CN101810705A - Content determination method of sanguisorbin I - Google Patents
Content determination method of sanguisorbin I Download PDFInfo
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Abstract
The invention discloses a content determination method of sanguisorbin I. The method comprises the following steps of: (1) respectively dissolving a standard product of sanguisorbin I and a sample containing sanguisorbin I in an ethanol aqueous solution to respectively prepare a standard solution and a sample solution; and (2) respectively injecting the standard solution and the sample solution which have equal volumes into a high-efficiency liquid-phase chromatograph, then, measuring and calculating the peak area of the standard solution and the sample solution to acquire the content of sanguisorbin I in the sample. The invention provides a rapid and accurate method for measuring the content of sanguisorbin I in a medical herb of garden burnet and extractives or preparations thereof.
Description
Technical field
The present invention relates to the content assaying method of Ziyuglycoside I in a kind of Chinese medicine Radix Sanguisorbae medical material and extract and the preparation, belong to technical field of Chinese medicines.
Background technology
The Chinese medicine Radix Sanguisorbae is the dry root of the rosaceous plant Radix Sanguisorbae Sanguisorba officinalis L. or Radix Sanguisorbae Sanguisorba officinalis L.var.longifolia (Bert.) the Y ü et Li that comes into leaves.
Chinese Pharmacopoeia 2005 editions records this kind, has wherein put down in writing the content assaying method of tannin, and content is under the concrete assay item: " get the about 0.4g of this product powder (crossing sieve No. four), accurate title is fixed, measures according to content of tannin algoscopy (appendix XB), promptly.Press dry product and calculate, must not be less than 10.0%.”。
The WS-11020 of National Drug Administration of the People's Republic of China (PRC) (ZD-1020)-2002 DIYU SHENGBAI PIAN standard (trying) has been put down in writing the Determination on content method of the gallic acid in the DIYU SHENGBAI PIAN: " measure according to high performance liquid chromatography (appendix VID of Chinese Pharmacopoeia version in 2000).Chromatographic condition and system suitability test are immobile phase with octadecylsilane chemically bonded silica; Methanol-acetonitrile-0.05mol/L phosphoric acid solution (5: 6: 89) is a mobile phase; The detection wavelength is 270nm.Theoretical cam curve is calculated by the gallic acid peak should be not less than 6000.It is an amount of that the preparation precision of reference substance solution takes by weighing the gallic acid reference substance, adds 50% methanol and make the solution that every 1ml contains 20 μ g, promptly.20 of this product are got in the preparation of need testing solution, remove coating, and accurate the title decides, porphyrize is got an amount of (being equivalent to 10 weight approximately), and accurate the title decides, the accurate 50% methanol 25ml that adds claims to decide weight, reflux 1 hour, put and be chilled to room temperature, claim again to decide weight, supply the weight that subtracts mistake with 50% methanol, shake up, filter, get subsequent filtrate, promptly.Accurate respectively reference substance solution and each the 5 μ l～10 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, promptly.Every of this product contains Radix Sanguisorbae with gallic acid (C
7H
5O
5) meter, must not be less than 24 μ g." above standard discloses Radix Sanguisorbae medical material and Rhizoma sanguisorbae preparation respectively---the assay of tannin and gallic acid in the DIYU SHENGBAI PIAN.
And the Radix Sanguisorbae medical material is widely used, indication is more, and its effective ingredient that is played a role of different indications also is different, particularly at Radix Sanguisorbae aspect the indication of leukocyte increasing level, only judge that by detecting tannin in the Rhizoma sanguisorbae preparation or gallic acid whether qualified preparation be unfavorable, former because existing research proves: sanguisorbin is the active component of Radix Sanguisorbae performance function of increasing leukocyte, Ziyuglycoside I particularly, and tannin and gallic acid do not play main effect [higher primary school's equality of leukocyte increasing level, the effective site screening of the short hemoposieis of Radix Sanguisorbae. Chinese natural drug, 2006 (2): 137-140; Chengdu Diao Pharmaceutical Group Co., Ltd. radix sanguisorbae total saponin extract and its production and use [P]. Chinese patent: 200310119248.1,2004-11-25; Chengdu Diao Pharmaceutical Group Co., Ltd. the application [P] of ursolic alkane triterpene glycosides in preparation leukocyte increasing and/or platelet medicine. Chinese patent: 03135776.8,20030908ZL03135776.8], therefore, then more reasonable by the content that detects Ziyuglycoside I in the Rhizoma sanguisorbae preparation, also quality that more can reactor product.Yet find in the practice that the detection method of existing tannin and/or gallic acid content is not suitable for the detection of Ziyuglycoside I.
Zou Shengqin etc. disclose maloic acid in the Radix Sanguisorbae and oleanolic acid content high performance liquid chromatography [Zou Shengqin, Chen Wu. the high-performance liquid chromatogram determination of maloic acid and oleanolic acid content in the Radix Sanguisorbae, the time precious traditional Chinese medical science traditional Chinese medicines, 2006,17 (8): 1373～1374]; Xie Junbo etc. disclose with 3 kinds of triterpenes in the HPLC-ELSD method Ilex purpurea Hassk.[I.chinensis Sims and saponin thereof, content assaying method [the Xie Junbo that comprises Ziyuglycoside I, Bi Zhiming, the content of triterpene and saponin thereof in the Li Ping .HPLC-ELSD method mensuration Ilex purpurea Hassk.[I.chinensis Sims], though also contain Ziyuglycoside I in the Ilex purpurea Hassk.[I.chinensis Sims, but because Ilex purpurea Hassk.[I.chinensis Sims is different plants with Radix Sanguisorbae, except that Ziyuglycoside I, therefore other chemical constituent differences that the two contains are applicable to the content assaying method of Ziyuglycoside I in the Ilex purpurea Hassk.[I.chinensis Sims and are not suitable for the Radix Sanguisorbae medical material, the assay of Ziyuglycoside I in Radix Sangusorbae extract or the Rhizoma sanguisorbae preparation; Sha Ming etc. disclose the HPLC finger printing of sanguisorbin constituents, flavones ingredient, wherein the HPLC chromatographic condition of sanguisorbin constituents is: chromatographic column: Shim-pack CLC ODS post (6.0mm * 150mm, 5 μ m), mobile phase: acetonitrile-water (30: 70) detects wavelength: 206nm, flow velocity: 1.0ml/min, column temperature: 25 ℃.[Sha Ming, Zhang Dongfang, .DNA dactylograms such as Meng Xiansheng and HPLC dactylogram are studied the quality evaluation of Chinese medicine Radix Sanguisorbae, Chinese Pharmaceutical Journal, 2002,37 (11): 815～818], but use this method appearance time long (20min), analysis efficiency is lower, and can not effectively separate by the saponin component that Ziyuglycoside I is close with its structure.More than Bao Dao analytical method all exists appearance time to reach 20min, and analysis efficiency is lower, and the effective isolating technological deficiency of saponin component that can not Ziyuglycoside I is close with its structure
Therefore, up to the present, still there is not the method that fast, accurately to measure Ziyuglycoside I content in Radix Sanguisorbae medical material and extract thereof or the preparation both at home and abroad.
