CN101813674A - 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 studies show 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, measure through conversion, 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 may take place, cause measurement result inaccurate; 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 described ginkgo leaf or its related preparations by external standard method as the described ginkgo leaf of test sample or the Kaempferol glucose rhamnoside in its related preparations.
In this method, before described determination step, earlier described reference substance and described test sample are dissolved in the solvent; The solvent that dissolves described reference substance is selected from the aqueous solution of methyl alcohol, methanol in water, ethanol, ethanol and in the moving phase any one; The solvent that dissolves described test sample is selected from the aqueous solution of methyl alcohol, methanol in water, ethanol, ethanol and in the moving phase any one.
In the described determination step, the detection wavelength is 190nm～400nm, preferred 360nm.Moving phase is any one among following solvents a～solvent c: described solvent a is a methanol in water, and described solvent b is the aqueous solution of acetonitrile, and described solvent c is the mixed liquor that methyl alcohol, acetonitrile and water are formed.Described 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, described solvent a is a methanol in water, and described solvent b is the aqueous solution of acetonitrile, and described solvent c is the mixed liquor that methyl alcohol, acetonitrile and water are formed; Described dressing agent is selected from any one in tetrahydrofuran, isopropyl alcohol and the dioxane; Described buffer solution is selected from any one in phosphate buffered solution, acetate buffer solution and the citrate buffer solution; Described ionic inhibitor is selected from any one in sodium dodecylsulphonate, undecyl sodium sulfonate, sodium hexanesulfonate, sodium heptanesulfonate, perfluorooctane sulfonate and the nonane sodium sulfonate; Described pH value correctives is selected from any one in formic acid, acetate, phosphoric acid and the citric acid.The addition of described dressing agent is 0.5%～10.0%, preferred 3.0%～5.0% of described solvent a or solvent b or a solvent c volume; The addition of described buffer solution is 0.5%～10.0%, preferred 3.0%～5.0% of described solvent a or solvent b or a solvent c volume; The addition of described ionic inhibitor is 0.1%～3.0%, preferred 0.5%～1.0% of described solvent a or solvent b or a solvent c volume; The addition of described pH value correctives is 0.05%～5.0%, preferred 0.2%～1.0% of described solvent a or solvent b or a solvent c volume.Described 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, described solid pharmaceutical preparation is pill, granule, tablet, capsule, pill, powder or medicinal tea, and described 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:
Described method a is:
1) solution of preparation reference substance: take by weighing Kaempferol glucose rhamnoside reference substance, add methyl alcohol and make the solution that every 1ml contains 0.04mg, obtain the solution of described 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, places the water-bath heating and refluxing extraction 40 minutes, take out, put coldly, claim to decide weight again, with mass percentage concentration is that 75% ethanol water is supplied the weight that subtracts mistake, shakes up, and filters, measure subsequent filtrate 25ml, evaporate to dryness, residue add water 2ml makes its dissolving, places the polyamide column of having handled well, water 50ml wash-out, discarding water liquid, is 75% ethanol water 100ml wash-out again with mass percentage concentration, collects eluent, evaporate to dryness, residue adds methyl alcohol makes its dissolving, quantitatively is transferred in the 5ml measuring bottle, is diluted to scale with methyl alcohol, shake up, miillpore filter with 0.45 μ m filters, and gets subsequent filtrate, obtains the solution of described ginkgo leaf; Wherein, described 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, it with the mass percentage concentration of 2～5 times of column volumes 2%～5% aqueous hydrochloric acid solution wash-out, being eluted to neutrality with deionized water again, then is 2%～5% aqueous hydrochloric acid solution wash-out with the mass percentage concentration of 2～5 times of column volumes, 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, with the aperture is the miillpore filter filtration of 0.45 μ m, get subsequent filtrate, obtain the solution of described ginkgo biloba p.e;
(3) solution of preparation Shu Xuening injection: with the aperture is the miillpore filter filtration Shu Xuening injection of 0.45 μ m, gets subsequent filtrate, obtains the solution of described Shu Xuening injection;
3) content of Kaempferol glucose rhamnoside in the described need testing solution of mensuration:
