CN103257191B - Method for assaying kidney tonifying and life lengthening capsule fingerprint - Google Patents
Method for assaying kidney tonifying and life lengthening capsule fingerprint Download PDFInfo
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- CN103257191B CN103257191B CN201310152331.2A CN201310152331A CN103257191B CN 103257191 B CN103257191 B CN 103257191B CN 201310152331 A CN201310152331 A CN 201310152331A CN 103257191 B CN103257191 B CN 103257191B
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Abstract
The invention discloses a method for assaying a kidney tonifying and life lengthening capsule fingerprint. The method comprises the following steps of: firstly preparing a kidney tonifying and life lengthening capsule solution, namely accurately weighing kidney tonifying and life lengthening capsule contents, adding a solvent into the contents, then performing ultrasonic treatment on the mixture for dissolving the mixture, cooling the dissolved mixture, supplementing the dissolved mixture by the solvent, and centrifuging and collecting supernate by centrifugation to obtain the kidney tonifying and life lengthening capsule solution; and then injecting the prepared kidney tonifying and life lengthening capsule solution into a liquid chromatography, performing gradient elution on an octadecyl silane chromatographic column by filling at the column temperature of 25-35 DEG C and the flow speed of 0.8-1.2 mL within the range of a flow phase at the volume ratio of acetonitrile to water of (2-100):(0-98), performing detection under the condition that the detection wavelength is 265-275 nm, thereby obtaining the kidney tonifying and life lengthening capsule fingerprint. The fingerprint measured by the method is high in precision, high in stability and high in reproducibility; and the simple and accurate method is supplied to quick identification for kidney tonifying and life lengthening capsules.
Description
Technical field
The invention belongs to Chinese traditional medicine identification field, relate to the assay method of BUSHEN YISHOU JIAONANG finger-print.
Background technology
BUSHEN YISHOU JIAONANG is compound Chinese medicinal preparation, by red ginseng, prepared fleece flower root, barrenwort, the red sage root, the fruit of Chinese wolfberry, pearl, glossy ganoderma, Radix Glycyrrhizae and sealwort totally nine taste medicines form, flavour of a drug are more, and quality control difficulty is larger.Proper mass standard is recorded in Drug Standard of Ministry of Public Health of the Peoples Republic of China 14, ginsenoside Rb1, Re, Rg1, icariin and protocatechualdehyde is adopted to be reference substance in quality standard respectively, TLC distinguish has been carried out to red ginseng wherein, barrenwort, the red sage root three taste medicinal material, but owing to identifying that composition kind is not enough to the quality reflecting this three tastes medicine less.And also quality monitoring is not carried out to other medicinal materials such as prepared fleece flower root, the fruit of Chinese wolfberry, glossy ganoderma, Radix Glycyrrhizae, sealworts, be not enough to control effectively to product quality.
Therefore set up a kind of comprehensively, macroscopic view, quantifiable discriminating and measure the developing direction that the method for active constituent content is Identification chinese herbs medicine compound preparation.Traditional Chinese medicine fingerprint refers to that certain Chinese medicine or some Chinese medicine preparation are after suitably processing, and adopt chromatographic process to obtain chromatogram or the spectrogram that can indicate its chemical feature.In recent years, a lot of Chinese medicine has all carried out the research of finger-print, but less to the finger-print research of large herbal mixture, and mainly because large herbal mixture flavour of a drug are many, quality of medicinal material fluctuation is large, and finished product similarity is low.BUSHEN YISHOU JIAONANG is the exclusive kind of our factory, for ensureing product quality, sets up BUSHEN YISHOU JIAONANG finger-print significant to control product quality.
Summary of the invention
In view of this, the object of the present invention is to provide a kind of assay method of BUSHEN YISHOU JIAONANG finger-print, solve the problem of prior art to BUSHEN YISHOU JIAONANG quality control difficulty.
For achieving the above object, the invention provides following technical scheme:
The assay method of BUSHEN YISHOU JIAONANG finger-print, comprises the steps:
A. prepare BUSHEN YISHOU JIAONANG solution: accurately take BUSHEN YISHOU JIAONANG content, add solvent, then carry out ultrasonic process dissolving, supply with solvent after cooling, then collected by centrifugation supernatant, obtain BUSHEN YISHOU JIAONANG solution; Described solvent is methyl alcohol, ethanol, methanol aqueous solution or ethanol water;
B. chromatographic condition: the filler of chromatographic column is octadecyl silane, and column temperature is 25-35 DEG C, and flow velocity is 0.8-1.2mL, and determined wavelength is 265-275nm, mobile phase with acetonitrile and water in volume ratio for 2-100:0-98 scope inside gradient wash-out;
C. measure: get BUSHEN YISHOU JIAONANG solution injection liquid chromatography prepared by step a, carry out high-performance liquid chromatogram determination according to the condition of step b, obtain BUSHEN YISHOU JIAONANG finger-print.
Preferably, described step a accurately takes BUSHEN YISHOU JIAONANG content, adds methyl alcohol or volume fraction is the ethanol of 50%, ultrasonic process 15 ~ 45 minutes, supply with the ethanol that methyl alcohol or volume fraction are 50% after cooling, then centrifugal at least 10 min under rotating speed is greater than 10000 r/min conditions.
Preferably, described step a accurately takes BUSHEN YISHOU JIAONANG content, adds the ethanol that volume fraction is 50%, ultrasonic process 30 minutes, cool rear volume fraction be 50% ethanol supply, then centrifugal 10 min under rotating speed is 10000 r/min conditions.
Preferred, frequency 40 kHz of described ultrasonic process.
Preferably, in described step b, chromatographic condition is: the filler of chromatographic column is octadecyl silane, and column temperature is 30 DEG C, and flow velocity is 1mL, and determined wavelength is 270nm, and mobile phase is acetonitrile and water, under the following conditions gradient elution:
When elution time is more than or equal to 0 minute and is less than 20 minutes, mobile phase A is the acetonitrile solution of 2%, and Mobile phase B is 98% aqueous solution; When elution time is more than or equal to 20 minutes and is less than 40 minutes when 20 minutes, mobile phase A is the acetonitrile solution of 10%, and Mobile phase B is 90% aqueous solution; When elution time is more than or equal to 40 minutes and is less than 55 minutes, mobile phase A is the acetonitrile solution of 20%, and Mobile phase B is 80% aqueous solution; When elution time is more than or equal to 55 minutes and is less than 80 minutes, mobile phase A is the acetonitrile solution of 25%, and Mobile phase B is 75% aqueous solution; When elution time is more than or equal to 80 minutes and is less than 85 minutes, mobile phase A is the acetonitrile solution of 30%, and Mobile phase B is 70% aqueous solution; When elution time is more than or equal to 85 minutes and is less than 90 minutes, mobile phase A is the acetonitrile solution of 90%, and Mobile phase B is 10% aqueous solution; When elution time is more than or equal to 90 minutes and is less than 92 minutes, mobile phase A is the acetonitrile solution of 90%, and Mobile phase B is 10% aqueous solution; When elution time is more than or equal to 92 minutes, mobile phase A is the acetonitrile solution of 2%, and Mobile phase B is 98% aqueous solution.
Preferably, in described step c, the BUSHEN YISHOU JIAONANG solution of injection liquid chromatography is 15-25 μ L.
Preferred, in described step c, the BUSHEN YISHOU JIAONANG solution of injection liquid chromatography is 20 μ L.
Beneficial effect of the present invention is: the assay method that the invention discloses BUSHEN YISHOU JIAONANG finger-print, pass through gradient elution method, can by the effective separation and detection of compositions multiple in compound preparation, the good separating effect at peak, zero lap peak, and peak shape is good, the characteristic component of nearly all flavour of a drug in compound preparation can be separated to, and the chemical composition that can realize BUSHEN YISHOU JIAONANG maximum possible detects, therefore can the quality of Efficient Characterization BUSHEN YISHOU JIAONANG, be conducive to comprehensive control of product quality; And the method have easy, good stability, precision are high, high repeatability and other advantages; Precision test display, after continuous sample introduction 6 times, the RSD of characteristic peak relative peak area is 0.23 ~ 1.60%, and the RSD of relative retention time is 0.08 ~ 0.34%; Stability test shows, and after different time sample introduction, the RSD of characteristic peak relative peak area is 0.20 ~ 1.75%, relative retention time RSD is 0.05 ~ 0.17%; Reappearance test display, same batch is carried out 6 and repeats experiment, and the RSD of Characteristic chromatographic peak (peak area/sample weighting amount) relative value obtained is 0.29 ~ 1.83%, relative retention time RSD is 0.02 ~ 0.44%; Compared with controlling with three simple thin layers in proper mass standard, quality control is more comprehensive.
