CN104020225B - Shengxuebao-medicine for improving hematopoitic function formulation characteristics collection of illustrative plates and method for building up thereof - Google Patents

Shengxuebao-medicine for improving hematopoitic function formulation characteristics collection of illustrative plates and method for building up thereof Download PDF

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CN104020225B
CN104020225B CN201410187475.6A CN201410187475A CN104020225B CN 104020225 B CN104020225 B CN 104020225B CN 201410187475 A CN201410187475 A CN 201410187475A CN 104020225 B CN104020225 B CN 104020225B
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retention time
peaks
relative
standard deviation
medicine
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CN104020225A (en
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谈发金
郑桂林
柳卓
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KANGSHOU PHARMACY Co Ltd HUNAN
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KANGSHOU PHARMACY Co Ltd HUNAN
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Abstract

The invention belongs to technical field of medicine quality control, the method for building up of disclosed Shengxuebao-medicine for improving hematopoitic function formulation characteristics collection of illustrative plates, comprise: get Paeoniflorin reference substance and to be dissolved in 50%-100% (volume) methyl alcohol to form Paeoniflorin reference substance concentration for 0.02mg/ml solution, after shaking up, obtain object of reference solution; Get the Shengxuebao-medicine for improving hematopoitic function formulation dissolution of set amount in 0%-100% methyl alcohol, 0%-50% ethanol or 25-125ml water, by refluxing extraction or ultrasonic process 10-60min after weighing, after cooling, weigh again, then mend heavy with homogeneous solvent, shake up, filter with miillpore filter, get subsequent filtrate as need testing solution; Draw object of reference solution and need testing solution injection high performance liquid chromatograph, detect according to high performance liquid chromatography, obtain the characteristic spectrum of Shengxuebao-medicine for improving hematopoitic function preparation.The present invention also discloses a kind of Shengxuebao-medicine for improving hematopoitic function formulation characteristics collection of illustrative plates.Above-mentioned characteristic spectrum achieves the comparatively complete detection to the Shengxuebao-medicine for improving hematopoitic function quality of the pharmaceutical preparations.

Description

Shengxuebao-medicine for improving hematopoitic function formulation characteristics collection of illustrative plates and method for building up thereof
Technical field
The present invention relates to technical field of medicine quality control, more specifically, relate to the method for building up of Shengxuebao-medicine for improving hematopoitic function formulation characteristics collection of illustrative plates.The invention still further relates to the Shengxuebao-medicine for improving hematopoitic function formulation characteristics collection of illustrative plates obtained by above-mentioned method for building up.
Background technology
Shengxuebao-medicine for improving hematopoitic function is by the Empirical formula of the famous traditional Chinese medicine and pharmacy expert of China, is prepared from through modern crafts.Shengxuebao-medicine for improving hematopoitic function is made primarily of this seven flavor medicine material of prepared fleece flower root, the fruit of glossy privet, mulberries, eclipta, the root of herbaceous peony, the Radix Astragali and rhizoma cibotii.The clinical practice of decades proves that Shengxuebao-medicine for improving hematopoitic function has nourishing liver and kidney, effect of replenishing qi and blood, for symptoms such as Neuroleptic Leukocytopenia caused by concurrent chemoradiotherapy of malignant tumor, spiritlessness and weakness, soreness and weakness of waist and knees, dizziness and tinnitus, palpitaition, insomnia, dry throat, poor appetite, food are few.
At present, according to the Shengxuebao-medicine for improving hematopoitic function preparation of the obtained multiple formulation of Shengxuebao-medicine for improving hematopoitic function prescription.Shengxuebao-medicine for improving hematopoitic function preparation on market is primarily of granule, mixture etc.Usually, the preparation technology of Shengxuebao-medicine for improving hematopoitic function preparation is: by the water extract of cataclasm for raw medicinal material rear extracting in water, make granule, tablet, pill, capsule, oral liquid or mixture according to the method for existing routine.Shengxuebao-medicine for improving hematopoitic function preparation is as Chinese patent drug, and its drug effect is the coefficient result of various active composition.Therefore, whether the various active component content of Shengxuebao-medicine for improving hematopoitic function preparation is up to standard is the key factor weighing the Shengxuebao-medicine for improving hematopoitic function quality of the pharmaceutical preparations.But the quality standard detecting method of current Shengxuebao-medicine for improving hematopoitic function preparation only measures the content of single index composition, does not detect other effective ingredient.This detection method has some limitations, and cannot reflect the quality level of Shengxuebao-medicine for improving hematopoitic function preparation more comprehensively.
Summary of the invention
The invention provides a kind of method for building up of Shengxuebao-medicine for improving hematopoitic function formulation characteristics collection of illustrative plates, to solve the problem that current Shengxuebao-medicine for improving hematopoitic function preparation detection method cannot reflect Shengxuebao-medicine for improving hematopoitic function quality of the pharmaceutical preparations level more comprehensively.
In order to solve the problems of the technologies described above, the invention provides following technical scheme:
The method for building up of Shengxuebao-medicine for improving hematopoitic function formulation characteristics collection of illustrative plates, comprises the following steps:
Getting Paeoniflorin reference substance to be dissolved in 50%-100% (volume) methyl alcohol to form Paeoniflorin reference substance concentration for 0.02mg/ml solution, obtains object of reference solution after shaking up;
Get the Shengxuebao-medicine for improving hematopoitic function formulation dissolution of set amount in 0%-100% methyl alcohol, 0%-50% ethanol or 25-125ml water, by refluxing extraction 10-60min after weighing, or ultrasonic process 10-60min after weighing, after cooling, weigh again, then mend heavy with homogeneous solvent, shake up, filter with miillpore filter, get subsequent filtrate as need testing solution, described Shengxuebao-medicine for improving hematopoitic function preparation is oral liquid or mixture, described set amount is 1.0-5.0ml, or described Shengxuebao-medicine for improving hematopoitic function preparation is granule, tablet, capsule, pill or Shengxuebao-medicine for improving hematopoitic function preparation intermediate, described set amount is the powder of 0.6-3.0g,
Draw described object of reference solution and need testing solution injection high performance liquid chromatograph, detect according to high performance liquid chromatography, obtain the characteristic spectrum of Shengxuebao-medicine for improving hematopoitic function preparation; Wherein, chromatographic column is C 18reverse-phase chromatographic column; Adopt gradient elution, and the flow velocity of the mobile phase of gradient eluent is 0.8ml/min-1.2ml/min; Determined wavelength is 200-230nm, and column temperature is 25 DEG C-40 DEG C; The gradient elution time is 60min-120min.
Preferably, in above-mentioned method for building up, the mobile phase of gradient eluent is acetonitrile-water, acetonitrile-(0.1%-0.2%) phosphoric acid solution, acetonitrile-0.1% formic acid solution, methyl alcohol-0.1% phosphoric acid solution, methyl alcohol-0.1% formic acid solution or methanol-water.
Preferably, in above-mentioned method for building up, the mobile phase of described gradient eluent is mobile phase A and Mobile phase B composition, and mobile phase A is acetonitrile, and Mobile phase B is 0.1% (volume) phosphoric acid solution.
Preferably, in above-mentioned method for building up, draw described object of reference solution and need testing solution and be 5 μ l and inject high performance liquid chromatographs, the time of described gradient elution is 60min, carries out gradient elution in the following way:
0-15min, the percent by volume of mobile phase A is 2%, and the percent by volume of Mobile phase B is 98%;
15-30min, the percent by volume of mobile phase A is 2%-15%, and the percent by volume of Mobile phase B is 98%-85%;
30-50min, the percent by volume of mobile phase A is 15%-25%, and the percent by volume of Mobile phase B is 85%-75%;
50-60min, the percent by volume of mobile phase A is 25%, and the percent by volume of Mobile phase B is 75%.
Preferably, in above-mentioned method for building up, the flow velocity of described mobile phase is 1.0ml/min, and the column length of described C18 reverse-phase chromatographic column is 250mm.
Shengxuebao-medicine for improving hematopoitic function formulation characteristics collection of illustrative plates, method for building up as above is adopted to obtain, described characteristic spectrum is detected by the need testing solution that the methanol solution got blood nourishing granules powder 0.6g and be dissolved in 25ml obtains and obtains, described characteristic spectrum has 7 characteristic peaks, wherein, No. 5 peaks take Paeoniflorin as the reference peak of reference, and each characteristic peak relative retention time and relative standard deviation are:
No. 1 peak: relative retention time 0.383 ± 0.019, relative standard deviation is 0.19%;
No. 2 peaks: relative retention time 0.672 ± 0.034, relative standard deviation is 0.07%;
No. 3 peaks: relative retention time 0.801 ± 0.040, relative standard deviation is 0.05%;
No. 4 peaks: relative retention time 0.956 ± 0.048, relative standard deviation is 0.01%;
No. 5 peaks: relative retention time 1.000 ± 0.000, relative standard deviation is 0.00%;
No. 6 peaks: relative retention time 1.143 ± 0.057, relative standard deviation is 0.01%;
No. 7 peaks: relative retention time 1.188 ± 0.059, relative standard deviation is 0.03%.
