CN1304840C - Method for quality control of injections for treating thromboangitis diseases - Google Patents

Abstract

The present invention discloses a Mailuoning injection for curing thrombus obdurate diseases and a quality control method, which comprises measuring of fingerprint pictures, and testing of content of total chlorogenic acid and cinnamic acid. Compared with the original quality control standard, the present invention has the advantages that the quality of the injection can be completely controlled, the product quality can be better controlled, and the stability and the curative effect of products are improved.

Description

A kind of method of quality control for the treatment of the parenteral solution of thrombosis

Technical field

The invention belongs to medical technical field, relating to a kind of is the method for quality control of the MAILUONING ZHUSHEYE of raw material for treating thrombosis with the vegetable crude drug root of bidentate achyranthes, radix scrophulariae, the stem of noble dendrobium, honeysuckle.

Background technology

The quality control standard of the MAILUONING ZHUSHEYE employing of using clinically at present, the discriminating aspect comprises the discriminating to chlorogenic acid and escoparone, the quality of product is only controlled in the assay aspect with the content of cinnamic acid, comprehensive inadequately to production quality control.The present invention has increased the mensuration of finger-print and to the assay of total chlorogenic acid, condition to the cinnamic acid assay is changed, and compares with the proper mass control criterion, can carry out total quality control to parenteral solution, therefore can better control product quality, improve the stability and the curative effect of product.

Summary of the invention

The object of the present invention is to provide a kind of method of quality control that can carry out whole and total quality control, comprise the mensuration of finger-print and the assay of total chlorogenic acid and cinnamic acid to MAILUONING ZHUSHEYE.

The objective of the invention is to be achieved through the following technical solutions:

Root of bidentate achyranthes 1-4 weight portion radix scrophulariae 1-4 weight portion stem of noble dendrobium 1-4 weight portion honeysuckle 1-4 weight portion

Above four traditional Chinese medicine material medicine materical crude slice adds medicinal material 4-8 times water gaging, soaks after 1-3 hour, and decoct and extract 2-3 time, each 1-3 hour, merge extract, filter, being evaporated to 65 ℃ of following relative densities is 1.25-1.35, cooling.Add ethanol and make and contain alcohol and measure, leave standstill to 70%-85%; Get supernatant, reclaim and eliminate ethanol, being concentrated into 65 ℃ of following relative densities is 1.05-1.15; Doubly measure the ethyl acetate counter-current extraction with 2-4, merge the ester extract, reclaim and eliminate ethyl acetate, add full dose 20%-60% water for injection, boil, cooling, filter, add an amount of water for injection, add Tween-80, stir evenly, transfer pH to 8.5-9.2, add or do not add the sodium chloride stirring and dissolving with 20%NaOH solution, add full dose water for injection, the miillpore filter with 0.22 μ m filters embedding at last, sterilization, lamp inspection, packing.

In the above-mentioned preparation process, regulate osmotic pressure as need, also can use glucose or glucose saline except that available sodium chloride, wherein the ratio commonly used of glucose and sodium chloride is in the glucose saline: glucose: sodium chloride=5: 0.9 or glucose: sodium chloride=10: 0.9.

The glucose saline detailed directions is:

50ml: glucose 2.5g and sodium chloride 0.45g

50ml: glucose 5g and sodium chloride 0.45g

100ml: glucose 5g and sodium chloride 0.9g

100ml: glucose 10g and sodium chloride 0.9g

250ml: glucose 12.5g and sodium chloride 2.25g

250ml: glucose 25g and sodium chloride 2.25g

500ml: glucose 25g and sodium chloride 4.5g

500ml: glucose 50g and sodium chloride 4.5g

In addition, the method for smart filter can also use the method for ultrafiltration to filter except the miillpore filter that uses 0.22 μ m filters before the embedding, and its molecular weight cutoff value is 50,000～100,000.

The method of quality control of parenteral solution:

[finger-print] high effective liquid chromatography for measuring.

With octadecylsilane chemically bonded silica is the C18 post of filling agent; 25 ℃ of column temperatures; With 0.1% aqueous formic acid is mobile phase A, is Mobile phase B with methyl alcohol, and two kinds of moving phases of A, B are carried out gradient elution by following condition of gradient elution: the proportionate relationship that adds 5% methanol solution by 95% 0.1% formic acid solution in the time of 0 to 5 minute is carried out wash-out; From 5 to 60 minutes, reduce to 50% by 0.1% formic acid solution by 95%, methyl alcohol is raised to 50% proportionate relationship by 5% and carries out wash-out; Flow velocity 1ml/min; The detection wavelength is 300nm; Number of theoretical plate calculates by the chlorogenic acid peak should be not less than 3000;

It is an amount of that the chlorogenic acid reference substance is got in the preparation of object of reference solution, and accurate the title decides, and adds 50% methyl alcohol and makes the solution that contains 160 μ g among every 1ml, promptly;

It is an amount of that parenteral solution is got in the preparation of need testing solution, filters with 0.45 μ m miillpore filter, promptly;

Accurate respectively object of reference solution and each the 20 μ l of need testing solution of drawing of determination method inject liquid chromatograph, measure by above-mentioned chromatographic condition; Be the S peak with the corresponding peak of object of reference in the test sample finger-print, calculate relative retention time and relative peak area ratio, the test sample finger-print should be similar to standard finger-print; The test sample finger-print has 10 total peaks;

No. 1 peak of relative retention time scope: 0.340～0.376, No. 2 peaks: 0.641～0.708,3 (S): 1.000, No. 4 peaks: 1.000～1.098, No. 5 peaks: 1.032～1.141, No. 6 peaks: 1.302～1.439, No. 7 peaks: 1.727～1.909, No. 8 peaks: 1.763～1.948, No. 9 peaks: 1.947～2.152, No. 10 peaks: 2.070～2.288;

No. 1 peak of relative peak area ratio range: 5.409～8.113, No. 2 peaks: 1.340～2.234, No. 4 peaks: 1.025～1.708, No. 5 peaks: 0.709～1.182;

Non-total peak area must not surpass 5% of total peak area;

[assay]

Cinnamic acid is according to high effective liquid chromatography for measuring.

Chromatographic condition and system suitability test are the C18 post of filling agent with octadecylsilane chemically bonded silica; Methyl alcohol (20～21%) one 0.1% phosphoric acid solution (55～60%)-acetonitrile (20～24%) is a moving phase, comparatively desirable moving phase is: 21: 55: 24 methyl alcohol-0.1% phosphoric acid solution-acetonitrile, and wherein phosphoric acid solution is also replaceable is formic acid solution, glacial acetic acid solution; The detection wavelength is 278nm.Number of theoretical plate calculates by the cinnamic acid peak should be not less than 1500;

It is an amount of that the cinnamic acid reference substance is got in the preparation of reference substance solution, and accurate the title decides, and adds 50% methyl alcohol and makes the solution that contains 2.0 μ m among every 1ml;

The preparation precision of need testing solution is measured parenteral solution 1.0ml, puts in the 10ml measuring bottle, adds 50% methyl alcohol and is diluted to scale, shakes up, promptly;

Accurate respectively reference substance solution and each the 20 μ l of need testing solution of drawing of determination method inject liquid chromatograph, measure, promptly;

The every 1ml of parenteral solution contains radix scrophulariae must not be less than 0.03mg in cinnamic acid (C9H8O2).

