CN1857589A - Method of measuring icariin and epimedin c content in Xianlinggubao preparation - Google Patents

Abstract

The present invention discloses the method of measuring icariin and epimedin C content in Xianlinggubao preparation, and the method is one high efficiency liquid chromatography method with icariin and epimedin C contrast and acetonitrile/water as flowing phase. Compared with available technology, the method adopts high efficiency separating column, replacement of the flowing phase component to separate icariin and epimedin C peaks completely, content limit redetermined based on experiment, increased epimedin C content limit with relatively greater peak area, and determined ratio range between icariin and epimedin C, so as to perfect the quality detection standard of Xianlinggubao preparation and ensure the medicine safe, effective and reliable.

Description

The content assaying method of icariin and epimedin in the XIANLING GUBAO ZHIJI

Technical field: the present invention relates to the content assaying method of icariin and epimedin in a kind of XIANLING GUBAO ZHIJI, belong to drug quality control technology field.

Background technology: the contained chemical constituent of Herba Epimedii is flavonoid, lignan's class, volatile oil, ceryl alcohol, hentriacontane, plant sterol, tannin and oils etc.Herba Epimedii has kidney-replenishing, bone and muscle strengthening, and the effect of wind-damp dispelling is mainly used in impotence and seminal emission, and the muscles and bones flaccidity is soft, rheumatic arthralgia, numbness contracture, diseases such as climacteric hypertension.XIANLING GUBAO ZHIJI is combined by Herba Epimedii, Radix Dipsaci, Radix Salviae Miltiorrhizae, the Rhizoma Anemarrhenae, Fructus Psoraleae and Radix Rehmanniae, has nourishing the liver and kidney, and reunion of bone is kept fit the function of strong bone, is mainly used in osteoporosis and osteoporosis, fracture, osteoarthritis, aseptic necrosis of bone etc.In XIANLING GUBAO ZHIJI, monarch drug in the Herba Epimedii side of being plays a part crucial reinforcing the kidney and supporting YANG, strengthening the tendons and bones, therefore can effectively control the quality of XIANLING GUBAO ZHIJI to the assay of epimedium herb in the preparation, thereby guarantee its clinical efficacy.

At present, the index composition of the epimedium herb that pharmacopeia requires only is an icariin, find through experimental study: owing to be subjected to condition restriction, icariin peak and epimedin peak overlapping caused content higher in the mensuration process when former content Determination of Icariin bioassay standard was set up.And learn after deliberation: the content of epimedin is higher than other flavones ingredients far away in the effective site in the Herba Epimedii (total flavones).So necessity of the quality standard that increases epimedium herb index composition is arranged, promptly increases the assay of epimedin.

Summary of the invention:

The objective of the invention is to: the content assaying method that icariin and epimedin in a kind of XIANLING GUBAO ZHIJI are provided, the present invention on the basis of existing technology, adopt the high efficiency separation post, the mobile phase component is changed, the icariin peak is thoroughly separated with the epimedin peak, and reformulated content limit through overtesting, increased the content limit of the epimedin that wherein peak area is bigger, thereby formulated proportion between the two, made the quality inspection standard of XIANLING GUBAO ZHIJI more perfect.

XIANLING GUBAO ZHIJI of the present invention is to be prepared from by Herba Epimedii, Radix Dipsaci, Radix Salviae Miltiorrhizae, the Rhizoma Anemarrhenae, Fructus Psoraleae and the Radix Rehmanniae relevant criterion [as WS-10269 (ZD-0269)-2002, WS-10289 (ZD-0289)-2002] according to China national Drug Administration.

The content assaying method of icariin and epimedin is to be contrast with icariin and epimedin reference substance in the XIANLING GUBAO ZHIJI of the present invention, and with acetonitrile: water is the high performance liquid chromatography of mobile phase.

