription; Remove and remove other later all flavour of a drug of ginkgo leaf, the negative sample solution that makes according to the preparation method of former processing technology and need testing solution is according to same chromatographic condition; Behind the sample introduction, in chromatogram, do not have and the consistent chromatographic peak of reference substance chromatographic peak retention time.Said external standard method is meant under the chromatographic condition of regulation; Get reference substance solution and need testing solution sample introduction respectively; Confirm the chromatographic peak of tested composition in the test sample chromatogram according to the retention time of reference substance chromatographic peak; Compare with the area of the area of this chromatographic peak or peak height and reference substance chromatographic peak or peak height and concentration thereof again, calculate the concentration of tested composition in the test sample, be the content that obtains tested composition in the test sample.
The method of Kaempferol glucose rhamnoside content in the mensuration gingko leaf preparation provided by the invention; When the preparation need testing solution; Do not need hydrolysis; Overcome and measured shortcomings such as the result is inaccurate, hydrolytic process wastes time and energy in the existing assay method, can accurately measure the real content of flavonoid glycoside composition in ginkgo leaf and the preparation thereof, helped effectively controlling the quality of Related product.
Description of drawings
Fig. 1 is the HPLC collection of illustrative plates of reference substance among the embodiment 1.
Fig. 2 is as the HPLC collection of illustrative plates of the ginkgo leaf medicinal material of test sample among the embodiment 1.
Fig. 3 is as the HPLC collection of illustrative plates of the ginkgo biloba p.e of test sample among the embodiment 1.
Fig. 4 is as the HPLC collection of illustrative plates of the Shu Xuening injection of test sample among the embodiment 1.
Fig. 5 is the HPLC collection of illustrative plates of reference substance among the embodiment 2.
Fig. 6 is as the HPLC collection of illustrative plates of the ginkgo leaf medicinal material of test sample among the embodiment 2.
Fig. 7 is as the HPLC collection of illustrative plates of the ginkgo biloba p.e of test sample among the embodiment 2.
Fig. 8 is as the HPLC collection of illustrative plates of the Shu Xuening injection of test sample among the embodiment 2.
Fig. 9 is the HPLC collection of illustrative plates of reference substance among the embodiment 3.
Figure 10 is as the HPLC collection of illustrative plates of the ginkgo biloba p.e of test sample among the embodiment 3.
Figure 11 is as the HPLC collection of illustrative plates of the Shu Xuening injection of test sample among the embodiment 3.
Embodiment
Below in conjunction with specific embodiment the present invention is described further, but the present invention is not limited to following examples.Concentration described in the present invention is mass percentage concentration if no special instructions.
The structural confirmation data of Kaempferol glucose rhamnoside:
English name: kaempferol 3-O-2 " glucosylrhamnoside
Structural formula:
Get dry ginkgo leaf 10kg, with 40 liters of refluxing extraction of ethanol 2 times, extract merges; Concentrating under reduced pressure, mix appearance with column chromatography silica gel (100 orders～200 orders) 20kg after, put in the apparatus,Soxhlet's; Use each 4 liters of refluxing extraction of sherwood oil, ethyl acetate and methyl alcohol successively; Ethyl acetate extract separates through silica gel (200 orders～300 orders) column chromatography, and using volume ratio is that 95: 5～50: 50 the methenyl choloride and the mixed liquor of methyl alcohol carry out gradient elution, collects corresponding cut; Through silicagel column chromatographic resolution, purifying repeatedly, promptly get.The nuclear-magnetism testing result of this compound is following:
1HNMR(300MHz，CD
3OD)δ：0.94(3H，d，J＝5.9Hz，H-6″)，3.19-3.43(6H，m，sugarprotons)，3.70(2H，m，H-6″′)，3.81(1H，dd，J
3，2＝3.5Hz，J
3?4″＝9.3Hz，H-3″)，4.29(1H，dd，J
2， 1＝1.3Hz，J
2，3＝3.5Hz，H-2″)，4.42(1H，d，J＝7.7Hz，H-1″′)，5.73(1H，d，J＝1.1Hz，H-1″)，6.20(1H，d，J＝1.9Hz，H-6)，6.37(1H，d，J＝1.9Hz，H-8)，6.94(2H，d，J＝8.9Hz，H-3′and?H-5′)，7.76(2H，d，J＝8.9Hz，H-2′and?H-6′)。
13CNMR(50MHz，DMSO-d
6)：δ17.7(C-6rha)，62.4(C-6glc)，72.0(C-5rha)，78.0(C-C-5glc)，73.5(C-4rha)，71.0(C-4glc)，7.18(C-3rha)，77.9(C-3glc)，82.7(C-2rha)，5.4(C-2glc)，102.6(C-1rha)，107.1(C-1glc)，106.0(C.10)，158.6(C-9)，94.9(C-8)，166.0(C-7)，100.0(C-6)，163.2(C-5)，179.6(C-4)，135.5(C-3)，159.4(C-2)，132.0(C-2′，C-6′)，116.6(C-3′，C-5′)，161.7(C-4′)，122.6(C-1′)。
By on can know that this compound structure is correct.