Summary of the invention
At above technological deficiency, the object of the present invention is to provide a kind of method that can fast, accurately measure the content of Ziyuglycoside I in Radix Sanguisorbae medical material, its extract or the preparation, a kind of method with the content of Ziyuglycoside I in high effective liquid chromatography for measuring Radix Sanguisorbae medical material, its extract or the preparation is provided particularly.
The method of the invention comprises:
1) test sample of getting the standard substance of Ziyuglycoside I respectively and containing Ziyuglycoside I is dissolved in the ethanol water, makes standard solution and need testing solution respectively;
2) get isopyknic standard solution and need testing solution respectively and inject high performance liquid chromatograph, the peak area of measurement standard product solution and need testing solution and calculating promptly gets the content of the Ziyuglycoside I in the test sample then;
Wherein, the immobile phase of described high performance liquid chromatograph is the carbon octadecyl silane; Mobile phase is methanol-water solution.
In the inventive method, as embodiment preferred, the particle diameter of described carbon octadecyl silane is≤5 μ m; Further the particle diameter of preferred described carbon octadecyl silane is 5 μ m, 4 μ m or 3.5 μ m.
Mobile phase of the present invention is the solution of methanol-water, is preferably: in percent by volume, the gradient solution of 60%～80% methanol in water or be 50%～80% methanol in water etc. the degree solution.
In the inventive method, when described mobile phase adopts the gradient solution of 60%～80% methanol in water; Preferred described gradient solution consists of: in percent by volume, and 0～17min, 60% → 70% methanol in water, 17～25min, 70% → 80% methanol in water, 25～35min, 80% methanol in water; Or
0～7min, 60% → 64% methanol in water, 7～12min, 64% → 66% methanol in water, 12～17min, 66% → 80% methanol in water, 17～25min, 80% methanol in water; Or 0～7min, 60% → 64% methanol in water, 7～15min, 64% methanol in water, 15～20min64% → 80% methanol in water, 20～25min, 80% methanol in water; Or
0～7min, 60% → 64% methanol in water, 7～12min, 64% methanol in water, 12～20min64% → 80% methanol in water, 20～27min, 80% methanol in water; Or
0～7min, 60% → 64% methanol in water, 7～13min, 64% methanol in water.
As embodiment preferred, described solution such as degree such as grade is: in percent by volume, and 64% methanol in water.
As embodiment preferred, the concentration of ethanol water of the present invention is the alcoholic acid aqueous solution of by volume 50%-60%.
The assay method of Ziyuglycoside I of the present invention can be used for the various aspects of field of medicaments, the present invention includes but be not limited in the Radix Sanguisorbae medical material, in the sanguisorbin extract, and various pharmaceutical preparatioies, as the content of the Ziyuglycoside I in the preparation of various gastrointestinal tract such as tablet, capsule, granule, injection or parenteral route administration and the mensuration of purity.
The Ziyuglycoside I standard substance can prepare Ziyuglycoside I with reference to method of the prior art: Cao Aimin, Zhang Dongfang, Sha Ming etc., saponins compound separation, evaluation and assay thereof in the Radix Sanguisorbae. and Chinese herbal medicine, 2003,5 (34): 397～399 are prepared.
As embodiment preferred, the content assaying method of Ziyuglycoside I comprises in the described Radix Sanguisorbae medical material:
1) preparation of need testing solution: get the Radix Sanguisorbae medical material, pulverize, cross sieve No. four, about 0.4g, the accurate title, decide, and puts in the 100ml measuring bottle, add 60% alcoholic acid aqueous solution 80ml, shake up, ultrasonic 15min is put to room temperature, be settled to scale, shake up, filter, get analysis of subsequent filtrate sample introduction or centrifuging and taking supernatant promptly;
The preparation of reference substance solution: get the about 50mg of Ziyuglycoside I reference substance, the accurate title, decide, and puts in the 50ml measuring bottle, adds 60% alcoholic acid aqueous solution dissolving and be settled to scale, shakes up; Precision is measured in 5.00ml and 25.00ml to the 50ml measuring bottle respectively, adds 60% alcoholic acid aqueous solution dilution and is settled to scale, shakes up, and promptly makes the reference substance solution that every 1ml contains 0.1mg and 0.5mg;
2) assay: accurate respectively reference substance solution and the need testing solution 10 μ l of drawing, inject high performance liquid chromatograph, measure the peak area of reference substance solution and need testing solution, peak area integrated value and concentration are got common logarithm respectively after, press the content of external standard two-point method calculating Ziyuglycoside I;
Wherein, high performance liquid chromatography is that the immobile phase particle diameter is less than or equal to 5 μ m with the carbon octadecyl silane, mobile phase is that methanol and water volume are 64%: 36% isocratic elution liquid or are the gradient eluent of the methanol-water solution of 60-64% for the methanol volumetric concentration, elution program is 0～7min, 60% → 64% methanol in water, 7～13min, 64% methanol in water flow velocity 0.8ml/min, 25 ℃ of column temperatures, the ELSD drift tube temperature is 80 ℃, gas flow rate 2.5L/min, shunt mode not, theoretical cam curve calculate by Ziyuglycoside I should be not less than 4 * 10
3
As embodiment preferred, the content assaying method of Ziyuglycoside I comprises in the described sanguisorbin extract:
1) preparation of need testing solution: get radix sanguisorbae total saponin powder 50mg, the accurate title, decide, and puts in the 100ml measuring bottle, adds 60% alcoholic acid aqueous solution 60ml, shake up, ultrasonic 5min is put to room temperature, is settled to scale, shake up, filter, get subsequent filtrate or the analysis of centrifuging and taking supernatant sample introduction;
The preparation of reference substance solution: get the about 50mg of Ziyuglycoside I reference substance, the accurate title, decide, and puts in the 50ml measuring bottle, adds 60% alcoholic acid aqueous solution dissolving and be settled to scale, shakes up; Precision is measured in 5.00ml and 25.00ml to the 50ml measuring bottle respectively, adds 60% alcoholic acid aqueous solution dilution and is settled to scale, shakes up and promptly makes the reference substance solution that every 1ml contains 0.1mg and 0.5mg;
2) assay: accurate respectively reference substance solution and the need testing solution 10 μ l of drawing, inject high performance liquid chromatograph, measure the peak area of reference substance solution and need testing solution, peak area integrated value and concentration are got common logarithm respectively after, press the content of external standard two-point method calculating Ziyuglycoside I;
