CN104198616A - Method for determining content of icariin in climacterium capsule - Google Patents
Method for determining content of icariin in climacterium capsule Download PDFInfo
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- CN104198616A CN104198616A CN201410475973.0A CN201410475973A CN104198616A CN 104198616 A CN104198616 A CN 104198616A CN 201410475973 A CN201410475973 A CN 201410475973A CN 104198616 A CN104198616 A CN 104198616A
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- icariin
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Abstract
The invention discloses a method for determining the content of icariin in a climacterium capsule. The method adopts a high performance liquid chromatography to determine the content of icariin in the climacterium capsule, and a sample pretreatment method comprises the following steps: porphyrizing the content of the climacterium capsule, precisely weighing 0.5-0.7g, adding 15-30ml of 25% methanol, ultrasonically extracting for 30 minutes, cooling and metering volume to 50ml, centrifuging for 10 minutes at the rotating speed of 10000 rpm, and taking supernatant fluid. The chromatographic conditions of icariin content determination are as follows: a chromatographic column is a Lichrospher C18 chromatographic column, a mobile phase is an aqueous solution of acetonitrile and trifluoroacetic acid with the mass fraction of 0.1% (the volume ratio is 28 to 72), the determining wavelength is 270 nm, and column temperature is 32.5 DEG C. The method aims at the climacterium capsule with special ingredients and can determine the content of icariin well.
Description
Technical field
The present invention relates to the content assaying method of icariin in Gengnianling Capsule, belong to medical product manufacturing technology field.
Background technology
Gengnianling Capsule is a kind of effect with warm kidney benefit the moon, adjusting and runing yang, is mainly used in gynaecology's medicine that women's climacteric syndrome belongs to yin-yang deficiency person.Gengnianling Capsule is mainly by barrenwort, Cobastab
1, the fruit of glossy privet, oryzanol, Cobastab
6form, the monarch drug in a prescription that wherein barrenwort is Gengnianling Capsule, in order to control the quality of Gengnianling Capsule, be necessary to provide a kind of method of quality control of Gengnianling Capsule, can be more quickly, whether the content that can measure comparatively exactly barrenwort meet index request simultaneously.In barrenwort, containing the flavonoid glycoside compound such as icariin, be its main effective constituent, therefore one of index components using icariin as Gengnianling Capsule assay conventionally.In prior art, the assay of icariin is mainly realized by thin layer chromatography scanning, high performance liquid chromatography, but for the specific Gengnianling Capsule of component, the content assaying method of icariin still awaits a progressive optimization.
Summary of the invention
The present invention, just for the demand of prior art existence, provides the content assaying method of icariin in a kind of Gengnianling Capsule, for the specific Gengnianling Capsule of component, can to icariin, carry out assay preferably.
For addressing the above problem, the technical solution used in the present invention is as follows:
The content assaying method of icariin in a kind of Gengnianling Capsule, adopt high performance liquid chromatography to carry out assay to the icariin in Gengnianling Capsule, and sample-pretreating method is: by the content porphyrize of Gengnianling Capsule, precision takes 0.5～0.7g, add massfraction and be methyl alcohol 15～30ml of 25%, ultrasonic extraction 30 minutes, the methanol constant volume that to let cool, take massfraction be 25%, to 50ml, with the rotating speed of 10000rpm centrifugal 10 minutes, is got supernatant.
Improvement as technique scheme, the chromatographic condition of Determination of Content of Icariin is: chromatographic column is Lichrospher C18 chromatographic column, mobile phase is acetonitrile: the trifluoroacetic acid aqueous solution that massfraction is 0.1% (volume ratio is 28: 72), mensuration wavelength is 270nm, and column temperature is 32.5 ℃.
Compared with prior art, implementation result of the present invention is as follows in the present invention:
The content assaying method of icariin is provided in Chinese Pharmacopoeia, wherein test sample pre-treating method is that precision takes Gengnianling Capsule powder and is dissolved in quantitative Diluted Alcohol, ultrasonic processing 1 hour, more weighed weight, with Diluted Alcohol, supply the weight of less loss, and specifically do not disclose the concentration of Diluted Alcohol; Because methyl alcohol has better dissolubility with respect to ethanol to the icariin of polarity, thereby the present invention selects methyl alcohol instead of ethanol to improve the pre-treating method of test sample, by hereinafter the experimental result shown in table 5 is known, take pure methyl alcohol as extracting solvent, to the extraction of icariin in Gengnianling Capsule preparation insufficient, and the methanol solution that massfraction provided by the invention is 25% can fully extract the icariin in Gengnianling Capsule; The content assaying method of the icariin that Chinese Pharmacopoeia is recorded, only disclose chromatographic column and take octadecylsilane chemically bonded silica as filling agent, and in fact the similar commercially available chromatographic column of different manufacturers also has different separating effects, the Lichrospher C18 chromatographic column that the present invention selects has preferably separating effect; In addition, the content assaying method of the icariin that Chinese Pharmacopoeia is recorded be take acetonitrile: water (volume ratio is 30: 70) is mobile phase, for by barrenwort, Cobastab
1, the fruit of glossy privet, oryzanol, Cobastab
6the Gengnianling Capsule forming, this mobile phase separating effect is unsatisfactory, thereby the present invention has attempted use acetonitrile: the trifluoroacetic acid aqueous solution that massfraction is 0.1% (volume ratio is 28: 72) is mobile phase, has good separating effect.