Embodiment 1,
Chromatographic condition and system suitability test: the model of chromatographic column is Agilent ZORBAX Eclipse Plus C18; Specification is 50mm * 4.6mm, and 1.8 μ m, filling agent are octadecylsilane chemically bonded silica; The mixed liquor that with acetonitrile and concentration is 0.4% phosphate aqueous solution is a moving phase; According to carrying out gradient elution shown in the table 1, the equilibration time of each wash-out is 10 minutes, and the flow velocity of moving phase is per minute 0.6ml; The detection wavelength is 360nm, and column temperature is 40 ℃, and number of theoretical plate calculates by Kaempferol glucose rhamnoside peak should be not less than 8000.In the table 1, A represents acetonitrile, and it is 0.4% phosphate aqueous solution that B represents concentration, and the number percent of listed A and B (%) is percent by volume.In the listed time period in the table 1, between the ascending or descending elution zone that changes of the percent by volume of moving phase, can directly set the endpoints thereof of inner volume number percent between this elution zone, the blas in the chromatograph can be adjusted automatically.
Table 1, condition of gradient elution
The preparation of reference substance solution: it is an amount of that precision takes by weighing Kaempferol glucose rhamnoside reference substance, adds methyl alcohol and process the solution that every 1ml contains 0.04mg, promptly gets.
The preparation of need testing solution:
(1) ginkgo leaf: get these article of ginkgo leaf, pulverize, precision takes by weighing 2g, and the accurate 75% ethanol 100ml that adds claims to decide weight; Put in the water-bath heating and refluxing extraction 40 minutes, and took out, put coldly, claim again to decide weight, supply the weight that subtracts mistake with 75% ethanol; Shake up, filter, precision is measured subsequent filtrate 25ml, and evaporate to dryness, residue add water 2ml makes dissolving; Place the polyamide column of having handled well (30～60 orders, 3g, internal diameter 1cm), water 50ml wash-out discards water liquid; Use 75% ethanol 100ml wash-out again, collect eluent, evaporate to dryness, residue add methyl alcohol makes dissolving in right amount, quantitatively is transferred in the 5ml measuring bottle; Be diluted to scale with methyl alcohol, shake up,, get subsequent filtrate, promptly get with the miillpore filter filtration of 0.45 μ m.
Wherein, used polyamide column is handled according to following method: get the polyamide granules of newly purchasing, screen out fine powder with 60 mesh sieves; The water logging bubble that adds 2～3 times of weight makes it wetting, in the chromatogram of packing into, with 2%～5% hydrochloric acid solution wash-out of 2～5 times of column volumes; Be eluted to neutrality with deionized water again; Then, be eluted to neutrality with deionized water again, get final product with 2%～5% sodium hydroxide solution wash-out of 2～5 times of column volumes.