Accompanying drawing explanation
In order to make object of the present invention, technical scheme and beneficial effect clearly, the invention provides following accompanying drawing and being described:
Fig. 1 is the HPLC collection of illustrative plates of the method detection BUSHEN YISHOU JIAONANG solution of embodiment 1.
Fig. 2 is the HPLC collection of illustrative plates of the method detection BUSHEN YISHOU JIAONANG solution of embodiment 4.
Fig. 3 is the HPLC collection of illustrative plates of the method detection BUSHEN YISHOU JIAONANG solution of embodiment 5.
Fig. 4 is the HPLC collection of illustrative plates of the method detection BUSHEN YISHOU JIAONANG solution of embodiment 6.
Fig. 5 is the HPLC collection of illustrative plates of the method detection BUSHEN YISHOU JIAONANG solution of embodiment 7.
Fig. 6 is the HPLC collection of illustrative plates of the method detection BUSHEN YISHOU JIAONANG solution of embodiment 8.
Fig. 7 is the HPLC collection of illustrative plates of the method detection BUSHEN YISHOU JIAONANG solution of embodiment 9.
Fig. 8 is the HPLC collection of illustrative plates of the method detection BUSHEN YISHOU JIAONANG solution of embodiment 10.
Fig. 9 is the HPLC collection of illustrative plates of the method detection BUSHEN YISHOU JIAONANG solution of embodiment 11.
Figure 10 is the HPLC collection of illustrative plates of the method detection BUSHEN YISHOU JIAONANG solution of embodiment 12.
Figure 11 is the HPLC collection of illustrative plates of the method detection BUSHEN YISHOU JIAONANG solution of embodiment 13.
Figure 12 is the HPLC collection of illustrative plates of the method detection BUSHEN YISHOU JIAONANG solution of embodiment 14.
Figure 13 is the HPLC collection of illustrative plates of the method detection BUSHEN YISHOU JIAONANG solution of embodiment 15.
Figure 14 is the HPLC collection of illustrative plates of the method detection BUSHEN YISHOU JIAONANG solution of embodiment 16.
Figure 15 is the HPLC collection of illustrative plates of the method detection BUSHEN YISHOU JIAONANG solution of embodiment 17.
Figure 16 is the HPLC collection of illustrative plates of the method detection BUSHEN YISHOU JIAONANG solution of embodiment 18.
Figure 17 is the HPLC collection of illustrative plates of the method detection BUSHEN YISHOU JIAONANG solution of embodiment 19.
Figure 18 is the HPLC collection of illustrative plates of the method detection BUSHEN YISHOU JIAONANG solution of embodiment 20.
Figure 19 is the HPLC collection of illustrative plates of the method detection BUSHEN YISHOU JIAONANG solution of embodiment 21.
Figure 20 is the HPLC collection of illustrative plates of the method detection BUSHEN YISHOU JIAONANG solution of embodiment 22.
Figure 21 is the HPLC collection of illustrative plates of the method detection BUSHEN YISHOU JIAONANG solution of embodiment 23.
Figure 22 is the HPLC collection of illustrative plates of the method detection BUSHEN YISHOU JIAONANG solution of embodiment 24.
Figure 23 is the HPLC collection of illustrative plates of the method detection BUSHEN YISHOU JIAONANG solution of embodiment 25.
Figure 24 is the HPLC collection of illustrative plates of extraction ofHerba Epimedii.
Figure 25 is the HPLC collection of illustrative plates of barrenwort standard items.
Figure 26 is the HPLC collection of illustrative plates of red sage root extract.
Figure 27 is the HPLC collection of illustrative plates of multiflorum extracting solution.
Figure 28 is the HPLC collection of illustrative plates of licorice extract.
Figure 29 is the HPLC collection of illustrative plates of fructus lycii extracted solution.
Figure 30 is the HPLC collection of illustrative plates of lucidum extracting liquid.
Figure 31 is the HPLC collection of illustrative plates of pearl powder extract.
Figure 32 is the HPLC collection of illustrative plates of sealwort extract.
Figure 33 is red ginseng extract HPLC collection of illustrative plates.
Figure 34 is the HPLC collection of illustrative plates of BUSHEN YISHOU JIAONANG solution.
Figure 35 is different batches BUSHEN YISHOU JIAONANG finger-print.
Embodiment
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.
Embodiment 1
The assay method of BUSHEN YISHOU JIAONANG finger-print, comprises the steps:
A. the preparation of BUSHEN YISHOU JIAONANG extract: accurately take 0.15 g BUSHEN YISHOU JIAONANG content, be placed in tool plug conical flask, then measuring 25 mL volume fractions is the ethanol of 50%, close plug, weighs, power be 250 W, frequency be 40 kHz conditions under ultrasonic process 30 minutes, weigh again after letting cool, weightlessness volume fraction be 50% ethanol supply, shaking up, is 10000 rmin at rotating speed
-1under condition, centrifugal 10 min, obtain BUSHEN YISHOU JIAONANG extract, for subsequent use;
B. chromatogram detects: the filler of chromatographic column is octadecyl silane, and column temperature is 30 DEG C, and flow velocity is 1mL/min, and sample size is 20 μ L, and determined wavelength is 270nm, and mobile phase acetonitrile and water carry out gradient elution by following condition;
Gradient elution order is as follows:
When elution time is more than or equal to 0 minute and is less than 20 minutes, mobile phase A is the acetonitrile solution of 2%, and Mobile phase B is 98% aqueous solution; When elution time is more than or equal to 20 minutes and is less than 40 minutes, mobile phase A is the acetonitrile solution of 10%, and Mobile phase B is 90% aqueous solution; When elution time is more than or equal to 40 minutes and is less than 55 minutes, mobile phase A is the acetonitrile solution of 20%, and Mobile phase B is 80% aqueous solution; When elution time is more than or equal to 55 minutes and is less than 80 minutes, mobile phase A is the acetonitrile solution of 25%, and Mobile phase B is 75% aqueous solution; When elution time is more than or equal to 80 minutes and is less than 85 minutes, mobile phase A is the acetonitrile solution of 30%, and Mobile phase B is 70% aqueous solution; When elution time is more than or equal to 85 minutes and is less than 92 minutes, mobile phase A is the acetonitrile solution of 90%, and Mobile phase B is 10% aqueous solution; When elution time is more than or equal to 92 minutes, mobile phase A is the acetonitrile solution of 2%, and Mobile phase B is 98% aqueous solution.
Obtain chromatogram as shown in Figure 1.
embodiment 2
solvent is investigated
The preparation of BUSHEN YISHOU JIAONANG solution: the BUSHEN YISHOU JIAONANG content (lot number: 11050088) accurately taking 4 part of 0.15 g, be placed in tool plug conical flask respectively, then 25 mL methyl alcohol are measured respectively, volume fraction is the methyl alcohol of 50%, volume fraction is the methyl alcohol of 50%, volume fraction be 95% ethanol and volume fraction be the ethanol of 50%, close plug, weigh, be 250 W at power, frequency is ultrasonic process 30 minutes under 40 kHz conditions, HPLC is carried out by the condition of embodiment 1, choosing retention time is 12.586 minutes, 16.990 minutes, 50.637 minutes, 71.667 minutes, 73.535 minutes, 75.082 minutes and 77.866 minutes totally 7 peaks as inspection target (7 chromatographic peak area sums account for more than 95% of total peak area), using peak area/sample weighting amount relative value as investigation foundation, result is as shown in table 1.
Complete list investigated by table 1, solvent
By table 1 with reference to the HPLC collection of illustrative plates peak type of Different solution, methyl alcohol and volume fraction be 50% ethanol be that solvent effect is better, and volume fraction is nontoxic, the environmental protection of ethanol of 50%, and therefore optimum selected volume fraction is that the ethanol of 50% is as solvent.
embodiment 3
ultrasonic time is investigated
The preparation of BUSHEN YISHOU JIAONANG solution: the BUSHEN YISHOU JIAONANG content (lot number: 11050088) accurately taking 3 part of 0.15 g, be placed in tool plug conical flask respectively, precision adds 50% ethanol 25 mL, and close plug, weighs, be 250 W at power, frequency is under 40 kHz conditions ultrasonic 15 minutes, 30 minutes and 45 minutes respectively, weighs after leaving standstill again, weightless part volume fraction be 50% ethanol supply, shake up, and then rotating speed is 10000 rmin
-1under condition, centrifugal 10 min, obtain BUSHEN YISHOU JIAONANG solution.Then according to the HPLC condition working sample of embodiment 1, choose retention time be 12.586 minutes, 16.990 minutes, 50.637 minutes, 71.667 minutes, 73.535 minutes, 75.082 minutes and 77.866 minutes totally 7 chromatographic peaks as inspection target (7 chromatographic peak area sums account for more than 95% of total peak area), using peak area/sample weighting amount relative value as investigation foundation, result is as shown in table 2.