Wherein, high performance liquid chromatograph is less than or equal to 5 μ m with the carbon octadecyl silane for the immobile phase particle diameter, with the methanol volumetric concentration is the methanol-water mixed solvent gradient elution of 60-64%, elution program is 0～7min, 60% → 64% methanol in water, 7～13min, 64% methanol in water, flow velocity 1.2ml/min, 25 ℃ of column temperatures, ELSD (Alltech 2000 ES) drift tube temperature is 100 ℃, gas flow rate 3.5L/min, shunt mode not, theoretical cam curve calculate by Ziyuglycoside I should be not less than 4 * 10
3
As embodiment preferred, the content assaying method of Ziyuglycoside I comprises in the described Radix Sanguisorbae tablet:
1) preparation of need testing solution: get 20 of DIYU SHENGBAI PIAN, the accurate title, decided porphyrize, precision takes by weighing the amount that is equivalent to the 2mg Ziyuglycoside I, put in the 150ml tool plug conical flask, accurately add 50% ethanol 20ml, shake up, weigh, ultrasonic 10min is put to room temperature, supplies the weight that subtracts mistake with 50% alcoholic acid aqueous solution, shake up, the centrifuging and taking supernatant promptly;
The preparation of reference substance solution: it is an amount of to be taken at 105 ℃ of Ziyuglycoside I reference substances that are dried to constant weight, adds 50% alcoholic acid aqueous solution dissolving and standardize solution and makes the reference substance solution that every 1ml contains 0.05mg and 0.2mg;
2) assay: accurate respectively reference substance solution and the need testing solution 20 μ l of drawing, inject chromatograph of liquid, measure the peak area of reference substance solution and need testing solution, peak area integrated value and concentration are got common logarithm respectively after, press the content of external standard two-point method calculating Ziyuglycoside I;
Wherein, high performance liquid chromatograph, the carbon octadecyl silane that is less than or equal to 5 μ m with particle diameter is an immobile phase, with the methanol-water volume ratio is that 64: 36 mixed solution is an isocratic elution liquid, or be the methanol-water mixed solvent gradient elution of 60-64% with the methanol volumetric concentration, elution program is 0～7min, 60% → 64% methanol in water, 7～13min, 64% methanol in water, flow velocity 1.0ml/min, 25 ℃ of column temperatures, ELSD drift tube temperature are 85 ℃, gas flow rate 2.8L/min, shunt mode not, theoretical cam curve calculate by Ziyuglycoside I should be not less than 4 * 10
3
As embodiment preferred, the method for detecting purity of described Ziyuglycoside I comprises:
1) preparation of sample solution: it is an amount of to get Ziyuglycoside I, adds dissolve with methanol and is diluted to the solution that every 1ml contains 0.5mg, promptly;
2) purity is measured: the accurate sample solution 10 μ l that draw, inject chromatograph of liquid, and measure, press the purity that the peak area normalization method is calculated Ziyuglycoside I;
Wherein, described high performance liquid chromatograph is an immobile phase with the carbon octadecyl silane, particle diameter is less than or equal to 5 μ m, with the methanol volumetric concentration is the methanol-water mixed solvent gradient elution of 60-64%, elution program is 0～7min, 60% → 64% methanol in water, 7～12min, 64% methanol in water, 12～20min, 64% → 80% methanol in water, 20～27min, 80% methanol in water, flow velocity 1.0ml/min, 25 ℃ of column temperatures, ELSD (Alltech 2000ES) drift tube temperature is 85 ℃, gas (air) flow velocity 2.8L/min, shunt mode not, theoretical cam curve is calculated by Ziyuglycoside I should be not less than 4 * 10
3
Compared with prior art, the present invention provides method fast and accurately for Ziyuglycoside I Determination on content in Radix Sanguisorbae medical material and extract or the preparation, Ziyuglycoside I and its chromatographic peak that is close to previously (isomers 3 β that structure is very close-0-α-L-arabopyranose-28-β-D glucopyranosyl ester-19 Alpha-hydroxies-oleanolic acid) can be realized baseline separation; And appearance time is short, only is 10min.
According to foregoing of the present invention,, under the prerequisite that does not break away from the above-mentioned basic fundamental thought of the present invention, can also make modification, replacement and the change of other various ways according to the ordinary skill knowledge and the conventional techniques means of this area.
Description of drawings
Fig. 1 a: embodiment 1 isocratic elution, (Synergi Hydro-RP C18 post (150 * 4.6mm, 4 μ m), acetonitrile-water (30: 70), flow velocity 1.0ml/min, 25 ℃ of column temperatures detect wavelength 210nm, analysis time 30min);
Fig. 1 b: embodiment 1 gradient elution, (Synergi Hydro-RP C18 post (150 * 4.6mm, 4 μ m), acetonitrile-water gradient elution (elution program: 0～20min, 30% acetonitrile → 50% acetonitrile; 20～25min, 50% acetonitrile → 80% acetonitrile), flow velocity 1.0ml/min, 25 ℃ of column temperatures detect wavelength 210nm, analysis time 30min);
Fig. 2-1: embodiment 2 chromatographic columns are selected (YMC-Pack proC
18Post (150 * 4.6mm, 3.5 μ m);
Fig. 2-2: embodiment 2 chromatographic columns are selected Synergi Hydro-RP C
18Post (150 * 4.6mm, 4 μ m);
Fig. 3-1: embodiment 3 Radix Sanguisorbae medical materials 62% methanol aqueous solution isocratic elution;
Fig. 3-2: embodiment 3 Radix Sanguisorbae total glycosides 62% methanol aqueous solution isocratic elution;
Fig. 3-3: embodiment 3 Radix Sanguisorbae medical materials 80% methanol aqueous solution isocratic elution;
Fig. 3-4: embodiment 3 Radix Sanguisorbae total glycosides 80% methanol aqueous solution isocratic elution;
Fig. 3-5: embodiment 3 Radix Sanguisorbae total glycosides 50% methanol aqueous solution isocratic elution;
Fig. 3-6: embodiment 3 Radix Sanguisorbae medical materials 50% methanol aqueous solution isocratic elution;
Fig. 4-1: embodiment 4 acetonitrile-water systems separate Ziyuglycoside I in the Radix Sanguisorbae medical material;
Fig. 4-2: embodiment 4 methanol-water systems separate Ziyuglycoside I in the Radix Sanguisorbae medical material;
Fig. 5-1: embodiment 5 medical material specificities are investigated (solvent);
Fig. 5-2: embodiment 5 medical material specificities are investigated (Ziyuglycoside I contrast solution);
Fig. 5-3: embodiment 5 medical material specificities are investigated (herbal extract);
Fig. 6-1: embodiment 6 DIYU SHENGBAI PIAN specificities are investigated (blank adjuvant);
Fig. 6-2: embodiment 6 DIYU SHENGBAI PIAN specificities are investigated (Ziyuglycoside I contrast solution);
Fig. 6-3: embodiment 6 DIYU SHENGBAI PIAN specificities are investigated (DIYU SHENGBAI PIAN sample liquid);
The Determination of Content Uniformity of Ziyuglycoside I in Fig. 7: embodiment 7 DIYU SHENGBAI PIAN;
The assay of Ziyuglycoside I in Fig. 8: the embodiment 8 Radix Sanguisorbae total glycosides extractives;
Fig. 9: embodiment 9 Ziyuglycoside I reference substance purity testings.