Specifically, the content assaying method of icariin and epimedin is in the XIANLING GUBAO ZHIJI:

According to an appendix VID of Chinese Pharmacopoeia version in 2005 high effective liquid chromatography for measuring:

Chromatographic condition and system suitability test are filler with octadecylsilane chemically bonded silica; Acetonitrile: water=25: 75 is mobile phase; The detection wavelength is 270nm; Number of theoretical plate calculates by the epimedin peak should be lower than 5000; Number of theoretical plate calculates by the icariin peak should be lower than 5000;

It is an amount of that the preparation precision of reference substance solution takes by weighing in the phosphorus pentoxide vacuum drying apparatus dry 36 hours icariin and epimedin reference substance, add methanol and make the mixed solution that every 1ml contains icariin and epimedin 50 μ g, 25 μ g respectively, product solution in contrast, promptly;

This preparation or its content under the content uniformity item got in the preparation of need testing solution, and porphyrize is got 0.5g, the accurate title, decide, and puts in the tool plug conical flask, the accurate Diluted Alcohol 50ml that adds, claim to decide weight, supersound process is 30～60 minutes under power 250W, frequency 25kHz, puts cold, claim again to decide weight, supply the weight that subtracts mistake, shake up with Diluted Alcohol, get supernatant, filter, get subsequent filtrate, promptly;

Algoscopy is drawn reference substance solution and each 10 μ l of need testing solution respectively, injects chromatograph of liquid, measures, promptly;

Every of this capsule contains epimedin and icariin must not be less than 1.0mg, 0.5mg respectively, and epimedin and icariin peak area ratio must not be less than 2: 1.

In order to ensure content assaying method science of the present invention, reasonable, feasible, the applicant has carried out following experimental study:

1. the selection of assay index components

Herba Epimedii is the monarch drug in the XIANLING GUBAO ZHIJI side, Herba Epimedii mainly contains flavone compound, comprise epimedin, icariin, also contain composition and lignin compositions such as icariin-I, icariin-C, hyperin, precious icariin-I, rhamanopyranosyl icariside-II, arrow icariin B, precious icariin-II, Flos Caryophylli icariin C and Flos Caryophylli icariin F.Has kidney-replenishing, bone and muscle strengthening, the effect of wind-damp dispelling.Modern pharmacology studies show that Herba Epimedii has the raising sexual function, enhancing immunity, and the protection heart improves myocardial ischemia, increases coronary flow, anti-arrhythmia, blood pressure lowering, antiplatelet aggregation, antiinflammatory, the anti-ageing effect of waiting for a long time.Through correlational study as can be known: the content of epimedin is higher than other flavones ingredients far away in the effective site in the Herba Epimedii (total flavones), therefore, is necessary to increase the assay of epimedin, further to improve the quality standard of Herba Epimedii index composition.

In order to finish the present invention, the applicant investigates the epimedium herb extraction process, determine optimum extraction process, and prepare a large amount of epimedins, the total flavones process of enriching is investigated, use macroporous resin and carry out epimedin and separate, purification, and formulated that specificity is strong, highly sensitive, the high performance liquid chromatography of stability and favorable reproducibility, reliable results.

2. instrument and reagent

Instrument: high performance liquid chromatograph Agilent1100, DAD detector, Agilent work station.

Chromatographic column: Agilent ODS 5 μ m 4.0 * 250mm

Reference substance: icariin, epimedin.

Reagent: acetonitrile (chromatographically pure, Fisher), WAHAHA pure water (Hangzhou Wahaha Group Co.,Ltd), ethanol (analytical pure, Beijing Chemical Plant).

3. method and result

3.1 chromatographiccondition

Mobile phase: acetonitrile-water (25: 75); Flow velocity: 1ml/min; Detect wavelength: 270nm; Number of theoretical plate calculates by epimedin and is not less than 5000.

3.2 selection of experiment conditions

3.2.1 measure the selection of wavelength:

With dissolve with methanol epimedin reference substance, make the solution that every 1ml contains 0.05mg, the U-2000 type visible-measure absorption spectrum on the uv-spectrophotometric instrument, the result has absorption maximum at 270nm place, so selection 270nm is as the detection wavelength.