Table 2. ultrasonic time is on the impact of BUSHEN YISHOU JIAONANG solution
as shown in Table 2, when ultrasonic time is 30 minutes, time length is suitable, and therefore best ultrasonic time is 30 minutes.
embodiment 4
The present embodiment is identical with embodiment 1, and its difference is that column temperature is 30 DEG C, and flow velocity is 1.0mL/min, and sample size is 20 μ L, and determined wavelength is 265nm, and mobile phase is that acetonitrile and water carry out gradient elution by following condition:
During elution time 0 minute, mobile phase A is the acetonitrile solution of 2.5%, and Mobile phase B is 97.5% aqueous solution; When 10 minutes, mobile phase A is the acetonitrile solution of 10%, and Mobile phase B is 90% aqueous solution; When 50 minutes, mobile phase A is the acetonitrile solution of 30%, and Mobile phase B is 70% aqueous solution; When 60 minutes, mobile phase A is the acetonitrile solution of 80%, and Mobile phase B is 20% aqueous solution; When 65 minutes, mobile phase A is the acetonitrile solution of 100%, and Mobile phase B is 0% aqueous solution; When 70 minutes, mobile phase A is the acetonitrile solution of 100%, and Mobile phase B is 0% aqueous solution; When 71 minutes, mobile phase A is the acetonitrile solution of 2.5%, and Mobile phase B is 97.5% aqueous solution; When 86 minutes, mobile phase A is the acetonitrile solution of 2.5%, and Mobile phase B is 97.5% aqueous solution.The chromatogram that the present embodiment obtains as shown in Figure 2.
embodiment 5
The present embodiment is identical with embodiment 1, and its difference is that column temperature is 30 DEG C, and flow velocity is 1.2mL/min, and sample size is 20 μ L, and determined wavelength is 265nm, and mobile phase is that acetonitrile and water carry out gradient elution by following condition:
When 0 minute, mobile phase A is the acetonitrile solution of 2.5%, and Mobile phase B is 97.5% aqueous solution; When 15 minutes, mobile phase A is the acetonitrile solution of 15%, and Mobile phase B is 85% aqueous solution; When 20 minutes, mobile phase A is the acetonitrile solution of 20%, and Mobile phase B is 80% aqueous solution; When 35 minutes, mobile phase A is the acetonitrile solution of 25%, and Mobile phase B is 75% aqueous solution;
When 40 minutes, mobile phase A is the acetonitrile solution of 65%, and Mobile phase B is 35% aqueous solution; When 55 minutes, mobile phase A is the acetonitrile solution of 80%, and Mobile phase B is 20% aqueous solution; When 65 minutes, mobile phase A is the acetonitrile solution of 90%, and Mobile phase B is 10% aqueous solution; When 70 minutes, mobile phase A is the acetonitrile solution of 90%, and Mobile phase B is 10% aqueous solution;
When 72 minutes, mobile phase A is the acetonitrile solution of 2.5%, and Mobile phase B is 97.5% aqueous solution; When 87 minutes, mobile phase A is the acetonitrile solution of 2.5%, and Mobile phase B is 97.5% aqueous solution.The chromatogram that the present embodiment obtains as shown in Figure 3.
embodiment 6
The present embodiment is identical with embodiment 1, and its difference is that column temperature is 30 DEG C, and flow velocity is 1.1mL/min, and sample size is 20 μ L, and determined wavelength is 265nm, and mobile phase is that acetonitrile and water carry out gradient elution by following condition:
When 0 minute, mobile phase A is the acetonitrile solution of 2.5%, and Mobile phase B is 97.5% aqueous solution; When 15 minutes, mobile phase A is the acetonitrile solution of 15%, and Mobile phase B is 85% aqueous solution; When 17 minutes, mobile phase A is the acetonitrile solution of 20%, and Mobile phase B is 80% aqueous solution; When 32 minutes, mobile phase A is the acetonitrile solution of 20%, and Mobile phase B is 80% aqueous solution;
When 34 minutes, mobile phase A is the acetonitrile solution of 55%, and Mobile phase B is 45% aqueous solution; When 49 minutes, mobile phase A is the acetonitrile solution of 65%, and Mobile phase B is 35% aqueous solution; When 59 minutes, mobile phase A is the acetonitrile solution of 90%, and Mobile phase B is 10% aqueous solution; When 64 minutes, mobile phase A is the acetonitrile solution of 90%, and Mobile phase B is 10% aqueous solution;
When 66 minutes, mobile phase A is the acetonitrile solution of 2.5%, and Mobile phase B is 97.5% aqueous solution; When 87 minutes, mobile phase A is the acetonitrile solution of 2.5%, and Mobile phase B is 97.5% aqueous solution.The chromatogram that the present embodiment obtains as shown in Figure 4.
embodiment 7
The present embodiment is identical with embodiment 1, and its difference is that column temperature is 30 DEG C, and flow velocity is 1.0mL/min, and sample size is 20 μ L, and determined wavelength is 265nm, and mobile phase is that acetonitrile and water carry out gradient elution by following condition:
When 0 minute, mobile phase A is the acetonitrile solution of 2.5%, and Mobile phase B is 97.5% aqueous solution; When 10 minutes, mobile phase A is the acetonitrile solution of 10%, and Mobile phase B is 90% aqueous solution; When 15 minutes, mobile phase A is the acetonitrile solution of 20%, and Mobile phase B is 80% aqueous solution; When 30 minutes, mobile phase A is the acetonitrile solution of 20%, and Mobile phase B is 80% aqueous solution;
When 35 minutes, mobile phase A is the acetonitrile solution of 45%, and Mobile phase B is 55% aqueous solution; When 55 minutes, mobile phase A is the acetonitrile solution of 55%, and Mobile phase B is 45% aqueous solution; When 65 minutes, mobile phase A is the acetonitrile solution of 90%, and Mobile phase B is 10% aqueous solution; When 70 minutes, mobile phase A is the acetonitrile solution of 90%, and Mobile phase B is 10% aqueous solution;
When 72 minutes, mobile phase A is the acetonitrile solution of 2.5%, and Mobile phase B is 97.5% aqueous solution; When 87 minutes, mobile phase A is the acetonitrile solution of 2.5%, and Mobile phase B is 97.5% aqueous solution.The chromatogram that the present embodiment obtains as shown in Figure 5.
embodiment 8
The present embodiment is identical with embodiment 1, and its difference is that column temperature is 30 DEG C, and flow velocity is 1.2mL/min, and sample size is 20 μ L, and determined wavelength is 265nm, and mobile phase is that acetonitrile and water carry out gradient elution by following condition:
When 0 minute, mobile phase A is the acetonitrile solution of 2.5%, and Mobile phase B is 97.5% aqueous solution; When 10 minutes, mobile phase A is the acetonitrile solution of 10%, and Mobile phase B is 90% aqueous solution; When 45 minutes, mobile phase A is the acetonitrile solution of 27.5%, and Mobile phase B is 72.5% aqueous solution; When 65 minutes, mobile phase A is the acetonitrile solution of 70%, and Mobile phase B is 30% aqueous solution; When 70 minutes, mobile phase A is the acetonitrile solution of 80%, and Mobile phase B is 20% aqueous solution;
When 75 minutes, mobile phase A is the acetonitrile solution of 80%, and Mobile phase B is 20% aqueous solution; When 77 minutes, mobile phase A is the acetonitrile solution of 2.5%, and Mobile phase B is 97.5% aqueous solution; When 92 minutes, mobile phase A is the acetonitrile solution of 2.5%, and Mobile phase B is 97.5% aqueous solution; The chromatogram that the present embodiment obtains as shown in Figure 6.
