CN104020225A - Specific chromatogram of blood-production capsule preparation and establishing method of specific chromatogram - Google Patents

Specific chromatogram of blood-production capsule preparation and establishing method of specific chromatogram Download PDF

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CN104020225A
CN104020225A CN201410187475.6A CN201410187475A CN104020225A CN 104020225 A CN104020225 A CN 104020225A CN 201410187475 A CN201410187475 A CN 201410187475A CN 104020225 A CN104020225 A CN 104020225A
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retention time
peaks
standard deviation
relative
peak
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CN104020225B (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 the technical field of medicament quality control and discloses an establishing method of a specific chromatogram of a blood-production capsule preparation. The establishing method comprises the following steps: dissolving a reference substance of paeoniflorin into 50%-100% (volume) methanol to form a solution of which the concentration of the reference substance of paeoniflorin is 0.02mg/ml, and uniformly shaking to obtain a reference solution; dissolving a quantified blood-production capsule preparation into 0%-100% methanol, 0%-50% ethanol or 25ml-125ml of water, weighing the solution, carrying out reflux extraction or ultrasonic treatment for 10-60 minutes, cooling, weighing the cooled solution, carrying out weight compensation by virtue of the same solvent, uniformly shaking, and filtering by virtue of a micro-hole filtering film, thereby obtaining subsequent filtrate as a test solution; absorbing the reference solution and the test solution, injecting the reference solution and the test solution into a high performance liquid chromatograph, and detecting by virtue of a high performance liquid chromatography so as to obtain the specific chromatogram of the blood-production capsule preparation. The invention further discloses the specific chromatogram of the blood-production capsule preparation. According to the specific chromatogram, the comprehensive detection on the quality of the blood-production capsule preparation is realized.

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 Control of drug quality technical field, 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 being obtained by above-mentioned method for building up.
Background technology
Shengxuebao-medicine for improving hematopoitic function be by the famous traditional Chinese medicine and pharmacy expert of China through proved recipe, be prepared from through modern crafts.Shengxuebao-medicine for improving hematopoitic function is mainly made up 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 practices of decades proves that Shengxuebao-medicine for improving hematopoitic function has nourishing liver and kidney, and effect of replenishing qi and blood, for symptoms such as leucocyte minimizing due to 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, made the Shengxuebao-medicine for improving hematopoitic function preparation of multiple formulation according to Shengxuebao-medicine for improving hematopoitic function prescription.Shengxuebao-medicine for improving hematopoitic function preparation on market is mainly by granule, mixture etc.Conventionally, the preparation technology of Shengxuebao-medicine for improving hematopoitic function preparation is: by the water extract of the cataclasm rear extracting in water of raw medicinal material, according to the agent of method granulation, tablet, pill, capsule, oral liquid or the mixture of 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 of 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 is only measured the content of single index composition, other effective ingredient is not detected.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, cannot reflect the problem of Shengxuebao-medicine for improving hematopoitic function quality of the pharmaceutical preparations level to solve current Shengxuebao-medicine for improving hematopoitic function preparation detection method 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:
Get Paeoniflorin reference substance and be dissolved in 50%-100% (volume) methyl alcohol to form Paeoniflorin reference substance concentration as 0.02mg/ml solution, after shaking up, obtain object of reference solution;
The Shengxuebao-medicine for improving hematopoitic function preparation of getting set amount is dissolved in 0%-100% methyl alcohol, 0%-50% ethanol or 25-125ml water, after weighing, pass through refluxing extraction or ultrasonic processing 10-60min, 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, the powder that described set amount is 0.6-3.0g;
Draw described object of reference solution and need testing solution and inject 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; Detection 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 injection high performance liquid chromatographs, the time of described gradient elution is 60min, carries out in the following way gradient elution:
0-15min, the percent by volume of mobile phase A is 2%, the percent by volume of Mobile phase B is 98%;
15-30min, the percent by volume of mobile phase A is 2%-15%, the percent by volume of Mobile phase B is 98%-85%;
30-50min, the percent by volume of mobile phase A is 15%-25%, the percent by volume of Mobile phase B is 85%-75%;
50-60min, the percent by volume of mobile phase A is 25%, 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, described C 18the column length of reverse-phase chromatographic column is 250nm.