(2) ginkgo biloba p.e: it is 070301 ginkgo biloba p.e 0.035g that precision takes by weighing the batch number of producing available from Zhejiang Conba Pharmaceutical Co., Ltd.; The accurate methyl alcohol 10ml that adds, sonicated 10 minutes, using the aperture is the miillpore filter filtration of 0.45 μ m; Get subsequent filtrate, promptly get.
(3) Shu Xuening injection: get parenteral solution, using the aperture is that 0.45 μ m miillpore filter filters, and gets subsequent filtrate, promptly gets.
Measure: accurate respectively reference substance solution and each 2 μ l of above-mentioned three kinds of need testing solutions of drawing, inject high performance liquid chromatograph and measure, gained high performance liquid chromatography result such as Fig. 1-shown in Figure 4.Wherein, Fig. 1 is the HPLC collection of illustrative plates of reference substance; The retention time of Kaempferol glucose rhamnoside is 18.934 minutes, and Fig. 2 is the HPLC collection of illustrative plates as the ginkgo leaf medicinal material of test sample, and the retention time of Kaempferol glucose rhamnoside is 18.718 minutes; Fig. 3 be in as the HPLC collection of illustrative plates of the ginkgo biloba p.e of test sample; The retention time of Kaempferol glucose rhamnoside is 18.921 minutes, and Fig. 4 is the HPLC collection of illustrative plates as the Shu Xuening injection of test sample, and the retention time of Kaempferol glucose rhamnoside is 18.738 minutes.
According to the content of external standard method calculating Kaempferol glucose rhamnoside, concrete computation process is following:
When obtaining said test sample and being ginkgo leaf, wherein the concentration of Kaempferol glucose rhamnoside is 0.023mg/g;
According to last identical method, calculate said test sample when being ginkgo biloba p.e, wherein the concentration of Kaempferol glucose rhamnoside is 8.02mg/g; When said test sample was Shu Xuening injection, wherein the concentration of Kaempferol glucose rhamnoside was 0.023mg/ml.
Embodiment 2,
Chromatographic condition and system suitability test:
The model of chromatographic column is HP 1100 type Inertsil
ODS-3 C18; Specification is 250mm * 4.6mm; 5 μ m; Filling agent is an octadecylsilane chemically bonded silica, and the mixed liquor of forming with mobile phase A and Mobile phase B is a moving phase, according to carrying out gradient elution shown in the table 2; The equilibration time of each wash-out is 10 minutes, and the flow velocity of moving phase is per minute 1.0ml; The detection wavelength is 360nm, and column temperature is 30 ℃, and number of theoretical plate calculates by Kaempferol glucose rhamnoside peak should be not less than 8000.Said mobile phase A be by water, acetonitrile, isopropyl alcohol and citric acid with volume ratio 1000: 470: 50: 6.08 mix the mixed liquor that get, and said Mobile phase B is water, acetonitrile, isopropyl alcohol and citric acid with volume ratio 1400: 200: 30: the mixed liquor that 6.88 mixing get.In the table 2, A represents said mobile phase A, and B represents said Mobile phase B, and the number percent of listed A and B (%) is percent by volume.In the listed time period in the table 2, between the ascending or descending elution zone that changes of the percent by volume of moving phase, can directly set the endpoints thereof of inner volume number percent between this elution zone, the blas in the chromatograph can be adjusted automatically.
Table 2, condition of gradient elution
The preparation of reference substance solution: it is an amount of that precision takes by weighing Kaempferol glucose rhamnoside reference substance, adds methyl alcohol and process the solution that every 1ml contains 0.04mg, promptly gets.
The preparation of need testing solution:
(1) ginkgo leaf: get these article of ginkgo leaf, pulverize, precision takes by weighing 2g, and the accurate 75% ethanol 100ml that adds claims to decide weight; Put in the water-bath heating and refluxing extraction 40 minutes, and took out, put coldly, claim again to decide weight, supply the weight that subtracts mistake with 75% ethanol; Shake up, filter, precision is measured subsequent filtrate 25ml, and evaporate to dryness, residue add water 2ml makes dissolving; Place the polyamide column (30～60 orders, 3g, internal diameter 1cm) of handling (this disposal route is with embodiment 1) well, water 50ml wash-out discards water liquid; Use 75% ethanol 100ml wash-out again, collect eluent, evaporate to dryness, residue add methyl alcohol makes dissolving in right amount, quantitatively is transferred in the 5ml measuring bottle; Be diluted to scale with methyl alcohol, shake up,, get subsequent filtrate, promptly get with the miillpore filter filtration of 0.45 μ m.