embodiment 9
The present embodiment is identical with embodiment 1, and its difference is that column temperature is 30 DEG C, and flow velocity is 1.2mL/min, and sample size is 20 μ L, and determined wavelength is 265nm, and mobile phase is that acetonitrile and water carry out gradient elution by following condition:
When 0 minute, mobile phase A is the acetonitrile solution of 2.5%, and Mobile phase B is 97.5% aqueous solution; When 10 minutes, mobile phase A is the acetonitrile solution of 10%, and Mobile phase B is 90% aqueous solution; When 12 minutes, mobile phase A is the acetonitrile solution of 20%, and Mobile phase B is 80% aqueous solution; When 24 minutes, mobile phase A is the acetonitrile solution of 22%, and Mobile phase B is 78% aqueous solution;
When 26 minutes, mobile phase A is the acetonitrile solution of 35%, and Mobile phase B is 65% aqueous solution; When 41 minutes, mobile phase A is the acetonitrile solution of 40%, and Mobile phase B is 60% aqueous solution; When 51 minutes, mobile phase A is the acetonitrile solution of 50%, and Mobile phase B is 50% aqueous solution; When 56 minutes, mobile phase A is the acetonitrile solution of 90%, and Mobile phase B is 10% aqueous solution;
When 61 minutes, mobile phase A is the acetonitrile solution of 90%, and Mobile phase B is 10% aqueous solution; When 63 minutes, mobile phase A is the acetonitrile solution of 2.5%, and Mobile phase B is 97.5% aqueous solution; When 78 minutes, mobile phase A is the acetonitrile solution of 2.5%, and Mobile phase B is 97.5% aqueous solution.The chromatogram that the present embodiment obtains as shown in Figure 7.
embodiment 10
The present embodiment is identical with embodiment 1, and its difference is that column temperature is 30 DEG C, and flow velocity is 1.2mL/min, and sample size is 20 μ L, and determined wavelength is 265nm, and mobile phase is that acetonitrile and water carry out gradient elution by following condition:
When 0 minute, mobile phase A is the acetonitrile solution of 2.5%, and Mobile phase B is 97.5% aqueous solution; When 10 minutes, mobile phase A is the acetonitrile solution of 10%, and Mobile phase B is 90% aqueous solution; When 12 minutes, mobile phase A is the acetonitrile solution of 20%, and Mobile phase B is 80% aqueous solution; When 24 minutes, mobile phase A is the acetonitrile solution of 22%, and Mobile phase B is 78% aqueous solution;
When 44 minutes, mobile phase A is the acetonitrile solution of 30%, and Mobile phase B is 70% aqueous solution; When 59 minutes, mobile phase A is the acetonitrile solution of 40%, and Mobile phase B is 60% aqueous solution; When 69 minutes, mobile phase A is the acetonitrile solution of 50%, and Mobile phase B is 50% aqueous solution; When 74 minutes, mobile phase A is the acetonitrile solution of 90%, and Mobile phase B is 10% aqueous solution;
When 79 minutes, mobile phase A is the acetonitrile solution of 90%, and Mobile phase B is 10% aqueous solution; When 81 minutes, mobile phase A is the acetonitrile solution of 2.5%, and Mobile phase B is 97.5% aqueous solution; When 96 minutes, mobile phase A is the acetonitrile solution of 2.5%, and Mobile phase B is 97.5% aqueous solution.The chromatogram that the present embodiment obtains as shown in Figure 8.
embodiment 11
The present embodiment is identical with embodiment 1, and its difference is that column temperature is 30 DEG C, and flow velocity is 1.1mL/min, and sample size is 20 μ L, and determined wavelength is 265nm, and mobile phase is that acetonitrile and water carry out gradient elution by following condition:
When 0 minute, mobile phase A is the acetonitrile solution of 2%, and Mobile phase B is 98% aqueous solution; When 15 minutes, mobile phase A is the acetonitrile solution of 10%, and Mobile phase B is 90% aqueous solution; When 20 minutes, mobile phase A is the acetonitrile solution of 20%, and Mobile phase B is 80% aqueous solution; When 32 minutes, mobile phase A is the acetonitrile solution of 22%, and Mobile phase B is 78% aqueous solution;
When 37 minutes, mobile phase A is the acetonitrile solution of 25%, and Mobile phase B is 75% aqueous solution; When 57 minutes, mobile phase A is the acetonitrile solution of 30%, and Mobile phase B is 70% aqueous solution; When 67 minutes, mobile phase A is the acetonitrile solution of 55%, and Mobile phase B is 45% aqueous solution; When 69 minutes, mobile phase A is the acetonitrile solution of 90%, and Mobile phase B is 10% aqueous solution;
When 74 minutes, mobile phase A is the acetonitrile solution of 90%, and Mobile phase B is 10% aqueous solution; When 76 minutes, mobile phase A is the acetonitrile solution of 2%, and Mobile phase B is 98% aqueous solution; When 91 minutes, mobile phase A is the acetonitrile solution of 2%, and Mobile phase B is 98% aqueous solution.The chromatogram that the present embodiment obtains as shown in Figure 9.
embodiment 12
The present embodiment is identical with embodiment 1, and its difference is that column temperature is 30 DEG C, and flow velocity is 1.1mL/min, and sample size is 20 μ L, and determined wavelength is 265nm, and mobile phase is that acetonitrile and water carry out gradient elution by following condition:
When 0 minute, mobile phase A is the acetonitrile solution of 2%, and Mobile phase B is 98% aqueous solution; When 15 minutes, mobile phase A is the acetonitrile solution of 10%, and Mobile phase B is 90% aqueous solution; When 20 minutes, mobile phase A is the acetonitrile solution of 20%, and Mobile phase B is 80% aqueous solution; When 35 minutes, mobile phase A is the acetonitrile solution of 20%, and Mobile phase B is 80% aqueous solution;
When 40 minutes, mobile phase A is the acetonitrile solution of 25%, and Mobile phase B is 75% aqueous solution; When 60 minutes, mobile phase A is the acetonitrile solution of 30%, and Mobile phase B is 70% aqueous solution; When 75 minutes, mobile phase A is the acetonitrile solution of 60%, and Mobile phase B is 40% aqueous solution; When 80 minutes, mobile phase A is the acetonitrile solution of 90%, and Mobile phase B is 10% aqueous solution;
When 85 minutes, mobile phase A is the acetonitrile solution of 90%, and Mobile phase B is 10% aqueous solution; When 87 minutes, mobile phase A is the acetonitrile solution of 2%, and Mobile phase B is 98% aqueous solution; When 102 minutes, mobile phase A is the acetonitrile solution of 2%, and Mobile phase B is 98% aqueous solution; When 109 minutes, mobile phase A is the acetonitrile solution of 2.5%, and Mobile phase B is 98% aqueous solution.The chromatogram that the present embodiment obtains as shown in Figure 10.
embodiment 13
The present embodiment is identical with embodiment 1, and its difference is that column temperature is 30 DEG C, and flow velocity is 1.1mL/min, and sample size is 20 μ L, and determined wavelength is 265nm, and mobile phase is that acetonitrile and water carry out gradient elution by following condition:
When 0 minute, mobile phase A is the acetonitrile solution of 2%, and Mobile phase B is 98% aqueous solution; When 15 minutes, mobile phase A is the acetonitrile solution of 10%, and Mobile phase B is 90% aqueous solution; When 25 minutes, mobile phase A is the acetonitrile solution of 20%, and Mobile phase B is 80% aqueous solution; When 35 minutes, mobile phase A is the acetonitrile solution of 20%, and Mobile phase B is 80% aqueous solution;
When 40 minutes, mobile phase A is the acetonitrile solution of 25%, and Mobile phase B is 75% aqueous solution; When 60 minutes, mobile phase A is the acetonitrile solution of 30%, and Mobile phase B is 70% aqueous solution; When 70 minutes, mobile phase A is the acetonitrile solution of 60%, and Mobile phase B is 40% aqueous solution; When 75 minutes, mobile phase A is the acetonitrile solution of 90%, and Mobile phase B is 10% aqueous solution;
When 80 minutes, mobile phase A is the acetonitrile solution of 90%, and Mobile phase B is 10% aqueous solution; When 82 minutes, mobile phase A is the acetonitrile solution of 2%, and Mobile phase B is 98% aqueous solution; When 97 minutes, mobile phase A is the acetonitrile solution of 2%, and Mobile phase B is 98% aqueous solution.The chromatogram that the present embodiment obtains as shown in figure 11.