Shengxuebao-medicine for improving hematopoitic function formulation characteristics collection of illustrative plates, adopt method for building up as above to obtain, described characteristic spectrum is dissolved in need testing solution that the methanol solution of 25ml obtains and detects and obtain by getting blood nourishing granules powder 0.6g, described characteristic spectrum has 7 characteristic peaks, wherein, No. 5 peaks taking Paeoniflorin as reference with reference to peak, 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, adopt method for building up as above to obtain, described characteristic spectrum is dissolved in need testing solution that the water of 125ml obtains and detects and obtain by getting 3.0g Shengxuebao-medicine for improving hematopoitic function preparation intermediate, described characteristic spectrum has 7 characteristic peaks, wherein, No. 5 peaks taking Paeoniflorin as reference with reference to peak, 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, adopt method for building up as above to obtain, described characteristic spectrum is dissolved in need testing solution that 50% methyl alcohol of 50ml obtains and detects and obtain by getting 1.2g Shengxuebao-medicine for improving hematopoitic function capsule powders, described characteristic spectrum has 7 characteristic peaks, wherein, No. 5 peaks taking Paeoniflorin as reference with reference to peak, 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, adopt method for building up as above to obtain, described characteristic spectrum is detected and is obtained by the need testing solution of getting 2.4g Shengxuebao-medicine for improving hematopoitic function tablet dissolved and obtaining in 25% ethanol of 100ml, described characteristic spectrum has 7 characteristic peaks, wherein, No. 5 peaks taking Paeoniflorin as reference with reference to peak, 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, adopt method for building up as above to obtain, described characteristic spectrum is dissolved in need testing solution that 50% ethanol of 25ml obtains and detects and obtain by getting 1ml Shengxuebao-medicine for improving hematopoitic function oral liquid, described characteristic spectrum has 7 characteristic peaks, wherein, No. 5 peaks taking Paeoniflorin as reference with reference to peak, 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 is 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 larger, has more completely retained the effective constituent in test liquid, clear and definite 3 index compositions, through relatively, adopt 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 preparation production run, the application of Shengxuebao-medicine for improving hematopoitic function formulation characteristics collection of illustrative plates can more comprehensively reflect whether stable uniform of technique, the effective constituent steady quality and the clinical efficacy that are conducive to ensure Shengxuebao-medicine for improving hematopoitic function preparation, method provided by the invention has advantageously ensured the whole process quality control of expecting terminal preparation from former.
Brief description of the drawings
In order to be illustrated more clearly in the technical scheme in the embodiment of the present invention, below the accompanying drawing of required use during embodiment is described is briefly described, apparently, for those of ordinary skills, do not paying under the prerequisite of creative work, can also obtain according to these accompanying drawings other accompanying drawing.
Fig. 1 object of reference stack 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 is green blood health-care capsule characteristic spectrum;
Fig. 7 is 10 batches of Shengxuebao-medicine for improving hematopoitic function capsule characteristic spectrums;
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 " Xuebao " oral liquid (blood medicine) characteristic spectrum of making a living;
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
The embodiment of the present invention provides a kind of method for building up of Shengxuebao-medicine for improving hematopoitic function formulation characteristics collection of illustrative plates, detects the problem that cannot more comprehensively reflect Shengxuebao-medicine for improving hematopoitic function quality of the pharmaceutical preparations level to solve current Shengxuebao-medicine for improving hematopoitic function preparation.
In order to make those skilled in the art person understand better the technical scheme in the embodiment of the present invention, and the above-mentioned purpose of the embodiment of the present invention, feature and advantage can be 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 are 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 mono-
The present embodiment one is set up Shengxuebao-medicine for improving hematopoitic function formulation characteristics collection of illustrative plates taking blood nourishing granules as experiment material.
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, obtain object of reference solution, object of reference stack chromatogram as shown in Figure 1.50%-100% in the present embodiment (volume) methyl alcohol refers to the methanol aqueous solution that the volume ratio of methyl alcohol is 50%-100%.
The preparation of S102, need testing solution.
Get blood nourishing granules powder 0.6g, accurately weighed being placed in tool plug conical flask, precision adds methyl alcohol 25ml, close plug, weighed weight.After ultrasonic processing 10min, let cool, more weighed weight, supply the weight cutting 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 according to high performance liquid chromatography, obtain blood nourishing granules characteristic spectrum.
(1) step S103 can select Agilent 1260 high performance liquid chromatographs, 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 that step S103 detects is as follows:
The selection of a, mobile phase.