Selecting model for use is Agilent ZORBAX Eclipse Plus C18, specification is 50mm * 4.6mm, the filling agent particle diameter is the performance liquid chromatographic column of 1.8 μ m, filling agent in the described 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, accurate respectively described reference substance solution and each 2 μ l of described need testing solution of drawing, inject described high performance liquid chromatograph respectively, according to carrying out gradient elution shown in the table 1, the equilibration time of each described gradient elution is 10 minutes, the flow velocity of described moving phase is per minute 0.6ml, described detection wavelength is 360nm, the column temperature of described chromatographic column is 40 ℃, number of theoretical plate calculates by Kaempferol glucose rhamnoside peak and is not less than 8000, in the described table 1, A represents acetonitrile, 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 described test sample of gained and reference substance calculates the peak area of described Kaempferol glucose rhamnoside and the peak area of described reference substance, calculates according to the external standard method shown in the following formula:
Obtain the concentration of Kaempferol glucose rhamnoside in the described need testing solution;
Described method b is:
1) solution of preparation reference substance: take by weighing Kaempferol glucose rhamnoside reference substance, add methyl alcohol and make the solution that every 1ml contains 0.04mg, obtain the solution of described 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, places the water-bath heating and refluxing extraction 40 minutes, take out, put coldly, claim to decide weight again, with mass percentage concentration is that 75% ethanol water is supplied the weight that subtracts mistake, shakes up, and filters, measure subsequent filtrate 25ml, evaporate to dryness, residue add water 2ml makes dissolving, places the polyamide column of having handled well, water 50ml wash-out, discarding water liquid, is 75% ethanol water 100ml wash-out again with mass percentage concentration, collects eluent, evaporate to dryness, residue adds methyl alcohol makes its dissolving, quantitatively is transferred in the 5ml measuring bottle, is diluted to scale with methyl alcohol, shake up, miillpore filter with 0.45 μ m filters, and gets subsequent filtrate, obtains the solution of described ginkgo leaf; Wherein, described 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, it with the mass percentage concentration of 2～5 times of column volumes 2%～5% aqueous hydrochloric acid solution wash-out, being eluted to neutrality with deionized water again, then is 2%～5% aqueous hydrochloric acid solution wash-out with the mass percentage concentration of 2～5 times of column volumes, 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, with the aperture is the miillpore filter filtration of 0.45 μ m, get subsequent filtrate, obtain the solution of described ginkgo biloba p.e;
(3) solution of preparation Shu Xuening injection: with the aperture is that 0.45 μ m miillpore filter filters Shu Xuening injection, gets subsequent filtrate, obtains the solution of described Shu Xuening injection.
3) content of Kaempferol glucose rhamnoside in the described need testing solution of mensuration:
Selecting model for use is HP 1100 type Inertsil
ODS-3 C18, specification is 250mm * 4.6mm, the filling agent particle diameter is the performance liquid chromatographic column of 5 μ m, filling agent in the described 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 described reference substance solution and described need testing solution respectively, inject described 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 described detection is 360nm, the column temperature of described chromatographic column is 30 ℃, and number of theoretical plate calculates by Kaempferol glucose rhamnoside peak and is not less than 8000; Described 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 described 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 described table 2, A represents described mobile phase A, and B represents described Mobile phase B, and % represents percent by volume;
Table 2, condition of gradient elution
High-efficient liquid phase chromatogram according to described test sample of gained and reference substance calculates the peak area of described Kaempferol glucose rhamnoside and the peak area of described reference substance, calculates according to the external standard method shown in the following formula:
Obtain the concentration of Kaempferol glucose rhamnoside in the described need testing solution;
Described 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 makes the solution that every 1ml contains 0.04mg, obtains the solution of described 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, with the aperture is the miillpore filter filtration of 0.45 μ m, get subsequent filtrate, obtain the solution of described ginkgo biloba p.e;
(2) solution of preparation Shu Xuening injection: with the aperture is that 0.45 μ m miillpore filter filters Shu Xuening injection, gets subsequent filtrate, obtains the solution of described Shu Xuening injection.