The specific embodiment
By the following examples the present invention is described in further detail again, but this should be interpreted as that the present invention only limits to following example, all technology that realizes based on foregoing of the present invention all belong to scope of the present invention.
The selection of embodiment 1 high performance liquid chromatography chromatographic column, mobile phase and elution program
Sanguisorbin belongs to triterpene saponin, and polarity is bigger, and experiment shows that it withs a hook at the end on the C18 chromatographic column, therefore selects the C18 analytical column for use.
The selection of mobile phase: Ziyuglycoside I is a neutral compound, need not to add soda acid additives or ion-pairing agent in the mobile phase.Therefore select flow phase system contrast screenings such as methanol-water, acetonitrile-water, the acetonitrile-water system is than methanol-water system baseline straightening in ultraviolet detection, peak shape is sharp-pointed, but it is still few than the methanol-water system through repeatedly adjusting the separation chromatography peak, and no matter adopting isocratic elution still is that the impurity peaks that is close to previously of gradient elution Ziyuglycoside I can't detect (seeing Fig. 1 a and 1b) fully, and the Ziyuglycoside I peak area obviously increases than the methanol-water system, and both can obtain certain separation (R=1.2) in the methanol-water system, therefore select the methanol-water system.According to the separating degree variation tendency, mobile phase is adjusted into gradient elution, see table 1 for details.
Table 1 eluent gradient elution program
| Numbering | Elution program |
| ??A | 0～7min, 47% methanol in water, 7～25min, 47% → 80% methanol in water, 25～35min, 80% methanol in water |
| ?? |
0～17min, 60% → 70% methanol in water, 17～25min, 70% → 80% methanol |
| ? | Aqueous solution, 25～35min, 80% methanol in water |
| ? |
0～7min, 60% → 64% methanol in water, 7～15min, 64% methanol in water, 15～20min, 64% → 80% methanol in water, 20～25min, 80% methanol in water |
| ? |
0～7min, 60% → 64% methanol in water, 7～12min, 64% → 66% methanol in water, 12～17min, 66% → 80% methanol in water, 17～25min, 80% methanol in water |
The result shows, in elution program B, C, D, Ziyuglycoside I and the impurity peaks separating effect basically identical (R=1.2) that is close to previously, Ziyuglycoside I goes out the peak at 7～12min of isocratic elution (64% methanol in water), retention time 10min, so elution requirement is optimized and revised and is 0～7min60% → 64% methanol in water, 7～12min, 64% methanol in water, 12～20min, 64% → 80% methanol in water, 20～27min, 80% methanol in water.And for assay, main peak eluting to be measured comes out to get final product, and therefore when the Ziyuglycoside I in the sample was carried out assay, elution requirement can be reduced to 0～7min, 60% → 64% methanol in water, 7～13min, 64% methanol in water.
The key that the Ziyuglycoside I analysis condition is selected is itself and a chromatographic peak that is close to are previously realized baseline separation, and the sample separation complexity of separate sources differs, and the medical material sample is better, Radix Sanguisorbae total glycosides and preparation takes second place, the Ziyuglycoside I reference substance is the most difficult; All be with C in front in the selection of mobile phase
18Post (4.6 * 150mm, 5 μ m) is the basis, has investigated conventional analysis post ZORBAX XDB-C again respectively under selected elution requirement
18Post (4.6 * 150mm, 5 μ m), Luna C
18Post (4.6 * 150mm, 5 μ m) and small particle diameter post YMC-Pack proC
18Post (4.6 * 150mm, 3.5 μ m), Synergi Hydro-RP C
18Post (4.6 * 150mm, 4 μ m) is to the separating effect of Ziyuglycoside I.The result shows, at particle diameter less 3.5 μ m and 4 μ m C
18On the analytical column in the reference substance Ziyuglycoside I and this impurity peaks all can reach baseline separation (R＞1.5).Under the above-mentioned condition of determining, the about 10min of Ziyuglycoside I chromatographic peak retention time; Each composition can all flow out chromatographic column (purity test) in the Radix Sanguisorbae sample in 27min, Ziyuglycoside I chromatographic peak tailing factor 1.1, and theoretical cam curve is greater than 1 * 10
4, the peak separating degree meets the requirement of HPLC assay method greater than 1.5.
When being used for assay, conventional analysis post (diameter 4.6mm * 150mm/250mm, 5 μ m) and 50～80% methanol isocratic elutions by changing chromatogram column length, flow velocity or column temperature, all can satisfy separation requirement.
(1) sample solution preparation: it is an amount of to get the Ziyuglycoside I crude product, adds dissolve with methanol and is diluted to the solution that every 1ml contains 0.5mg, promptly;
(2) chromatographic condition: with the methanol-water system is the eluent gradient eluting, elution program is 0～7min, 60% → 64% methanol in water, 7～12min, 64% methanol in water, 12～20min, 64% → 80% methanol in water, 20～27min, 80% methanol in water, flow velocity 1.0ml/min, 25 ℃ of column temperatures detect wavelength 210nm, ELSD:85 ℃, 2.8L/min, gainl; Investigated YMC-Pack proC
18Post (150 * 4.6mm, 3.5 μ m) (ultraviolet detection) and SynergiHydro-RP C
18Post (150 * 4.6mm, 4 μ m) (ELSD detection) to the separating effect of Ziyuglycoside I, the results are shown in Figure 2.
Fig. 2 result shows, is less than or equal at particle diameter that the Ziyuglycoside I main peak is separated fully with front next-door neighbour's impurity peaks peak energy on the chromatographic column of 5 μ m.
Embodiment 3 methanol-water system isocratic elutions separate Ziyuglycoside I in Radix Sanguisorbae medical material and the Radix Sanguisorbae total glycosides
(1) medical material sample solution preparation: get the about 0.4g of Radix Sanguisorbae medicinal powder (crossing sieve No. four), the accurate title, decide, put in the 100ml measuring bottle, add 60% alcoholic acid aqueous solution 80ml, shake up ultrasonic 15min, put to room temperature, be settled to scale, shake up, filter and to get the analysis of subsequent filtrate sample introduction or centrifuging and taking supernatant promptly.
(2) preparation of Radix Sanguisorbae total glycosides sample solution: it is an amount of to get the Radix Sanguisorbae medicinal material coarse powder, adding 10 times of volume decoctings boils, each 1h boils three times altogether, filters, merging filtrate, last macroporous adsorbent resin, distilled water thorough washing, back volumetric concentration 70% alcoholic acid aqueous solution eluting, merge eluent and concentrated, be drying to obtain the radix sanguisorbae total saponin powder.Get the about 50mg of Radix Sanguisorbae total glycosides powder, accurate claim surely, put in the 100ml measuring bottle, add 60% ethanol 60ml, shake up, ultrasonic 5min is put to room temperature, is settled to scale, shakes up, and filters to get the analysis of subsequent filtrate sample introduction or centrifuging and taking supernatant promptly;
(3) chromatographic condition: with the methanol-water system is the mobile phase isocratic elution, flow velocity 0.8～1.5ml/min, 25 ℃～40 ℃ of column temperatures, ELSD:85 ℃, 2.8L/min, gain1; Investigated Synergi Hydro-RP C respectively
18Post (150 * 4.6mm, 4 μ m), Gemini C
18Post (150 * 4.6mm, 5 μ m) and luna C
18Post (250 * 4.6mm, 5 μ m) the results are shown in Figure 3 to the separating effect of Ziyuglycoside I and its front next-door neighbour's impurity peaks.