In sum, the present invention adopts HPLC analytical method to measure the content of icariin, by developing applicable pre-treating method and chromatographiccondition, the content assaying method detection sensitivity of icariin provided by the present invention is high, the minimum of sample detection limited the quantity of as 0.0002ng, and the accuracy of measurement of Icariin content is high, and average recovery rate is 99.42%, RSD=2.36%, precision and repeatability are also better.
Accompanying drawing explanation
Fig. 1 is that icariin reference substance detects the HPLC figure that limits the quantity of;
Fig. 2 is icariin purity test HPLC figure;
Fig. 3 is icariin purity test HPLC figure (retention time that changes the mutual-assistance icariin that flows reduces 1 times);
Fig. 4 is icariin purity test HPLC figure (retention time that changes the mutual-assistance icariin that flows increases by 1 times);
Fig. 5 is for lacking barrenwort negative sample HPLC figure;
Fig. 6 is formulation samples HPLC figure;
Fig. 7 is icariin reference substance HPLC figure;
Fig. 8 is epimedium herb HPLC figure.
Embodiment
Content of the present invention is described below in conjunction with specific embodiments.
In Gengnianling Capsule of the present invention, the content assaying method of icariin is as follows:
One, instrument and reagent: Waters 510 pumps, ultraviolet 486 detecting devices, Rheodyne injector, acetonitrile is chromatographically pure (CALEDON LABORATORIES LTD), trifluoroacetic acid is chromatographically pure (Tedia company), and water is sub-boiling distillation water, and it is pure that all the other reagent are analysis.
Two, reference substance source and purity test: icariin reference substance is provided by Chinese biological drug inspection office.As shown in hereinafter accompanying drawing 2 to 4, with concentration (0.676mg/ml), carry out standard items purity test, collection of illustrative plates is integration in 2 times of retention times, and result shows that icariin standard items purity is more than 98%; Change mobile phase ratio and make retention time increase and decrease more than 1 times, in chromatogram, do not occur impurity peaks.
Three, the selection of chromatographic condition:
Chromatographic column: Huaiyin Han Bang company, anti-phase C18 post: Lichrospher C18 (4.6 * 250mm, 5 μ m); Dalian chemical physics institute of the Chinese Academy of Sciences, anti-phase C18 post: Kromasil C18 (4.6 * 250mm, 5 μ m).Result demonstration, better with Lichrospher C18 chromatographic column, therefore select.
Mobile phase: acetonitrile-sour water system of take is carried out stratographic analysis as mobile phase, and mobile phase is acetonitrile: the trifluoroacetic acid aqueous solution that mass percent is 0.1% (volume ratio is 28: 72).
Measure wavelength: according to ultraviolet spectrum, the maximum absorption wavelength of icariin is 270nm, measure wavelength and be defined as 270nm, column temperature is 32.5 ℃.
Under these conditions, icariin and other peak are good separation, and the HPLC chromatogram of sample is as shown in hereinafter accompanying drawing 6 to 8.With icariin, calculate post effect, theoretical cam curve is 3024, reaches baseline separation, in order to ensure quantitative test accurately, in system experimentation, icariin theoretical cam curve must not be lower than 2000.Repeat two Lichrospher C18 chromatographic columns, all can reach above-mentioned Separation Indexes, meet the requirements.
Sample is measured in accordance with the law, and continuous sample introduction 6 times is measured peak area and be the results are shown in following table 1.
Table 1 icariin precision is investigated test findings
| Sequence number | Icariin peak area |
| 1 | 3345000 |
| 2 | 3320000 |
| 3 | 3348000 |
| 4 | 3360000 |
| 5 | 3358000 |
| 6 | 3347000 |
| RSD％ | 0.43 |
By upper table, shown, it is that RSD equals 0.43% (<2.0%) that icariin precision is investigated test findings, meets the requirements.