3) content of Kaempferol glucose rhamnoside in the described need testing solution of mensuration:
Selecting model for use is Inertsil ODS-3 C18, specification is 250mm * 4.6mm, the filling agent particle diameter is the performance liquid chromatographic column of 5 μ m, filling agent in the described 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, draw each 10 μ l of described reference substance solution and described need testing solution respectively, inject described 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, the flow velocity of moving phase is per minute 1.0ml, the wavelength of described detection is 360nm, the column temperature of described chromatographic column is 30 ℃, and number of theoretical plate calculates by Kaempferol glucose rhamnoside peak and is not less than 8000; In the described 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 described test sample of gained and reference substance calculates the peak area of described Kaempferol glucose rhamnoside and the peak area of described reference substance, calculates according to the external standard method shown in the following formula:
Obtain the concentration of Kaempferol glucose rhamnoside in the described need testing solution.
In the said determination method, the described 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, 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.Described external standard method is meant under the chromatographic condition of regulation, get reference substance solution and need testing solution sample introduction respectively, determine 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, shortcomings such as measurement result is inaccurate in the existing assay method, hydrolytic process wastes time and energy have been overcome, can accurately measure the real content of flavonoid glycoside composition in ginkgo leaf and the preparation thereof, help 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
The invention will be further described below in conjunction with specific embodiment, 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, after mixing sample with column chromatography silica gel (100 orders～200 orders) 20kg, 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, with volume ratio is that 95: 5～50: 50 the methenyl choloride and the mixed liquor of methyl alcohol carry out gradient elution, collect corresponding cut, through silicagel column chromatographic resolution, purifying repeatedly, promptly.The nuclear-magnetism testing result of this compound is as follows:
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′)。
As from the foregoing, this compound structure is correct.
Chromatographic condition and system suitability test: the model of chromatographic column is Agilent ZORBAX Eclipse Plus C18, specification is 50mm * 4.6mm, 1.8 μ m, filling agent is an 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 make the solution that every 1ml contains 0.04mg, promptly.
The preparation of need testing solution:
(1) ginkgo leaf: get ginkgo leaf this product, pulverize, precision takes by weighing 2g, the accurate 75% ethanol 100ml that adds claims to decide weight, puts in the water-bath heating and refluxing extraction 40 minutes, take out, put coldly, claim to decide weight again, 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, discard water liquid, use 75% ethanol 100ml wash-out again, collect eluent, evaporate to dryness, residue adds methyl alcohol makes dissolving in right amount, quantitatively is transferred in the 5ml measuring bottle, is diluted to scale with methyl alcohol, shake up, miillpore filter with 0.45 μ m filters, and gets subsequent filtrate, promptly.
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 is the miillpore filter filtration of 0.45 μ m with the aperture, get subsequent filtrate, promptly.
(3) Shu Xuening injection: getting parenteral solution, is that 0.45 μ m miillpore filter filters with the aperture, gets subsequent filtrate, promptly.
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, Fig. 2 is the HPLC collection of illustrative plates as the ginkgo leaf medicinal material of test sample, 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, 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 as follows:
When obtaining described test sample and being ginkgo leaf, wherein the concentration of Kaempferol glucose rhamnoside is 0.023mg/g;
According to last identical method, calculate described test sample when being ginkgo biloba p.e, wherein the concentration of Kaempferol glucose rhamnoside is 8.02mg/g; When described 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, 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.Described 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 described 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 described mobile phase A, and B represents described 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 make the solution that every 1ml contains 0.04mg, promptly.
The preparation of need testing solution:
(1) ginkgo leaf: get ginkgo leaf this product, pulverize, precision takes by weighing 2g, the accurate 75% ethanol 100ml that adds claims to decide weight, puts in the water-bath heating and refluxing extraction 40 minutes, take out, put coldly, claim to decide weight again, 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, discard water liquid, use 75% ethanol 100ml wash-out again, collect eluent, evaporate to dryness, residue adds methyl alcohol makes dissolving in right amount, quantitatively is transferred in the 5ml measuring bottle, is diluted to scale with methyl alcohol, shake up, miillpore filter with 0.45 μ m filters, and gets subsequent filtrate, promptly.