The result shows, for samples such as Radix Sanguisorbae medical material and radix sanguisorbae total saponin extracts, particle diameter less than the chromatographic column of 5 μ m on 50%～80% methanol in water isocratic elution Ziyuglycoside I and the impurity peaks that is close to previously can be realized baseline separation, the methanol-water isocratic elution can be realized baseline separation too on the chromatographic column of 5 conventional μ m.
The separation of Ziyuglycoside I in the embodiment 4 Radix Sanguisorbae medical materials
(1) sample solution preparation: get the Radix Sanguisorbae medical material, pulverize, cross sieve No. four, about 0.4g, precision claims fixed, put in the 100ml measuring bottle, add 60% alcoholic acid aqueous solution 80ml, shake up ultrasonic 15min, put to room temperature, be settled to scale, shake up, filter and to get the analysis of subsequent filtrate sample introduction or centrifuging and taking supernatant promptly.
(2) chromatographic condition: ZORBAX XDB-C
18Post (4.6 * 150mm, 5 μ m) on, flow velocity 1.0ml/min, 25 ℃ of column temperatures detect wavelength 210nm, and (elution program is 0～7min, 60% → 64% methanol in water to use methanol-water system gradient elution respectively, 7～12min, 64% methanol in water, 12～20min, 64% → 80% methanol in water, 20～27min, 80% methanol in water) with the acetonitrile-water system Ziyuglycoside I in the medical material is separated, the results are shown in Figure 4.
Fig. 4 result shows that the methanol-water system can separate the assorted peak of Ziyuglycoside I and its next-door neighbour in the Radix Sanguisorbae medical material fully, and acetonitrile-water then can not separate the two.
The assay and the method validation of Ziyuglycoside I in the embodiment 5 Radix Sanguisorbae medical materials
Content assaying method:
(1) chromatographic condition and system suitability test: with the carbon octadecyl silane is immobile phase (4.6 * 150mm, particle diameter 4 μ m), methanol-water mixed solvent gradient elution, elution program are (0～7min, 60% → 64% methanol in water, 7～13min, 64% methanol in water), flow velocity 0.8ml/min, 25 ℃ of column temperatures, ELSD drift tube temperature are 80 ℃, gas (air) flow velocity 2.5L/min, shunt mode not, theoretical cam curve calculate by Ziyuglycoside I should be not less than 4 * 10
3
(2) preparation of need testing solution: get the about 0.4g of Radix Sanguisorbae medicinal powder (crossing sieve No. four), the accurate title, decide, put in the 100ml measuring bottle, add 60% alcoholic acid aqueous solution 80ml, shake up ultrasonic 15min, put to room temperature, be settled to scale, shake up, filter and to get the analysis of subsequent filtrate sample introduction or centrifuging and taking supernatant promptly.
(3) preparation of reference substance solution: get the about 50mg of Ziyuglycoside I reference substance, the accurate title, decide, and puts in the 50ml measuring bottle, adds 60% alcoholic acid aqueous solution dissolving and be settled to scale, shakes up; Precision is measured in 5.00ml and 25.00ml to the 50ml measuring bottle respectively, adds 60% alcoholic acid aqueous solution dilution and is settled to scale, shakes up and promptly makes the reference substance solution that every 1ml contains 0.1mg and 0.5mg;
(4) content assaying method: accurate respectively reference substance solution and the need testing solution 10 μ l of drawing, inject chromatograph of liquid, measure the peak area of reference substance solution and need testing solution, after peak area integrated value and concentration got common logarithm respectively, the content that calculates Ziyuglycoside I by the external standard two-point method was (in dry product) 3.340%.
Method validation:
(1) under selected chromatographic condition, Ziyuglycoside I has stronger chromatographic peak and separating degree preferably in the Radix Sanguisorbae medical material, and solvent is interference measurement not, sees Fig. 5.The result shows that this method has higher specificity.
(2) linearity
Accurate compound concentration is 0.0998mg/ml, 0.1996mg/ml, 0.2
Each 10ml of Ziyuglycoside I reference substance solution of mg/ml, 0.3992mg/ml, 0.4990mg/ml, accurately draw 10 μ l and inject chromatograph of liquid, measure by above-mentioned condition, common logarithm with the peak area integrated value is a vertical coordinate, the common logarithm of Ziyuglycoside I solution concentration is that abscissa carries out rectilinear regression, linear equation is logA=1.5846logC+7.1419 (r=0.9998), shows that Ziyuglycoside I has the good linear relation in 0.998 μ g～4.990 μ g scopes.
Table 2 linear relationship measurement result
(3) precision
Repeatability
Get Radix Sanguisorbae medicinal powder (crossing sieve No. four) 0.4g respectively, the accurate title, decide, and presses 6 parts of test sample preparation method preparations, the accurate subsequent filtrate 10 μ l sample introductions of drawing, measure the peak area of Ziyuglycoside I, calculate content with the external standard two-point method after peak area integrated value and concentration are got common logarithm, the results are shown in Table 3.
The repeated measurement result of table 3
The result shows that this law repeatability is good.
Withinday precision
Get Ziyuglycoside I contrast solution A (0.0998mg/ml), B (0.499mg/ml) and Radix Sanguisorbae medical material need testing solution, by 0,2,4,6,8 hours intervals are measured the Ziyuglycoside I peak area respectively, calculate content with the external standard two-point method after peak area integrated value and concentration got common logarithm, the results are shown in Table 4.
Table 4 withinday precision measurement result
The result shows that this law withinday precision is good.
Day to day precision
Get the contrast liquid of withinday precision and test liquid respectively at the 0th, 1,2,3 day (d) sample introduction, measure the Ziyuglycoside I peak area, calculate content with the external standard two-point method after peak area integrated value and concentration are got common logarithm, the results are shown in Table 5.
Table 5 day to day precision measurement result
The result shows that the day to day precision of this law is good.
(4) application of sample recovery test
Get Radix Sanguisorbae medicinal powder (the crossing sieve No. four) 0.2g of known content, the accurate title, decide, put in the measuring bottle of 100ml, accurate respectively Ziyuglycoside I reference substance solution 1,2, the 3ml that adds 5.020mg/ml, reuse 60% alcoholic acid aqueous solution is supplied 80ml, by 3.7 following need testing solution preparation method preparations, promptly.Precision is measured subsequent filtrate 10 μ l sample introductions, and the record peak area calculates content with the external standard two-point method after peak area integrated value and concentration got common logarithm, the results are shown in Table 6.