Four, the preparation of need testing solution: get tablet under this product content uniformity item, porphyrize, gets 0.5g, accurately weighed, put in 50ml measuring bottle, add 25% methyl alcohol appropriate, ultrasonic extraction 30 minutes, lets cool, constant volume, high speed centrifugation (10000rpm) 10 minutes, gets supernatant, obtains.
Five, the investigation of linear relationship: the Specification Curve of Increasing of icariin: precision takes the icariin reference substance 6.76mg that is dried to constant weight in 60 ℃, be placed in 10ml volumetric flask, add mobile phase and be diluted to scale, make icariin standard stock solution (676 μ g/ml).Adopt stepwise dilution method to be made into respectively again the serial reference substance liquid of 338 μ g/ml, 169 μ g/ml, 84.5 μ g/ml, 42.25 μ g/ml, 21.12 μ g/ml, draw respectively above-mentioned solution 20 μ l, liquid chromatograph injector, measure, take peak area as ordinate (Y), reference substance amount (μ g/ml) is horizontal ordinate (X), and drawing standard curve, obtains regression equation:
Y＝(4.871×10
3)X+(23340.678) (R＝0.9998)
Icariin concentration is good linear relationship between 676-21.12 μ g/ml, the results are shown in following table 2.
Table 2 icariin typical curve
Sample is measured in accordance with the law, enters a pin every 2 hours, and totally 6 times, measurement result sees the following form 3.
Table 3 icariin study on the stability test findings
| Time (hour) | Icariin peak area |
| 0 | 3350000 |
| 2 | 3352000 |
| 4 | 3351000 |
| 6 | 3343000 |
| 8 | 3353000 |
| 10 | 3365000 |
| RSD％ | 0.21 |
By above-mentioned table 3, shown, icariin study on the stability test findings is that RSD equals 0.21% (<2.0%), meets the requirements.
Six, blank test: former side removes epimedium herb, is prepared to obtain the blank preparation of scarce barrenwort by preparation process method.Get blank preparation powder 0.5g, accurately weighed, put in 50ml measuring bottle, add 25% methyl alcohol appropriate, ultrasonic extraction 30min, lets cool, constant volume, high speed centrifugation (10000rpm) 10 minutes, gets supernatant 1ml, to 5ml measuring bottle, uses mobile phase constant volume, mixes, and obtains.Press content assaying method analysis, blank sample chromatogram does not have Interference Peaks in the corresponding retention time of icariin.It is noiseless that Icariin content is measured in explanation under this condition, and this method is accurate, feasible, sees below shown in civilian accompanying drawing 5.
Seven, stability test: same sample (lot number 030406) test liquid, every 2 hours, survey once, to survey altogether 6 times, measurement result sees the following form.Result shows, in test liquid, icariin is stable in 10 hours, and RSD is 1.55%, meets the requirements.The results are shown in following table 4.
The study on the stability result of icariin in table 4 preparation
Eight, extracting method test: in order to propose as much as possible the icariin in preparation, to extracting solvent, investigate, get lot number and be 030409 preparation 0.5g, accurately weighed, according to the form below operation, ultrasonic extraction 30min, let cool, constant volume, high speed centrifugation (10000rpm) 10 minutes, gets supernatant and get final product.The results are shown in following table 5.
In table 5 preparation, icariin extracts solvent selection
| Sample number | Sample weight | Methanol concentration (%) | Quantity of solvent (ml) | Icariin peak average area | Content (mg/g) |
| 1 | 0.669 | 0 | 25 | 175600 | 3.291 |
| 2 | 0.667 | 25 | 25 | 178600 | 3.304 |
| 3 | 0.604 | 50 | 25 | 145500 | 3.248 |
| 4 | 0.608 | 75 | 25 | 141600 | 3.254 |
| 5 | 0.608 | 100 | 25 | 132600 | 3.182 |
| 6 | 0.608 | 0 | 50 | 79280 | 3.348 |
| 7 | 0.665 | 25 | 50 | 94420 | 3.371 |
| 8 | 0.623 | 50 | 50 | 71920 | 3.200 |
| 9 | 0.623 | 75 | 50 | 66740 | 3.204 |
| 10 | 0.625 | 100 | 50 | 65830 | 3.247 |
Above-mentioned table 5 result shows, the ultrasonic extraction of methyl alcohol 50ml with 25% can make the icariin in preparation extract completely.
Nine, precision test: same test liquid (lot number 030406), to measure in accordance with the law, continuous sample introduction 6 times, measures peak area and the results are shown in following table 6.
In table 6 preparation, the precision of icariin is investigated test findings
| Sequence number | Icariin peak area |
| 1 | 103700 |
| 2 | 102700 |
| 3 | 100800 |
| 4 | 104700 |
| 5 | 99910 |
| 6 | 102700 |
| RSD％ | 1.74 |
Above-mentioned table 6 result shows, in test liquid, the RSD of icariin is 1.74%, meets the requirements.