Mobile phase is made up of mobile phase A and Mobile phase B.Concrete, can select 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. to test for mobile phase (be mobile phase A, after be Mobile phase B) above.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 separates 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, 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 select as mobile phase.
Table 1
The selection of b, detection wavelength.
Application DAD detecting device has carried out long mensuration of all-wave to test sample and has compared, and the response of result main chromatographic peak in 200-230nm surveys characteristic spectrum is larger, and each chromatographic peak feature is outstanding, is 200-230nm therefore preferably adopt detection wavelength.
The selection of c, chromatographic column.
In the present embodiment, chromatographic column is C 18reverse-phase chromatographic column can.By comparing, Agilent ZORBAX XDB-C 18(5 μ m, 4.6 × 250mm), Thermo Syncronis-C 18(5 μ m, 4.6 × 250mm), Yi Lite HypersilODS2-C 18(5 μ m, 4.6 × 250mm), enlightening horse Diamonsil-C 18the chromatographic column of (5 μ m, 4.6 × 250mm) 4 kinds of different model specifications, result shows, and 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 25 DEG C of different column temperatures, separating effect to same sample 30 DEG C, 35 DEG C, 40 DEG C time.Result shows, different column temperatures are less on the separation impact of chromatographic peak, and during with 25 DEG C-40 DEG C, separating effect is all more satisfactory.
The selection of e, flow rate of mobile phase.
The present embodiment one is under the identical condition of other chromatographic condition, and having investigated respectively flow velocity is the impact that 0.8ml/min, 1.0ml/min, 1.2ml/min separate same sample chromatographic peak.Result shows, in the time that 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 the present embodiment one provides detects 120min under above-mentioned chromatographic condition, and result shows, in 60 minutes, in sample, all peaks all can be eluted completely, therefore be 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, investigate the relative retention time of main chromatographic peak and the consistance of relative peak area.The RSD < 3% of the relative retention time of the main chromatographic peak of result and relative peak area, shows 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, investigate the relative retention time of main chromatographic peak and the consistance of relative peak area.The RSD < 3% of the relative retention time of the main chromatographic peak of result and relative peak area, shows 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 RSD < 3% of the relative retention time of the main chromatographic peak of result and relative peak area, shows that the method is reproducible.
In the method for building up that the present embodiment one provides, accurate above-mentioned object of reference solution and the each 5 μ l of need testing solution of drawing, inject respectively high performance liquid chromatograph, according to high effective liquid chromatography for measuring, 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 that Paeoniflorin peak is that the relative retention time of each characteristic peak and relative standard deviation are with reference to peak:
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 and 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 that Paeoniflorin peak is that the relative standard deviation of each characteristic peak is as shown in table 2 with reference to peak.Table 2 is 10 batches of blood nourishing granules characteristic spectrum relative deviation result tables.
Table 2
(5) evaluation of the 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 blood nourishing granules characteristic spectrum feature relative retention time and formulation is compared, its relative retention time relative deviation should setting ± 5% in.
Embodiment bis-
The present embodiment two is set up Shengxuebao-medicine for improving hematopoitic function formulation characteristics collection of illustrative plates taking Shengxuebao-medicine for improving hematopoitic function preparation intermediate as experiment material.
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, obtain object of reference solution, object of reference stack chromatogram as shown in Figure 1.50%-100% in the present embodiment (volume) methyl alcohol refers to the methanol aqueous solution that the volume ratio of methyl alcohol is 50%-100%.
The preparation of S202, need testing solution.
Get Shengxuebao-medicine for improving hematopoitic function preparation intermediate 3.0g, accurately weighed being placed in tool plug conical flask, precision adds 125ml water, close plug, weighed weight.After ultrasonic processing 30min, let cool, more weighed weight, water is supplied the weight cutting, and shakes 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 according to high performance liquid chromatography, obtain Shengxuebao-medicine for improving hematopoitic function preparation intermediate characteristic spectrum.
(1) step S203 can select Agilent 1260 high performance liquid chromatographs, 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 that step S203 detects is as follows:
The selection of a, mobile phase.
Mobile phase is made up of mobile phase A and Mobile phase B.Concrete, can select 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. to test for mobile phase (be mobile phase A, after be Mobile phase B) above.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 separates 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, 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 select as mobile phase.
Table 3
The selection of b, detection wavelength.
In the present embodiment, detection wavelength is 200nm.