Shengxuebao-medicine for improving hematopoitic function formulation characteristics collection of illustrative plates, method for building up as above is adopted to obtain, described characteristic spectrum is detected by the need testing solution that the water got 3.0g Shengxuebao-medicine for improving hematopoitic function preparation intermediate and be dissolved in 125ml obtains and obtains, described characteristic spectrum has 7 characteristic peaks, wherein, No. 5 peaks take Paeoniflorin as the reference peak of reference, and each characteristic peak relative retention time and relative standard deviation are:
No. 1 peak: relative retention time 0.383 ± 0.019, relative standard deviation is 0.14%;
No. 2 peaks: relative retention time 0.672 ± 0.034, relative standard deviation is 0.00%;
No. 3 peaks: relative retention time 0.801 ± 0.040, relative standard deviation is 0.05%;
No. 4 peaks: relative retention time 0.956 ± 0.048, relative standard deviation is 0.00%;
No. 5 peaks: relative retention time 1.000 ± 0.000, relative standard deviation is 0.00%;
No. 6 peaks: relative retention time 1.143 ± 0.057, relative standard deviation is 0.00%;
No. 7 peaks: relative retention time 1.188 ± 0.059, relative standard deviation is 0.00%.
Shengxuebao-medicine for improving hematopoitic function formulation characteristics collection of illustrative plates, method for building up as above is adopted to obtain, described characteristic spectrum is detected by the need testing solution that 50% methyl alcohol got 1.2g Shengxuebao-medicine for improving hematopoitic function capsule powders and be dissolved in 50ml obtains and obtains, described characteristic spectrum has 7 characteristic peaks, wherein, No. 5 peaks take Paeoniflorin as the reference peak of reference, and each characteristic peak relative retention time and relative standard deviation are:
No. 1 peak: relative retention time 0.383 ± 0.019, relative standard deviation is 1.19%;
No. 2 peaks: relative retention time 0.672 ± 0.034, relative standard deviation is 0.43%;
No. 3 peaks: relative retention time 0.801 ± 0.040, relative standard deviation is 0.19%;
No. 4 peaks: relative retention time 0.956 ± 0.048, relative standard deviation is 0.05%;
No. 5 peaks: relative retention time 1.000 ± 0.000, relative standard deviation is 0.00%;
No. 6 peaks: relative retention time 1.143 ± 0.057, relative standard deviation is 0.28%;
No. 7 peaks: relative retention time 1.188 ± 0.059, relative standard deviation is 0.12%.
Shengxuebao-medicine for improving hematopoitic function formulation characteristics collection of illustrative plates, method for building up as above is adopted to obtain, described characteristic spectrum to detect in the need testing solution that 25% ethanol of 100ml obtains obtain by getting 2.4g Shengxuebao-medicine for improving hematopoitic function tablet dissolved, described characteristic spectrum has 7 characteristic peaks, wherein, No. 5 peaks take Paeoniflorin as the reference peak of reference, and each characteristic peak relative retention time and relative standard deviation are:
No. 1 peak: relative retention time 0.383 ± 0.019, relative standard deviation is 0.19%;
No. 2 peaks: relative retention time 0.672 ± 0.034, relative standard deviation is 0.07%;
No. 3 peaks: relative retention time 0.801 ± 0.040, relative standard deviation is 0.05%;
No. 4 peaks: relative retention time 0.956 ± 0.048, relative standard deviation is 0.01%;
No. 5 peaks: relative retention time 1.000 ± 0.000, relative standard deviation is 0.00%;
No. 6 peaks: relative retention time 1.143 ± 0.057, relative standard deviation is 0.01%;
No. 7 peaks: relative retention time 1.188 ± 0.059, relative standard deviation is 0.03%.
Shengxuebao-medicine for improving hematopoitic function formulation characteristics collection of illustrative plates, method for building up as above is adopted to obtain, described characteristic spectrum is detected by the need testing solution that 50% ethanol got 1ml Shengxuebao-medicine for improving hematopoitic function oral liquid and be dissolved in 25ml obtains and obtains, described characteristic spectrum has 7 characteristic peaks, wherein, No. 5 peaks take Paeoniflorin as the reference peak of reference, and each characteristic peak relative retention time and relative standard deviation are:
No. 1 peak: relative retention time 0.383 ± 0.019, relative standard deviation is 0.21%;
No. 2 peaks: relative retention time 0.672 ± 0.034, relative standard deviation is 0.08%;
No. 3 peaks: relative retention time 0.801 ± 0.040, relative standard deviation is 0.07%;
No. 4 peaks: relative retention time 0.956 ± 0.048, relative standard deviation is 0.05%;
No. 5 peaks: relative retention time 1.000 ± 0.000, relative standard deviation is 0.00%;
No. 6 peaks: relative retention time 1.143 ± 0.057, relative standard deviation is 0.00%;
No. 7 peaks: relative retention time 1.188 ± 0.059, relative standard deviation is 0.04%
Beneficial effect of the present invention is as follows:
The characteristic spectrum method for building up of A, Shengxuebao-medicine for improving hematopoitic function preparation provided by the invention is simple to the pre-treating method of test sample, and characteristic chemical constituent retains complete, and need testing solution has good stability.
The precision of B, efficient liquid-phase chromatography method is higher, reappearance good, and analysis time is shorter, has certain specificity; In gained characteristic spectrum, each characteristic peak separating effect is better, and Shengxuebao-medicine for improving hematopoitic function formulation characteristics collection of illustrative plates and intermediate characteristic spectrum have good correlativity.
The characteristic spectrum that C, the present invention set up has 7 characteristic peaks, and quantity of information is comparatively large, more completely remains the effective constituent in test liquid, specify that 3 index compositions, through comparing, adopting retention time moderate, degree of separation is better, and the larger Paeoniflorin object of reference of peak area is as with reference to peak.
The application of D, Shengxuebao-medicine for improving hematopoitic function preparation intermediate characteristic spectrum can increase the controllability of Shengxuebao-medicine for improving hematopoitic function formulation manufacturing processes, the application of Shengxuebao-medicine for improving hematopoitic function formulation characteristics collection of illustrative plates more comprehensively can reflect technique whether stable uniform, be conducive to the effective constituent steady quality and the clinical efficacy that ensure Shengxuebao-medicine for improving hematopoitic function preparation, method provided by the invention has advantageously ensured the whole process quality control from raw material to terminal preparation.
Accompanying drawing explanation
In order to be illustrated more clearly in the technical scheme in the embodiment of the present invention, below the accompanying drawing used required in describing embodiment is briefly described, apparently, for those of ordinary skills, under the prerequisite not paying creative work, other accompanying drawing can also be obtained according to these accompanying drawings.
Fig. 1 object of reference superposition chromatogram;
Fig. 2 is blood nourishing granules characteristic spectrum;
Fig. 3 is 10 batches of blood nourishing granules characteristic spectrums;
Fig. 4 is Shengxuebao-medicine for improving hematopoitic function preparation intermediate characteristic spectrum;
Fig. 5 is 10 batches of Shengxuebao-medicine for improving hematopoitic function preparation intermediate characteristic spectrums;
Fig. 6 makes a living Xue-bao capsules characteristic spectrum;
Fig. 7 is 10 batches of Shengxuebao-medicine for improving hematopoitic function capsule characteristics collection of illustrative plates;
Fig. 8 is Shengxuebao-medicine for improving hematopoitic function particle characteristic collection of illustrative plates;
Fig. 9 is 10 batches of Shengxuebao-medicine for improving hematopoitic function particle characteristic collection of illustrative plates;
Figure 10 makes a living " Xuebao " oral liquid (blood medicine) characteristic spectrum;
Figure 11 is 10 batches of Shengxuebao-medicine for improving hematopoitic function oral liquid characteristic spectrums;
Figure 12 is the ownership collection of illustrative plates of Shengxuebao-medicine for improving hematopoitic function formulation characteristics peak and each medicinal material characteristic peak.
Embodiment
Embodiments provide a kind of method for building up of Shengxuebao-medicine for improving hematopoitic function formulation characteristics collection of illustrative plates, detect the problem that more comprehensively cannot reflect Shengxuebao-medicine for improving hematopoitic function quality of the pharmaceutical preparations level to solve current Shengxuebao-medicine for improving hematopoitic function preparation.
Technical scheme in the embodiment of the present invention is understood better in order to make those skilled in the art person, and enable the above-mentioned purpose of the embodiment of the present invention, feature and advantage become apparent more, below in conjunction with accompanying drawing, the technical scheme in the embodiment of the present invention is described in further detail.
In the embodiment of the present invention, medicinal material, medicine source and reference substance source is as follows:
Prepared fleece flower root medicinal material (producer: Anhui Shao Huatang pharmaceutcal corporation, Ltd, lot number: JY100202); Fructus Ligustri Lucidi (producer: Anhui Shao Huatang pharmaceutcal corporation, Ltd, lot number: ZY1402002); Mulberry fruit medicinal material (producer: Anhui Shao Huatang pharmaceutcal corporation, Ltd, lot number: ZY1401022); Eclipta medicinal material (producer: Anhui Shao Huatang pharmaceutcal corporation, Ltd, lot number: ZY1402007); White Peony Root (producer: Anhui Shao Huatang pharmaceutcal corporation, Ltd, lot number: JY140101); Milkvetch Root (producer: Anhui Shao Huatang pharmaceutcal corporation, Ltd, lot number: ZY1401021); Rhizoma cibotii medicinal material (producer: Anhui Shao Huatang pharmaceutcal corporation, Ltd, lot number: ZY1401020); Paeoniflorin reference substance (National Institute for Food and Drugs Control, 110738-201337).
Shengxuebao-medicine for improving hematopoitic function particle (Hu'nan Kangshou Pharmaceutical Co., Ltd., lot number is 20120501,20121105,20121207,20121211,20130701,20130704,20130801,130701,130803,130805); Shengxuebao-medicine for improving hematopoitic function capsule, tablet, oral liquid, pill (laboratory self-control); Shengxuebao-medicine for improving hematopoitic function intermediate (laboratory self-control).