(2) ginkgo biloba p.e: it is 070301 ginkgo biloba p.e 0.035g that precision takes by weighing the batch number of producing available from Zhejiang Conba Pharmaceutical Co., Ltd.; The accurate methyl alcohol 10ml that adds, sonicated 10 minutes is with the miillpore filter filtration of 0.45 μ m; Get subsequent filtrate, promptly get.
(3) Shu Xuening injection is got parenteral solution, with the miillpore filter filtration of 0.45 μ m, gets subsequent filtrate, promptly gets.
Measure: accurate respectively reference substance solution and each 10 μ l of above-mentioned three kinds of need testing solutions of drawing, inject high performance liquid chromatograph and measure, gained high performance liquid chromatography result such as Fig. 5-shown in Figure 8.Wherein, Fig. 5 is the HPLC collection of illustrative plates of reference substance; The retention time of Kaempferol glucose rhamnoside is 52.635 minutes, and Fig. 6 is the HPLC collection of illustrative plates as the ginkgo leaf medicinal material of test sample, and the retention time of Kaempferol glucose rhamnoside is 51.722 minutes; Fig. 7 be in as the HPLC collection of illustrative plates of the ginkgo biloba p.e of test sample; The retention time of Kaempferol glucose rhamnoside is 50.846 minutes, and Fig. 8 is the HPLC collection of illustrative plates as the Shu Xuening injection of test sample, and the retention time of Kaempferol glucose rhamnoside is 50.565 minutes.
According to the content of external standard method calculating Kaempferol glucose rhamnoside, concrete computation process is following:
When obtaining said test sample and being ginkgo leaf, wherein the concentration of Kaempferol glucose rhamnoside is 0.023mg/g;
According to last identical method, calculate said test sample when being ginkgo biloba p.e, wherein the concentration of Kaempferol glucose rhamnoside is 8.02mg/g; When said test sample was Shu Xuening injection, wherein the concentration of Kaempferol glucose rhamnoside was 0.023mg/ml.
Embodiment 3,
Chromatographic condition and system suitability test:
The model of chromatographic column is Inertsil ODS-3C18; Specification is 250mm * 4.6mm, and 5 μ m, filling agent are octadecylsilane chemically bonded silica; The mixed liquor that with acetonitrile and concentration is 0.4% phosphate aqueous solution is a moving phase; According to carrying out gradient elution shown in the table 3, the equilibration time of each wash-out is 10 minutes, and the flow velocity of moving phase is per minute 1.0ml; The detection wavelength is 360nm, and column temperature is 30 ℃, and number of theoretical plate calculates by Kaempferol glucose rhamnoside peak should be not less than 8000.In the table 3, A represents acetonitrile, and it is 0.4% phosphate aqueous solution that B represents concentration, and the number percent of listed A and B (%) is percent by volume.In the listed time period in the table 3, between the ascending or descending elution zone that changes of the percent by volume of moving phase, can directly set the endpoints thereof of inner volume number percent between this elution zone, the blas in the chromatograph can be adjusted automatically.
Table 3, condition of gradient elution
The preparation of reference substance solution: it is an amount of that precision takes by weighing Kaempferol glucose rhamnoside reference substance, adds methyl alcohol and process the solution that every 1ml contains 0.04mg, promptly gets.
The preparation of need testing solution
(1) ginkgo biloba p.e: it is 070301 ginkgo biloba p.e 0.035g that precision takes by weighing the batch number of producing available from Zhejiang Conba Pharmaceutical Co., Ltd.; The accurate methyl alcohol 10ml that adds, sonicated 10 minutes is with the miillpore filter filtration of 0.45 μ m; Get subsequent filtrate, promptly get.