3.2.2 the selection of mobile phase:

Acetonitrile-water (30: 70) on probation, the peak type is bad, and separating effect is very undesirable.

Change acetonitrile-water (24: 76) into, reach preferably and separate.

3.2.3 blank assay

The preparation of blank solution is the ratio in the prescription taste of Chinese medicine, autogamy does not contain group's medicine of Herba Epimedii, make blank preparation by its technology, press need testing solution preparation method preparation again and measure, blank solution is not being seen chromatographic peak with epimedin, retention time place that the icariin reference substance is identical as a result.

3.2.4 extracting solvent selects:

Take by weighing XIANLING GUBAO ZHIJI 0.5g (lot number 051209), the accurate title, decide, and puts in the tool plug conical flask, adds methanol, Diluted Alcohol, each 50ml of ethanol respectively, and supersound process 1 hour is measured its icariin and epimedin content.The results are shown in Table 1:

The different influences of extracting solvent of table 1 to epimedin, icariin content

Extract solvent	Methanol	Diluted Alcohol	Ethanol
				Epimedin content (mg/g) icariin content (mg/g)	3.2642 1.5642	4.0537 1.7577	3.7135 1.4128

The result shows that the effect of Diluted Alcohol do extraction solvent is best.Therefore, select for use Diluted Alcohol to extract as extracting solvent.

3.2.5 extraction time is determined:

Take by weighing XIANLING GUBAO ZHIJI 0.5g (lot number 051209), the accurate title, decide, and puts in the tool plug conical flask, add Diluted Alcohol 50ml respectively, supersound process (power 250W, frequency 25KHz) each 0.5,1.0,1.5 hour, measure the content of its icariin and epimedin, the results are shown in Table 2:

Table 2 different extraction times are to the influence of epimedin, icariin content

Extraction time (min)

0.5

1.0

1.5

Epimedin content (mg/g) icariin content (mg/g)

3.1243 1.6507

4.1584 1.7385

4.2069 1.7315

According to above result, select supersound extraction 1 hour comparatively suitable.

3.3 linear relationship is investigated

Accurate epimedin reference substance solution (0.0404mg/ml) 2,4,8,12, the 16 μ l that draw, inject chromatograph of liquid, the record peak area, (μ g) is abscissa with reference substance epimedin sample size, is vertical coordinate with the peak area, the drawing standard curve, its regression equation is A=1509.3524X+13.7157 (r=0.9992), brings the disconnected minimum point value of linearity into following formula and calculates, and relative deviation is less than 1% as a result, think that working curve was similar to initial point, therefore adopt the appearance one point method to calculate.The result shows that the epimedin sample size is the good linear relation in 0.0808～0.6464 μ g scope.

In like manner, accurate icariin reference substance solution (0.1004mg/ml) 1,3,5,8, the 10 μ l that draw, inject chromatograph of liquid, the record peak area, (μ g) is abscissa with reference substance icariin sample size, with the peak area is vertical coordinate, the drawing standard curve, its regression equation is Y=2109.8x+4.6272 (r=0.9998), fitting to former point equation is: Y=2115.063X, and bring the short minimum point value of linearity into above-mentioned two formulas and calculate, relative deviation is 0.96% as a result, think that working curve was similar to initial point, therefore adopt one point external standard method to calculate.The result shows that the icariin sample size is the good linear relation in 0.10～1.2 μ g scope.The results are shown in Table 3, table 4.

Table 3 epimedin linear relationship is investigated

Sampling volume (μ l)	2	4	8	12	16
						Sample size (μ g)	0.0808	0.1616	0.3232	0.4848	0.6464
Peak area (Mau*s)	125.24849	257.19345	511.98727	762.33679	972.88281

Table 4 icariin linear relationship is investigated

The amount of icariin (μ g)	0.1004	0.3012	0.5020	0.8032	1.004	1.2048
							Peak area (Mau*s)	200.660	639.502	1076.194	1727.701	2112.015	2532.912

3.4 stability test

Get same test sample (lot number 051214) 0.5g, the need testing solution preparation method is prepared in the reference standard, respectively at preparation back 0,2,4,6,8,10 hours, measure epimedin and icariin peak area according to last method, the record peak area value, the result shows that need testing solution is good at 10 hours internal stabilities, sees Table 5,6.