embodiment 14
The present embodiment is identical with embodiment 1, and its difference is that column temperature is 30 DEG C, and flow velocity is 1.1mL/min, and sample size is 20 μ L, and determined wavelength is 265nm, and mobile phase is that acetonitrile and water carry out gradient elution by following condition:
When 0 minute, mobile phase A is the acetonitrile solution of 2%, and Mobile phase B is 98% aqueous solution; When 15 minutes, mobile phase A is the acetonitrile solution of 10%, and Mobile phase B is 90% aqueous solution; When 20 minutes, mobile phase A is the acetonitrile solution of 15%, and Mobile phase B is 85% aqueous solution; When 40 minutes, mobile phase A is the acetonitrile solution of 20%, and Mobile phase B is 80% aqueous solution;
When 45 minutes, mobile phase A is the acetonitrile solution of 25%, and Mobile phase B is 75% aqueous solution; When 70 minutes, mobile phase A is the acetonitrile solution of 30%, and Mobile phase B is 70% aqueous solution; When 80 minutes, mobile phase A is the acetonitrile solution of 65%, and Mobile phase B is 35% aqueous solution; When 82 minutes, mobile phase A is the acetonitrile solution of 90%, and Mobile phase B is 10% aqueous solution;
When 87 minutes, mobile phase A is the acetonitrile solution of 90%, and Mobile phase B is 10% aqueous solution; When 89 minutes, mobile phase A is the acetonitrile solution of 2%, and Mobile phase B is 98% aqueous solution; When 104 minutes, mobile phase A is the acetonitrile solution of 2%, and Mobile phase B is 98% aqueous solution.The chromatogram that the present embodiment obtains as shown in figure 12.
embodiment 15
The present embodiment is identical with embodiment 1, and its difference is that column temperature is 30 DEG C, and flow velocity is 1mL/min, and sample size is 20 μ L, and determined wavelength is 265nm, and mobile phase is that acetonitrile and water carry out gradient elution by following condition:
When 0 minute, mobile phase A is the acetonitrile solution of 2%, and Mobile phase B is 98% aqueous solution; When 15 minutes, mobile phase A is the acetonitrile solution of 10%, and Mobile phase B is 90% aqueous solution; When 20 minutes, mobile phase A is the acetonitrile solution of 17%, and Mobile phase B is 83% aqueous solution; When 35 minutes, mobile phase A is the acetonitrile solution of 20%, and Mobile phase B is 80% aqueous solution;
When 40 minutes, mobile phase A is the acetonitrile solution of 27%, and Mobile phase B is 73% aqueous solution; When 55 minutes, mobile phase A is the acetonitrile solution of 30%, and Mobile phase B is 70% aqueous solution; When 60 minutes, mobile phase A is the acetonitrile solution of 60%, and Mobile phase B is 40% aqueous solution; When 70 minutes, mobile phase A is the acetonitrile solution of 90%, and Mobile phase B is 10% aqueous solution;
When 75 minutes, mobile phase A is the acetonitrile solution of 90%, and Mobile phase B is 10% aqueous solution; When 77 minutes, mobile phase A is the acetonitrile solution of 2%, and Mobile phase B is 98% aqueous solution; When 92 minutes, mobile phase A is the acetonitrile solution of 2%, and Mobile phase B is 98% aqueous solution.The chromatogram that the present embodiment obtains as shown in figure 13.
embodiment 16
The present embodiment is identical with embodiment 1, and its difference is that column temperature is 30 DEG C, and flow velocity is 1mL/min, and sample size is 20 μ L, and determined wavelength is 265nm, and mobile phase is that acetonitrile and water carry out gradient elution by following condition:
When 0 minute, mobile phase A is the acetonitrile solution of 2%, and Mobile phase B is 98% aqueous solution; When 15 minutes, mobile phase A is the acetonitrile solution of 10%, and Mobile phase B is 90% aqueous solution; When 20 minutes, mobile phase A is the acetonitrile solution of 16%, and Mobile phase B is 84% aqueous solution; When 40 minutes, mobile phase A is the acetonitrile solution of 20%, and Mobile phase B is 80% aqueous solution; When 45 minutes, mobile phase A is the acetonitrile solution of 26%, and Mobile phase B is 74% aqueous solution; When 65 minutes, mobile phase A is the acetonitrile solution of 30%, and Mobile phase B is 70% aqueous solution; When 70 minutes, mobile phase A is the acetonitrile solution of 65%, and Mobile phase B is 35% aqueous solution; When 80 minutes, mobile phase A is the acetonitrile solution of 75%, and Mobile phase B is 25% aqueous solution;
When 82 minutes, mobile phase A is the acetonitrile solution of 90%, and Mobile phase B is 10% aqueous solution; When 87 minutes, mobile phase A is the acetonitrile solution of 90%, and Mobile phase B is 10% aqueous solution; When 89 minutes, mobile phase A is the acetonitrile solution of 2%, and Mobile phase B is 98% aqueous solution; When 104 minutes, mobile phase A is the acetonitrile solution of 2%, and Mobile phase B is 98% aqueous solution.The chromatogram that the present embodiment obtains as shown in figure 14.
embodiment 17
The present embodiment is identical with embodiment 1, and its difference is that column temperature is 30 DEG C, and flow velocity is 1mL/min, and sample size is 20 μ L, and determined wavelength is 265nm, and mobile phase is that acetonitrile and water carry out gradient elution by following condition:
When 0 minute, mobile phase A is the acetonitrile solution of 2 %, and Mobile phase B is 98% aqueous solution; When 15 minutes, mobile phase A is the acetonitrile solution of 10%, and Mobile phase B is 90% aqueous solution; When 20 minutes, mobile phase A is the acetonitrile solution of 16%, and Mobile phase B is 84% aqueous solution; When 40 minutes, mobile phase A is the acetonitrile solution of 20%, and Mobile phase B is 80% aqueous solution;
When 45 minutes, mobile phase A is the acetonitrile solution of 26%, and Mobile phase B is 74% aqueous solution; When 65 minutes, mobile phase A is the acetonitrile solution of 30%, and Mobile phase B is 70% aqueous solution; When 85 minutes, mobile phase A is the acetonitrile solution of 65%, and Mobile phase B is 35% aqueous solution; When 95 minutes, mobile phase A is the acetonitrile solution of 75%, and Mobile phase B is 25% aqueous solution;
When 97 minutes, mobile phase A is the acetonitrile solution of 90%, and Mobile phase B is 10% aqueous solution; When 102 minutes, mobile phase A is the acetonitrile solution of 90%, and Mobile phase B is 10% aqueous solution; When 104 minutes, mobile phase A is the acetonitrile solution of 2%, and Mobile phase B is 98% aqueous solution; When 119 minutes, mobile phase A is the acetonitrile solution of 2%, and Mobile phase B is 98% aqueous solution.The chromatogram that the present embodiment obtains as shown in figure 15.
embodiment 18
The present embodiment is identical with embodiment 1, and its difference is that column temperature is 30 DEG C, and flow velocity is 1mL/min, and sample size is 20 μ L, and determined wavelength is 265nm, and mobile phase is that acetonitrile and water carry out gradient elution by following condition:
When 0 minute, mobile phase A is the acetonitrile solution of 2%, and Mobile phase B is 98% aqueous solution; When 15 minutes, mobile phase A is the acetonitrile solution of 10%, and Mobile phase B is 90% aqueous solution; When 20 minutes, mobile phase A is the acetonitrile solution of 16%, and Mobile phase B is 84% aqueous solution; When 40 minutes, mobile phase A is the acetonitrile solution of 20%, and Mobile phase B is 80% aqueous solution;
When 45 minutes, mobile phase A is the acetonitrile solution of 26%, and Mobile phase B is 74% aqueous solution; When 65 minutes, mobile phase A is the acetonitrile solution of 30%, and Mobile phase B is 70% aqueous solution; When 70 minutes, mobile phase A is the acetonitrile solution of 40%, and Mobile phase B is 60% aqueous solution; When 90 minutes, mobile phase A is the acetonitrile solution of 65%, and Mobile phase B is 35% aqueous solution;
When 92 minutes, mobile phase A is the acetonitrile solution of 90%, and Mobile phase B is 10% aqueous solution; When 97 minutes, mobile phase A is the acetonitrile solution of 90%, and Mobile phase B is 10% aqueous solution; When 99 minutes, mobile phase A is the acetonitrile solution of 2 %, and Mobile phase B is 98% aqueous solution; When 114 minutes, mobile phase A is the acetonitrile solution of 2 %, and Mobile phase B is 98% aqueous solution.The chromatogram that the present embodiment obtains as shown in figure 16.