Total chlorogenic acid

It is an amount of that the chlorogenic acid reference substance is got in the preparation of reference substance solution, and accurate the title decides, and adds water and makes the solution that contains 0.4mg among every 1ml, promptly;

Typical curve prepares precision and measures reference substance solution 0.5ml, 1.0ml, 1.5ml, 2.0ml, 2.5ml, puts respectively in the 50ml measuring bottle, adds water to scale, shakes up; With water is blank, according to spectrophotometric method, measures absorbance log at the wavelength place of 347nm, is ordinate with the absorbance log, is horizontal ordinate with concentration, the drawing standard curve;

The determination method precision is measured parenteral solution 1.0ml, puts in the 100ml measuring bottle, adds water to scale, shakes up, and measures absorbance log at 347nm wavelength place, reads chlorogenic acid weight the need testing solution (μ g) from typical curve, calculates, promptly;

The every 1ml of parenteral solution contain honeysuckle in total chlorogenic acid by chlorogenic acid (C16H18O9), must not be less than 0.80mg.

The alleged parenteral solution of the present invention comprises that specification is the injection formulation of 50ml, 100ml, 250ml, 500ml, 1ml, 2ml, 5ml, 10ml, 20ml.

The purpose of setting up traditional Chinese medicine fingerprint is kind, quantity and the proportionate relationship that comprehensively reflects inherent chemical constitution contained in the Chinese medicine, thereby the quality that comprehensively reflects Chinese medicine, especially in present stage, Effective Components of Chinese Herb is most not clear and definite, and the method for employing traditional Chinese medicine fingerprint more can effectively reflect the inherent quality of Chinese medicine.Therefore, the chromatographic fingerprinting that adopts in this method of quality control can be controlled on the whole to the product quality of MAILUONING ZHUSHEYE, only the content of single composition is controlled and be not limited to, thereby has been guaranteed the effective and steady quality of this product safety.The method of quality control of former MAILUONING ZHUSHEYE only adopts single cinnamic acid content assaying method, and as seen weak point is arranged.

Adopt 347nm as detecting wavelength in this method of quality control in the total chlorogenic acid assay method, other composition is to the interference of total chlorogenic acid when having avoided conventional employing 326nm wavelength place to measure.Total chlorogenic acid and cinnamic acid in the MAILUONING ZHUSHEYE total solid matters proportion greater than 25%, the foundation of this assay method has solved the problem that total content that the Chinese medicine intravenous injection requires to measure composition is not less than total solid 25%, illustrates that total chlorogenic acid and cinnamic acid are one of MAILUONING ZHUSHEYE principal ingredients.

Following experimental example and embodiment are used to further specify the present invention.

Experimental example 1: the methodological study of finger-print

Stability test: get with a need testing solution, detect at different time respectively, investigate the consistance of chromatographic peak retention time and peak area ratio, the results are shown in Table 1 and table 2, the result shows that the MAILUONING ZHUSHEYE test sample is stable in 12h.

Table 1 MAILUONING ZHUSHEYE stability test result (relative retention time)

Peak number	0h	2h	4.5h	6h	8h	12h	Average	RSD％
									1	0.364	0.367	0.365	0.365	0.364	0.360	0.364	0.634
2	0.681	0.683	0.681	0.681	0.681	0.679	0.681	0.182
									3(S)	1.000	1.000	1.000	1.000	1.000	1.000	1.000	0.000
4	1.045	1.046	1.046	1.047	1.046	1.044	1.046	0.076
									5	1.087	1.087	1.087	1.087	1.087	1.088	1.087	0.040
6	1.365	1.364	1.365	1.366	1.365	1.365	1.365	0.044
									7	1.802	1.799	1.803	1.800	1.804	1.812	1.803	0.271
8	1.838	1.835	1.839	1.836	1.840	1.849	1.840	0.270
									9	2.027	2.024	2.029	2.026	2.031	2.041	2.030	0.286
10	2.152	2.150	2.156	2.152	2.155	2.163	2.155	0.203

Table 2 MAILUONING ZHUSHEYE stability test result (relative peak area ratio)

Peak number	0h	2h	4.5h	6h	8h	12h	Average	RSD％
									1	6.766	6.791	6.811	6.830	6.976	6.946	6.853	1.261
2	1.871	1.858	1.867	1.868	1.865	1.868	1.866	0.226
									3(S)	1.000	1.000	1.000	1.000	1.000	1.000	1.000	0.000
4	1.458	1.451	1.445	1.441	1.411	1.421	1.438	1.258
									5	0.981	0.981	0.981	0.981	0.980	0.980	0.981	0.056
6	-	-	-	-		-	-	-
									7	-	-	-	-		-	-	-
8	-	-	-	-		-	-	-
									9	-	-	-	-		-	-	-
10	-	-	-	-		-	-	-

The precision test: get same need testing solution, continuous sample introduction 6 times is investigated the relative retention time of chromatographic peak and the consistance of peak area ratio.The results are shown in Table 3 and table 4.Each chromatographic peak of the total peak area ratio of regulation, the relative standard deviation of its peak area ratio is all less than 3%.

Table 3 MAILUONING ZHUSHEYE Precision test result (relative retention time)

Peak number	1	2	3	4	5	6	Average	RSD％
									1	0.364	0.363	0.367	0.363	0.365	0.365	0.364	0.377
2	0.681	0.681	0.683	0.681	0.681	0.681	0.681	0.106
									3(S)	1.000	1.000	1.000	1.000	1.000	1.000	1.000	0.000
4	1.045	1.045	1.046	1.046	1.046	1.047	1.046	0.065
									5	1.087	1.086	1.087	1.087	1.087	1.087	1.087	0.044
6	1.365	1.361	1.364	1.365	1.365	1.366	1.364	0.117
									7	1.802	1.796	1.799	1.802	1.803	1.800	1.800	0.144
8	1.838	1.832	1.835	1.839	1.839	1.836	1.837	0.144
									9	2.027	2.022	2.024	2.029	2.029	2.026	2.026	0.143
10	2.152	2.147	2.150	2.156	2.156	2.152	2.152	0.160

Table 4 MAILUONING ZHUSHEYE Precision test result (relative peak area ratio)

Peak number	1	2	3	4	5	6	Average	RSD％
									1	6.766	6.779	6.791	6.810	6.811	6.830	6.798	0.345
2	1.871	1.854	1.858	1.857	1.867	1.868	1.862	0.369
									3(S)	1.000	1.000	1.000	1.000	1.000	1.000	1.000	0.000
4	1.458	1.454	1.451	1.448	1.445	1.441	1.449	0.431
									5	0.981	0.981	0.981	0.981	0.981	0.981	0.981	0.026
6	-	-	-	-	-	-	-	-
									7	-	-	-	-	-	-	-	-
8	-	-	-	-	-	-	-	-
									9	-	-	-	-	-	-	-	-
10	-	-	-	-	-	-	-	-

The reappearance experiment: get the parenteral solution of same lot number, 6 parts of parallel preparation test samples, and detect.The results are shown in Table 5 and table 6.Each chromatographic peak of the total peak area ratio of regulation in finger-print, the relative standard deviation of its relative peak area ratio is all less than 3%.