Table 5 epimedin stability experiment

Time	Peak area (Mau*s)	Average	RSD(％)
				0	725.74371	712.788	1.131
2	719.83386
		4	705.53790
6	707.99670
		8	708.32776
10	709.28516

Table 6 icariin stability experiment

Time	Peak area (Mau*s)	Average	RSD(％)
				0	1012.313	1025.003	1.29％
2	1022.716
		4	1020.080
6	1047.485
		8	1022.419

3.5 precision test

Accurate same epimedin (C=0.0404mg/ml), each 10 μ l of icariin (C=0.1004mg/ml) reference substance solution of drawing repeat sample introduction 6 times, measure peak area and write down collection of illustrative plates, the results are shown in Table 7,8.The result shows that precision meets the requirements.

The test of table 7 epimedin precision

	Peak area (Mau*s)	Average	RSD(％)
				1	639.66168	641.371	0.188
2	641.82422
		3	641.55096
4	640.18201
		5	642.76141
6	642.24707

The test of table 8 icariin precision

	Peak area (Mau*s)	Average	RSD(％)
				1	2161.172
2	2118.171
				3	2166.837	2133.01	1.36
4	2115.738
				5	2103.102

3.6 replica test

Same lot number (051214) sample is taken by weighing 5 parts, prepare test liquid, measure its epimedin and icariin content in accordance with the law, the results are shown in Table 9,10 by the method under the assay item of the present invention.Experimental result shows that this method repeatability is good.

Epimedin measurement result in table 9 replica test

	Sample size (g)	Chao Huoding C content (mg/g)	Average content (mg/g)	RSD(％)
					1	0.5042	4.4074	4.4561	1.78
2	0.5025	4.4805
			3	0.5023	4.5336
4	0.5128	4.3439
			5	0.5007	4.5151

Icariin measurement result in table 10 replica test

	Sample size (g)	Icariin content (mg/g)	Average content (mg/g)	RSD(％)
					1	0.5042	1.9856	1.9909	0.79
2	0.5025	1.9892
			3	0.5023	1.9692
4	0.5128	2.0115
			5	0.5007	1.9988

3.7 recovery test

Adopt the application of sample absorption method.Precision takes by weighing the same lot number (lot number 051214 of known content, known epimedin and icariin content are respectively 4.4561mg/g, 1.9909mg/g) the about 0.25g of sample each 5 parts, the accurate title, decide, accurate respectively adding epimedin and icariin mix each 50ml of reference substance solution and (get epimedin, icariin reference substance, add Diluted Alcohol and make the solution that concentration is respectively 0.024mg/ml, 7.52 μ g/ml), preparation method preparation and above-mentioned chromatographic condition by test liquid under the assay item of the present invention are measured calculate recovery rate.The results are shown in Table 11,12.

Epimedin recovery test and result in the celestial Ling Neibao capsule of table 11

Sequence number	Sample sample weighting amount (g)	Sample epimedin amount (mg)	Add epimedin amount (mg)	The epimedin amount of recording (mg)	The response rate (%)	Average recovery rate (%)	RSD (％)
								1	0.2505	1.1163	1.20	2.3135	99.77	100.11	1.99
2	0.2486	1.1078	1.20	2.3340	102.18
						3	0.2517	1.1216	1.20	2.3442	101.88
4	0.2609	1.1630	1.20	2.3308	97.32
						5	0.2526	1.1256	1.20	2.3182	99.38

Icariin recovery test and result in the celestial Ling Neibao capsule of table 12