Embodiment one
The present embodiment one is that experiment material sets up Shengxuebao-medicine for improving hematopoitic function formulation characteristics collection of illustrative plates with blood nourishing granules.
The method for building up of the Shengxuebao-medicine for improving hematopoitic function formulation characteristics collection of illustrative plates that the embodiment of the present invention one provides, comprises the steps.
The preparation of S101, object of reference solution.
Take Paeoniflorin reference substance, be placed in volumetric flask, add 50%-100% (volume) methyl alcohol and dissolve and be diluted to the solution of 0.02mg/ml, shake up, namely obtain object of reference solution, object of reference superposition chromatogram as shown in Figure 1.In the present embodiment, 50%-100% (volume) methyl alcohol refers to the volume ratio of methyl alcohol is the methanol aqueous solution of 50%-100%.
The preparation of S102, need testing solution.
Get blood nourishing granules powder 0.6g, to be accurately weighedly placed in tool plug conical flask, precision adds methyl alcohol 25ml, close plug, weighed weight.Let cool after ultrasonic process 10min, more weighed weight, supply the weight cut with methyl alcohol, shake up rear filtration.Get subsequent filtrate as need testing solution.
S103, absorption object of reference solution and need testing solution inject high performance liquid chromatograph, detect, obtain blood nourishing granules characteristic spectrum according to high performance liquid chromatography.
(1) step S103 can select Agilent 1260 high performance liquid chromatograph, is contained in the degassed machine of line vacuum (G-1311C), binary pump (G-1311C), standard automatic sampler (G-1329B), intelligent column oven (G-1316A), DAD detecting device (G-1314B), Agilent1260Infinity chromatographic work station (Anjelen Sci. & Tech. Inc of the U.S.); SB-5200D ultrasonic washing instrument (NingBo XinZhi Biology Science Co., Ltd); FAZ004B analytical balance (section is helped in Shanghai); BT125D electronic balance (Sai Duolisi scientific instrument (Beijing) company limited).
(2) chromatographic condition of step S103 detection is as follows:
The selection of a, mobile phase.
Mobile phase is made up of mobile phase A and Mobile phase B.Concrete, acetonitrile-water, acetonitrile-(0.1%-0.2%) phosphoric acid solution, acetonitrile-0.1% formic acid solution, methyl alcohol-0.1% phosphoric acid solution, methyl alcohol-0.1% formic acid solution, methanol-water etc. can be selected to test for mobile phase (above for mobile phase A, after be Mobile phase B).Result surface, when acetonitrile-0.1% phosphoric acid solution, acetonitrile-0.2% phosphoric acid solution are mobile phase, the peak shape of chromatographic peak is good, each peak is separated more complete, when wherein acetonitrile-0.1% phosphoric acid solution is mobile phase, effect is best, preferably select acetonitrile-0.1% phosphoric acid solution as optimal flow phase, namely mobile phase A is acetonitrile, and Mobile phase B is 0.1% (volume) phosphoric acid solution.In a, % all represents percent by volume.
Due to complicated component in blood nourishing granules, under constant current conditions, each component separating degree in need testing solution is poor, the overall permanence of more difficult embodiment characteristic spectrum; Mobile phase is with gradient elution as shown in table 1 below, and in sample, each component all has good separation, and baseline is steady, therefore selects as mobile phase.
Table 1
The selection of b, determined wavelength.
Application DAD detecting device has carried out the long mensuration of all-wave to test sample and has compared, and the response that result surveys main chromatographic peak in characteristic spectrum in 200-230nm institute is comparatively large, and each chromatographic peak feature is given prominence to, therefore preferred employing determined wavelength is 200-230nm.
The selection of c, chromatographic column.
In the present embodiment, chromatographic column is C 18reverse-phase chromatographic column can.By comparing, AgilentZORBAXXDB-C 18(5 μm, 4.6 × 250mm), ThermoSyncronis-C 18(5 μm, 4.6 × 250mm), Yi Lite HypersilODS2-C 18(5 μm, 4.6 × 250mm), enlightening horse Diamonsil-C 18the chromatographic column of (5 μm, 4.6 × 250mm) 4 kinds of different model specifications, result shows, it is moderate that the chromatographic column of above 4 kinds of brands all can reach retention time, and peak number order is more, the comparatively ideal chromatogram effect that degree of separation is good.
The selection of d, column temperature.
The present embodiment one investigated different column temperature 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C time separating effect to same sample.Result shows, the separation impact of different column temperature on chromatographic peak is less, all more satisfactory with separating effect when 25 DEG C-40 DEG C.
The selection of e, flow rate of mobile phase.
The present embodiment one is under the condition that other chromatographic condition is identical, and having investigated flow velocity is respectively the impact that 0.8ml/min, 1.0ml/min, 1.2ml/min are separated same sample chromatographic peak.Result shows, when flow rate of mobile phase is 1.0ml/min, chromatographic peak retention time is moderate, and degree of separation is best, and repeatability is best, and therefore, flow rate of mobile phase is preferably 1.0ml/min.
(3) the characteristic spectrum method for building up that provides of the present embodiment one, under above-mentioned chromatographic condition, detect 120min, result shows, in 60 minutes, in sample, all peaks all can be eluted completely, therefore are defined as 60 minutes analysis time.
(4) methodological study
1. stability test.
Get same need testing solution, under above-mentioned liquid phase chromatogram condition, detect respectively at 0,2,4,6,12,24 hour, each sample introduction 5 μ l, investigates the relative retention time of main chromatographic peak and the consistance of relative peak area.The relative retention time of the main chromatographic peak of result and the RSD < 3% of relative peak area, show in need testing solution 24 hours more stable.
2. precision test.
Get with a need testing solution, under above-mentioned liquid phase chromatogram condition, repeat sample introduction 6 times, each sample introduction 5 μ l, investigates the relative retention time of main chromatographic peak and the consistance of relative peak area.The relative retention time of the main chromatographic peak of result and the RSD < 3% of relative peak area, show that instrument precision is good.
3. replica test
Get same batch sample, prepare 6 parts of need testing solutions by test sample preparation method, under above-mentioned liquid phase chromatogram condition, sample introduction analysis, investigates the relative retention time of main chromatographic peak and the consistance of relative peak area.The relative retention time of the main chromatographic peak of result and the RSD < 3% of relative peak area, show that the method is reproducible.
In the method for building up that the present embodiment one provides, the above-mentioned object of reference solution of accurate absorption and each 5 μ l of need testing solution, inject high performance liquid chromatograph respectively, according to high effective liquid chromatography for measuring, and recording feature collection of illustrative plates, as shown in Figure 2.
Feature spectrogram shown in Fig. 2 has 7 characteristic peaks, and wherein No. 5 peaks are Paeoniflorin peak for reference to peak, and the relative retention time of each characteristic peak and relative standard deviation are:
No. 1 peak: relative retention time 0.383 ± 0.019, relative standard deviation is 0.19%;
No. 2 peaks: relative retention time 0.672 ± 0.034, relative standard deviation is 0.07%;
No. 3 peaks: relative retention time 0.801 ± 0.040, relative standard deviation is 0.05%;
No. 4 peaks: relative retention time 0.956 ± 0.048, relative standard deviation is 0.01%;
No. 5 peaks: relative retention time 1.000 ± 0.000, relative standard deviation is 0.00%;
No. 6 peaks: relative retention time 1.143 ± 0.057, relative standard deviation is 0.01%;
No. 7 peaks: relative retention time 1.188 ± 0.059, relative standard deviation is 0.03%.
Get 10 batches of blood nourishing granules to detect, formulate characteristic spectrum as shown in Figure 3,10 batches of Shengxuebao-medicine for improving hematopoitic function particle characteristic of correspondence collection of illustrative plates all have 7 characteristic peaks, and wherein No. 5 peaks are Paeoniflorin peak for reference to peak, and the relative standard deviation of each characteristic peak is as shown in table 2.Table 2 is 10 batches of blood nourishing granules characteristic spectrum relative deviation result tables.
Table 2
(5) evaluation of each characteristic peak of characteristic spectrum.
Blood nourishing granules characteristic spectrum has and all has 7 characteristic peaks, each characteristic peak standard relative retention time of each for blood nourishing granules characteristic spectrum feature relative retention time and formulation is compared, its relative retention time relative deviation should setting ± 5% within.
Embodiment two
The present embodiment two with Shengxuebao-medicine for improving hematopoitic function preparation intermediate for experiment material sets up Shengxuebao-medicine for improving hematopoitic function formulation characteristics collection of illustrative plates.
The method for building up of the Shengxuebao-medicine for improving hematopoitic function formulation characteristics collection of illustrative plates that the embodiment of the present invention two provides, comprises the steps.
The preparation of S201, object of reference solution.
Take Paeoniflorin reference substance, be placed in volumetric flask, add 50%-100% (volume) methyl alcohol and dissolve and be diluted to the solution of 0.02mg/ml, shake up, namely obtain object of reference solution, object of reference superposition chromatogram as shown in Figure 1.In the present embodiment, 50%-100% (volume) methyl alcohol refers to the volume ratio of methyl alcohol is the methanol aqueous solution of 50%-100%.
The preparation of S202, need testing solution.
Get Shengxuebao-medicine for improving hematopoitic function preparation intermediate 3.0g, to be accurately weighedly placed in tool plug conical flask, precision adds 125ml water, close plug, weighed weight.Let cool after ultrasonic process 30min, more weighed weight, supply the weight cut with water, shake up rear filtration.Get subsequent filtrate as need testing solution.