Table 5 MAILUONING ZHUSHEYE reproducible test results (relative retention time)

Peak number	1	2	3	4	5	6	Average	RSD％
									1	0.363	0.363	0.363	0.363	0.362	0.364	0.363	0.201
2	0.680	0.680	0.680	0.680	0.678	0.681	0.680	0.149
									3(S)	1.000	1.000	1.000	1.000	1.000	1.000	1.000	0.000
4	1.046	1.046	1.046	1.025	1.041	1.045	1.041	0.807
									5	1.087	1.087	1.087	1.087	1.083	1.087	1.087	0.163
6	1.365	1.365	1.365	1.365	1.359	1.364	1.364	0.193
									7	1.804	1.804	1.804	1.805	1.795	1.800	1.802	0.216
8	1.841	1.840	1.841	1.842	1.831	1.837	1.839	0.223
									9	2.031	2.031	2.031	2.033	2.020	2.026	2.029	0.232
10	2.156	2.156	2.156	2.158	2.145	2.151	2.154	0.231

Table 6 MAILUONING ZHUSHEYE reproducible test results (relative peak area ratio)

Peak number	1	2	3	4	5	6	Average	RSD％
									1	6.690	6.462	6.672	6.495	6.636	6.529	6.581	1.481
2	1.870	1.830	1.814	1.815	1.813	1.821	1.827	1.210
									3(S)	1.000	1.000	1.000	1.000	1.000	1.000	1.000	0.000
4	1.483	1.461	1.421	1.450	1.424	1.443	1.447	1.603
									5	0.986	0.968	0.965	0.965	0.964	0.965	0.969	0.864
6	-	-	-	-	-	-	-	-
									7	-	-	-	-	-	-	-	-
8	-	-	-	-	-	-	-	-
									9	-	-	-	-	-	-	-	-
10	-	-	-	-	-	-	-	-

Experimental example 2: the research of finger-print and every technical parameter

Finger-print: Fig. 1 is finger-print and every technical parameter of MAILUONING ZHUSHEYE, and Fig. 2 is blank baseline collection of illustrative plates, and Fig. 3 is the object of reference collection of illustrative plates, and Fig. 4 is the finger-print of 2 hours MAILUONING ZHUSHEYE of record.

The demarcation at total peak: according to the testing result of 10 batches of test samples, selected each batch sample total, retention time and peak area metastable 10 chromatographic peaks are as the total peak of MAILUONING ZHUSHEYE finger-print, its peak area sum accounts for peak area greater than 95%.Analyze through HPLC/MS, preliminary definite wherein 4 peaks are respectively 5 hydroxymethyl furfural (No. 1 peak), chlorogenic acid (No. 3 peaks), caffeic acid (No. 4 peaks), cinnamic acid (No. 10 peaks).

The ratio of total peak area: the relative retention time testing result at 10 batches of each total peaks of test sample sees Table 7, and the ratio testing result of each total peak area sees Table 8.

Non-total peak area: the number percent testing result of each non-total peak area sees Table 9 in the collection of illustrative plates of 10 batches of test samples.

10 batches of MAILUONING ZHUSHEYE testing results of table 7 (relative retention time)

Peak number	200112081	200112091	200302162	200303061	200303201	200303202	200303211	200304021	200304112	200304121	Average
												1	0.358	0.358	0.355	0.354	0.360	0.360	0.360	0.358	0.358	0.358	0.358
2	0.674	0.674	0.673	0.671	0.674	0.674	0.675	0.676	0.676	0.676	0.674
												3(S)	1.000	1.000	1.000	1.000	1.000	1.000	1.000	1.000	1.000	1.000	1.000
4	1.044	1.044	1.045	1.045	1.046	1.046	1.046	1.046	1.046	1.046	1.046
												5	1.087	1.087	1.087	1.087	1.087	1.087	1.087	1.087	1.086	1.086	1.087
6	1.369	1.369	1.372	1.373	1.373	1.372	1.372	1.371	1.369	1.369	1.371
												7	1.816	1.817	1.821	1.825	1.822	1.820	1.820	1.815	1.811	1.811	1.818
8	1.854	1.855	1.858	1.862	1.860	1.858	1.859	1.852	1.849	1.848	1.856
												9	2.047	2.048	2.054	2.059	2.054	2.052	2.053	2.047	2.043	2.042	2.050
10	2.177	2.178	2.185	2.191	2.183	2.182	2.182	2.175	2.170	2.169	2.179

10 batches of MAILUONING ZHUSHEYE testing results of table 8 (relative peak area ratio)

Peak number	200112081	200112091	200302162	200303061	200303201	200303202	200303211	200304021	200304112	200304121	Average
												1	7.659	7.791	5.993	6.338	6.668	6.756	6.582	6.631	6.735	6.456	6.761
2	1.757	1.751	1.745	1.849	1.754	1.743	1.753	1.788	1.841	1.887	1.787
												3(S)	1.000	1.000	1.000	1.000	1.000	1.000	1.000	1.000	1.000	1.000	1.000
4	1.238	1.297	1.195	1.323	1.236	1.154	1.370	1.496	1.689	1.662	1.366
												5	0.950	0.951	0.929	0.950	0.947	0.944	0.956	0.965	0.922	0.941	0.945
6	0.682	0.714	0.323	0.366	0.338	0.334	0.387	0.364	0.426	0.448	0.438
												7	1.054	1.139	1.025	1.008	0.943	0.970	1.002	1.018	1.064	1.158	1.038
8	0.478	0.522	0.526	0.533	0.484	0.495	0.522	0.502	0.562	0.614	0.524
												9	0.824	0.885	0.772	0.736	0.697	0.722	0.727	0.752	0.784	0.836	0.773
10	2.393	2.424	0.917	1.056	0.958	0.940	0.978	0.999	1.213	1.203	1.308

10 batches of MAILUONING ZHUSHEYE testing results of table 9 (non-total peak area)

Lot number	200112081	200112091	200302162	200303061	200303201	200303202	200303211	200304021	200304112	200304121	Average
												Non-total peak area number percent (%)	4.24	4.52	2.71	2.27	2.39	2.43	2.40	2.20	1.74	1.82	2.67

Experimental example 3: the correlation research of parenteral solution, intermediate and Chinese crude drug finger-print

Utilize the HPLC/DAD/MS technology, we investigate the correlativity of MAILUONING ZHUSHEYE, intermediate and medicinal material, the result shows, between MAILUONING ZHUSHEYE and its intermediate correlativity is preferably arranged, and the stem of noble dendrobium, the root of bidentate achyranthes, radix scrophulariae and honeysuckle four traditional Chinese medicine material all have corresponding embodiment in parenteral solution and intermediate, the results are shown in Table 10.