(2) Shu Xuening injection: get parenteral solution,, get subsequent filtrate, promptly get with the miillpore filter filtration of 0.45 μ m.
Measure: accurate respectively reference substance solution and each 10 μ l of above-mentioned three kinds of need testing solutions of drawing, inject high performance liquid chromatograph and measure, gained high performance liquid chromatography result is shown in Fig. 9-11.Wherein, Fig. 9 is the HPLC collection of illustrative plates of reference substance; The retention time of Kaempferol glucose rhamnoside is 44.452 minutes, and Figure 10 is the HPLC collection of illustrative plates as the ginkgo biloba p.e of test sample, and the retention time of Kaempferol glucose rhamnoside is 44.332 minutes; Figure 11 is the HPLC collection of illustrative plates as the Shu Xuening injection of test sample, and the retention time of Kaempferol glucose rhamnoside is 44.335 minutes.
According to the content of external standard method calculating Kaempferol glucose rhamnoside, concrete computation process is following:
When obtaining said test sample and being ginkgo biloba p.e, wherein the concentration of Kaempferol glucose rhamnoside is 8.02mg/g; When said test sample was Shu Xuening injection, wherein the concentration of Kaempferol glucose rhamnoside was 8.02mg/ml.
Claims (1)
1. method of measuring Kaempferol glucose rhamnoside content in ginkgo leaf or its related preparations is to measure according to the arbitrary method among following method a, method b and the method c:
Said related preparations is ginkgo leaf, ginkgo biloba p.e or Shu Xuening injection;
Said method a is:
1) solution of preparation reference substance: take by weighing Kaempferol glucose rhamnoside reference substance, add methyl alcohol and process the solution that every 1ml contains 0.04mg, obtain the solution of said reference substance;
2) preparation need testing solution:
(1) solution of preparation ginkgo leaf: get ginkgo leaf, pulverize, take by weighing 2g, the adding mass percentage concentration is 75% ethanol water 100ml, claims to decide weight; Placed the water-bath heating and refluxing extraction 40 minutes, and took out, put coldly, claim decide weight again, the use mass percentage concentration is that 75% ethanol water is supplied the weight that subtracts mistake; Shake up, filter, measure subsequent filtrate 25ml, evaporate to dryness, residue add water 2ml makes its dissolving; Place the polyamide column of having handled well, water 50ml wash-out discards water liquid, and using mass percentage concentration again is 75% ethanol water 100ml wash-out, collects eluent; Evaporate to dryness, residue add methyl alcohol makes its dissolving, quantitatively is transferred in the 5ml measuring bottle, is diluted to scale with methyl alcohol; Shake up,, get subsequent filtrate, obtain the solution of said ginkgo leaf with the miillpore filter filtration of 0.45 μ m; Wherein, Said polyamide column is handled according to following method: get polyamide granules and screen out fine powder with 60 mesh sieves, the water logging bubble that adds 2～3 times of weight makes it wetting, in the chromatographic column of packing into; The mass percentage concentration of using 2～5 times of column volumes is 2%～5% aqueous hydrochloric acid solution wash-out; Be eluted to neutrality with deionized water again, the mass percentage concentration of then using 2～5 times of column volumes is 2%～5% aqueous hydrochloric acid solution wash-out, is eluted to neutrality with deionized water again;
(2) solution of preparation ginkgo biloba p.e: take by weighing the batch number of producing available from Zhejiang Conba Pharmaceutical Co., Ltd. and be 070301 ginkgo biloba p.e 0.035g; Add methyl alcohol 10ml; Sonicated 10 minutes; Using the aperture is the miillpore filter filtration of 0.45 μ m, gets subsequent filtrate, obtains the solution of said ginkgo biloba p.e;
(3) solution of preparation Shu Xuening injection: using the aperture is the miillpore filter filtration Shu Xuening injection of 0.45 μ m, gets subsequent filtrate, obtains the solution of said Shu Xuening injection;
3) content of Kaempferol glucose rhamnoside in the said need testing solution of mensuration:
Selecting model for use is that Agilent ZORBAX Eclipse Plus C18, specification are that 50mm * 4.6mm, filling agent particle diameter are the performance liquid chromatographic column of 1.8 μ m, and the filling agent in the said chromatographic column is an octadecylsilane chemically bonded silica, is that the mixed liquor that 0.4% phosphate aqueous solution is formed is a moving phase with acetonitrile and mass percentage concentration; Accurate respectively said reference substance solution and each 2 μ l of said need testing solution of drawing inject high performance liquid chromatograph respectively, according to carrying out gradient elution shown in the table 1; The equilibration time of each said gradient elution is 10 minutes; The flow velocity of said moving phase is per minute 0.6ml, and the detection wavelength is 360nm, and the column temperature of said chromatographic column is 40 ℃; Number of theoretical plate calculates by Kaempferol glucose rhamnoside peak and is not less than 8000; In the said table 1, A represents acetonitrile, and B representation quality percentage concentration is 0.4% phosphate aqueous solution; % represents percent by volume, and the unit of time is minute;
Table 1, condition of gradient elution
High-efficient liquid phase chromatogram according to said test sample of gained and reference substance calculates the peak area of said Kaempferol glucose rhamnoside and the peak area of said reference substance, calculates according to the external standard method shown in the following formula:
Obtain the concentration of Kaempferol glucose rhamnoside in the said need testing solution;
Said method b is:
1) solution of preparation reference substance: take by weighing Kaempferol glucose rhamnoside reference substance, add methyl alcohol and process the solution that every 1ml contains 0.04mg, obtain the solution of said reference substance;
2) preparation need testing solution:
(1) solution of preparation ginkgo leaf: get ginkgo leaf, pulverize, take by weighing 2g, the adding mass percentage concentration is 75% ethanol water 100ml, claims to decide weight; Placed the water-bath heating and refluxing extraction 40 minutes, and took out, put coldly, claim decide weight again, the use mass percentage concentration is that 75% ethanol water is supplied the weight that subtracts mistake; Shake up, filter, measure subsequent filtrate 25ml, evaporate to dryness, residue add water 2ml makes dissolving; Place the polyamide column of having handled well, water 50ml wash-out discards water liquid, and using mass percentage concentration again is 75% ethanol water 100ml wash-out, collects eluent; Evaporate to dryness, residue add methyl alcohol makes its dissolving, quantitatively is transferred in the 5ml measuring bottle, is diluted to scale with methyl alcohol; Shake up,, get subsequent filtrate, obtain the solution of said ginkgo leaf with the miillpore filter filtration of 0.45 μ m; Wherein, Said polyamide column is handled according to following method: get polyamide granules and screen out fine powder with 60 mesh sieves, the water logging bubble that adds 2～3 times of weight makes it wetting, in the chromatographic column of packing into; The mass percentage concentration of using 2～5 times of column volumes is 2%～5% aqueous hydrochloric acid solution wash-out; Be eluted to neutrality with deionized water again, the mass percentage concentration of then using 2～5 times of column volumes is 2%～5% aqueous hydrochloric acid solution wash-out, is eluted to neutrality with deionized water again;
(2) solution of preparation ginkgo biloba p.e: take by weighing the batch number of producing available from Zhejiang Conba Pharmaceutical Co., Ltd. and be 070301 ginkgo biloba p.e 0.035g; Add methyl alcohol 10ml; Sonicated 10 minutes; Using the aperture is the miillpore filter filtration of 0.45 μ m, gets subsequent filtrate, obtains the solution of said ginkgo biloba p.e;
(3) solution of preparation Shu Xuening injection: using the aperture is that 0.45 μ m miillpore filter filters Shu Xuening injection, gets subsequent filtrate, obtains the solution of said Shu Xuening injection;
3) content of Kaempferol glucose rhamnoside in the said need testing solution of mensuration:
Selecting model for use is that HP 1100 types