embodiment 19
The present embodiment is identical with embodiment 1, and its difference is that column temperature is 30 DEG C, and flow velocity is 1mL/min, and sample size is 20 μ L, and determined wavelength is 265nm, and mobile phase is that acetonitrile and water carry out gradient elution by following condition:
When 0 minute, mobile phase A is the acetonitrile solution of 2%, and Mobile phase B is 98% aqueous solution; When 15 minutes, mobile phase A is the acetonitrile solution of 10%, and Mobile phase B is 90% aqueous solution; When 20 minutes, mobile phase A is the acetonitrile solution of 16%, and Mobile phase B is 84% aqueous solution; When 40 minutes, mobile phase A is the acetonitrile solution of 20%, and Mobile phase B is 80% aqueous solution;
When 45 minutes, mobile phase A is the acetonitrile solution of 26%, and Mobile phase B is 74% aqueous solution; When 65 minutes, mobile phase A is the acetonitrile solution of 30%, and Mobile phase B is 70% aqueous solution; When 95 minutes, mobile phase A is the acetonitrile solution of 65%, and Mobile phase B is 35% aqueous solution; When 97 minutes, mobile phase A is the acetonitrile solution of 90%, and Mobile phase B is 10% aqueous solution;
When 102 minutes, mobile phase A is the acetonitrile solution of 90%, and Mobile phase B is 10% aqueous solution; When 104 minutes, mobile phase A is the acetonitrile solution of 2%, and Mobile phase B is 98% aqueous solution; When 119 minutes, mobile phase A is the acetonitrile solution of 2%, and Mobile phase B is 98% aqueous solution.The chromatogram that the present embodiment obtains as shown in figure 17.
embodiment 20
The present embodiment is identical with embodiment 1, and its difference is that column temperature is 30 DEG C, and flow velocity is 1mL/min, and sample size is 20 μ L, and determined wavelength is 265nm, and mobile phase is that acetonitrile and water carry out gradient elution by following condition:
When 0 minute, mobile phase A is the acetonitrile solution of 2%, and Mobile phase B is 98% aqueous solution; When 15 minutes, mobile phase A is the acetonitrile solution of 10%, and Mobile phase B is 90% aqueous solution; When 20 minutes, mobile phase A is the acetonitrile solution of 16%, and Mobile phase B is 84% aqueous solution; When 40 minutes, mobile phase A is the acetonitrile solution of 20%, and Mobile phase B is 80% aqueous solution;
When 45 minutes, mobile phase A is the acetonitrile solution of 26%, and Mobile phase B is 74% aqueous solution; When 65 minutes, mobile phase A is the acetonitrile solution of 30%, and Mobile phase B is 70% aqueous solution; When 100 minutes, mobile phase A is the acetonitrile solution of 60%, and Mobile phase B is 40% aqueous solution; When 102 minutes, mobile phase A is the acetonitrile solution of 90%, and Mobile phase B is 10% aqueous solution; When 107 minutes, mobile phase A is the acetonitrile solution of 90%, and Mobile phase B is 10% aqueous solution; When 109 minutes, mobile phase A is the acetonitrile solution of 2%, and Mobile phase B is 98% aqueous solution; When 124 minutes, mobile phase A is the acetonitrile solution of 2%, and Mobile phase B is 98% aqueous solution.The chromatogram that the present embodiment obtains as shown in figure 18.
embodiment 21
The present embodiment is identical with embodiment 1, and its difference is that column temperature is 30 DEG C, and flow velocity is 0.9mL/min, and sample size is 20 μ L, and determined wavelength is 265nm, and mobile phase is that acetonitrile and water carry out gradient elution by following condition:
When 0 minute, mobile phase A is the acetonitrile solution of 2%, and Mobile phase B is 98% aqueous solution; When 15 minutes, mobile phase A is the acetonitrile solution of 10%, and Mobile phase B is 90% aqueous solution; When 20 minutes, mobile phase A is the acetonitrile solution of 16%, and Mobile phase B is 84% aqueous solution; When 40 minutes, mobile phase A is the acetonitrile solution of 20%, and Mobile phase B is 80% aqueous solution; When 45 minutes, mobile phase A is the acetonitrile solution of 25%, and Mobile phase B is 75% aqueous solution; When 65 minutes, mobile phase A is the acetonitrile solution of 28%, and Mobile phase B is 72% aqueous solution; When 75 minutes, mobile phase A is the acetonitrile solution of 45%, and Mobile phase B is 55% aqueous solution; When 85 minutes, mobile phase A is the acetonitrile solution of 50%, and Mobile phase B is 50% aqueous solution; When 90 minutes, mobile phase A is the acetonitrile solution of 90%, and Mobile phase B is 10% aqueous solution; When 95 minutes, mobile phase A is the acetonitrile solution of 90%, and Mobile phase B is 10% aqueous solution; When 97 minutes, mobile phase A is the acetonitrile solution of 2%, and Mobile phase B is 98% aqueous solution; When 112 minutes, mobile phase A is the acetonitrile solution of 2%, and Mobile phase B is 98% aqueous solution.The chromatogram that the present embodiment obtains as shown in figure 19.
embodiment 22
The present embodiment is identical with embodiment 1, and its difference is that column temperature is 35 DEG C, and flow velocity is 0.9mL/min, and sample size is 20 μ L, and determined wavelength is 265nm, and mobile phase is that acetonitrile and water carry out gradient elution by following condition:
When 0 minute, mobile phase A is the acetonitrile solution of 2%, and Mobile phase B is 98% aqueous solution; When 15 minutes, mobile phase A is the acetonitrile solution of 10%, and Mobile phase B is 90% aqueous solution; When 20 minutes, mobile phase A is the acetonitrile solution of 15%, and Mobile phase B is 85% aqueous solution; When 40 minutes, mobile phase A is the acetonitrile solution of 20%, and Mobile phase B is 80% aqueous solution;
When 50 minutes, mobile phase A is the acetonitrile solution of 25%, and Mobile phase B is 75% aqueous solution; When 70 minutes, mobile phase A is the acetonitrile solution of 28%, and Mobile phase B is 72% aqueous solution; When 80 minutes, mobile phase A is the acetonitrile solution of 42%, and Mobile phase B is 58% aqueous solution; When 95 minutes, mobile phase A is the acetonitrile solution of 55%, and Mobile phase B is 45% aqueous solution;
When 100 minutes, mobile phase A is the acetonitrile solution of 90%, and Mobile phase B is 10% aqueous solution; When 105 minutes, mobile phase A is the acetonitrile solution of 90%, and Mobile phase B is 10% aqueous solution; When 107 minutes, mobile phase A is the acetonitrile solution of 2%, and Mobile phase B is 98% aqueous solution; When 122 minutes, mobile phase A is the acetonitrile solution of 2%, and Mobile phase B is 98% aqueous solution.The chromatogram that the present embodiment obtains as shown in figure 20.
embodiment 23
The present embodiment is identical with embodiment 1, and its difference is that column temperature is 35 DEG C, and flow velocity is 0.9mL/min, and sample size is 20 μ L, and determined wavelength is 265nm, and mobile phase is that acetonitrile and water carry out gradient elution by following condition:
When 0 minute, mobile phase A is the acetonitrile solution of 2%, and Mobile phase B is 98% aqueous solution; When 20 minutes, mobile phase A is the acetonitrile solution of 10%, and Mobile phase B is 90% aqueous solution; When 40 minutes, mobile phase A is the acetonitrile solution of 20%, and Mobile phase B is 80% aqueous solution; When 50 minutes, mobile phase A is the acetonitrile solution of 25%, and Mobile phase B is 75% aqueous solution; When 70 minutes, mobile phase A is the acetonitrile solution of 28%, and Mobile phase B is 72% aqueous solution; When 80 minutes, mobile phase A is the acetonitrile solution of 40%, and Mobile phase B is 60% aqueous solution; When 85 minutes, mobile phase A is the acetonitrile solution of 90%, and Mobile phase B is 10% aqueous solution; When 90 minutes, mobile phase A is the acetonitrile solution of 90%, and Mobile phase B is 10% aqueous solution;
When 92 minutes, mobile phase A is the acetonitrile solution of 2%, and Mobile phase B is 98% aqueous solution; When 107 minutes, mobile phase A is the acetonitrile solution of 2 %, and Mobile phase B is 98% aqueous solution.The chromatogram that the present embodiment obtains as shown in figure 21.