The correlativity of table 10 MAILUONING ZHUSHEYE, intermediate and medicinal materials fingerprint

t R(min)	m/z	Infer structure	The source medicinal material	Mass spectrogram
					10.37	127[M+H] +，109[M+H-H 2O] +	5 hydroxymethyl furfural	The root of bidentate achyranthes	Figure 19
26.97	355[M+H] +，377[M+Na] +	Chlorogenic acid	Honeysuckle	Figure 20
					28.16	181[M+H] +，163[M+H-H 2O] +	Caffeic acid	Honeysuckle	Figure 21
29.80	264[M+H] +	Dendrobine	The stem of noble dendrobium	Figure 22
					47.29	515[M-H] -	Isochlorogenic acid	Honeysuckle	Figure 23
56.57	149[M+H] +，131[M+H-H 2O] +	Cinnamic acid	Radix scrophulariae	Figure 24

Extract respectively according to injection preparation technology and to have prepared the parenteral solution that only contains the single medicinal material, measure respectively in parenteral solution 1.0ml to the 10ml measuring bottle of single medicinal material, thin up shakes up to scale, filters with the 0.45um miillpore filter, as need testing solution.

Condition determination is pressed parenteral solution finger-print chromatographic condition, and promptly moving phase is the A:0.1% aqueous formic acid, B: methyl alcohol, flow velocity 1ml/min; Detect wavelength 300nm; Reference wavelength: 390nm; Column temperature: 25 ℃, sample size 20ul, be 90min writing time.The gradient elution program is:

	A％(V/V)	B％(V/V)
			0min 5min 60min	95 95 50	5 5 50

Detecting wavelength is the detection wavelength 300nm of MAILUONING ZHUSHEYE finger-print and the detection wavelength of each flavor medicinal materials fingerprint.The results are shown in Table 11.

The correlativity of table 11 MAILUONING ZHUSHEYE, intermediate and medicinal materials fingerprint

Total peak number in the parenteral solution	The root of bidentate achyranthes	Dendrobium	Stem of Eyeshaped Dendrobium	Radix scrophulariae	Flos Lonicerae	Honeysuckle
								1	+	-	-	+	+	+
2	-	-	-	-	+	+
							3(s)	-	-	-	-	+	+
4	-	-	-	-	+	+
							5	-	-	-	-	+	+
6	-	-	-	-	+	+
							7	-	-	-	-	+	+
8	-	-	-	-	+	+
							9	-	-	-	-	+	+
10	-	-	-	+	-	-

+: in the HPLC figure of single medicinal material test liquid, this peak is arranged ,-: in the HPLC figure of single medicinal material test liquid, there is not this peak

Experimental example 4: the research of cinnamic acid assay

1. instrument and reagent

515 type high performance liquid chromatographs (production of U.S. Waters company), 996 type diode array detector, M32 chromatographic work station.

Methyl alcohol, acetonitrile are chromatographically pure (production of U.S. world company), and water is ultrapure water, and it is pure that other reagent are analysis.

The cinnamic acid reference substance is available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute, lot number 786-9001.

2. chromatographic condition

Octadecylsilane chemically bonded silica is filling agent C18 post (Phenomenex company produces for 4.6 * 250mm, granularity 5 μ m), moving phase is methyl alcohol-acetonitrile-0.1% phosphoric acid solution (21: 24: 55), and flow velocity 1ml/min, column temperature are room temperature, detect wavelength 278nm, sample size 20 μ l.

3. test method

3.1 chromatographic condition is selected

Cinnamic acid assay moving phase is methyl alcohol-0.1% phosphoric acid solution (55: 45) in MAILUONING ZHUSHEYE (10ml) the proper mass standard, through adjusting and optimizing, determine that methyl alcohol-acetonitrile-0.1% phosphoric acid solution (21: 24: 55) is to measure the comparatively desirable moving phase of cinnamic acid content on this basis.

3.2 the range of linearity

Precision takes by weighing cinnamic acid reference substance 6.40mg and puts in the 100ml measuring bottle, add 50% dissolve with methanol and be diluted to scale, precision is measured 0.1ml, 0.2ml, 0.4ml, 0.6ml, 0.8ml, 1.0ml respectively, 1.5ml puts in the 10ml measuring bottle, add 50% methyl alcohol to scale, making concentration is the cinnamic acid reference substance solution of 0.64 μ g/ml, 1.28 μ g/ml, 2.56 μ g/ml, 3.84 μ g/ml, 5.12 μ g/ml, 6.40 μ g/ml, 9.60 μ g/ml, sample introduction 20 μ l the results are shown in Table 12.

Table 12 cinnamic acid typical curve

Sequence number

	1	2	3	4	5	6	7
								Concentration (μ g/ml) peak area	0.64 96346	1.28 188979	2.56 382978	3.84 589017	5.12 788208	6.40 998991	9.60 1484455

With reference substance concentration is horizontal ordinate, is ordinate drawing standard curve with the cinnamic acid peak area, gets regression equation Y=155918X-8750.1, and r=0.9999 shows that cinnamic acid has favorable linearity in the concentration range of 0.64-9.60 μ g/ml.

3.3 system suitability test

Press the described preparation test sample of text, sample introduction 20 μ l, the number of theoretical plate and the tailing factor at calculating cinnamic acid peak.

3.3.1 number of theoretical plate

+n＝5.54(tR/Wh/2)2＝5.54×(12.514÷0.267)2＝12169

3.3.2 tailing factor

T＝W0.05h/2d1＝0.521÷(2×0.253)＝1.03

3.4 precision test

Get need testing solution, continuous sample introduction 5 times, sample size 20 μ l measure peak area, calculate the RSD value, the results are shown in Table 13.

The test of table 13 precision

The sample introduction number of times	1	2	3	4	5
						Peak area RSD	1095052 0.19％	1098044	1094911	1096006	1099597

The result shows that this method has good precision.

3.5 replica test

Get same batch sample, prepare 5 parts of need testing solutions by the text method, sample introduction 20 μ l measure the cinnamic acid peak area, calculate the RSD value, the results are shown in Table 14.

Table 14 replica test

Sequence number	Peak area	Content (mg/ml)	Average content (mg/ml)	RSD
					1 2 3 4 5	1097802 1086589 1089769 1084842 1081195	0.05884 0.05824 0.05841 0.05815 0.05795	0.05832	0.58％

The result shows that this law has good repeatability.

3.6 stability test

Get above-mentioned need testing solution,, measure peak area, calculate the RSD value, the results are shown in Table 15 respectively at 0,2,4,8,12 hour sample introduction.

Table 15 stability test

Time (hour)	0	2	4	8	12
						Peak area mean value RSD	1096006 0.9％	1073610	1085133 1088430	1090504	1096895

The result shows in 12 hours and to measure that cinnamic acid content is comparatively stable in the test sample.

3.7 recovery test

It is that the sample 0.5ml of 43.44 μ g/ml puts in the 10ml measuring bottle that precision is measured known cinnamic acid content, and accurate cinnamic acid reference substance solution (the 6.4 μ g/ml) 4.0ml that adds adds 50% methyl alcohol and is diluted to scale, measures content, and calculate recovery rate the results are shown in Table 16.