ODS-3C18, specification are that 250mm * 4.6mm, filling agent particle diameter are the performance liquid chromatographic column of 5 μ m; Filling agent in the said chromatographic column is an octadecylsilane chemically bonded silica; The mixed liquor of forming with mobile phase A and Mobile phase B is a moving phase; Draw each 10 μ l of said reference substance solution and said need testing solution respectively; Inject high performance liquid chromatograph respectively, according to carrying out gradient elution shown in the table 2, the equilibration time of each wash-out is 10 minutes; The flow velocity of moving phase is per minute 1.0ml; The wavelength that detects is 360nm, and the column temperature of said chromatographic column is 30 ℃, and number of theoretical plate calculates by Kaempferol glucose rhamnoside peak and is not less than 8000; Said mobile phase A be by water, acetonitrile, isopropyl alcohol and citric acid with volume ratio 1000: 470: 50: 6.08 mix the mixed liquor that get, and said Mobile phase B is water, acetonitrile, isopropyl alcohol and citric acid with volume ratio 1400: 200: 30: the mixed liquor that 6.88 mixing get; In the said table 2, A represents said mobile phase A, and B represents said Mobile phase B, and % represents percent by volume, and the unit of time is minute;
Table 2, condition of gradient elution
High-efficient liquid phase chromatogram according to said test sample of gained and reference substance calculates the peak area of said Kaempferol glucose rhamnoside and the peak area of said reference substance, calculates according to the external standard method shown in the following formula:
Obtain the concentration of Kaempferol glucose rhamnoside in the said need testing solution;
Said method c is:
1) solution of preparation reference substance: it is an amount of to take by weighing Kaempferol glucose rhamnoside reference substance, adds methyl alcohol and processes the solution that every 1ml contains 0.04mg, obtains the solution of said reference substance;
2) preparation need testing solution:
(1) solution of preparation ginkgo biloba p.e: take by weighing the batch number of producing available from Zhejiang Conba Pharmaceutical Co., Ltd. and be 070301 ginkgo biloba p.e 0.035g; Add methyl alcohol 10ml; Sonicated 10 minutes; Using the aperture is the miillpore filter filtration of 0.45 μ m, gets subsequent filtrate, obtains the solution of said ginkgo biloba p.e;
(2) solution of preparation Shu Xuening injection: using the aperture is that 0.45 μ m miillpore filter filters Shu Xuening injection, gets subsequent filtrate, obtains the solution of said Shu Xuening injection;
3) content of Kaempferol glucose rhamnoside in the said need testing solution of mensuration:
Selecting model for use is that Inertsil ODS-3C18, specification are that 250mm * 4.6mm, filling agent particle diameter are the performance liquid chromatographic column of 5 μ m; Filling agent in the said chromatographic column is an octadecylsilane chemically bonded silica; With acetonitrile and mass percentage concentration is that the mixed liquor that 0.4% phosphate aqueous solution is formed is a moving phase, draws respectively 10 μ l of said reference substance solution and said need testing solution respectively, injects high performance liquid chromatograph respectively; According to carrying out gradient elution shown in the table 3; The equilibration time of each wash-out is 10 minutes, and the flow velocity of moving phase is per minute 1.0ml, and the wavelength of detection is 360nm; The column temperature of said chromatographic column is 30 ℃, and number of theoretical plate calculates by Kaempferol glucose rhamnoside peak and is not less than 8000; In the said table 3, A represents acetonitrile, and B representation quality percentage concentration is 0.4% phosphate aqueous solution, and % represents percent by volume, and the unit of time is minute;
Table 3, condition of gradient elution
High-efficient liquid phase chromatogram according to said test sample of gained and reference substance calculates the peak area of said Kaempferol glucose rhamnoside and the peak area of said reference substance, calculates according to the external standard method shown in the following formula:
Obtain the concentration of Kaempferol glucose rhamnoside in the said need testing solution.
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| CN106905393B (en) * | 2017-03-01 | 2020-03-03 | 中国药科大学 | Ginkgo flavone compound |
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