Sequence number	Sample sample weighting amount (g)	The icariin amount of recording (mg)	Sample icariin amount (mg)	Add icariin amount (mg)	The response rate (%)	Average recovery rate (%)	RSD (％)
								1	0.2505	0.8755	0.4987	0.376	100.21	97.47	2.19
2	0.2486	0.8591	0.4949	96.86
					3	0.2517	0.8608	0.5011	95.66
4	0.2609	0.8923	0.5194	99.18
					5	0.2526	0.8617	0.5029	95.42

Above result shows that epimedin and icariin average recovery rate are respectively 100.11%, 97.47%, and RSD is respectively 1.99%, 2.19%.Illustrate that this method is feasible.

According to above result of the test, the condition of content assaying method of the present invention is defined as: with octadecylsilane chemically bonded silica is filler; Acetonitrile-water (25: 75) is a mobile phase; Flow velocity: 1.0ml/min; The detection wavelength is 270nm.Number of theoretical plate calculates by the epimedin peak should be not less than 5000.

Need testing solution preparation method of the present invention is defined as: get this preparation or its content under the content uniformity item, porphyrize is got 0.5g, the accurate title, decide, and puts in the tool plug conical flask, the accurate Diluted Alcohol 50ml that adds, claim to decide weight, supersound process (power 250W, frequency 25kHz) 1 hour, put cold, claim again to decide weight, supply the weight that subtracts mistake, shake up with ethanol, filter, promptly.

3.8 this algoscopy of assay computational methods adopts one point external standard method to calculate content, precision takes by weighing test sample 0.5g, prepares test liquid by quality standard, and sample introduction is measured, and computing formula is as follows.

(1) need testing solution concentration is calculated:

$C_S=\frac{C_R\×A_S}{A_R}---\left(1\right)$

In the formula: C _S: be need testing solution concentration (mg/ml).

C _R: be reference substance solution concentration (mg/ml).

A _s: be need testing solution peak area value (mAu*s)

A _R: be reference substance solution peak area value (mAu*s)

(2) formulation content computing formula:

In the formula: C _s: need testing solution concentration (mg/ml)

V _S: be the volume (ml) of need testing solution.

W ₁: be the weighing (g) of sample.

W ₂: be average particle heavy (g).

Merge (1) (2) formula, its computing formula is (3):

3.9 sample determination and content limit are formulated us to ten batches of our company " XIANLING GUBAO JIAONANG ", by its epimedin of above-mentioned high effective liquid chromatography for measuring and icariin content, the results are shown in Table 13.

Table 13 epimedin measurement result table

Lot number	Epimedin average peak area (Mau*s) (n=2)		Chao Huoding C average content (mg/ grain) (n=2)		Icariin average peak area (Mau*s) (n=2)		Icariin average content (mg/ grain) (n=2)
									040905 041001 041010 041013 050718 051204 051207 051208 051210 051212	469.256	473.598	0.958	0.979	244.278	248.595	0.486	0.477
546.859	551.105	1.561	1.529	319.742	308.815	0.569	0.575
								915.716		922.924	2.407	2.384	419.598	403.426	0.671	0.658
745.802	740.298	1.655	1.591	268.069	257.332	0.516	0.528
								I380.982		1389.703	2.964	2.896	347.301	354.459	0.612	0.606
941.804	940.704	2.451	2.440	334.781	337.916	0.715	0.728
								983.635		992.806	2.615	2.528	297.723	290.498	0.551	0.548
834.491	849.264	1.726	1.735	479.682	469.251	0.865	0.837
								1024.562		1025.796	2.685	2.706	311.879	309.765	0.914	0.925
974.672	949.236	2.562	2.511	318.255	313.206	0.584	0.576

According to above epimedin and ten batches of peak areas of icariin and assay result, every contains epimedin and icariin must not be less than 1.0mg, 0.5mg respectively in tentative " XIANLING GUBAO JIAONANG ", and epimedin and icariin peak area ratio must not be less than 2: 1.