S203, absorption object of reference solution and need testing solution inject high performance liquid chromatograph, detect, obtain Shengxuebao-medicine for improving hematopoitic function preparation intermediate characteristic spectrum according to high performance liquid chromatography.
(1) step S203 can select Agilent 1260 high performance liquid chromatograph, is contained in the degassed machine of line vacuum (G-1311C), binary pump (G-1311C), standard automatic sampler (G-1329B), intelligent column oven (G-1316A), DAD detecting device (G-1314B), Agilent1260Infinity chromatographic work station (Anjelen Sci. & Tech. Inc of the U.S.); SB-5200D ultrasonic washing instrument (NingBo XinZhi Biology Science Co., Ltd); FAZ004B analytical balance (section is helped in Shanghai); BT125D electronic balance (Sai Duolisi scientific instrument (Beijing) company limited).
(2) chromatographic condition of step S203 detection is as follows:
The selection of a, mobile phase.
Mobile phase is made up of mobile phase A and Mobile phase B.Concrete, acetonitrile-water, acetonitrile-(0.1%-0.2%) phosphoric acid solution, acetonitrile-0.1% formic acid solution, methyl alcohol-0.1% phosphoric acid solution, methyl alcohol-0.1% formic acid solution, methanol-water etc. can be selected to test for mobile phase (above for mobile phase A, after be Mobile phase B).Result surface, when acetonitrile-0.1% phosphoric acid solution, acetonitrile-0.2% phosphoric acid solution are mobile phase, the peak shape of chromatographic peak is good, each peak is separated more complete, when wherein acetonitrile-0.1% phosphoric acid solution is mobile phase, effect is best, preferably select acetonitrile-0.1% phosphoric acid solution as optimal flow phase, namely mobile phase A is acetonitrile, and Mobile phase B is 0.1% (volume) phosphoric acid solution.In a, % all represents percent by volume.
Due to complicated component in Shengxuebao-medicine for improving hematopoitic function preparation intermediate, under constant current conditions, each component separating degree in need testing solution is poor, the overall permanence of more difficult embodiment characteristic spectrum; Mobile phase is with gradient elution as shown in table 3 below, and in sample, each component all has good separation, and baseline is steady, therefore selects as mobile phase.
Table 3
The selection of b, determined wavelength.
In the present embodiment, determined wavelength is 200nm.
The selection of c, chromatographic column.
In the present embodiment, chromatographic column can be C 18reverse-phase chromatographic column.By comparing, AgilentZORBAXXDB-C 18(5 μm, 4.6 × 250mm), ThermoSyncronis-C 18(5 μm, 4.6 × 250mm), Yi Lite HypersilODS2-C 18(5 μm, 4.6 × 250mm), enlightening horse Diamonsil-C 18the chromatographic column of (5 μm, 4.6 × 250mm) 4 kinds of different model specifications, result shows, it is moderate that the chromatographic column of above 4 kinds of brands all can reach retention time, and peak number order is more, the comparatively ideal chromatogram effect that degree of separation is good.In the present embodiment, two adopt chromatographic column to be AgilentZORBAXXDB-C 18(5 μm, 4.6 × 250mm) reverse-phase chromatographic column.
The selection of d, column temperature.
The present embodiment two investigated different column temperature 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C time separating effect to same sample.Result shows, the separation impact of different temperatures on chromatographic peak is less.In the present embodiment two, column temperature is 25 DEG C.
The selection of e, flow rate of mobile phase.
The present embodiment two is under the condition that other chromatographic condition is identical, and having investigated flow velocity is respectively the impact that 0.8ml/min, 1.0ml/min, 1.2ml/min are separated same sample chromatographic peak.Result shows, when the flow velocity of mobile phase is 1.0ml/min, chromatographic peak retention time is moderate, and degree of separation is best, and repeatability is best, and in the present embodiment two, flow rate of mobile phase is 1.0ml/min.
(3) the characteristic spectrum method for building up that provides of the present embodiment, under above-mentioned chromatographic condition, detect 120min, result shows, in 60 minutes, in sample, all peaks all can be eluted completely, therefore in the present embodiment two analysis time be defined as 60 minutes.
(4) methodological study
1. stability test.
Get same need testing solution, under above-mentioned liquid phase chromatogram condition, detect respectively at 0,2,4,6,12,24 hour, each sample introduction 5 μ l, investigates the relative retention time of main chromatographic peak and the consistance of relative peak area.The relative retention time of the main chromatographic peak of result and the RSD < 3% of relative peak area, show in need testing solution 24 hours more stable.
2. precision test.
Get with a need testing solution, under above-mentioned liquid phase chromatogram condition, repeat sample introduction 6 times, each sample introduction 5 μ l, investigates the relative retention time of main chromatographic peak and the consistance of relative peak area.The relative retention time of the main chromatographic peak of result and the RSD < 3% of relative peak area, show that instrument precision is good.
3. replica test
Get same batch sample, prepare 6 parts of need testing solutions by test sample preparation method, under above-mentioned liquid phase chromatogram condition, sample introduction analysis, investigates the relative retention time of main chromatographic peak and the consistance of relative peak area.The relative retention time of the main chromatographic peak of result and the RSD < 3% of relative peak area, show that the method is reproducible.
In the method for building up that the present embodiment two provides, the above-mentioned object of reference solution of accurate absorption and each 5 μ l of need testing solution, inject high performance liquid chromatograph respectively, according to high effective liquid chromatography for measuring, and recording feature collection of illustrative plates, as shown in Figure 4.
Feature spectrogram shown in Fig. 4 has 7 characteristic peaks, and wherein No. 5 peaks are Paeoniflorin peak for reference to peak, and the relative retention time of each characteristic peak and relative standard deviation are:
No. 1 peak: relative retention time 0.383 ± 0.019, relative standard deviation is 0.14%;
No. 2 peaks: relative retention time 0.672 ± 0.034, relative standard deviation is 0.00%;
No. 3 peaks: relative retention time 0.801 ± 0.040, relative standard deviation is 0.05%;
No. 4 peaks: relative retention time 0.956 ± 0.048, relative standard deviation is 0.00%;
No. 5 peaks: relative retention time 1.000 ± 0.000, relative standard deviation is 0.00%;
No. 6 peaks: relative retention time 1.143 ± 0.057, relative standard deviation is 0.00%;
No. 7 peaks: relative retention time 1.188 ± 0.059, relative standard deviation is 0.00%.
Get 10 batches of Shengxuebao-medicine for improving hematopoitic function preparation intermediates to detect, formulate characteristic spectrum as shown in Figure 5,10 batches of Shengxuebao-medicine for improving hematopoitic function preparation intermediate characteristic of correspondence collection of illustrative plates all have 7 characteristic peaks, and wherein No. 5 peaks are Paeoniflorin peak for reference to peak, and the relative standard deviation of each characteristic peak is as shown in table 4.Table 4 is 10 batches of Shengxuebao-medicine for improving hematopoitic function preparation intermediate characteristic spectrum relative deviation result tables.
Table 4
(5) evaluation of each characteristic peak of characteristic spectrum.
Shengxuebao-medicine for improving hematopoitic function preparation intermediate characteristic spectrum has and all has 7 characteristic peaks, each characteristic peak standard relative retention time of each for blood nourishing granules characteristic spectrum feature relative retention time and formulation is compared, its relative retention time relative deviation should setting ± 5% within.
Embodiment three
The present embodiment three with Shengxuebao-medicine for improving hematopoitic function capsule for experiment material sets up Shengxuebao-medicine for improving hematopoitic function formulation characteristics collection of illustrative plates.
The method for building up of the Shengxuebao-medicine for improving hematopoitic function formulation characteristics collection of illustrative plates that the embodiment of the present invention three provides, comprises the steps.
The preparation of S301, object of reference solution.
Take Paeoniflorin reference substance, be placed in volumetric flask, add 50%-100% (volume) methyl alcohol and dissolve and be diluted to the solution of 0.02mg/ml, shake up, namely obtain object of reference solution, object of reference superposition chromatogram as shown in Figure 1.In the present embodiment, 50%-100% (volume) methyl alcohol refers to the volume ratio of methyl alcohol is the methanol aqueous solution of 50%-100%.
The preparation of S302, need testing solution.
Get Shengxuebao-medicine for improving hematopoitic function capsule powders 1.2g, be accurately weighedly placed in tool plug conical flask, precision adds 50% methyl alcohol 50ml, close plug, weighed weight.Let cool after adding hot reflux 10min, more weighed weight, supply the weight cut with 50% methyl alcohol, shake up rear filtration.Get subsequent filtrate as need testing solution.
S303, absorption object of reference solution and need testing solution inject high performance liquid chromatograph, detect, obtain blood nourishing granules characteristic spectrum according to high performance liquid chromatography.
(1) step S303 can select Agilent 1260 high performance liquid chromatograph, is contained in the degassed machine of line vacuum (G-1311C), binary pump (G-1311C), standard automatic sampler (G-1329B), intelligent column oven (G-1316A), DAD detecting device (G-1314B), Agilent1260Infinity chromatographic work station (Anjelen Sci. & Tech. Inc of the U.S.); SB-5200D ultrasonic washing instrument (NingBo XinZhi Biology Science Co., Ltd); FAZ004B analytical balance (section is helped in Shanghai); BT125D electronic balance (Sai Duolisi scientific instrument (Beijing) company limited).
(2) chromatographic condition of step S303 detection is as follows:
The selection of a, mobile phase.