Table 6 determination of recovery rates result
The result shows that this law average recovery meets the requirements, and accuracy is higher.
The assay of Ziyuglycoside I in embodiment 6 DIYU SHENGBAI PIAN
Content assaying method:
(1) chromatographic condition and system suitability test: with the carbon octadecyl silane is immobile phase (4.6 * 150mm, particle diameter is less than 5 μ m), methanol-water (64: 36) isocratic elution, flow velocity 1.0ml/min, 25 ℃ of column temperatures, ELSD (Alltech 2000) drift tube temperature is 85 ℃, gas (air) flow velocity 2.8L/min, shunt mode not, theoretical cam curve calculate by Ziyuglycoside I should be not less than 4 * 10
3
(2) preparation of need testing solution: get 20 of DIYU SHENGBAI PIAN (Chengdu Diao Pharmaceutical Group self-sufficient and strategically located region Pharmaceutical is produced, lot number 070301), the accurate title, decide, porphyrize, precision take by weighing in right amount (being equivalent to Ziyuglycoside I 2mg approximately), put in the 150ml tool plug conical flask, accurately add 50% ethanol 20.00ml, shake up, weigh, ultrasonic 10min, put to room temperature, supply the weight that subtracts mistake with 50% ethanol, shake up, the centrifuging and taking supernatant promptly;
(3) preparation of reference substance solution: get the about 25mg of Ziyuglycoside I reference substance, the accurate title, decide, and puts in the 50ml measuring bottle, adds 50% alcoholic acid aqueous solution dissolving and be settled to scale, shakes up; Precision is measured in 5.00ml and 20.00ml to the 50ml measuring bottle respectively, adds 50% alcoholic acid aqueous solution dilution and is settled to scale, shakes up and promptly makes the reference substance solution that every 1ml contains 0.05mg and 0.2mg;
(4) content assaying method: accurate respectively reference substance solution and the need testing solution 20 μ l of drawing, inject chromatograph of liquid, measure the peak area of reference substance solution and need testing solution, after peak area integrated value and concentration got common logarithm respectively, the content that calculates Ziyuglycoside I by the external standard two-point method was the 0.18mg/ sheet.
Method validation:
(1) specificity: under selected chromatographic condition, Ziyuglycoside I has stronger chromatographic peak and separating degree preferably in the DIYU SHENGBAI PIAN, and solvent and adjuvant be interference measurement not, sees Fig. 6.The result shows that this method has higher specificity.
(2) linearity
Accurate compound concentration is each 10ml of Ziyuglycoside I reference substance solution of 0.05014mg/ml, 0.1003mg/ml, 0.2006mg/ml, 0.4011mg/ml, 0.5014mg/ml, accurately draw 20 μ l and inject chromatograph of liquid, measure by above-mentioned condition, common logarithm with the peak area integrated value is a vertical coordinate, the common logarithm of Ziyuglycoside I solution concentration is that abscissa carries out rectilinear regression, linear equation is logA=1.6549logC+7.0118 (r=1), shows that Ziyuglycoside I has the good linear relation in 1.003 μ g～10.03 μ g scopes.
Table 7 linear relationship measurement result
(3) repeatability
Get Radix Sanguisorbae medical material SHENGBAI PIAN powder (lot number 070301 respectively, be equivalent to Ziyuglycoside I 2mg approximately) about 1g, the accurate title, decide, press 5 parts of test sample preparation method preparations, the accurate subsequent filtrate 20 μ l sample introductions of drawing, measure the peak area of Ziyuglycoside I, calculate content with the external standard two-point method after peak area integrated value and concentration are got common logarithm, the results are shown in Table 8.
The repeated measurement result of table 8
The result shows that this law repeatability is good.
(4) application of sample recovery test
Get the DIYU SHENGBAI PIAN powder (lot number 070301 of known content (Ziyuglycoside I 0.18%), be equivalent to Ziyuglycoside I 1mg approximately) 0.5g, the accurate title, decide, put in the conical flask bottle of 150ml, accurate respectively Ziyuglycoside I reference substance solution 1,3, the 10ml that adds 0.3008mg/ml, reuse 50% ethanol is supplied 20ml, presses the preparation of need testing solution preparation method, promptly.Precision is measured subsequent filtrate 20 μ l sample introductions, and the record peak area calculates content with the external standard two-point method after peak area integrated value and concentration got common logarithm, the results are shown in Table 9.
Table 9 determination of recovery rates result
The result shows that this law average recovery meets the requirements, and accuracy is higher.
The Determination of Content Uniformity of Ziyuglycoside I in embodiment 7 DIYU SHENGBAI PIAN
(1) chromatographic condition and system suitability test: with the carbon octadecyl silane is immobile phase (4.6 * 150mm, particle diameter is less than 5 μ m), methanol-water (64: 36) isocratic elution, flow velocity 1.0ml/min, 25 ℃ of column temperatures, ELSD (Alltech 2000) drift tube temperature is 85 ℃, gas (air) flow velocity 2.8L/min, shunt mode not, theoretical cam curve calculate by Ziyuglycoside I should be not less than 4 * 10
3
(2) preparation of need testing solution: (Chengdu Diao Pharmaceutical Group self-sufficient and strategically located region Pharmaceutical is produced to get DIYU SHENGBAI PIAN, lot number 070301) 10, accurate respectively title is fixed, puts in the 5ml centrifuge tube, accurately add 50% ethanol 2.00ml, shake up, weigh, ultrasonic 10min, put to room temperature, supply the weight that subtracts mistake with 50% ethanol, shake up, the centrifuging and taking supernatant promptly;
(3) preparation of reference substance solution: get the about 25mg of Ziyuglycoside I reference substance, the accurate title, decide, and puts in the 50ml measuring bottle, adds 50% dissolve with ethanol and be settled to scale, shakes up; Precision is measured in 5.00ml and 20.00ml to the 50ml measuring bottle respectively, adds 50% ethanol dilution and is settled to scale, shakes up and promptly makes the reference substance solution that every 1ml contains 0.05mg and 0.2mg;
(4) content assaying method: accurate respectively reference substance solution 20 μ l and the need testing solution 20 μ l of drawing, inject chromatograph of liquid, measure the peak area of reference substance solution and need testing solution, after peak area integrated value and concentration got common logarithm respectively, calculate the content of 10 middle Ziyuglycoside I respectively by the external standard two-point method, its RSD value should be no more than 7.0% (table 10, Fig. 7).