embodiment 24
The present embodiment is identical with embodiment 1, and its difference is that column temperature is 35 DEG C, and flow velocity is 0.85mL/min, and sample size is 20 μ L, and determined wavelength is 265nm, and mobile phase is that acetonitrile and water carry out gradient elution by following condition:
When 0 minute, mobile phase A is the acetonitrile solution of 2%, and Mobile phase B is 98% aqueous solution; When 20 minutes, mobile phase A is the acetonitrile solution of 10%, and Mobile phase B is 90% aqueous solution; When 40 minutes, mobile phase A is the acetonitrile solution of 20%, and Mobile phase B is 80% aqueous solution; When 55 minutes, mobile phase A is the acetonitrile solution of 25%, and Mobile phase B is 75% aqueous solution; When 80 minutes, mobile phase A is the acetonitrile solution of 30%, and Mobile phase B is 70% aqueous solution; When 85 minutes, mobile phase A is the acetonitrile solution of 90%, and Mobile phase B is 10% aqueous solution; When 90 minutes, mobile phase A is the acetonitrile solution of 90%, and Mobile phase B is 10% aqueous solution; When 92 minutes, mobile phase A is the acetonitrile solution of 2%, and Mobile phase B is 98% aqueous solution; When 107 minutes, mobile phase A is the acetonitrile solution of 2%, and Mobile phase B is 98% aqueous solution.The chromatogram that the present embodiment obtains as shown in figure 22.
embodiment 25
The present embodiment is identical with embodiment 1, and its difference is that column temperature is 25 DEG C, and flow velocity is 0.8mL/min, and sample size is 25 μ L, and determined wavelength is 275nm, and mobile phase is that acetonitrile and water carry out gradient elution by following condition:
When 0 minute, mobile phase A is the acetonitrile solution of 2%, and Mobile phase B is 98% aqueous solution; When 20 minutes, mobile phase A is the acetonitrile solution of 10%, and Mobile phase B is 90% aqueous solution; When 40 minutes, mobile phase A is the acetonitrile solution of 20%, and Mobile phase B is 80% aqueous solution; When 65 minutes, mobile phase A is the acetonitrile solution of 25%, and Mobile phase B is 75% aqueous solution; When 85 minutes, mobile phase A is the acetonitrile solution of 30%, and Mobile phase B is 70% aqueous solution; When 90 minutes, mobile phase A is the acetonitrile solution of 90%, and Mobile phase B is 10% aqueous solution; When 95 minutes, mobile phase A is the acetonitrile solution of 90%, and Mobile phase B is 10% aqueous solution; When 97 minutes, mobile phase A is the acetonitrile solution of 2%, and Mobile phase B is 98% aqueous solution;
When 112 minutes, mobile phase A is the acetonitrile solution of 2%, and Mobile phase B is 98% aqueous solution.The chromatogram that the present embodiment obtains as shown in figure 23.
embodiment 26
The extract of barrenwort, the red sage root, the fleece-flower root (system), Radix Glycyrrhizae, matrimony vine, glossy ganoderma, pearl powder, sealwort and red ginseng is prepared respectively according to the method for embodiment 1, then HPLC detection is carried out, obtain chromatogram (Figure 24-33) respectively, BUSHEN YISHOU JIAONANG is detected liquid by the preparation of embodiment 1 method simultaneously, and then carry out HPLC according to the condition of embodiment 22, obtain chromatogram as shown in figure 34.Analyze the ownership situation of each chromatographic peak in BUSHEN YISHOU JIAONANG HPLC collection of illustrative plates, in the HPLC collection of illustrative plates of result display BUSHEN YISHOU JIAONANG, there is the characteristic peak representing its medicinal material and have 16, specifically as shown in table 3.Wherein 16 feature coneincones numbers 8, the peak area of 9 and 11 3 characteristic peaks is less, fingerprint spectrum method is investigated defective, and the medicinal material that three characteristic peaks are corresponding respectively, in HPLC collection of illustrative plates, separately there is further feature peak more can represent its corresponding medicinal material, as peak numbers 12 ~ 16, corresponding medicinal material is barrenwort, peak numbers 5, 6 corresponding medicinal materials are Radix Glycyrrhizae, according to bibliographical information, No. 16 peaks are icariin, 12 ~ No. 14 peaks are respectively Epimedin A, Epimedin B, epimedin C (see: Zhang Huafeng, Gao Xiang, Epimedin A in the .HPLC method Simultaneously test barrenwort such as Lu great Yan, B, the content of C and icariin. analytical test journal, 2007, 26(2): 198-201. Pei Li is wide, Guo Baolin, yellow Wenhua etc. the HPLC Fingerprints of Epimedium Main Resources kind and Identification of Species [J]. CHINA JOURNAL OF CHINESE MATERIA MEDICA, 2008, 33 (14): 1662-1668. Chen Yan, Zhao Yanhong, the content of 5 kinds of Main Flavonoids constituents in the .RP-HPLC method Simultaneously test different cultivars epimedium herbs such as Jia Xiaobin. China Dispensary, 2008, 19(6): 431-433.).By above analysis, in final selected BUSHEN YISHOU JIAONANG HPLC collection of illustrative plates, 13 chromatographic peaks are as the characteristic peak (delete peak number 8,9,11) of its finger-print.
Characteristic peak ownership situation in table 3. BUSHEN YISHOU JIAONANG HPLC collection of illustrative plates
embodiment 27
fingerprint map analyzing
The similarity evaluation 2004A version using Chinese Pharmacopoeia Commission to recommend has carried out similarity analysis to the finger-print of the BUSHEN YISHOU JIAONANG of 14 batches, first the data of chromatographic work station are led in people's traditional Chinese medicine fingerprint Similarity Measure software, selected above-mentioned 13 characteristic peaks carry out Peak tracking, and be with reference to spectrum with lot number 10060078, the common pattern of sample finger-print is calculated by averaging method, and common pattern is standard according to this, carry out overall similarity evaluation, Figure 35 is shown in by each batch of BUSHEN YISHOU JIAONANG finger-print, similarity evaluation the results are shown in Table 4.
Table 4. BUSHEN YISHOU JIAONANG sample similarity result
As known from Table 4, the Similarity value of different batches BUSHEN YISHOU JIAONANG sample is all more than 97%, show that similarity is good, each batch sample has identical chromatographic characteristics peak, therefore chemical composition consistance is better, the finger-print set up has stability and controllability, can be used as the Appreciation gist controlling BUSHEN YISHOU JIAONANG quality.
In the BUSHEN YISHOU JIAONANG finger-print measured according to the inventive method, compare by the chromatographic peak in the HPLC collection of illustrative plates to different batches BUSHEN YISHOU JIAONANG sample and number, and in conjunction with the chromatographic peak information in the HPLC collection of illustrative plates of medicinal raw material, final selected 13 representative chromatographic peaks are as total peak, total peak accounts for total peak area more than 95%, is respectively:
No. 1 peak, Average residence time RT is 12.812min, RSD is 0.23%, and peak area and sample weighting amount ratio are 759674, RSD is 29.09%;
No. 2 peaks, Average residence time RT is 17.403min, RSD is 0.52%, and peak area and sample weighting amount ratio are 7325599, RSD is 47.85%;
No. 3 peaks, Average residence time RT is 20.813min, RSD is 0.41%, and peak area and sample weighting amount ratio are 445658, RSD is 26.02%;
No. 4 peaks, Average residence time RT is 31.006min, RSD is 0.28%, and peak area and sample weighting amount ratio are 403201, RSD is 14.20%;
No. 5 peaks, Average residence time RT is 49.220min, RSD is 0.15%, and peak area and sample weighting amount ratio are 1831518, RSD is 20.87%;
No. 6 peaks, Average residence time RT is 49.925min, RSD is 0.19%, and peak area and sample weighting amount ratio are 845317, RSD is 52.84%;
No. 7 peaks, Average residence time RT is 51.365min, RSD is 0.20%, and peak area and sample weighting amount ratio are 4420173, RSD is 24.35%;
No. 8 peaks, Average residence time RT is 61.770min, RSD is 0.08%, and peak area and sample weighting amount ratio are 1081885, RSD is 26.07%;
No. 9 peaks, Average residence time RT is 71.595min, RSD is 0.11%, and peak area and sample weighting amount ratio are 2279481, RSD is 18.89%;
No. 10 peaks, Average residence time RT is 73.443min, RSD is 0.12%, and peak area and sample weighting amount ratio are 2775409, RSD is 23.88%;
No. 11 peaks, Average residence time RT is 74.949min, RSD is 0.13%, and peak area and sample weighting amount ratio are 20039687, RSD is 20.39%;
No. 12 peaks, Average residence time RT is 76.208min, RSD is 0.14%, and peak area and sample weighting amount ratio are 549428, RSD is 44.39%;
No. 13 peaks, Average residence time RT is 77.800min, RSD is 0.12%, and peak area and sample weighting amount ratio 14924163 are that RSD is 14.09%;
In above-mentioned finger-print, the peak that the ratio that unimodal area accounts for total peak area is greater than 5% has four, is respectively:
No. 2 peaks, Average residence time RT is 17.403min, RSD is 0.52%, and peak area and sample weighting amount ratio are 7325599, RSD is 47.85%;
No. 7 peaks, Average residence time RT is 51.365min, RSD is 0.20%, and peak area and sample weighting amount ratio are 4420173, RSD is 24.35%;
No. 11 peaks, Average residence time RT is 74.949min, RSD is 0.13%, and peak area and sample weighting amount ratio are 20039687, RSD is 20.39%;
No. 13 peaks, Average residence time RT is 77.800min, RSD is 0.12%, and peak area and sample weighting amount ratio 14924163 are that RSD is 14.09%;
embodiment 28
methodological study
1. system suitability test
Get icariin standard items, carry out HPLC according to the chromatographic condition of embodiment 1, calculate the theoretical pedal number of icariin, the theoretical cam curve of result display icariin is 179488.