Mobile phase is made up of mobile phase A and Mobile phase B.Concrete, acetonitrile-water, acetonitrile-(0.1%-0.2%) phosphoric acid solution, acetonitrile-0.1% formic acid solution, methyl alcohol-0.1% phosphoric acid solution, methyl alcohol-0.1% formic acid solution, methanol-water etc. can be selected to test for mobile phase (above for mobile phase A, after be Mobile phase B).Result surface, when acetonitrile-0.1% phosphoric acid solution, acetonitrile-0.2% phosphoric acid solution are mobile phase, the peak shape of chromatographic peak is good, each peak is separated more complete, when wherein acetonitrile-0.1% phosphoric acid solution is mobile phase, effect is best, preferably select acetonitrile-0.1% phosphoric acid solution as optimal flow phase, namely mobile phase A is acetonitrile, and Mobile phase B is 0.1% (volume) phosphoric acid solution.In a, % all represents percent by volume.
Due to complicated component in Shengxuebao-medicine for improving hematopoitic function capsule, under constant current conditions, each component separating degree in need testing solution is poor, the overall permanence of more difficult embodiment characteristic spectrum; Mobile phase is with gradient elution as shown in table 5 below, and in sample, each component all has good separation, and baseline is steady, therefore selects as mobile phase.
Table 5
The selection of b, determined wavelength.
In the present embodiment three, determined wavelength is 210nm.
The selection of c, chromatographic column.
By comparing, AgilentZORBAXXDB-C 18(5 μm, 4.6 × 250mm), ThermoSyncronis-C 18(5 μm, 4.6 × 250mm), Yi Lite HypersilODS2-C 18(5 μm, 4.6 × 250mm), enlightening horse Diamonsil-C 18the chromatographic column of (5 μm, 4.6 × 250mm) 4 kinds of different model specifications, result shows, it is moderate that the chromatographic column of above 4 kinds of brands all can reach retention time, and peak number order is more, the comparatively ideal chromatogram effect that degree of separation is good.In the present embodiment, chromatographic column is C 18reverse-phase chromatographic column.In the present embodiment, three adopt chromatographic column to be AgilentZORBAXXDB-C 18(5 μm, 4.6 × 250mm) reverse-phase chromatographic column.
The selection of d, column temperature.
The present embodiment three investigated different column temperature 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C time separating effect to same sample.Result shows, the separation impact of different temperatures on chromatographic peak is less.In the present embodiment three, column temperature is 30 DEG C.
The selection of e, flow rate of mobile phase.
The present embodiment three is under the condition that other chromatographic condition is identical, and having investigated flow velocity is respectively the impact that 0.8ml/min, 1.0ml/min, 1.2ml/min are separated same sample chromatographic peak.Result shows, when flow rate of mobile phase is 0.8ml/min, chromatographic peak retention time is moderate, and degree of separation is best, and repeatability is best, and in the present embodiment three, flow rate of mobile phase is 0.8ml/min.
(3) the characteristic spectrum method for building up that provides of the present embodiment three, under above-mentioned chromatographic condition, detect 120min, result shows, in 60 minutes, in sample, all peaks all can be eluted completely, therefore in the present embodiment three analysis time be defined as 60 minutes.
(4) methodological study
1. stability test.
Get same need testing solution, under above-mentioned liquid phase chromatogram condition, detect respectively at 0,2,4,6,12,24 hour, each sample introduction 5 μ l, investigates the relative retention time of main chromatographic peak and the consistance of relative peak area.The relative retention time of the main chromatographic peak of result and the RSD < 3% of relative peak area, show in need testing solution 24 hours more stable.
2. precision test.
Get with a need testing solution, under above-mentioned liquid phase chromatogram condition, repeat sample introduction 6 times, each sample introduction 5 μ l, investigates the relative retention time of main chromatographic peak and the consistance of relative peak area.The relative retention time of the main chromatographic peak of result and the RSD < 3% of relative peak area, show that instrument precision is good.
3. replica test
Get same batch sample, prepare 6 parts of need testing solutions by test sample preparation method, under above-mentioned liquid phase chromatogram condition, sample introduction analysis, investigates the relative retention time of main chromatographic peak and the consistance of relative peak area.The relative retention time of the main chromatographic peak of result and the RSD < 3% of relative peak area, show that the method is reproducible.
In the method for building up that the present embodiment three provides, the above-mentioned object of reference solution of accurate absorption and each 5 μ l of need testing solution, inject high performance liquid chromatograph respectively, according to high effective liquid chromatography for measuring, and recording feature collection of illustrative plates, as shown in Figure 6.
Feature spectrogram shown in Fig. 6 has 7 characteristic peaks, and wherein No. 5 peaks are Paeoniflorin peak for reference to peak, and the relative retention time of each characteristic peak and relative standard deviation are:
No. 1 peak: relative retention time 0.383 ± 0.019, relative standard deviation is 1.19%;
No. 2 peaks: relative retention time 0.672 ± 0.034, relative standard deviation is 0.43%;
No. 3 peaks: relative retention time 0.801 ± 0.040, relative standard deviation is 0.19%;
No. 4 peaks: relative retention time 0.956 ± 0.048, relative standard deviation is 0.05%;
No. 5 peaks: relative retention time 1.000 ± 0.000, relative standard deviation is 0.00%;
No. 6 peaks: relative retention time 1.143 ± 0.057, relative standard deviation is 0.28%;
No. 7 peaks: relative retention time 1.188 ± 0.059, relative standard deviation is 0.12%.
Get 10 batches of Shengxuebao-medicine for improving hematopoitic function capsules to detect, formulate characteristic spectrum as shown in Figure 7,10 batches of Shengxuebao-medicine for improving hematopoitic function capsule characteristic of correspondence collection of illustrative plates all have 7 characteristic peaks, and wherein No. 5 peaks are Paeoniflorin peak for reference to peak, and the relative standard deviation of each characteristic peak is as shown in table 6.Table 6 is 10 batches of Shengxuebao-medicine for improving hematopoitic function capsule characteristics collection of illustrative plates relative deviation result tables.
Table 6
(5) evaluation of each characteristic peak of characteristic spectrum.
Shengxuebao-medicine for improving hematopoitic function capsule characteristics collection of illustrative plates has and all has 7 characteristic peaks, each characteristic peak standard relative retention time of each for blood nourishing granules characteristic spectrum feature relative retention time and formulation is compared, its relative retention time relative deviation should setting ± 5% within.
Embodiment four
The present embodiment four with Shengxuebao-medicine for improving hematopoitic function tablet for experiment material sets up Shengxuebao-medicine for improving hematopoitic function formulation characteristics collection of illustrative plates.
The method for building up of the Shengxuebao-medicine for improving hematopoitic function formulation characteristics collection of illustrative plates that the embodiment of the present invention four provides, comprises the steps.
The preparation of S401, object of reference solution.
Take Paeoniflorin reference substance, be placed in volumetric flask, add 50%-100% (volume) methyl alcohol and dissolve and be diluted to the solution of 0.02mg/ml, shake up, namely obtain object of reference solution, object of reference superposition chromatogram as shown in Figure 1.In the present embodiment, 50%-100% (volume) methyl alcohol refers to the volume ratio of methyl alcohol is the methanol aqueous solution of 50%-100%.
The preparation of S402, need testing solution.
Get Shengxuebao-medicine for improving hematopoitic function tablet powder 2.4g, be accurately weighedly placed in tool plug conical flask, precision adds 25% ethanol 100ml, close plug, weighed weight.Let cool after ultrasonic process 60min, more weighed weight, supply the weight cut with 25% ethanol, shake up rear filtration.Get subsequent filtrate as need testing solution.
S403, absorption object of reference solution and need testing solution inject high performance liquid chromatograph, detect, obtain Shengxuebao-medicine for improving hematopoitic function Tablet characteristics collection of illustrative plates according to high performance liquid chromatography.
(1) step S403 can select Agilent 1260 high performance liquid chromatograph, is contained in the degassed machine of line vacuum (G-1311C), binary pump (G-1311C), standard automatic sampler (G-1329B), intelligent column oven (G-1316A), DAD detecting device (G-1314B), Agilent1260Infinity chromatographic work station (Anjelen Sci. & Tech. Inc of the U.S.); SB-5200D ultrasonic washing instrument (NingBo XinZhi Biology Science Co., Ltd); FAZ004B analytical balance (section is helped in Shanghai); BT125D electronic balance (Sai Duolisi scientific instrument (Beijing) company limited).
(2) chromatographic condition of step S403 detection is as follows:
The selection of a, mobile phase.
Mobile phase is made up of mobile phase A and Mobile phase B.Concrete, acetonitrile-water, acetonitrile-(0.1%-0.2%) phosphoric acid solution, acetonitrile-0.1% formic acid solution, methyl alcohol-0.1% phosphoric acid solution, methyl alcohol-0.1% formic acid solution, methanol-water etc. can be selected to test for mobile phase (above for mobile phase A, after be Mobile phase B).Result surface, when acetonitrile-0.1% phosphoric acid solution, acetonitrile-0.2% phosphoric acid solution are mobile phase, the peak shape of chromatographic peak is good, each peak is separated more complete, when wherein acetonitrile-0.1% phosphoric acid solution is mobile phase, effect is best, preferably select acetonitrile-0.1% phosphoric acid solution as optimal flow phase, namely mobile phase A is acetonitrile, and Mobile phase B is 0.1% (volume) phosphoric acid solution.In a, % all represents percent by volume.
Due to complicated component in Shengxuebao-medicine for improving hematopoitic function tablet, under constant current conditions, each component separating degree in need testing solution is poor, the overall permanence of more difficult embodiment characteristic spectrum; Mobile phase is with gradient elution as shown in table 7 below, and in sample, each component all has good separation, and baseline is steady, therefore selects as mobile phase.