Table 10 Determination of Content Uniformity result
The assay of Ziyuglycoside I in embodiment 8 radix sanguisorbae total saponin extracts
(1) chromatographic condition and system suitability test: with the carbon octadecyl silane is immobile phase (4.6 * 150mm, particle diameter is less than 3.5 μ m), methanol-water mixed solvent gradient elution, elution program is 0～7min, 60% → 64% methanol in water, 7～13min, 64% methanol in water, flow velocity 1.2ml/min, 25 ℃ of column temperatures, ELSD (Alltech 2000ES) drift tube temperature is 100 ℃, gas (air) flow velocity 3.5L/min, shunt mode not, theoretical cam curve calculate by Ziyuglycoside I should be not less than 4 * 10
3
(2) preparation of need testing solution: it is an amount of to get the Radix Sanguisorbae medicinal material coarse powder, adding 10 times of volume decoctings boils, each 1h boils three times altogether, filters, merging filtrate, last macroporous adsorbent resin, distilled water thorough washing, back 70% ethanol elution, merge eluent and concentrated, be drying to obtain radix sanguisorbae total saponin powder (080604).Get the about 50mg of Radix Sanguisorbae total glycosides powder, accurate claim surely, put in the 100ml measuring bottle, add 60% ethanol 60ml, shake up, ultrasonic 5min is put to room temperature, is settled to scale, shakes up, and filters to get the analysis of subsequent filtrate sample introduction or centrifuging and taking supernatant promptly;
(3) preparation of reference substance solution: get the about 50mg of Ziyuglycoside I reference substance, the accurate title, decide, and puts in the 50ml measuring bottle, adds 60% alcoholic acid aqueous solution dissolving and be settled to scale, shakes up; Precision is measured in 5.00ml and 25.00ml to the 50ml measuring bottle respectively, adds 60% alcoholic acid aqueous solution dilution and is settled to scale, shakes up and promptly makes the reference substance solution that every 1ml contains 0.1mg and 0.5mg;
(4) content assaying method: accurate respectively reference substance solution and the need testing solution 10 μ l of drawing, inject chromatograph of liquid, measure the peak area of reference substance solution and need testing solution, after peak area integrated value and concentration got common logarithm respectively, the content that calculates Ziyuglycoside I by the external standard two-point method was 50% (Fig. 8).
The purity testing of embodiment 9 Ziyuglycoside I reference substances
(1) chromatographic condition and system suitability test: with the carbon octadecyl silane is immobile phase (4.6 * 150mm, particle diameter is less than 5 μ m), methanol-water mixed solvent gradient elution, elution program is 0～7min, 60% → 64% methanol in water, 7～12min, 64% methanol in water, 12～20min64% → 80% methanol in water, 20～27min, 80% methanol in water, flow velocity 1.0ml/min, 25 ℃ of column temperatures, ELSD (Alltech 2000ES) drift tube temperature is 85 ℃, gas (air) flow velocity 2.8L/min, shunt mode not, theoretical cam curve calculate by Ziyuglycoside I should be not less than 9000;
(2) preparation of sample solution: it is an amount of to get Ziyuglycoside I, adds dissolve with methanol and is diluted to the solution that every 1ml contains 0.5mg, promptly;
(3) purity test method: the accurate sample solution 10 μ l that draw, inject chromatograph of liquid, measure, press the peak area normalization method and calculate the purity of Ziyuglycoside I greater than 99.0% (Fig. 9).
The preparation of embodiment 10 Ziyuglycoside I reference substances
It is an amount of to get the Radix Sanguisorbae medicinal material coarse powder, adds 10 times of volume decoctings and boils, and each 1h boils three times altogether, filter, and merging filtrate, last macroporous adsorbent resin, the distilled water thorough washing, back 70% ethanol elution merges eluent and also concentrates, and is drying to obtain the radix sanguisorbae total saponin powder.With methanol (1g: 5ml) reflux 10min, filtered while hot treats that filtrate is chilled to room temperature, stirs to drip water down, and is muddy to occurring, and do not disappear, stop, leaving standstill 12h, sucking filtration precipitates with 30% washing with alcohol, collect, vacuum drying is pulverized, time recrystallized product of winning.Recrystallization one to twice repeatedly again, Ziyuglycoside I.
Claims (11)
1. the content assaying method of a Ziyuglycoside I is characterized in that, described method comprises:
1) test sample of getting the standard substance of Ziyuglycoside I respectively and containing Ziyuglycoside I is dissolved in the ethanol water, makes standard solution and need testing solution respectively;
2) get isopyknic standard solution and need testing solution respectively and inject high performance liquid chromatograph, the peak area of measurement standard product solution and need testing solution and calculating promptly gets the content of the Ziyuglycoside I in the test sample then;
Wherein, the immobile phase of described high performance liquid chromatograph is the carbon octadecyl silane; Mobile phase is methanol-water solution.
2. method according to claim 1 is characterized in that, the particle diameter of described carbon octadecyl silane is≤5 μ m.
3. method according to claim 2 is characterized in that, the particle diameter of described carbon octadecyl silane is 5 μ m, 4 μ m or 3.5 μ m.
4. method according to claim 3 is characterized in that, described mobile phase is the solution of methanol-water; Be preferably in percent by volume, the gradient solution of 60%～80% methanol in water or 50%～80% methanol in water etc. the degree solution.
5. method according to claim 4, it is characterized in that, described gradient solution is: in percent by volume, and 0～17min, 60% → 70% methanol in water, 17～25min, 70% → 80% methanol in water, 25～35min, 80% methanol in water; Or
0～7min, 60% → 64% methanol in water, 7～12min, 64% → 66% methanol in water, 12～17min, 66% → 80% methanol in water, 17～25min, 80% methanol in water; Or
0～7min, 60% → 64% methanol in water, 7～15min, 64% methanol in water, 15～20min64% → 80% methanol in water, 20～25min, 80% methanol in water; Or
0～7min, 60% → 64% methanol in water, 7～12min, 64% methanol in water, 12～20min64% → 80% methanol in water, 20～27min, 80% methanol in water; Or
0～7min, 60% → 64% methanol in water, 7～13min, 64% methanol in water.
6. method according to claim 5 is characterized in that, described solution such as degree such as grade is in percent by volume, 64% methanol in water.
7. method according to claim 6 is characterized in that, described alcoholic acid aqueous solution is the alcoholic acid aqueous solution of by volume 50%-60%.