The selection of c, chromatographic column.
In the present embodiment, chromatographic column can be C 18reverse-phase chromatographic column.By comparing, Agilent ZORBAX XDB-C 18(5 μ m, 4.6 × 250mm), Thermo Syncronis-C 18(5 μ m, 4.6 × 250mm), Yi Lite Hypersil ODS2-C 18(5 μ m, 4.6 × 250mm), enlightening horse Diamonsil-C 18the chromatographic column of (5 μ m, 4.6 × 250mm) 4 kinds of different model specifications, result shows, and 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 employing chromatographic columns are Agilent ZORBAX XDB-C 18(5 μ m, 4.6 × 250mm) reverse-phase chromatographic column.
The selection of d, column temperature.
The present embodiment two investigated 25 DEG C of different column temperatures, separating effect to same sample 30 DEG C, 35 DEG C, 40 DEG C time.Result shows, different temperatures is less on the separation impact of chromatographic peak.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 identical condition of other chromatographic condition, and having investigated respectively flow velocity is the impact that 0.8ml/min, 1.0ml/min, 1.2ml/min separate same sample chromatographic peak.Result shows, in the time that 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 the present embodiment provides detects 120min under above-mentioned chromatographic condition, and result shows, in 60 minutes, in sample, all peaks all can be eluted completely, therefore be defined as 60 minutes analysis time in the present embodiment two.
(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, investigate the relative retention time of main chromatographic peak and the consistance of relative peak area.The RSD < 3% of the relative retention time of the main chromatographic peak of result and relative peak area, shows 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, investigate the relative retention time of main chromatographic peak and the consistance of relative peak area.The RSD < 3% of the relative retention time of the main chromatographic peak of result and relative peak area, shows 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 RSD < 3% of the relative retention time of the main chromatographic peak of result and relative peak area, shows that the method is reproducible.
In the method for building up that the present embodiment two provides, accurate above-mentioned object of reference solution and the each 5 μ l of need testing solution of drawing, inject respectively high performance liquid chromatograph, according to high effective liquid chromatography for measuring, 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 that Paeoniflorin peak is that the relative retention time of each characteristic peak and relative standard deviation are with reference to peak:
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%.
Getting 10 batches of Shengxuebao-medicine for improving hematopoitic function preparation intermediates detects, 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 that Paeoniflorin peak is that the relative standard deviation of each characteristic peak is as shown in table 4 with reference to peak.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 the 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 blood nourishing granules characteristic spectrum feature relative retention time and formulation is compared, its relative retention time relative deviation should setting ± 5% in.
Embodiment tri-
The present embodiment three is set up Shengxuebao-medicine for improving hematopoitic function formulation characteristics collection of illustrative plates taking Shengxuebao-medicine for improving hematopoitic function capsule as experiment material.
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, obtain object of reference solution, object of reference stack chromatogram as shown in Figure 1.50%-100% in the present embodiment (volume) methyl alcohol refers to the methanol aqueous solution that the volume ratio of methyl alcohol is 50%-100%.
The preparation of S302, need testing solution.
Get Shengxuebao-medicine for improving hematopoitic function capsule powders 1.2g, accurately weighed being placed in tool plug conical flask, precision adds 50% methyl alcohol 50ml, close plug, weighed weight.After adding hot reflux 10min, let cool, more weighed weight, supply the weight cutting 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 according to high performance liquid chromatography, obtain blood nourishing granules characteristic spectrum.
(1) step S303 can select Agilent 1260 high performance liquid chromatographs, 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 that step S303 detects is as follows:
The selection of a, mobile phase.
Mobile phase is made up of mobile phase A and Mobile phase B.Concrete, can select 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. to test for mobile phase (be mobile phase A, after be Mobile phase B) above.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 separates 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, 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 select as mobile phase.
Table 5
The selection of b, detection wavelength.
In the present embodiment three, detection wavelength is 210nm.
The selection of c, chromatographic column.
By comparing, Agilent ZORBAX XDB-C 18(5 μ m, 4.6 × 250mm), Thermo Syncronis-C 18(5 μ m, 4.6 × 250mm), Yi Lite Hypersil ODS2-C 18(5 μ m, 4.6 × 250mm), enlightening horse Diamonsil-C 18the chromatographic column of (5 μ m, 4.6 × 250mm) 4 kinds of different model specifications, result shows, and 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 employing chromatographic columns are Agilent ZORBAX XDB-C 18(5 μ m, 4.6 × 250mm) reverse-phase chromatographic column.