Table 7
The selection of b, determined wavelength.
In the present embodiment, determined wavelength is 215nm.
The selection of c, chromatographic column.
In the present embodiment, chromatographic column can be C 18reverse-phase chromatographic column.By comparing, AgilentZORBAXXDB-C 18(5 μm, 4.6 × 250mm), ThermoSyncronis-C 18(5 μm, 4.6 × 250mm), Yi Lite HypersilODS2-C 18(5 μm, 4.6 × 250mm), enlightening horse Diamonsil-C 18the chromatographic column of (5 μm, 4.6 × 250mm) 4 kinds of different model specifications, result shows, it is moderate that the chromatographic column of above 4 kinds of brands all can reach retention time, and peak number order is more, the comparatively ideal chromatogram effect that degree of separation is good.In the present embodiment, four adopt chromatographic column to be AgilentZORBAXXDB-C 18(5 μm, 4.6 × 250mm) reverse-phase chromatographic column.
The selection of d, column temperature.
The present embodiment four investigated different column temperature 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C time separating effect to same sample.Result shows, the separation impact of different temperatures on chromatographic peak is less.In the present embodiment four, column temperature is 35 DEG C.
The selection of e, flow rate of mobile phase.
The present embodiment four is under the condition that other chromatographic condition is identical, and having investigated flow velocity is respectively the impact that 0.8ml/min, 1.0ml/min, 1.2ml/min are separated same sample chromatographic peak.Result shows, when flow rate of mobile phase is 0.8ml/min, chromatographic peak retention time is moderate, and degree of separation is best, and repeatability is best, and in the present embodiment four, flow rate of mobile phase is 1.0ml/min.
(3) the characteristic spectrum method for building up that provides of the present embodiment, under above-mentioned chromatographic condition, detect 120min, result shows, in 60 minutes, in sample, all peaks all can be eluted completely, therefore in the present embodiment four analysis time be defined as 60 minutes.
(4) methodological study
1. stability test.
Get same need testing solution, under above-mentioned liquid phase chromatogram condition, detect respectively at 0,2,4,6,12,24 hour, each sample introduction 5 μ l, investigates the relative retention time of main chromatographic peak and the consistance of relative peak area.The relative retention time of the main chromatographic peak of result and the RSD < 3% of relative peak area, show in need testing solution 24 hours more stable.
2. precision test.
Get with a need testing solution, under above-mentioned liquid phase chromatogram condition, repeat sample introduction 6 times, each sample introduction 5 μ l, investigates the relative retention time of main chromatographic peak and the consistance of relative peak area.The relative retention time of the main chromatographic peak of result and the RSD < 3% of relative peak area, show that instrument precision is good.
3. replica test
Get same batch sample, prepare 6 parts of need testing solutions by test sample preparation method, under above-mentioned liquid phase chromatogram condition, sample introduction analysis, investigates the relative retention time of main chromatographic peak and the consistance of relative peak area.The relative retention time of the main chromatographic peak of result and the RSD < 3% of relative peak area, show that the method is reproducible.
In the method for building up that the present embodiment four provides, the above-mentioned object of reference solution of accurate absorption and each 5 μ l of need testing solution, inject high performance liquid chromatograph respectively, according to high effective liquid chromatography for measuring, and recording feature collection of illustrative plates, as shown in Figure 8.
Feature spectrogram shown in Fig. 8 has 7 characteristic peaks, and wherein No. 5 peaks are Paeoniflorin peak for reference to peak, and the relative retention time of each characteristic peak and relative standard deviation are:
No. 1 peak: relative retention time 0.383 ± 0.019, relative standard deviation is 0.19%;
No. 2 peaks: relative retention time 0.672 ± 0.034, relative standard deviation is 0.07%;
No. 3 peaks: relative retention time 0.801 ± 0.040, relative standard deviation is 0.05%;
No. 4 peaks: relative retention time 0.956 ± 0.048, relative standard deviation is 0.01%;
No. 5 peaks: relative retention time 1.000 ± 0.000, relative standard deviation is 0.00%;
No. 6 peaks: relative retention time 1.143 ± 0.057, relative standard deviation is 0.01%;
No. 7 peaks: relative retention time 1.188 ± 0.059, relative standard deviation is 0.03%.
Get 10 batches of Shengxuebao-medicine for improving hematopoitic function tablets to detect, formulate characteristic spectrum as shown in Figure 9,10 batches of Shengxuebao-medicine for improving hematopoitic function tablet characteristic of correspondence collection of illustrative plates all have 7 characteristic peaks, and wherein No. 5 peaks are Paeoniflorin peak for reference to peak, and the relative standard deviation of each characteristic peak is as shown in table 8.Table 8 is 10 batches of Shengxuebao-medicine for improving hematopoitic function Tablet characteristics collection of illustrative plates relative deviation result tables.
Table 8
(5) evaluation of each characteristic peak of characteristic spectrum.
Shengxuebao-medicine for improving hematopoitic function Tablet characteristics collection of illustrative plates has and all has 7 characteristic peaks, each characteristic peak standard relative retention time of Shengxuebao-medicine for improving hematopoitic function Tablet characteristics collection of illustrative plates each feature relative retention time and formulation is compared, its relative retention time relative deviation should setting ± 5% within.
Embodiment five
The present embodiment five with Shengxuebao-medicine for improving hematopoitic function preparation oral liquid for experiment material sets up Shengxuebao-medicine for improving hematopoitic function formulation characteristics collection of illustrative plates.
The method for building up of the Shengxuebao-medicine for improving hematopoitic function formulation characteristics collection of illustrative plates that the embodiment of the present invention five provides, comprises the steps.
The preparation of S501, object of reference solution.
Take Paeoniflorin reference substance, be placed in volumetric flask, add 50%-100% (volume) methyl alcohol and dissolve and be diluted to the solution of 0.02mg/ml, shake up, namely obtain object of reference solution, object of reference superposition chromatogram as shown in Figure 1.In the present embodiment, 50%-100% (volume) methyl alcohol refers to the volume ratio of methyl alcohol is the methanol aqueous solution of 50%-100%.
The preparation of S302, need testing solution.
Get Shengxuebao-medicine for improving hematopoitic function oral liquid 1ml, be accurately weighedly placed in tool plug conical flask, precision adds 50% ethanol 25ml, close plug, weighed weight.Let cool after adding hot reflux 30min, more weighed weight, supply the weight cut with 50% ethanol, shake up rear filtration.Get subsequent filtrate as need testing solution.
S303, absorption object of reference solution and need testing solution inject high performance liquid chromatograph, detect, obtain Shengxuebao-medicine for improving hematopoitic function oral liquid characteristic spectrum according to high performance liquid chromatography.
(1) step S303 can select Agilent 1260 high performance liquid chromatograph, is contained in the degassed machine of line vacuum (G-1311C), binary pump (G-1311C), standard automatic sampler (G-1329B), intelligent column oven (G-1316A), DAD detecting device (G-1314B), Agilent1260Infinity chromatographic work station (Anjelen Sci. & Tech. Inc of the U.S.); SB-5200D ultrasonic washing instrument (NingBo XinZhi Biology Science Co., Ltd); FAZ004B analytical balance (section is helped in Shanghai); BT125D electronic balance (Sai Duolisi scientific instrument (Beijing) company limited).
(2) chromatographic condition of step S303 detection is as follows:
The selection of a, mobile phase.
Mobile phase is made up of mobile phase A and Mobile phase B.Concrete, acetonitrile-water, acetonitrile-(0.1%-0.2%) phosphoric acid solution, acetonitrile-0.1% formic acid solution, methyl alcohol-0.1% phosphoric acid solution, methyl alcohol-0.1% formic acid solution, methanol-water etc. can be selected to test for mobile phase (above for mobile phase A, after be Mobile phase B).Result surface, when acetonitrile-0.1% phosphoric acid solution, acetonitrile-0.2% phosphoric acid solution are mobile phase, the peak shape of chromatographic peak is good, each peak is separated more complete, when wherein acetonitrile-0.1% phosphoric acid solution is mobile phase, effect is best, preferably select acetonitrile-0.1% phosphoric acid solution as optimal flow phase, namely mobile phase A is acetonitrile, and Mobile phase B is 0.1% (volume) phosphoric acid solution.In a, % all represents percent by volume.
Due to complicated component in Shengxuebao-medicine for improving hematopoitic function oral liquid, under constant current conditions, each component separating degree in need testing solution is poor, the overall permanence of more difficult embodiment characteristic spectrum; Mobile phase is with gradient elution as shown in table 9 below, and in sample, each component all has good separation, and baseline is steady, therefore selects as mobile phase.
Table 9
The selection of b, determined wavelength.
In the present embodiment, determined wavelength is 210nm.
The selection of c, chromatographic column.
By comparing, AgilentZORBAXXDB-C 18(5 μm, 4.6 × 250mm), ThermoSyncronis-C 18(5 μm, 4.6 × 250mm), Yi Lite HypersilODS2-C 18(5 μm, 4.6 × 250mm), enlightening horse Diamonsil-C 18the chromatographic column of (5 μm, 4.6 × 250mm) 4 kinds of different model specifications, result shows, it is moderate that the chromatographic column of above 4 kinds of brands all can reach retention time, and peak number order is more, the comparatively ideal chromatogram effect that degree of separation is good.In the present embodiment, chromatographic column is C 18reverse-phase chromatographic column.In the present embodiment, five adopt chromatographic column to be AgilentZORBAXXDB-C 18(5 μm, 4.6 × 250mm) reverse-phase chromatographic column.
The selection of d, column temperature.