Table 16 recovery test

Sequence number	Test sample content (μ g/ml)	Reference substance addition (μ g/ml)	Measured value (μ g/ml)	The recovery (%)	Average recovery rate (%)	RSD
							1 2 3 4 5	2.172 2.172 2.172 2.172 2.172	2.560 2.560 2.560 2.560 2.560	47.35 47.44 46.48 46.83 46.88	100.00 100.50 96.72 98.08 98.28	98.72	1.55％

Annotate:

The recovery (%)=(measured value-test sample content) ÷ reference substance addition * 100%

The result shows that this law has the good recovery.

3.8 detectability

Precision is measured cinnamic acid reference substance solution (64 μ g/ml) 0.05ml and is put in the 50ml measuring bottle, add 50% methyl alcohol and be diluted to scale, make the cinnamic acid reference substance solution of 0.064 μ g/ml, sample introduction 20 μ l, noise is 0.00012, and response 0.00031, signal to noise ratio (S/N ratio) are 2.6, therefore determine to detect to be limited to 15 * 10-10g, quantitatively be limited to 49 * 10-10g.

3.9 blank test

Prepare blank sample (lacking radix scrophulariae) by production technology, and press the described preparation test sample of text and measure, the result does not see the cinnamic acid peak, illustrates that cinnamic acid is the exclusive composition of radix scrophulariae, and is blank to its not interference.

3.10 sample determination

Press the described content assaying method of text, measure cinnamic acid content in 3 batches of MAILUONING ZHUSHEYEs (100ml), the results are shown in Table 17.

Cinnamic acid content in table 17 MAILUONING ZHUSHEYE (100ml)

Lot number	20020306	20020307	20020308
				Cinnamic acid content (mg/ml)	0.04420	0.04408	0.04325

The result shows that cinnamic acid content is than increasing to some extent in the MAILUONING ZHUSHEYE (10ml) in every bottle of MAILUONING ZHUSHEYE (100ml).

The research of experimental example 5, total chlorogenic acid assay

Be rich in chlorogenic acid and isomeride thereof (isochlorogenic acid, neochlorogenic acid, Cryptochlorogenic acid etc.) in many bibliographical information honeysuckles.They have identical parent nucleus, and therefore also having extremely similarly, ultraviolet spectrum absorbs.Parenteral solution technology extracting method can effectively extract such organic acid, therefore total chlorogenic acid in the parenteral solution is carried out quality controling research and is very important.

Because the content of isomer of chlorogenic acid is few, character is unstable, reference substance is difficult to preparation, only chlorogenic acid has been carried out assay in most quality standards, does not relate to other isomeride.Parenteral solution is in finger-print research, and finding has the ultraviolet absorption curve of 7 chromatographic peaks consistent with chlorogenic acid.Through contrast, can determine that one of them peak is a chlorogenic acid.By to the discovering of traditional Chinese medicine honeysuckle, these chromatographic peaks be in the honeysuckle chlorogenic acid in extracting subtractive process progressively development produce, therefore these compositions ownership can be total chlorogenic acid, decide content with the chlorogenic acid instrumentation.

Through the sample spectrogram is carried out three-dimensional UV scanning, gather the ultra-violet absorption spectrum of all chromatographic peaks, discovery is except total chlorogenic acid, all the other compositions in the above uv absorption of 340nm especially 347nm seldom or do not have, so can choose the uv absorption of 347nm place working sample, with total chlorogenic acid in the determined by ultraviolet spectrophotometry parenteral solution, see accompanying drawing 5.

1. specificity is investigated

1.1 the chromatographic condition octadecylsilane chemically bonded silica is filling agent AngilentExtentC-18 chromatographic column (5 μ m, 4.6 * 250mm); The Waters2690 chromatograph; The Waters996PDA diode array detector; Waters M32 workstation: moving phase: methyl alcohol-0.1% phosphoric acid solution-acetonitrile, gradient elution; Flow velocity 1ml/min; Column temperature: 35 ℃: the detection wavelength is 327nm.

Table 18 gradient elution proportion of mobile phase changes program

Time (minute)	Methyl alcohol (%)	0.1% phosphoric acid solution (%)	Acetonitrile (%)	Linear
					0 30 50 60 62	1 5 5 5 1	95 80 75 65 95	4 15 20 30 4	5 5 6 6

1.2 honeysuckle prepares by technology, each step process point sample collection:

1.2.1 crude drug (handling by method under honeysuckle item of Chinese Pharmacopoeia version in 2000) is got Flos Lonicerae and honeysuckle each 0.5g of medicinal powder, the accurate title, decide, put in the 100ml tool plug conical flask, the accurate 50% methyl alcohol 50.0ml that adds claims to decide weight, 1 hour (250W of Extraction by Ultrasound, 40KHz), put and supply weight after cold, filter, discard filtrate just, get subsequent filtrate as test sample.

1.2.2 decocting boils

Get Flos Lonicerae and honeysuckle each 100g of medicinal powder, add 6 times water, decoct secondary, each 1.5 hours, merge decoction liquor, filter, get subsequent filtrate as test sample.

1.2.3 alcohol precipitation

Get above-mentioned filtrate and concentrate, cooling adds ethanol and makes and contain the alcohol amount to 80%.Leave standstill, get supernatant as test sample.

1.2.4 ethyl acetate extracts

Get above-mentioned supernatant, the ethyl acetate extraction with 3 times of amount volumes merges the ester extract, and heating eliminates ethyl acetate, medicinal extract adds water boil, and cooling is filtered, and transfers pH to 9.0-9.2 with 20%NaOH solution, be transferred in the 50ml measuring bottle, thin up is to scale, as test sample.

1.2.5 preparation

Precision is measured MAILUONING ZHUSHEYE (100ml) 5.0ml and is put in the 25ml measuring bottle, and thin up shakes up to scale, as the preparation test sample.

1.3 reference substance solution preparation

It is an amount of to get the chlorogenic acid reference substance, makes the 0.05mg/ml reference substance solution with 50% methyl alcohol.

1.4 determination method

Accurate reference substance solution and each 20ul of need testing solution of drawing injects liquid chromatograph by above-mentioned chromatographic condition, measures, and writes down 60 minutes chromatograms.

1.5 test findings

Chromatogram to each need testing solution compares, chlorogenic acid reduces in process of production gradually in the honeysuckle, development produces other compositions, and the ultra-violet absorption spectrum of these compositions is almost identical with chlorogenic acid, can tentatively be defined as the isomeride or the decomposition product of chlorogenic acid with reference to interrelated data, its ultraviolet spectrum behavior is consistent, can be used as the element of the first species and measures.

Because used detecting device is a diode array detector, can carry out full wavelength scanner to each chromatographic peak, find except total chlorogenic acid by contrast, all the other compositions in the above uv absorption of 340nm especially 347nm seldom or do not have, so parenteral solution is selected total chlorogenic acid in the 347nm place determined by ultraviolet spectrophotometry parenteral solution for use.