Ten, replica test: same lot number (lot number 030406) preparation, processing sample is 6 parts in accordance with the law, measures in accordance with the law, and the average content of repeated result icariin is 1.31mg/g, and RSD=1.93% sees the following form 7.
The replica test result of icariin in table 7 preparation
11, detection sensitivity and detection lower limit: when this product detects, detection signal (mv) is about 1428 with the ratio of reference substance concentration (ng), when icariin sample size 0.0007ng, presents the chromatographic peak of 1.05mv intensity, sees below shown in civilian accompanying drawing 1.The minimum of sample detection limited the quantity of as 0.0002ng.
12, recovery test: precision takes totally 6 parts of this product (030406), 2 parts one group, each group adds respectively icariin reference substance solution (0.0816mg/ml) 0.6ml, 0.7ml, 0.8ml.Measure, calculating icariin average recovery rate is 99.42%, RSD=2.36% in accordance with the law.The results are shown in following table 8.
The average recovery test findings of icariin in table 8 preparation
Above content is detailed description made for the present invention in conjunction with specific embodiments, can not assert that the concrete enforcement of the present invention only limits to these explanations.For those skilled in the art, without departing from the inventive concept of the premise, can also make some simple deduction or replace, all should be considered as belonging to the scope of protection of the invention.
Claims (2)
1. the content assaying method of icariin in a kind of Gengnianling Capsule as claimed in claim 1, it is characterized in that, adopt high performance liquid chromatography to carry out assay to the icariin in Gengnianling Capsule, and sample-pretreating method is: by the content porphyrize of Gengnianling Capsule, precision takes 0.5～0.7g, add massfraction and be methyl alcohol 15～30ml of 25%, ultrasonic extraction 30 minutes, the methanol constant volume that to let cool, take massfraction be 25%, to 50ml, with the rotating speed of 10000rpm centrifugal 10 minutes, is got supernatant.
2. the content assaying method of icariin in a kind of Gengnianling Capsule as claimed in claim 2, it is characterized in that, the chromatographic condition of Determination of Content of Icariin is: chromatographic column is Lichrospher C18 chromatographic column, mobile phase is acetonitrile: the trifluoroacetic acid aqueous solution that massfraction is 0.1% (volume ratio is 28: 72), mensuration wavelength is 270nm, and column temperature is 32.5 ℃.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104950068A (en) * | 2015-06-03 | 2015-09-30 | 贵阳中医学院第二附属医院 | Quality detection method for kidney-replenishing and wind-dispelling climacterium mixture |
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| CN1586610A (en) * | 2004-09-23 | 2005-03-02 | 内蒙古伊泰医药科技开发有限责任公司 | Process for preparing granular powder for treating neurosism and quality control method |
| CN1657093A (en) * | 2004-09-23 | 2005-08-24 | 内蒙古伊泰医药科技开发有限责任公司 | Preparation method of granular agent for raising leucocyte and its quality control method |
| CN101856381A (en) * | 2009-04-09 | 2010-10-13 | 凌沛学 | Preparation method and quality control method for medicinal preparation for treating women climacteric syndrome |
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2014
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Patent Citations (3)
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| CN1586610A (en) * | 2004-09-23 | 2005-03-02 | 内蒙古伊泰医药科技开发有限责任公司 | Process for preparing granular powder for treating neurosism and quality control method |
| CN1657093A (en) * | 2004-09-23 | 2005-08-24 | 内蒙古伊泰医药科技开发有限责任公司 | Preparation method of granular agent for raising leucocyte and its quality control method |
| CN101856381A (en) * | 2009-04-09 | 2010-10-13 | 凌沛学 | Preparation method and quality control method for medicinal preparation for treating women climacteric syndrome |
Non-Patent Citations (2)
| Title |
|---|
| OLGA N. POZHARITSKAYA等: "Determination of icariin in rat plasma by reverse-phase high-performance liquid chromatography after oral administration of a lipid-based suspension of Epimedium koreanum extract", 《BIOMEDICAL CHROMATOGRAPHY》, vol. 22, 31 December 2008 (2008-12-31), pages 625 - 629 * |
| 李树立: "高效液相色谱法测定更年灵胶囊中淫羊藿苷的含量", 《中国现代药物应用》, vol. 3, no. 12, 30 June 2009 (2009-06-30), pages 128 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104950068A (en) * | 2015-06-03 | 2015-09-30 | 贵阳中医学院第二附属医院 | Quality detection method for kidney-replenishing and wind-dispelling climacterium mixture |
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Application publication date: 20141210 |