(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.
(3) Shu Xuening injection is got parenteral solution, with the miillpore filter filtration of 0.45 μ m, gets subsequent filtrate, promptly.
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, Fig. 6 is the HPLC collection of illustrative plates as the ginkgo leaf medicinal material of test sample, 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, 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 as follows:
When obtaining described test sample and being ginkgo leaf, wherein the concentration of Kaempferol glucose rhamnoside is 0.023mg/g;
According to last identical method, calculate described test sample when being ginkgo biloba p.e, wherein the concentration of Kaempferol glucose rhamnoside is 8.02mg/g; When described 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, 5 μ m, filling agent is an 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 make the solution that every 1ml contains 0.04mg, promptly.
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.
(2) Shu Xuening injection: get parenteral solution,, get subsequent filtrate, promptly 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, Figure 10 is the HPLC collection of illustrative plates as the ginkgo biloba p.e of test sample, 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 as follows:
When obtaining described test sample and being ginkgo biloba p.e, wherein the concentration of Kaempferol glucose rhamnoside is 8.02mg/g; When described test sample was Shu Xuening injection, wherein the concentration of Kaempferol glucose rhamnoside was 8.02mg/ml.
Claims (10)
1. a method of measuring Kaempferol glucose rhamnoside content in ginkgo leaf or its related preparations comprises the steps:
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 described ginkgo leaf or its related preparations by external standard method as the described ginkgo leaf of test sample or the Kaempferol glucose rhamnoside in its related preparations.
2. method according to claim 1 is characterized in that: before described mensuration, earlier described reference substance and described test sample are dissolved in respectively in the solvent, obtain reference substance solution and need testing solution respectively; The solvent that dissolves described reference substance is selected from the aqueous solution of methyl alcohol, methanol in water, ethanol, ethanol and in the moving phase any one; The solvent that dissolves described test sample is selected from the aqueous solution of methyl alcohol, methanol in water, ethanol, ethanol and in the moving phase any one.
3. method according to claim 1 and 2, it is characterized in that: the concentration of described reference substance solution is to contain the described reference substance of 0.001mg～1.00mg in the described reference substance solution of every 1ml, contains the described reference substance of 0.04mg in the described reference substance solution of preferred every 1ml.
4. according to the arbitrary described method of claim 1-3, it is characterized in that: described moving phase is any one among the following solvents a-solvent c:
Described solvent a is a methanol in water, and described solvent b is the aqueous solution of acetonitrile, and described solvent c is the mixed liquor that methyl alcohol, acetonitrile and water are formed.
5. according to the arbitrary described method of claim 2-4, it is characterized in that: described moving phase is 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;
Described solvent a is a methanol in water, and described solvent b is the aqueous solution of acetonitrile, and described solvent c is the mixed liquor that methyl alcohol, acetonitrile and water are formed;
Described dressing agent is selected from any one in tetrahydrofuran, isopropyl alcohol and the dioxane;
Described buffer solution is selected from any one in phosphate buffered solution, acetate buffer solution and the citrate buffer solution;
Described ionic inhibitor is selected from any one in sodium dodecylsulphonate, undecyl sodium sulfonate, sodium hexanesulfonate, sodium heptanesulfonate, perfluorooctane sulfonate and the nonane sodium sulfonate;
Described pH value correctives is selected from any one in formic acid, acetate, phosphoric acid and the citric acid.
6. method according to claim 5 is characterized in that: the addition of described dressing agent is 0.5%～10.0%, preferred 3.0%～5.0% of described solvent a or solvent b or a solvent c volume;
The addition of described buffer solution is 0.5%～10.0%, preferred 3.0%～5.0% of described solvent a or solvent b or a solvent c volume;
The addition of described ionic inhibitor is 0.1%～3.0%, preferred 0.5%～1.0% of described solvent a or solvent b or a solvent c volume;
The addition of described pH value correctives is 0.05%～5.0%, preferred 0.2%～1.0% of described solvent a or solvent b or a solvent c volume.