The present embodiment five investigated different column temperature 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C time separating effect to same sample.Result shows, the separation impact of different temperatures on chromatographic peak is less.In the present embodiment five, column temperature is 30 DEG C.
The selection of e, flow rate of mobile phase.
The present embodiment is under the condition that other chromatographic condition is identical, and having investigated flow velocity is respectively the impact that 0.8ml/min, 1.0ml/min, 1.2ml/min are separated same sample chromatographic peak.Result shows, as υ=0.8ml/min, chromatographic peak retention time is moderate, and degree of separation is best, and repeatability is best, and in the present embodiment five, flow rate of mobile phase is 0.8ml/min.
(3) the characteristic spectrum method for building up that provides of the present embodiment, under above-mentioned chromatographic condition, detect 120min, result shows, in 60 minutes, in sample, all peaks all can be eluted completely, therefore in the present embodiment five analysis time be defined as 60 minutes.
(4) methodological study
1. stability test.
Get same need testing solution, under above-mentioned liquid phase chromatogram condition, detect respectively at 0,2,4,6,12,24 hour, each sample introduction 5 μ l, investigates the relative retention time of main chromatographic peak and the consistance of relative peak area.The relative retention time of the main chromatographic peak of result and the RSD < 3% of relative peak area, show in need testing solution 24 hours more stable.
2. precision test.
Get with a need testing solution, under above-mentioned liquid phase chromatogram condition, repeat sample introduction 6 times, each sample introduction 5 μ l, investigates the relative retention time of main chromatographic peak and the consistance of relative peak area.The relative retention time of the main chromatographic peak of result and the RSD < 3% of relative peak area, show that instrument precision is good.
3. replica test
Get same batch sample, prepare 6 parts of need testing solutions by test sample preparation method, under above-mentioned liquid phase chromatogram condition, sample introduction analysis, investigates the relative retention time of main chromatographic peak and the consistance of relative peak area.The relative retention time of the main chromatographic peak of result and the RSD < 3% of relative peak area, show that the method is reproducible.
In the method for building up that the present embodiment five provides, the above-mentioned object of reference solution of accurate absorption and each 5 μ l of need testing solution, inject high performance liquid chromatograph respectively, according to high effective liquid chromatography for measuring, and recording feature collection of illustrative plates, as shown in Figure 2.
Feature spectrogram shown in Figure 10 has 7 characteristic peaks, and wherein No. 5 peaks are Paeoniflorin peak for reference to peak, and the relative retention time of each characteristic peak and relative standard deviation are:
No. 1 peak: relative retention time 0.383 ± 0.019, relative standard deviation is 0.21%;
No. 2 peaks: relative retention time 0.672 ± 0.034, relative standard deviation is 0.08%;
No. 3 peaks: relative retention time 0.801 ± 0.040, relative standard deviation is 0.07%;
No. 4 peaks: relative retention time 0.956 ± 0.048, relative standard deviation is 0.05%;
No. 5 peaks: relative retention time 1.000 ± 0.000, relative standard deviation is 0.00%;
No. 6 peaks: relative retention time 1.143 ± 0.057, relative standard deviation is 0.00%;
No. 7 peaks: relative retention time 1.188 ± 0.059, relative standard deviation is 0.04%.
Get 10 batches of Shengxuebao-medicine for improving hematopoitic function capsules to detect, formulate characteristic spectrum as shown in figure 11,10 batches of Shengxuebao-medicine for improving hematopoitic function capsule characteristic of correspondence collection of illustrative plates all have 7 characteristic peaks, and wherein No. 5 peaks are Paeoniflorin peak for reference to peak, and the relative standard deviation of each characteristic peak is as shown in table 10.Table 10 is 10 batches of Shengxuebao-medicine for improving hematopoitic function oral liquid characteristic spectrum relative deviation result tables.
Table 10
(5) evaluation of each characteristic peak of characteristic spectrum.
Shengxuebao-medicine for improving hematopoitic function oral liquid characteristic spectrum has and all has 7 characteristic peaks, each characteristic peak standard relative retention time of Shengxuebao-medicine for improving hematopoitic function oral liquid characteristic spectrum each feature relative retention time and formulation is compared, its relative retention time relative deviation should setting ± 5% within.
Embodiment six
What the present embodiment six detected is that the correlativity of Shengxuebao-medicine for improving hematopoitic function preparation and each raw medicinal material and characteristic peak belong to.
(1) instrument and reagent
Agilent 1260 high performance liquid chromatograph, is contained in the degassed machine of line vacuum (G-1311C), binary pump (G-1311C), standard automatic sampler (G-1329B), intelligent column oven (G-1316A), DAD detecting device (G-1314B), Agilent1260Infinity chromatographic work station (Anjelen Sci. & Tech. Inc of the U.S.); SB-5200D ultrasonic washing instrument (NingBo XinZhi Biology Science Co., Ltd); FAZ004B analytical balance (section is helped in Shanghai); BT125D electronic balance (Sai Duolisi scientific instrument (Beijing) company limited).
(2) chromatographic condition
Chromatographic column is ThermoSyncronis-C 18(5 μm, 4.6 × 250mm) reverse-phase chromatographic column; Column temperature is 30 DEG C; Determined wavelength is 230nm; Mobile phase A is acetonitrile, and Mobile phase B is 0.1% (volume) phosphoric acid solution, and overall flow rate is 1.0ml/min; 60 minutes analysis times; The gradient elution that mobile phase is as shown in table 12 below.
Table 12
(3) preparation of object of reference solution
Take Paeoniflorin object of reference, be placed in volumetric flask, add 75% methyl alcohol and dissolve and dilute the solution made every 1ml and contain 0.02mg, shake up, obtain object of reference solution.
(4) preparation of need testing solution
Get Shengxuebao-medicine for improving hematopoitic function pill 0.6g, put in tool plug conical flask, precision adds methyl alcohol 25ml, close plug, weighed weight, reflow treatment 60 minutes, lets cool, more weighed weight, supplies the weight of less loss with methyl alcohol, shake up, filter, get subsequent filtrate, obtain Shengxuebao-medicine for improving hematopoitic function pill need testing solution.
The preparation method of medicinal material solution: get each single medicinal material respectively, become dry cream by formula preparation, beat powder, get the amount of converting suitable Shengxuebao-medicine for improving hematopoitic function preparation intermediate, corresponding single medicinal material solution is prepared, totally 7 taste medicinal material solution by Shengxuebao-medicine for improving hematopoitic function preparation intermediate need testing solution preparation method.
The preparation method of negative medicinal material solution: get other medicinal materials removing wherein certain taste medicinal material respectively, dry cream is become by formula preparation, beat powder, get the amount of converting suitable Shengxuebao-medicine for improving hematopoitic function preparation intermediate, corresponding negative medicinal material solution is prepared, the negative medicinal material solution of totally 7 tastes by Shengxuebao-medicine for improving hematopoitic function preparation intermediate need testing solution preparation method.
(5) formulation of single medicinal material, feminine gender, each characteristic peak collection of illustrative plates ownership of finished product.
The above-mentioned object of reference solution of accurate absorption and each 5 μ l of need testing solution, inject high performance liquid chromatograph respectively, according to high effective liquid chromatography for measuring, and record chromatogram; According to the collection of illustrative plates of gained, formulate the ownership of each characteristic peak, in table 13 and accompanying drawing 12.
Table 13
The HPLC of described Shengxuebao-medicine for improving hematopoitic function preparation has 14 total peaks at the characteristic spectrum of 230nm, and the total peak wherein belonging to the root of herbaceous peony is: peak 2, peak 4, peak 7, peak 8, peak 9, peak 13, peak 14; The total peak belonging to the fruit of glossy privet is: peak 2, peak 3, peak 6, peak 12; The total peak belonging to rhizoma cibotii is: peak 1, peak 2, peak 5; The total peak belonging to eclipta is: peak 1, peak 3, peak 5; The total peak belonging to prepared fleece flower root is: peak 1, peak 11; The total peak belonging to the Radix Astragali is: peak 2, peak 10; The total peak belonging to mulberry fruit is: peak 1, peak 2, peak 3, peak 5.Wherein choosing the suitable total peak of retention time and peak area is that characteristic peak is set up characteristic spectrum and wherein had: peak 4, peak 5, peak 7, peak 8, peak 9 (S), peak 11, peak 12.
By embodiment one and embodiment six known, beneficial effect of the present invention is as follows:
The characteristic spectrum method for building up of A, Shengxuebao-medicine for improving hematopoitic function preparation provided by the invention is simple to the pre-treating method of test sample, and characteristic chemical constituent retains complete, and need testing solution has good stability.
The precision of B, this efficient liquid-phase chromatography method is higher, reappearance good, and analysis time is shorter, has certain specificity; In gained characteristic spectrum, each characteristic peak separating effect is better, and Shengxuebao-medicine for improving hematopoitic function formulation characteristics collection of illustrative plates and intermediate characteristic spectrum have good correlativity;
The characteristic spectrum that C, the present invention set up has 7 characteristic peaks, quantity of information is comparatively large, more completely remains the effective constituent in test liquid, specify that (No. 6 peaks are 2 to 3 index compositions, 3,5,4'-tetrahydroxystilbene-2-O-β-D-Glucose glycosides, No. 7 peaks are Specnuezhenide), through comparing, employing retention time is moderate, and degree of separation is better, and the larger Paeoniflorin object of reference of peak area is as with reference to peak;
The application of D, Shengxuebao-medicine for improving hematopoitic function preparation intermediate characteristic spectrum can increase the controllability of Shengxuebao-medicine for improving hematopoitic function formulation manufacturing processes, the application of Shengxuebao-medicine for improving hematopoitic function formulation characteristics collection of illustrative plates more comprehensively can reflect technique whether stable uniform, be conducive to the effective constituent steady quality and the clinical efficacy that ensure Shengxuebao-medicine for improving hematopoitic function preparation, method provided by the invention has advantageously ensured the whole process quality control from raw material to terminal preparation.