The selection of d, column temperature.
The present embodiment three investigated 25 DEG C of different column temperatures, separating effect to same sample 30 DEG C, 35 DEG C, 40 DEG C time.Result shows, different temperatures is less on the separation impact of chromatographic peak.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 identical condition of other chromatographic condition, and having investigated respectively flow velocity is the impact that 0.8ml/min, 1.0ml/min, 1.2ml/min separate same sample chromatographic peak.Result shows, in the time that 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 the present embodiment three provides detects 120min under above-mentioned chromatographic condition, and result shows, in 60 minutes, in sample, all peaks all can be eluted completely, therefore be defined as 60 minutes analysis time in the present embodiment three.
(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, investigate the relative retention time of main chromatographic peak and the consistance of relative peak area.The RSD < 3% of the relative retention time of the main chromatographic peak of result and relative peak area, shows 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, investigate the relative retention time of main chromatographic peak and the consistance of relative peak area.The RSD < 3% of the relative retention time of the main chromatographic peak of result and relative peak area, shows 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 RSD < 3% of the relative retention time of the main chromatographic peak of result and relative peak area, shows that the method is reproducible.
In the method for building up that the present embodiment three provides, accurate above-mentioned object of reference solution and the each 5 μ l of need testing solution of drawing, inject respectively high performance liquid chromatograph, according to high effective liquid chromatography for measuring, 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 that Paeoniflorin peak is that the relative retention time of each characteristic peak and relative standard deviation are with reference to peak:
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 and 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 that Paeoniflorin peak is that the relative standard deviation of each characteristic peak is as shown in table 6 with reference to peak.Table 6 is 10 batches of Shengxuebao-medicine for improving hematopoitic function capsule characteristic spectrum relative deviation result tables.
Table 6
(5) evaluation of the each characteristic peak of characteristic spectrum.
Shengxuebao-medicine for improving hematopoitic function capsule characteristic spectrum has and all has 7 characteristic peaks, each characteristic peak standard relative retention time of each blood nourishing granules characteristic spectrum feature relative retention time and formulation is compared, its relative retention time relative deviation should setting ± 5% in.
Embodiment tetra-
The present embodiment four is set up Shengxuebao-medicine for improving hematopoitic function formulation characteristics collection of illustrative plates taking Shengxuebao-medicine for improving hematopoitic function tablet as experiment material.
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, obtain object of reference solution, object of reference stack chromatogram as shown in Figure 1.50%-100% in the present embodiment (volume) methyl alcohol refers to the methanol aqueous solution that the volume ratio of methyl alcohol is 50%-100%.
The preparation of S402, need testing solution.
Get Shengxuebao-medicine for improving hematopoitic function tablet powder 2.4g, accurately weighed being placed in tool plug conical flask, precision adds 25% ethanol 100ml, close plug, weighed weight.After ultrasonic processing 60min, let cool, more weighed weight, supply the weight cutting 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 according to high performance liquid chromatography, obtain Shengxuebao-medicine for improving hematopoitic function tablet characteristic spectrum.
(1) step S403 can select Agilent 1260 high performance liquid chromatographs, 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 that step S403 detects is as follows:
The selection of a, mobile phase.
Mobile phase is made up of mobile phase A and Mobile phase B.Concrete, can select 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. to test for mobile phase (be mobile phase A, after be Mobile phase B) above.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 separates 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, 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 select as mobile phase.
Table 7
The selection of b, detection wavelength.
In the present embodiment, detection wavelength is 215nm.
The selection of c, chromatographic column.
In the present embodiment, chromatographic column can be C 18reverse-phase chromatographic column.By comparing, Agilent ZORBAX XDB-C 18(5 μ m, 4.6 × 250mm), Thermo Syncronis-C 18(5 μ m, 4.6 × 250mm), Yi Lite Hypersil ODS2-C 18(5 μ m, 4.6 × 250mm), enlightening horse Diamonsil-C 18the chromatographic column of (5 μ m, 4.6 × 250mm) 4 kinds of different model specifications, result shows, and 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 employing chromatographic columns are Agilent ZORBAX XDB-C 18(5 μ m, 4.6 × 250mm) reverse-phase chromatographic column.
The selection of d, column temperature.