7. according to the arbitrary described method of claim 1-6, it is characterized in that: in the described determination step, the detection wavelength is 190nm～400nm, preferred 360nm.
8. according to the arbitrary described method of claim 1-7, it is characterized in that: described 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.
9. method according to claim 8 is characterized in that: described solid pharmaceutical preparation is pill, granule, tablet, capsule, pill, powder or medicinal tea, and described liquid preparation is injection, oral liquid, tincture or soft extract.
10. according to the arbitrary described method of claim 1-9, it is characterized in that: the method for Kaempferol glucose rhamnoside content in described mensuration ginkgo leaf or its related preparations is to measure according to the arbitrary method among the following method a-method c:
Described method a is:
1) solution of preparation reference substance: take by weighing Kaempferol glucose rhamnoside reference substance, add methyl alcohol and make the solution that every 1ml contains 0.04mg, obtain the solution of described 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, places the water-bath heating and refluxing extraction 40 minutes, take out, put coldly, claim to decide weight again, with mass percentage concentration is that 75% ethanol water is supplied the weight that subtracts mistake, shakes up, and filters, measure subsequent filtrate 25ml, evaporate to dryness, residue add water 2ml makes its dissolving, places the polyamide column of having handled well, water 50ml wash-out, discarding water liquid, is 75% ethanol water 100ml wash-out again with mass percentage concentration, collects eluent, evaporate to dryness, residue adds methyl alcohol makes its dissolving, quantitatively is transferred in the 5ml measuring bottle, is diluted to scale with methyl alcohol, shake up, miillpore filter with 0.45 μ m filters, and gets subsequent filtrate, obtains the solution of described ginkgo leaf; Wherein, described 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, it with the mass percentage concentration of 2～5 times of column volumes 2%～5% aqueous hydrochloric acid solution wash-out, being eluted to neutrality with deionized water again, then is 2%～5% aqueous hydrochloric acid solution wash-out with the mass percentage concentration of 2～5 times of column volumes, 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, with the aperture is the miillpore filter filtration of 0.45 μ m, get subsequent filtrate, obtain the solution of described ginkgo biloba p.e;
(3) solution of preparation Shu Xuening injection: with the aperture is the miillpore filter filtration Shu Xuening injection of 0.45 μ m, gets subsequent filtrate, obtains the solution of described Shu Xuening injection;
3) content of Kaempferol glucose rhamnoside in the described need testing solution of mensuration:
Selecting model for use is Agilent ZORBAX Eclipse Plus C 18, specification is 50mm * 4.6mm, the filling agent particle diameter is the performance liquid chromatographic column of 1.8 μ m, filling agent in the described 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, accurate respectively described reference substance solution and each 2 μ l of described need testing solution of drawing, inject described high performance liquid chromatograph respectively, according to carrying out gradient elution shown in the table 1, the equilibration time of each described gradient elution is 10 minutes, the flow velocity of described moving phase is per minute 0.6ml, described detection wavelength is 360nm, the column temperature of described chromatographic column is 40 ℃, number of theoretical plate calculates by Kaempferol glucose rhamnoside peak and is not less than 8000, in the described table 1, A represents acetonitrile, 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 described test sample of gained and reference substance calculates the peak area of described Kaempferol glucose rhamnoside and the peak area of described reference substance, calculates according to the external standard method shown in the following formula:
Obtain the concentration of Kaempferol glucose rhamnoside in the described need testing solution;
Described method b is:
1) solution of preparation reference substance: take by weighing Kaempferol glucose rhamnoside reference substance, add methyl alcohol and make the solution that every 1ml contains 0.04mg, obtain the solution of described 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, places the water-bath heating and refluxing extraction 40 minutes, take out, put coldly, claim to decide weight again, with mass percentage concentration is that 75% ethanol water is supplied the weight that subtracts mistake, shakes up, and filters, measure subsequent filtrate 25ml, evaporate to dryness, residue add water 2ml makes dissolving, places the polyamide column of having handled well, water 50ml wash-out, discarding water liquid, is 75% ethanol water 100ml wash-out again with mass percentage concentration, collects eluent, evaporate to dryness, residue adds methyl alcohol makes its dissolving, quantitatively is transferred in the 5ml measuring bottle, is diluted to scale with methyl alcohol, shake up, miillpore filter with 0.45 μ m filters, and gets subsequent filtrate, obtains the solution of described ginkgo leaf; Wherein, described 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, it with the mass percentage concentration of 2～5 times of column volumes 2%～5% aqueous hydrochloric acid solution wash-out, being eluted to neutrality with deionized water again, then is 2%～5% aqueous hydrochloric acid solution wash-out with the mass percentage concentration of 2～5 times of column volumes, 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, with the aperture is the miillpore filter filtration of 0.45 μ m, get subsequent filtrate, obtain the solution of described ginkgo biloba p.e;
(3) solution of preparation Shu Xuening injection: with the aperture is that 0.45 μ m miillpore filter filters Shu Xuening injection, gets subsequent filtrate, obtains the solution of described Shu Xuening injection;
3) content of Kaempferol glucose rhamnoside in the described need testing solution of mensuration:
Selecting model for use is HP 1100 type Inertsil
ODS-3C18, specification is 250mm * 4.6mm, the filling agent particle diameter is the performance liquid chromatographic column of 5 μ m, filling agent in the described 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 described reference substance solution and described need testing solution respectively, inject described 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 described detection is 360nm, the column temperature of described chromatographic column is 30 ℃, and number of theoretical plate calculates by Kaempferol glucose rhamnoside peak and is not less than 8000; Described 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 described 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 described table 2, A represents described mobile phase A, and B represents described Mobile phase B, and % represents percent by volume;
Table 2, condition of gradient elution
High-efficient liquid phase chromatogram according to described test sample of gained and reference substance calculates the peak area of described Kaempferol glucose rhamnoside and the peak area of described reference substance, calculates according to the external standard method shown in the following formula:
Obtain the concentration of Kaempferol glucose rhamnoside in the described need testing solution;
Described 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 makes the solution that every 1ml contains 0.04mg, obtains the solution of described 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, with the aperture is the miillpore filter filtration of 0.45 μ m, get subsequent filtrate, obtain the solution of described ginkgo biloba p.e;
(2) solution of preparation Shu Xuening injection: with the aperture is that 0.45 μ m miillpore filter filters Shu Xuening injection, gets subsequent filtrate, obtains the solution of described Shu Xuening injection;
3) content of Kaempferol glucose rhamnoside in the described need testing solution of mensuration:
Selecting model for use is Inertsil ODS-3C18, specification is 250mm * 4.6mm, the filling agent particle diameter is the performance liquid chromatographic column of 5 μ m, filling agent in the described 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, draw each 10 μ l of described reference substance solution and described need testing solution respectively, inject described 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, the flow velocity of moving phase is per minute 1.0ml, the wavelength of described detection is 360nm, the column temperature of described chromatographic column is 30 ℃, and number of theoretical plate calculates by Kaempferol glucose rhamnoside peak and is not less than 8000; In the described 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 described test sample of gained and reference substance calculates the peak area of described Kaempferol glucose rhamnoside and the peak area of described reference substance, calculates according to the external standard method shown in the following formula:
Obtain the concentration of Kaempferol glucose rhamnoside in the described need testing solution.
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| CN105319333A (en) * | 2015-11-11 | 2016-02-10 | 广西中医药大学 | Quality detection method for Jingxuening capsule |
| CN105319333B (en) * | 2015-11-11 | 2017-04-26 | 广西中医药大学 | Quality detection method for Jingxuening capsule |
| CN106905393A (en) * | 2017-03-01 | 2017-06-30 | 中国药科大学 | A kind of gingkgo flavonoidses |
| CN106905393B (en) * | 2017-03-01 | 2020-03-03 | 中国药科大学 | Ginkgo flavone compound |
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