2. instrument and reagent

Tianjin, island UV-1601PC ultraviolet spectrophotometer, Tianjin, island UVPC workstation, 1cm quartz cuvette.

Water is pure water, and it is pure that agents useful for same is analysis.

The chlorogenic acid reference substance is available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute, lot number 0753-9607.

3. linear relationship

Precision takes by weighing chlorogenic acid reference substance 8.54mg and puts in the 25ml measuring bottle, and thin up shakes up to scale, promptly gets reference substance solution (containing chlorogenic acid 0.3416mg among every 1ml).

Typical curve prepares precision measures reference substance solution 0.5ml, 1.0ml, 1.5ml, 2.0ml, 2.5ml, 3.0ml and puts respectively in the 50ml measuring bottle, adds water to scale, shakes up.Making chlorogenic acid content is the solution of 3.416 μ g/ml, 6.832 μ g/ml, 10.248 μ g/ml, 13.664 μ g/ml, 17.080 μ g/ml, 20.496 μ g/ml, with water is blank, according to spectrophotometric method (Chinese Pharmacopoeia version appendix in 2000 VB), measure absorbance log at the 347nm place.With the absorbance log is ordinate, is horizontal ordinate with concentration, the drawing standard curve.The results are shown in Table 19.

Table 19 linear relationship is measured

Concentration (μ g/ml)	3.416	6.832	10.248	13.664	17.080	20.496
							Absorption value	0.104	0.203	0.299	0.393	0.485	0.583

The test findings statistics, chlorogenic acid reference substance concentration and ultraviolet absorptivity regression equation are A=0.0279 * C+0.011, r=0.9999.Show that chlorogenic acid is good linear relationship with ultraviolet absorptivity in 3.416～20.496 μ g/ml concentration ranges.

4. precision test

Get need testing solution and measure ultraviolet absorptivity, measure 6 times, the results are shown in Table 20.

The test of table 20 precision

Sequence number

	1	2	3	4	5
						Measured value mean value RSD	0.388 0.388 0.21％	0.388	0.387	0.389	0.389

The result shows that this method has good precision.

5. replica test

Get same batch sample, prepare 6 parts of need testing solutions, measure ultraviolet absorptivity, calculate the RSD value, the results are shown in Table 21 by the text method.

Table 21 replica test

Sequence number	Ultraviolet absorptivity	Content (mg/ml)	Average content (mg/ml)	RSD
					1 2 3 4 5 6	0.388 0.388 0.388 0.390 0.392 0.395	1.351 1.351 1.351 1.358 1.365 1.376	1.359	0.75％

The result shows that this law has good repeatability.

6 stability tests

Get above-mentioned need testing solution,, calculate the RSD value, the results are shown in Table 22 respectively at 0,1,2,4,8,12 hour mensuration ultraviolet absorptivity.

Table 22 stability test

Time (hour)	0	1	2	4	8	12
							Measured value mean value RSD	0.388 0.388 0.27％	0.388	0.389	0.387	0.387	0.386

The result shows, measures in 12 hours that total chlorogenic acid content is stable in the test sample.

7 recovery tests

It is that the sample 0.5ml of 1.359mg/ml places the 100ml measuring bottle that precision is measured known content, accurate respectively chlorogenic acid reference substance solution (0.3416mg/ml) 1.5ml, 2.0ml, the 2.5ml of adding, thin up is measured uv absorption to scale at the 347nm place, calculate recovery rate the results are shown in Table 23.

Table 23 recovery test (n=6)

Sequence number	Test sample content (μ g/ml)	Reference substance addition (μ g/ml)	Actual measurement content (μ g/ml)	The recovery (%)	Mean value (%)	RSD
							1 2 3 4 5 6	6.795 6.795 6.795 6.795 6.795 6.795	5.124 5.124 6.832 6.832 8.540 8.540	12.043 11.953 13.513 13.423 15.341 15.394	101.04 100.29 99.16 98.50 100.04 100.39	99.90	0.92％

Annotate:

The recovery (%)=(measured value-test sample content) ÷ reference substance addition * 100%

The result shows that this law has the good recovery.

8 blank tests

Prepare blank sample (lacking honeysuckle) by production technology, and to measure the 347nm uv absorption by the described preparation test sample of text be 0.004, be lower than 5% of range of linearity least concentration (3.416 μ g/ml) absorbance log, blank not interference is described, this method has specificity.

9 sample determinations

Measure total chlorogenic acid content in 3 batch samples according to the method described above, the results are shown in Table 24.

Table 24 total chlorogenic acid assay

Lot number	20020306	20020307	20020308
				Content (mg/ml)	0.949	0.867	1.153

Experimental example 6, principal component accounts for the research of total solids

" with clean medicinal material is that the injection that component is prepared should be studied mensuration effective constituent, index components or total constituents to regulation in " technical requirement of traditional Chinese medicine research ", selects the method for favorable reproducibility, and does the methodological study test.The total content of the composition of measuring should be not less than 20% (vein is with being no less than 25%) of total solid.The additives of adjusting osmotic pressure etc. by actual addition deduction, should not count." 3 batch samples are measured total solid, total chlorogenic acid content and cinnamic acid content, the total content that calculates the composition of measuring accounts for the ratio of total solid, the results are shown in Table 25.

Table 25 principal component accounts for the content (former table 20) of total solid matters

Lot number	Actual measurement total solid (mg/ml)	Deduction additives total solids (mg/ml)	Chlorogenic acid organic acid content (mg/ml)	Cinnamic acid content (mg/ml)	Principal component accounts for the content (%) of total solid matters
						20020306 20020307 20020308	11.56 11.78 13.08	2.35 2.54 3.80	0.949 0.867 1.153	0.04420 0.04408 0.04325	42.26 35.87 31.48

Annotate:

Deduction additives amount of solid=actual measurement total solid-NaCl content-Tween 80 content-NaOH content

Test findings can illustrate that the ratio that total chlorogenic acid and cinnamic acid content in the parenteral solution account for total solid has reached more than 25%, and is up to specification.

Description of drawings:

Fig. 1: be finger-print and every technical parameter of MAILUONING ZHUSHEYE of the present invention;

Fig. 2: be blank baseline collection of illustrative plates;

Fig. 3: be object of reference chromatographic peak collection of illustrative plates;

Fig. 4: for writing down the finger-print of 2 hours MAILUONING ZHUSHEYE of the present invention;

Fig. 5: be MAILUONING ZHUSHEYE uv-spectrogram of the present invention.

Embodiment 1: the mensuration of finger-print

[finger-print] high performance liquid chromatography (Chinese Pharmacopoeia version appendix in 2000 VID) is measured.