The present embodiment four investigated 25 DEG C of different column temperatures, separating effect to same sample 30 DEG C, 35 DEG C, 40 DEG C time.Result shows, different temperatures is less on the separation impact of chromatographic peak.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 identical condition of other chromatographic condition, and having investigated respectively flow velocity is the impact that 0.8ml/min, 1.0ml/min, 1.2ml/min separate same sample chromatographic peak.Result shows, in the time that 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 the present embodiment provides detects 120min under above-mentioned chromatographic condition, and result shows, in 60 minutes, in sample, all peaks all can be eluted completely, therefore be defined as 60 minutes analysis time in the present embodiment four.
(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, investigate the relative retention time of main chromatographic peak and the consistance of relative peak area.The RSD < 3% of the relative retention time of the main chromatographic peak of result and relative peak area, shows 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, investigate the relative retention time of main chromatographic peak and the consistance of relative peak area.The RSD < 3% of the relative retention time of the main chromatographic peak of result and relative peak area, shows 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 RSD < 3% of the relative retention time of the main chromatographic peak of result and relative peak area, shows that the method is reproducible.
In the method for building up that the present embodiment four provides, accurate above-mentioned object of reference solution and the each 5 μ l of need testing solution of drawing, inject respectively high performance liquid chromatograph, according to high effective liquid chromatography for measuring, 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 that Paeoniflorin peak is that the relative retention time of each characteristic peak and relative standard deviation are with reference to peak:
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 and 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 that Paeoniflorin peak is that the relative standard deviation of each characteristic peak is as shown in table 8 with reference to peak.Table 8 is 10 batches of Shengxuebao-medicine for improving hematopoitic function tablet characteristic spectrum relative deviation result tables.
Table 8
(5) evaluation of the each characteristic peak of characteristic spectrum.
Shengxuebao-medicine for improving hematopoitic function tablet characteristic spectrum has and all has 7 characteristic peaks, each characteristic peak standard relative retention time of each Shengxuebao-medicine for improving hematopoitic function tablet characteristic spectrum feature relative retention time and formulation is compared, its relative retention time relative deviation should setting ± 5% in.
Embodiment five
The present embodiment five is set up Shengxuebao-medicine for improving hematopoitic function formulation characteristics collection of illustrative plates taking Shengxuebao-medicine for improving hematopoitic function preparation oral liquid as experiment material.
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, obtain object of reference solution, object of reference stack chromatogram as shown in Figure 1.50%-100% in the present embodiment (volume) methyl alcohol refers to the methanol aqueous solution that the volume ratio of methyl alcohol is 50%-100%.
The preparation of S302, need testing solution.
Get Shengxuebao-medicine for improving hematopoitic function oral liquid 1ml, accurately weighed being placed in tool plug conical flask, precision adds 50% ethanol 25ml, close plug, weighed weight.After adding hot reflux 30min, let cool, more weighed weight, supply the weight cutting 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 according to high performance liquid chromatography, obtain Shengxuebao-medicine for improving hematopoitic function oral liquid characteristic spectrum.
(1) step S303 can select Agilent 1260 high performance liquid chromatographs, 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 that step S303 detects is as follows:
The selection of a, mobile phase.
Mobile phase is made up of mobile phase A and Mobile phase B.Concrete, can select 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. to test for mobile phase (be mobile phase A, after be Mobile phase B) above.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 separates 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, 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 select as mobile phase.
Table 9
The selection of b, detection wavelength.
In the present embodiment, detection wavelength is 210nm.
The selection of c, chromatographic column.
By comparing, Agilent ZORBAX XDB-C 18(5 μ m, 4.6 × 250mm), Thermo Syncronis-C 18(5 μ m, 4.6 × 250mm), Yi Lite Hypersil ODS2-C 18(5 μ m, 4.6 × 250mm), enlightening horse Diamonsil-C 18the chromatographic column of (5 μ m, 4.6 × 250mm) 4 kinds of different model specifications, result shows, and 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 employing chromatographic columns are Agilent ZORBAX XDB-C 18(5 μ m, 4.6 × 250mm) reverse-phase chromatographic column.
The selection of d, column temperature.