precision test
Prepare BUSHEN YISHOU JIAONANG solution by embodiment 1 method and carry out HPLC analysis, continuous sample introduction 6 times, record chromatogram, the RSD calculating Characteristic chromatographic peak relative peak area is 0.23 ~ 1.60%, the RSD of relative retention time is 0.08 ~ 0.34%, and result shows that precision is good.
stability test
prepare BUSHEN YISHOU JIAONANG solution by embodiment 1 method and carry out HPLC analysis, respectively at 0 hour, 3 hours, 6 hours, 9 hours, 12 hours and 24 hours sample introductions, record chromatogram, the RSD calculating each characteristic peak relative peak area is 0.20 ~ 1.75%, relative retention time RSD is 0.05 ~ 0.17%.Result shows that need testing solution is basicly stable in 24 hours.
reappearance is tested
With a collection of BUSHEN YISHOU JIAONANG 6 parts of (lot numbers: 12110125), prepare BUSHEN YISHOU JIAONANG solution by embodiment 1 method and carry out HPLC analysis, sample introduction respectively, record chromatogram, the RSD calculating each Characteristic chromatographic peak (peak area/sample weighting amount) relative value is 0.29 ~ 1.83%, relative retention time RSD is 0.02 ~ 0.44%, and result shows that reappearance is good.
What finally illustrate is, above preferred embodiment is only in order to illustrate technical scheme of the present invention and unrestricted, although by above preferred embodiment to invention has been detailed description, but those skilled in the art are to be understood that, various change can be made to it in the form and details, and not depart from claims of the present invention limited range.
Claims (5)
1. the assay method of BUSHEN YISHOU JIAONANG finger-print, is characterized in that, comprises the steps:
Step a. prepares BUSHEN YISHOU JIAONANG solution: accurately take BUSHEN YISHOU JIAONANG content, add methyl alcohol or volume fraction is the ethanol of 50%, ultrasonic process 15 ~ 45 minutes, supplies with the ethanol that methyl alcohol or volume fraction are 50% after cooling, is then greater than 10000 r/min at rotating speed
/centrifugal at least 10 min under condition;
Step b. chromatographic condition: the filler of chromatographic column is octadecyl silane, and column temperature is 30 DEG C, and flow velocity is 1ml/min, and determined wavelength is 270nm, and mobile phase is acetonitrile and water, under the following conditions gradient elution:
When elution time is more than or equal to 0 minute and is less than 20 minutes, mobile phase A is the acetonitrile solution of 2%, and Mobile phase B is 98% aqueous solution;
When elution time is more than or equal to 20 minutes and is less than 40 minutes, mobile phase A is the acetonitrile solution of 10%, and Mobile phase B is 90% aqueous solution;
When elution time is more than or equal to 40 minutes and is less than 55 minutes, mobile phase A is the acetonitrile solution of 20%, and Mobile phase B is 80% aqueous solution;
When elution time is more than or equal to 55 minutes and is less than 80 minutes, mobile phase A is the acetonitrile solution of 25%, and Mobile phase B is 75% aqueous solution;
When elution time is more than or equal to 80 minutes and is less than 85 minutes, mobile phase A is the acetonitrile solution of 30%, and Mobile phase B is 70% aqueous solution;
When elution time is more than or equal to 85 minutes and is less than 90 minutes, mobile phase A is the acetonitrile solution of 90%, and Mobile phase B is 10% aqueous solution;
When elution time is more than or equal to 90 minutes and is less than 92 minutes, mobile phase A is the acetonitrile solution of 90%, and Mobile phase B is 10% aqueous solution;
When elution time is more than or equal to 92 minutes, mobile phase A is the acetonitrile solution of 2%, and Mobile phase B is 98% aqueous solution;
Step c measures: get BUSHEN YISHOU JIAONANG solution injection liquid chromatography prepared by step a, carry out high-performance liquid chromatogram determination, obtain BUSHEN YISHOU JIAONANG finger-print according to the condition of step b.
2. the assay method of BUSHEN YISHOU JIAONANG finger-print according to claim 1, it is characterized in that: described step a accurately takes BUSHEN YISHOU JIAONANG content, add the ethanol that volume fraction is 50%, ultrasonic process 30 minutes, cool rear volume fraction be 50% ethanol supply, then centrifugal 10 min under rotating speed is 10000 r/min conditions.
3. the assay method of BUSHEN YISHOU JIAONANG finger-print according to any one of claim 1-2, is characterized in that: frequency 40 kHz of described ultrasonic process.
4. the assay method of BUSHEN YISHOU JIAONANG finger-print according to claim 1, it is characterized in that: in described step c, the BUSHEN YISHOU JIAONANG solution of injection liquid chromatography is 15-25 μ L.
5. the assay method of BUSHEN YISHOU JIAONANG finger-print according to claim 4, it is characterized in that: in described step c, the BUSHEN YISHOU JIAONANG solution of injection liquid chromatography is 20 μ L.
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| CN107064320B (en) * | 2016-10-21 | 2019-09-24 | 广东省中医院 | A kind of method of quality control and its finger-print of reinforcing spleen and kidney side |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1907429A (en) * | 2005-08-05 | 2007-02-07 | 北京益芝堂现代制药有限公司 | Method for preparing and controlling the quality of Chinese medicinal soft capsule |
| CN101011480A (en) * | 2007-02-13 | 2007-08-08 | 美晨集团股份有限公司 | Method for overall monitoring kidney-tonifying body-strengthening tablet quality |
| CN102818861A (en) * | 2012-05-10 | 2012-12-12 | 丽珠医药集团股份有限公司 | Quality control method and application of Qingdu Anshen capsule |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1907429A (en) * | 2005-08-05 | 2007-02-07 | 北京益芝堂现代制药有限公司 | Method for preparing and controlling the quality of Chinese medicinal soft capsule |
| CN101011480A (en) * | 2007-02-13 | 2007-08-08 | 美晨集团股份有限公司 | Method for overall monitoring kidney-tonifying body-strengthening tablet quality |
| CN102818861A (en) * | 2012-05-10 | 2012-12-12 | 丽珠医药集团股份有限公司 | Quality control method and application of Qingdu Anshen capsule |
Non-Patent Citations (3)
| Title |
|---|
| 《补肾方分煎和合煎提取物的指纹图谱比较》;史万忠,徐德生,刘力,石印玉;《中成药》;20040125;第26卷(第1期);全文 * |
| 姜琳琳,李晓晶,于涛,赵陆华.《肾通颗粒HPLC指纹图谱的研究》.《中成药》.2007,第29卷(第5期),第1页左栏第1行至第2页右栏第13行. * |
| 赵陆华,屠颖,吴孟华,谭喜莹,梅玲华.《肾宝合剂HPLC指纹图谱的研究》.《中国药科大学学报》.2005,第36卷(第2期),全文. * |
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