8. method of utilizing the arbitrary described method of claim 1-7 to measure the content of Ziyuglycoside I in the Radix Sanguisorbae medical material is characterized in that described method comprises:
1) preparation of need testing solution: get the Radix Sanguisorbae medical material, pulverize, cross sieve No. four, about 0.4g, the accurate title, decide, and puts in the 100ml measuring bottle, add 60% alcoholic acid aqueous solution 80ml, shake up, ultrasonic 15min is put to room temperature, be settled to scale, shake up, filter, get analysis of subsequent filtrate sample introduction or centrifuging and taking supernatant promptly;
The preparation of reference substance solution: get the about 50mg of Ziyuglycoside I reference substance, the accurate title, decide, and puts in the 50ml measuring bottle, adds 60% alcoholic acid water dissolution and be settled to scale, shakes up; Precision is measured in 5.00ml and 25.00ml to the 50ml measuring bottle respectively, adds 60% alcoholic acid aqueous solution dilution and is settled to scale, shakes up and promptly makes the reference substance solution that every 1ml contains 0.1mg and 0.5mg;
2) assay: accurate respectively reference substance solution and the need testing solution 10 μ l of drawing, inject high performance liquid chromatograph, measure the peak area of reference substance solution and need testing solution, peak area integrated value and concentration are got common logarithm respectively after, press the content of external standard two-point method calculating Ziyuglycoside I;
Wherein, high performance liquid chromatography is an immobile phase with the carbon octadecyl silane, particle diameter is less than or equal to 5 μ m, mobile phase is that methanol and water volume are the gradient eluent of 64%: 36% isocratic elution liquid or the methanol volumetric concentration methanol-water mixed solution that is 60-64%, elution program is 0～7min, 60% → 64% methanol in water, 7～13min, 64% methanol in water, flow velocity 0.8ml/min, 25 ℃ of column temperatures, the ELSD drift tube temperature is 80 ℃, gas flow rate 2.5L/min, shunt mode not, theoretical cam curve is calculated by Ziyuglycoside I should be not less than 4 * 10
3
9. method of utilizing the arbitrary described method of claim 1-7 to measure the content of Ziyuglycoside I in the sanguisorbin extract is characterized in that described method comprises:
1) preparation of need testing solution: get radix sanguisorbae total saponin powder 50mg, the accurate title, decide, and puts in the 100ml measuring bottle, adds 60% ethanol 60ml, shakes up, and ultrasonic 5min is put to room temperature, is settled to scale, shakes up, and filters, and gets subsequent filtrate or the analysis of centrifuging and taking supernatant sample introduction;
The preparation of reference substance solution: get the about 50mg of Ziyuglycoside I reference substance, the accurate title, decide, and puts in the 50ml measuring bottle, adds 60% alcoholic acid aqueous solution dissolving and be settled to scale, shakes up; Precision is measured in 5.00ml and 25.00ml to the 50ml measuring bottle respectively, adds 60% alcoholic acid aqueous solution dilution and is settled to scale, shakes up and promptly makes the reference substance solution that every 1ml contains 0.1mg and 0.5mg;
2) assay: accurate respectively reference substance solution and the need testing solution 10 μ l of drawing, inject high performance liquid chromatograph, measure the peak area of reference substance solution and need testing solution, peak area integrated value and concentration are got common logarithm respectively after, press the content of external standard two-point method calculating Ziyuglycoside I;
Wherein, high performance liquid chromatograph is an immobile phase with the carbon octadecyl silane, and particle diameter is less than or equal to 5 μ m, is the methanol-water mixed solvent gradient elution of 60-64% with the methanol volumetric concentration, elution program is 0～7min, 60% → 64% methanol in water, 7～13min, 64% methanol in water, flow velocity 1.2ml/min, 25 ℃ of column temperatures, the ELSD drift tube temperature is 100 ℃, gas flow rate 3.5L/min, shunt mode not, theoretical cam curve is calculated by Ziyuglycoside I should be not less than 4 * 10
3
10. method of utilizing the arbitrary described method of claim 1-7 to measure the content of Ziyuglycoside I in the Radix Sanguisorbae tablet is characterized in that described method comprises:
1) preparation of need testing solution: get 20 of DIYU SHENGBAI PIAN, the accurate title, decided porphyrize, precision takes by weighing the amount that is equivalent to the 2mg Ziyuglycoside I, put in the 150ml tool plug conical flask, accurately add 50% alcoholic acid aqueous solution 20ml, shake up, weigh, ultrasonic 10min is put to room temperature, supplies the weight that subtracts mistake with 50% alcoholic acid aqueous solution, shake up, the centrifuging and taking supernatant promptly;
The preparation of reference substance solution: it is an amount of to be taken at 105 ℃ of Ziyuglycoside I reference substances that are dried to constant weight, adds 50% alcoholic acid aqueous solution dissolving and standardize solution and makes the reference substance solution that every 1ml contains 0.05mg and 0.2mg;
2) assay: accurate respectively reference substance solution and the need testing solution 20 μ l of drawing, inject colleges and universities' chromatograph of liquid, measure the peak area of reference substance solution and need testing solution, peak area integrated value and concentration are got common logarithm respectively after, press the content of external standard two-point method calculating Ziyuglycoside I;
Wherein, high performance liquid chromatograph, the carbon octadecyl silane that is less than or equal to 5 μ m with particle diameter is an immobile phase, with the methanol-water volume ratio is that 64: 36 mixed solution is an isocratic elution liquid, or be the methanol-water mixed solvent gradient elution of 60-64% with the methanol volumetric concentration, elution program is 0～7min, 60% → 64% methanol in water, 7～13min, 64% methanol in water, flow velocity 1.0ml/min, 25 ℃ of column temperatures, ELSD drift tube temperature are 85 ℃, air velocity 2.8L/min, shunt mode not, theoretical cam curve calculate by Ziyuglycoside I should be not less than 4 * 10
3
11. the method for detecting purity of a Ziyuglycoside I is characterized in that, described method comprises:
1) preparation of sample solution: it is an amount of to get Ziyuglycoside I, adds dissolve with methanol and is diluted to the solution that every 1ml contains 0.5mg, promptly;
2) purity is measured: the accurate sample solution 10 μ l that draw, inject chromatograph of liquid, and measure, press the purity that the peak area normalization method is calculated Ziyuglycoside I;
Wherein, described high performance liquid chromatograph is an immobile phase with the carbon octadecyl silane, particle diameter is less than or equal to 5 μ m, the methanol volumetric concentration is the methanol-water mixed solvent gradient elution of 60-64%, elution program is 0～7min, 60% → 64% methanol in water, 7～12min, 64% methanol in water, 12～20min, 64% → 80% methanol in water, 20～27min, 80% methanol in water, flow velocity 1.0ml/min, 25 ℃ of column temperatures, the ELSD drift tube temperature is 85 ℃, gas (air) flow velocity 2.8L/min, shunt mode not, theoretical cam curve is calculated by Ziyuglycoside I should be not less than 4 * 10
3
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| CN107064322A (en) * | 2016-12-01 | 2017-08-18 | 成都中医药大学 | Gallic acid and the HPLC wavelength of sanguisorbin I contents switching method in garden burnet or garden burnet class preparation are determined simultaneously |
| CN107064322B (en) * | 2016-12-01 | 2019-12-31 | 成都中医药大学 | HPLC wavelength switching method for simultaneously measuring contents of gallic acid and sanguisorbin I in sanguisorba or sanguisorba preparations |
| CN109975437A (en) * | 2017-12-27 | 2019-07-05 | 四川科瑞德制药股份有限公司 | The analysis method of sanguisorbin I in garden burnet or product containing garden burnet |
| CN116359355A (en) * | 2021-12-27 | 2023-06-30 | 江阴天江药业有限公司 | Application of sanguisorbin I in quality detection of holly leaf samples and corresponding quality detection method |
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