The present embodiment five investigated 25 DEG C of different column temperatures, separating effect to same sample 30 DEG C, 35 DEG C, 40 DEG C time.Result shows, different temperatures is less on the separation impact of chromatographic peak.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 identical condition of other chromatographic condition, and having investigated respectively flow velocity is the impact that 0.8ml/min, 1.0ml/min, 1.2ml/min separate same sample chromatographic peak.Result shows, in the time of 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 the present embodiment provides detects 120min under above-mentioned chromatographic condition, and result shows, in 60 minutes, in sample, all peaks all can be eluted completely, therefore be defined as 60 minutes analysis time in the present embodiment five.
(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, investigate the relative retention time of main chromatographic peak and the consistance of relative peak area.The RSD < 3% of the relative retention time of the main chromatographic peak of result and relative peak area, shows 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, investigate the relative retention time of main chromatographic peak and the consistance of relative peak area.The RSD < 3% of the relative retention time of the main chromatographic peak of result and relative peak area, shows 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 RSD < 3% of the relative retention time of the main chromatographic peak of result and relative peak area, shows that the method is reproducible.
In the method for building up that the present embodiment five provides, accurate above-mentioned object of reference solution and the each 5 μ l of need testing solution of drawing, inject respectively high performance liquid chromatograph, according to high effective liquid chromatography for measuring, 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 that Paeoniflorin peak is that the relative retention time of each characteristic peak and relative standard deviation are with reference to peak:
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 and 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 that Paeoniflorin peak is that the relative standard deviation of each characteristic peak is as shown in table 10 with reference to peak.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 the 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 each Shengxuebao-medicine for improving hematopoitic function oral liquid characteristic spectrum feature relative retention time and formulation is compared, its relative retention time relative deviation should setting ± 5% in.
Embodiment six
What the present embodiment six detected is correlativity and the characteristic peak ownership of Shengxuebao-medicine for improving hematopoitic function preparation and each raw medicinal material.
(1) instrument and reagent
Agilent 1260 high performance liquid chromatographs, are 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 Thermo Syncronis-C 18(5 μ m, 4.6 × 250mm) reverse-phase chromatographic column; Column temperature is 30 DEG C; Detection 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 and make every 1ml containing the solution of 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 respectively each single medicinal material, become dry cream by formula preparation, beat powder, get the amount of the suitable Shengxuebao-medicine for improving hematopoitic function preparation intermediate of conversion, by Shengxuebao-medicine for improving hematopoitic function preparation intermediate need testing solution, preparation method prepares corresponding single medicinal material solution, totally 7 taste medicinal material solution.
The preparation method of negative medicinal material solution: get respectively and remove wherein other medicinal materials of certain taste medicinal material, become dry cream by formula preparation, beat powder, get the amount of the suitable Shengxuebao-medicine for improving hematopoitic function preparation intermediate of conversion, by Shengxuebao-medicine for improving hematopoitic function preparation intermediate need testing solution, preparation method prepares corresponding negative medicinal material solution, the negative medicinal material solution of totally 7 tastes.
(5) formulation of single medicinal material, feminine gender, the each characteristic peak collection of illustrative plates ownership of finished product.
Accurate above-mentioned object of reference solution and the each 5 μ l of need testing solution of drawing, inject respectively high performance liquid chromatograph, according to high effective liquid chromatography for measuring, 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 that wherein belongs to the root of herbaceous peony is: peak 2, peak 4, peak 7, peak 8, peak 9, peak 13, peak 14; The total peak that belongs to the fruit of glossy privet is: peak 2, peak 3, peak 6, peak 12; The total peak that belongs to rhizoma cibotii is: peak 1, peak 2, peak 5; The total peak that belongs to eclipta is: peak 1, peak 3, peak 5; The total peak that belongs to prepared fleece flower root is: peak 1, peak 11; The total peak that belongs to the Radix Astragali is: peak 2, peak 10; The total peak that belongs to mulberry fruit is: peak 1, peak 2, peak 3, peak 5.The suitable total peak of wherein choosing 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.
Known by embodiment mono-and embodiment six, 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 is 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 larger, has more completely retained the effective constituent in test liquid, and (No. 6 peak is 2 to clear and definite 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 preparation production run, the application of Shengxuebao-medicine for improving hematopoitic function formulation characteristics collection of illustrative plates can more comprehensively reflect whether stable uniform of technique, the effective constituent steady quality and the clinical efficacy that are conducive to ensure Shengxuebao-medicine for improving hematopoitic function preparation, method provided by the invention has advantageously ensured the whole process quality control of expecting terminal preparation from former.