Chromatographic condition and system suitability test are the C18 post (4.6 * 250mm, granularity 5 μ m) of filling agent with octadecylsilane chemically bonded silica; 25 ℃ of column temperatures; With 0.1% aqueous formic acid is mobile phase A, with methyl alcohol is Mobile phase B, two kinds of moving phases of A, B are carried out gradient elution by following condition of gradient elution: the proportionate relationship that adds 5% methanol solution by 95% 0.1% formic acid solution in the time of 0 to 5 minute is carried out wash-out, from 5 to 60 minutes, reduce to 50% by 0.1% formic acid solution by 95%, methyl alcohol is raised to 50% proportionate relationship by 5% and carries out wash-out; Flow velocity 1ml/min; The detection wavelength is 300nm; Number of theoretical plate calculates by the chlorogenic acid peak should be not less than 3000;

It is an amount of that the chlorogenic acid reference substance is got in the preparation of object of reference solution, and accurate the title decides, and adds 50% methyl alcohol and makes the solution that contains 160 μ g among every 1ml, promptly;

It is an amount of that parenteral solution is got in the preparation of need testing solution, filters with 0.45 μ m miillpore filter, promptly; Accurate respectively object of reference solution and each the 20 μ l of need testing solution of drawing of determination method inject liquid chromatograph, measure by above-mentioned chromatographic condition.Be the S peak with the corresponding peak of object of reference in the test sample finger-print, calculate relative retention time and relative peak area ratio, should meet the following requirements:

The test sample finger-print should be similar to standard finger-print;

The finger-print Should Be 10 has the peak;

No. 1 peak of relative retention time: 0.364, No. 2 peaks: 0.678,3 (S): 1.000, No. 4 peaks: 1.043, No. 5 peaks: 1.083, No. 6 peaks: 1.362, No. 7 peaks: 1.794, No. 8 peaks: 1.833, No. 9 peaks: 2.020, No. 10 peaks: 2.149;

No. 1 peak of relative peak area ratio: 6.780, No. 2 peaks: 1.363, No. 4 peaks: 1.220, No. 5 peaks: 0.932;

Non-total peak area: must not cross 5% of total peak area.

Embodiment 2: the assay of cinnamic acid

Measure according to high performance liquid chromatography (Chinese Pharmacopoeia version appendix in 2000 VID).

Chromatographic condition and system suitability test are the C18 post (4.6 * 250mm, granularity 5 μ m) of filling agent with octadecylsilane chemically bonded silica; 21: 55: 24 methyl alcohol-0.1% formic acid solution-acetonitrile is a moving phase; The detection wavelength is 278nm.Number of theoretical plate calculates by the cinnamic acid peak should be not less than 1500;

It is an amount of that the cinnamic acid reference substance is got in the preparation of reference substance solution, and accurate the title decides, and adds 50% methyl alcohol and makes the solution that contains 2.0 μ g among every 1ml;

The preparation precision of need testing solution is measured parenteral solution 1.0ml, puts in the 10ml measuring bottle, adds 50% methyl alcohol and is diluted to scale, shakes up, promptly;

Accurate respectively reference substance solution and each the 20 μ l of need testing solution of drawing of determination method inject liquid chromatograph, measure, promptly;

The every 1ml of parenteral solution contains radix scrophulariae must not be less than 0.03mg in cinnamic acid (C9H8O2).

Embodiment 3: the assay of total chlorogenic acid

It is an amount of that the chlorogenic acid reference substance is got in the preparation of reference substance solution, and accurate the title decides, and adds water and makes the solution that contains 0.4mg among every 1ml, promptly.

Typical curve prepares precision and measures reference substance solution 0.5ml, 1.0ml, 1.5ml, 2.0ml, 2.5ml, puts respectively in the 50ml measuring bottle, adds water to scale, shakes up.With water is blank, according to spectrophotometric method (Chinese Pharmacopoeia version appendix in 2000 VB), measures absorbance log at the wavelength place of 347nm.With the absorbance log is ordinate, is horizontal ordinate with concentration, the drawing standard curve.

The determination method precision is measured parenteral solution 1.0ml, puts in the 100ml measuring bottle, adds water to scale, shakes up, and measures absorbance log at 347nm wavelength place, reads chlorogenic acid weight the need testing solution (μ g) from typical curve, calculates, promptly.

The every 1ml of parenteral solution contain honeysuckle in total chlorogenic acid by chlorogenic acid (C16H18O9), must not be less than 0.80mg.

Embodiment 4: the method for quality control of MAILUONING ZHUSHEYE

[finger-print] high performance liquid chromatography (Chinese Pharmacopoeia version appendix in 2000 VID) is measured.

Chromatographic condition and system suitability test are the C18 post (4.6 * 250mm, granularity 5 μ m) of filling agent with octadecylsilane chemically bonded silica; 25 ℃ of column temperatures; With 0.1% aqueous formic acid is mobile phase A, with methyl alcohol is Mobile phase B, two kinds of moving phases of A, B are carried out gradient elution by following condition of gradient elution: the proportionate relationship that adds 5% methanol solution by 95% 0.1% formic acid solution in the time of 0 to 5 minute is carried out wash-out, from 5 to 60 minutes, reduce to 50% by 0.1% formic acid solution by 95%, methyl alcohol is raised to 50% proportionate relationship by 5% and carries out wash-out; Flow velocity 1ml/min; The detection wavelength is 300nm; Number of theoretical plate calculates by the chlorogenic acid peak should be not less than 3000;

It is an amount of that the chlorogenic acid reference substance is got in the preparation of object of reference solution, and accurate the title decides, and adds 50% methyl alcohol and makes the solution that contains 160 μ g among every 1ml, promptly;

It is an amount of that parenteral solution is got in the preparation of need testing solution, filters with 0.45 μ m miillpore filter, promptly;

Accurate respectively object of reference solution and each the 20 μ l of need testing solution of drawing of determination method inject liquid chromatograph, measure by above-mentioned chromatographic condition.Be the S peak with the corresponding peak of object of reference in the test sample finger-print, calculate relative retention time and peak area ratio, should meet the following requirements:

The test sample finger-print should be similar to standard finger-print;

The finger-print Should Be 10 has the peak;

No. 1 peak of relative retention time: 0.364, No. 2 peaks: 0.678,3 (S): 1.000, No. 4 peaks: 1.043, No. 5 peaks: 1.083, No. 6 peaks: 1.362, No. 7 peaks: 1.794, No. 8 peaks: 1.833, No. 9 peaks: 2.020, No. 10 peaks: 2.149;

No. 1 peak of relative peak area ratio: 6.780, No. 2 peaks: 1.363, No. 4 peaks: 1.220, No. 5 peaks: 0.932;

Non-total peak area: must not cross 5% of total peak area.

[assay]

Cinnamic acid is measured according to high performance liquid chromatography (Chinese Pharmacopoeia version appendix in 2000 VID).

Chromatographic condition and system suitability test are the C18 post (4.6 * 250mm, granularity 5 μ m) of filling agent with octadecylsilane chemically bonded silica; 21: 55: 24 methyl alcohol-0.1% phosphoric acid solution-acetonitrile is a moving phase; The detection wavelength is 278nm.Number of theoretical plate calculates by the cinnamic acid peak should be not less than 1500;

It is an amount of that the cinnamic acid reference substance is got in the preparation of reference substance solution, and accurate the title decides, and adds 50% methyl alcohol and makes the solution that contains 2.0 μ g among every 1ml;

The preparation precision of need testing solution is measured parenteral solution 1.0ml, puts in the 10ml measuring bottle, adds 50% methyl alcohol and is diluted to scale, shakes up, promptly;

Accurate respectively reference substance solution and each the 20 μ l of need testing solution of drawing of determination method inject liquid chromatograph, measure, promptly;

The every 1ml of parenteral solution contains radix scrophulariae must not be less than 0.03mg in cinnamic acid (C9H8O2).