Compared with prior art, content assaying method of the present invention adopts the high efficiency separation post, the mobile phase component is changed, the icariin peak is thoroughly separated with the epimedin peak, and reformulated content limit through overtesting, increase the content limit of the epimedin that wherein peak area is bigger, thereby formulated proportion between the two, make the quality inspection standard of XIANLING GUBAO ZHIJI more perfect, thereby guaranteed safety, effectiveness, the reliability of medicine.

The specific embodiment:

Embodiments of the invention 1: the content assaying method of icariin and epimedin is in the XIANLING GUBAO ZHIJI:

According to an appendix VID of Chinese Pharmacopoeia version in 2005 high effective liquid chromatography for measuring:

Chromatographic condition and system suitability test are filler with octadecylsilane chemically bonded silica; Acetonitrile: water=25: 75 is mobile phase; The detection wavelength is 270nm; Number of theoretical plate calculates by the epimedin peak should be lower than 5000;

It is an amount of that the preparation precision of reference substance solution takes by weighing in the phosphorus pentoxide vacuum drying apparatus dry 36 hours icariin and epimedin reference substance, add methanol and make the mixed solution that every 1ml contains icariin and epimedin 50 μ g, 25 μ g respectively, product solution in contrast, promptly;

This preparation or its content under the content uniformity item got in the preparation of need testing solution, and porphyrize is got 0.5g, the accurate title, decide, and puts in the tool plug conical flask, the accurate Diluted Alcohol 50ml that adds, claim to decide weight, supersound process is 60 minutes under power 250W, frequency 25kHz, puts cold, claim again to decide weight, supply the weight that subtracts mistake, shake up with ethanol, get supernatant, filter, get subsequent filtrate, promptly;

Algoscopy is drawn reference substance solution and each 10 μ l of need testing solution respectively, injects chromatograph of liquid, measures, promptly;

Every of this capsule contains epimedin and icariin must not be less than 1.0mg, 0.5mg respectively, and epimedin and icariin peak area ratio must not be less than 2: 1.

Claims (2)

1. the content assaying method of icariin and epimedin in the XIANLING GUBAO ZHIJI is characterized in that: it is to be contrast with icariin and epimedin reference substance, and with acetonitrile: water is the high performance liquid chromatography of mobile phase.

2. according to the content assaying method of icariin and epimedin in the described XIANLING GUBAO ZHIJI of claim 1, it is characterized in that: concrete assay method is:

According to an appendix VID of Chinese Pharmacopoeia version in 2005 high effective liquid chromatography for measuring:

Chromatographic condition and system suitability test are filler with octadecylsilane chemically bonded silica; Acetonitrile: water=25: 75 is mobile phase; The detection wavelength is 270nm; Number of theoretical plate calculates by the epimedin peak should be lower than 5000; Number of theoretical plate calculates by the icariin peak should be lower than 5000;

It is an amount of that the preparation precision of reference substance solution takes by weighing in the phosphorus pentoxide vacuum drying apparatus dry 36 hours icariin and epimedin reference substance, add methanol and make the mixed solution that every 1ml contains icariin and epimedin 50 μ g, 25 μ g respectively, product solution in contrast, promptly;

This preparation or its content under the content uniformity item got in the preparation of need testing solution, and porphyrize is got 0.5g, the accurate title, decide, and puts in the tool plug conical flask, the accurate Diluted Alcohol 50ml that adds, claim to decide weight, supersound process is 30～60 minutes under power 250W, frequency 25kHz, puts cold, claim again to decide weight, supply the weight that subtracts mistake, shake up with Diluted Alcohol, get supernatant, filter, get subsequent filtrate, promptly;

Algoscopy is drawn reference substance solution and each 10 μ l of need testing solution respectively, injects chromatograph of liquid, measures, promptly;

Every of this capsule contains epimedin and icariin must not be less than 1.0mg, 0.5mg respectively, and epimedin and icariin peak area ratio must not be less than 2: 1.