Above-described embodiment one-embodiment six is some specific embodiments that the present invention announces, between parts different between each embodiment only otherwise contradiction, combination in any can form new embodiment, and these embodiments are all in category disclosed in the embodiment of the present invention.
Above-described embodiment of the present invention, does not form limiting the scope of the present invention.Any amendment done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.

Claims (7)

1. the method for building up of Shengxuebao-medicine for improving hematopoitic function formulation characteristics collection of illustrative plates, is characterized in that, comprises the following steps:
Getting Paeoniflorin reference substance to be dissolved in 50%-100% volumes methanol to form Paeoniflorin reference substance concentration for 0.02mg/ml solution, obtains object of reference solution after shaking up;
Get the Shengxuebao-medicine for improving hematopoitic function formulation dissolution of set amount in 0%-100% methyl alcohol, 0%-50% ethanol or 25-125ml water, by refluxing extraction 10-60min after weighing, or ultrasonic process 10-60min after weighing, after cooling, weigh again, then mend heavy with homogeneous solvent, shake up, filter with miillpore filter, get subsequent filtrate as need testing solution, described Shengxuebao-medicine for improving hematopoitic function preparation is oral liquid, and described set amount is 1.0-5.0ml, or described Shengxuebao-medicine for improving hematopoitic function preparation is granule, tablet, capsule, pill or Shengxuebao-medicine for improving hematopoitic function preparation intermediate, and described set amount is the powder of 0.6-3.0g;
Draw described object of reference solution and need testing solution injection high performance liquid chromatograph, detect according to high performance liquid chromatography, obtain the characteristic spectrum of Shengxuebao-medicine for improving hematopoitic function preparation; Wherein, chromatographic column is C18 reverse-phase chromatographic column; Adopt gradient elution, and the flow velocity of the mobile phase of gradient eluent is 0.8ml/min-1.2ml/min; Determined wavelength is 200-230nm, and column temperature is 25 DEG C-40 DEG C;
Wherein, the mobile phase of described gradient eluent is mobile phase A and Mobile phase B composition, and mobile phase A is acetonitrile, and Mobile phase B is 0.1% volume phosphoric acid solution;
Draw described object of reference solution and need testing solution to be 5 μ l and to inject high performance liquid chromatographs, the time of described gradient elution is 60min, carries out gradient elution in the following way:
0-15min, the percent by volume of mobile phase A is 2%, and the percent by volume of Mobile phase B is 98%;
15-30min, the percent by volume of mobile phase A is 2%-15%, and the percent by volume of Mobile phase B is 98%-85%;
30-50min, the percent by volume of mobile phase A is 15%-25%, and the percent by volume of Mobile phase B is 85%-75%;
50-60min, the percent by volume of mobile phase A is 25%, and the percent by volume of Mobile phase B is 75%.
2. method for building up according to claim 1, is characterized in that, the flow velocity of described mobile phase is 1.0ml/min, and the column length of described C18 reverse-phase chromatographic column is 250mm.
3. Shengxuebao-medicine for improving hematopoitic function formulation characteristics collection of illustrative plates, it is characterized in that, method for building up as claimed in claim 1 is adopted to obtain, described characteristic spectrum is detected by the need testing solution that the methanol solution got blood nourishing granules powder 0.6g and be dissolved in 25ml obtains and obtains, described characteristic spectrum has 7 characteristic peaks, wherein, No. 5 peaks take Paeoniflorin as the reference peak of reference, and each characteristic peak relative retention time and relative standard deviation are:
No. 1 peak: relative retention time 0.383 ± 0.019, relative standard deviation is 0.19%;
No. 2 peaks: relative retention time 0.672 ± 0.034, relative standard deviation is 0.07%;
No. 3 peaks: relative retention time 0.801 ± 0.040, relative standard deviation is 0.05%;
No. 4 peaks: relative retention time 0.956 ± 0.048, relative standard deviation is 0.01%;
No. 5 peaks: relative retention time 1.000 ± 0.000, relative standard deviation is 0.00%;
No. 6 peaks: relative retention time 1.143 ± 0.057, relative standard deviation is 0.01%;
No. 7 peaks: relative retention time 1.188 ± 0.059, relative standard deviation is 0.03%.
4. Shengxuebao-medicine for improving hematopoitic function formulation characteristics collection of illustrative plates, it is characterized in that, method for building up as claimed in claim 1 is adopted to obtain, described characteristic spectrum is detected by the need testing solution that the water got 3.0g Shengxuebao-medicine for improving hematopoitic function preparation intermediate and be dissolved in 125ml obtains and obtains, described characteristic spectrum has 7 characteristic peaks, wherein, No. 5 peaks take Paeoniflorin as the reference peak of reference, and each characteristic peak relative retention time and relative standard deviation are:
No. 1 peak: relative retention time 0.383 ± 0.019, relative standard deviation is 0.14%;
No. 2 peaks: relative retention time 0.672 ± 0.034, relative standard deviation is 0.00%;
No. 3 peaks: relative retention time 0.801 ± 0.040, relative standard deviation is 0.05%;
No. 4 peaks: relative retention time 0.956 ± 0.048, relative standard deviation is 0.00%;
No. 5 peaks: relative retention time 1.000 ± 0.000, relative standard deviation is 0.00%;
No. 6 peaks: relative retention time 1.143 ± 0.057, relative standard deviation is 0.00%;
No. 7 peaks: relative retention time 1.188 ± 0.059, relative standard deviation is 0.00%.
5. Shengxuebao-medicine for improving hematopoitic function formulation characteristics collection of illustrative plates, it is characterized in that, method for building up as claimed in claim 1 is adopted to obtain, described characteristic spectrum is detected by the need testing solution that 50% methyl alcohol got 1.2g Shengxuebao-medicine for improving hematopoitic function capsule powders and be dissolved in 50ml obtains and obtains, described characteristic spectrum has 7 characteristic peaks, wherein, No. 5 peaks take Paeoniflorin as the reference peak of reference, and each characteristic peak relative retention time and relative standard deviation are:
No. 1 peak: relative retention time 0.383 ± 0.019, relative standard deviation is 1.19%;
No. 2 peaks: relative retention time 0.672 ± 0.034, relative standard deviation is 0.43%;
No. 3 peaks: relative retention time 0.801 ± 0.040, relative standard deviation is 0.19%;
No. 4 peaks: relative retention time 0.956 ± 0.048, relative standard deviation is 0.05%;
No. 5 peaks: relative retention time 1.000 ± 0.000, relative standard deviation is 0.00%;
No. 6 peaks: relative retention time 1.143 ± 0.057, relative standard deviation is 0.28%;
No. 7 peaks: relative retention time 1.188 ± 0.059, relative standard deviation is 0.12%.
6. Shengxuebao-medicine for improving hematopoitic function formulation characteristics collection of illustrative plates, it is characterized in that, method for building up as claimed in claim 1 is adopted to obtain, described characteristic spectrum to detect in the need testing solution that 25% ethanol of 100ml obtains obtain by getting 2.4g Shengxuebao-medicine for improving hematopoitic function tablet dissolved, described characteristic spectrum has 7 characteristic peaks, wherein, No. 5 peaks take Paeoniflorin as the reference peak of reference, and each characteristic peak relative retention time and relative standard deviation are:
No. 1 peak: relative retention time 0.383 ± 0.019, relative standard deviation is 0.19%;
No. 2 peaks: relative retention time 0.672 ± 0.034, relative standard deviation is 0.07%;
No. 3 peaks: relative retention time 0.801 ± 0.040, relative standard deviation is 0.05%;
No. 4 peaks: relative retention time 0.956 ± 0.048, relative standard deviation is 0.01%;
No. 5 peaks: relative retention time 1.000 ± 0.000, relative standard deviation is 0.00%;
No. 6 peaks: relative retention time 1.143 ± 0.057, relative standard deviation is 0.01%;
No. 7 peaks: relative retention time 1.188 ± 0.059, relative standard deviation is 0.03%.
7. Shengxuebao-medicine for improving hematopoitic function formulation characteristics collection of illustrative plates, it is characterized in that, method for building up as claimed in claim 1 is adopted to obtain, described characteristic spectrum is detected by the need testing solution that 50% ethanol got 1ml Shengxuebao-medicine for improving hematopoitic function oral liquid and be dissolved in 25ml obtains and obtains, described characteristic spectrum has 7 characteristic peaks, wherein, No. 5 peaks take Paeoniflorin as the reference peak of reference, and each characteristic peak relative retention time and relative standard deviation are:
No. 1 peak: relative retention time 0.383 ± 0.019, relative standard deviation is 0.21%;
No. 2 peaks: relative retention time 0.672 ± 0.034, relative standard deviation is 0.08%;
No. 3 peaks: relative retention time 0.801 ± 0.040, relative standard deviation is 0.07%;
No. 4 peaks: relative retention time 0.956 ± 0.048, relative standard deviation is 0.05%;
No. 5 peaks: relative retention time 1.000 ± 0.000, relative standard deviation is 0.00%;
No. 6 peaks: relative retention time 1.143 ± 0.057, relative standard deviation is 0.00%;
No. 7 peaks: relative retention time 1.188 ± 0.059, relative standard deviation is 0.04%.
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