Above-described embodiment one-embodiment six some specific embodiments that just the present invention announces, between each embodiment between different part only otherwise contradiction, can combination in any form new embodiment, and these embodiment are all in the disclosed category of 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 of doing within the spirit and principles in the present invention, be equal to and replace and improvement etc., within all should being included in protection scope of the present invention.

Claims (10)

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:
Get Paeoniflorin reference substance and be dissolved in 50%-100% (volume) methyl alcohol to form Paeoniflorin reference substance concentration as 0.02mg/ml solution, after shaking up, obtain object of reference solution;
The Shengxuebao-medicine for improving hematopoitic function preparation of getting set amount is dissolved in 0%-100% methyl alcohol, 0%-50% ethanol or 25-125ml water, after weighing, pass through refluxing extraction or ultrasonic processing 10-60min, 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, the powder that described set amount is 0.6-3.0g;
Draw described object of reference solution and need testing solution and inject 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; Detection wavelength is 200-230nm, and column temperature is 25 DEG C-40 DEG C; The gradient elution time is 60min-120min.
2. method for building up according to claim 1, it is characterized in that, 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.
3. method for building up according to claim 2, is characterized in that, 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.
4. method for building up according to claim 3, is characterized in that, draws described object of reference solution and need testing solution and is 5 μ l injection high performance liquid chromatographs, and the time of described gradient elution is 60min, carries out in the following way gradient elution:
0-15min, the percent by volume of mobile phase A is 2%, the percent by volume of Mobile phase B is 98%;
15-30min, the percent by volume of mobile phase A is 2%-15%, the percent by volume of Mobile phase B is 98%-85%;
30-50min, the percent by volume of mobile phase A is 15%-25%, the percent by volume of Mobile phase B is 85%-75%;
50-60min, the percent by volume of mobile phase A is 25%, the percent by volume of Mobile phase B is 75%.
5. method for building up according to claim 4, is characterized in that, the flow velocity of described mobile phase is 1.0ml/min, described C 18the column length of reverse-phase chromatographic column is 250nm.
6. Shengxuebao-medicine for improving hematopoitic function formulation characteristics collection of illustrative plates, it is characterized in that, adopt method for building up as claimed in claim 1 to obtain, described characteristic spectrum is dissolved in need testing solution that the methanol solution of 25ml obtains and detects and obtain by getting blood nourishing granules powder 0.6g, described characteristic spectrum has 7 characteristic peaks, wherein, No. 5 peaks taking Paeoniflorin as reference with reference to peak, 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, adopt method for building up as claimed in claim 1 to obtain, described characteristic spectrum is dissolved in need testing solution that the water of 125ml obtains and detects and obtain by getting 3.0g Shengxuebao-medicine for improving hematopoitic function preparation intermediate, described characteristic spectrum has 7 characteristic peaks, wherein, No. 5 peaks taking Paeoniflorin as reference with reference to peak, 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%.
8. Shengxuebao-medicine for improving hematopoitic function formulation characteristics collection of illustrative plates, it is characterized in that, adopt method for building up as claimed in claim 1 to obtain, described characteristic spectrum is dissolved in need testing solution that 50% methyl alcohol of 50ml obtains and detects and obtain by getting 1.2g Shengxuebao-medicine for improving hematopoitic function capsule powders, described characteristic spectrum has 7 characteristic peaks, wherein, No. 5 peaks taking Paeoniflorin as reference with reference to peak, 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%.
9. Shengxuebao-medicine for improving hematopoitic function formulation characteristics collection of illustrative plates, it is characterized in that, adopt method for building up as claimed in claim 1 to obtain, described characteristic spectrum is detected and is obtained by the need testing solution of getting 2.4g Shengxuebao-medicine for improving hematopoitic function tablet dissolved and obtaining in 25% ethanol of 100ml, described characteristic spectrum has 7 characteristic peaks, wherein, No. 5 peaks taking Paeoniflorin as reference with reference to peak, 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%.
10. Shengxuebao-medicine for improving hematopoitic function formulation characteristics collection of illustrative plates, it is characterized in that, adopt method for building up as claimed in claim 1 to obtain, described characteristic spectrum is dissolved in need testing solution that 50% ethanol of 25ml obtains and detects and obtain by getting 1ml Shengxuebao-medicine for improving hematopoitic function oral liquid, described characteristic spectrum has 7 characteristic peaks, wherein, No. 5 peaks taking Paeoniflorin as reference with reference to peak, 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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