Total chlorogenic acid

It is an amount of that the chlorogenic acid reference substance is got in the preparation of reference substance solution, and accurate the title decides, and adds water and makes the solution that contains 0.4mg among every 1ml, promptly;

Typical curve prepares precision and measures reference substance solution 0.5ml, 1.0ml, 1.5ml, 2.0ml, 2.5ml, puts respectively in the 50ml measuring bottle, adds water to scale, shakes up.With water is blank, according to spectrophotometric method (Chinese Pharmacopoeia version appendix in 2000 VB), measures absorbance log at the wavelength place of 347nm.With the absorbance log is ordinate, is horizontal ordinate with concentration, the drawing standard curve;

The determination method precision is measured parenteral solution 1.0ml, puts in the 100ml measuring bottle, adds water to scale, shakes up, and measures absorbance log at 347nm wavelength place, reads chlorogenic acid weight the need testing solution (μ g) from typical curve, calculates, promptly;

The every 1ml of parenteral solution contain honeysuckle in total chlorogenic acid by chlorogenic acid (C16H18O9), must not be less than 0.80mg.

Claims (6)

1, a kind of method of quality control that is used for the treatment of the MAILUONING ZHUSHEYE of thrombosis is characterized in that containing in this method the mensuration of finger-print, and the chromatographic condition of determining fingerprint pattern is:

With octadecylsilane chemically bonded silica is the C of filling agent ₁₈Post; 25 ℃ of column temperatures; With 0.1% aqueous formic acid is mobile phase A, with methyl alcohol is Mobile phase B, two kinds of moving phases of A, B are carried out gradient elution by following condition of gradient elution: the proportionate relationship that adds 5% methanol solution by 95% 0.1% formic acid solution in the time of 0 to 5 minute is carried out wash-out, from 5 to 60 minutes, reduce to 50% by 0.1% formic acid solution by 95%, methyl alcohol is raised to 50% proportionate relationship by 5% and carries out wash-out; Flow velocity 1ml/min; The detection wavelength is 300nm; Number of theoretical plate calculates by the chlorogenic acid peak should be not less than 3000;

The preparation of object of reference solution: it is an amount of to get the chlorogenic acid reference substance, and accurate the title decides, and adds 50% methyl alcohol and makes the solution that contains 160 μ g among every 1ml, promptly;

The preparation of need testing solution: it is an amount of to get parenteral solution, filters with 0.45 μ m miillpore filter, promptly;

Determination method: accurate respectively object of reference solution and each 20 μ l of need testing solution of drawing, inject liquid chromatograph, measure by above-mentioned chromatographic condition; Be the S peak with the corresponding peak of object of reference in the test sample finger-print, calculate relative retention time and relative peak area ratio, the test sample finger-print should be similar to standard finger-print.

2, a kind of method of quality control for the treatment of the MAILUONING ZHUSHEYE of thrombosis as claimed in claim 1 is characterized in that the test sample finger-print is in this method:

The test sample finger-print has 10 total peaks;

No. 1 peak of relative retention time scope: 0.340～0.376, No. 2 peaks: 0.641～0.708, No. 3 peaks: 1.000, No. 4 peaks: 1.000～1.098, No. 5 peaks: 1.032～1.141, No. 6 peaks: 1.302～1.439, No. 7 peaks: 1.727～1.909, No. 8 peaks: 1.763～1.948, No. 9 peaks: 1.947～2.152, No. 10 peaks: 2.070～2.288;

No. 1 peak of relative peak area ratio range: 5.409～8.113, No. 2 peaks: 1.340～2.234, No. 4 peaks: 1.025～1.708, No. 5 peaks: 0.709～1.182;

Non-total peak area must not surpass 5% of total peak area.

3, a kind of method of quality control for the treatment of the MAILUONING ZHUSHEYE of thrombosis as claimed in claim 2 is characterized in that the test sample finger-print is in this method:

The test sample finger-print has 10 total peaks;

No. 1 peak of relative retention time: 0.364, No. 2 peaks: 0.678, No. 3 peaks: 1.00, No. 4 peaks: 1.043, No. 5 peaks: 1.083, No. 6 peaks: 1.362, No. 7 peaks: 1.794, No. 8 peaks: 1.833, No. 9 peaks: 2.020, No. 10 peaks: 2.149;

No. 1 peak of relative peak area ratio: 6.780, No. 2 peaks: 1.363, No. 4 peaks: 1.220, No. 5 peaks: 0.932.

4, a kind of method of quality control for the treatment of the MAILUONING ZHUSHEYE of thrombosis is characterized in that this method is:

A. the mensuration of finger-print

With octadecylsilane chemically bonded silica is the C of filling agent ₁₈Post; 25 ℃ of column temperatures; With 0.1% aqueous formic acid is mobile phase A, is Mobile phase B with methyl alcohol, and two kinds of moving phases of A, B are carried out gradient elution by following condition of gradient elution: the proportionate relationship that adds 5% methanol solution by 95% 0.1% formic acid solution in the time of 0 to 5 minute is carried out wash-out; From 5 to 60 minutes, reduce to 50% by 0.1% formic acid solution by 95%, methyl alcohol is raised to 50% proportionate relationship by 5% and carries out wash-out; Flow velocity 1ml/min; The detection wavelength is 300nm; Number of theoretical plate calculates by the chlorogenic acid peak should be not less than 3000;

The preparation of object of reference solution: it is an amount of to get the chlorogenic acid reference substance, and accurate the title decides, and adds 50% methyl alcohol and makes the solution that contains 160 μ g among every 1ml, promptly;

The preparation of need testing solution: it is an amount of to get parenteral solution, filters with 0.45 μ m miillpore filter, promptly; Determination method: accurate respectively object of reference solution and each 20 μ l of need testing solution of drawing, inject liquid chromatograph, measure by above-mentioned chromatographic condition; Be the S peak with the corresponding peak of object of reference in the test sample finger-print, calculate relative retention time and relative peak area ratio, the test sample finger-print should be similar to standard finger-print; The test sample finger-print has 10 total peaks;

No. 1 peak of relative retention time scope: 0.340～0.376, No. 2 peaks: 0.641～0.708, No. 3 peaks: 1.000, No. 4 peaks: 1.000～1.098, No. 5 peaks: 1.032～1.141, No. 6 peaks: 1.302～1.439, No. 7 peaks: 1.727～1.909, No. 8 peaks: 1.763～1.948, No. 9 peaks: 1.947～2.152, No. 10 peaks: 2.070～2.288;

No. 1 peak of relative peak area ratio range: 5.409～8.113, No. 2 peaks: 1.340～2.234, No. 4 peaks: 1.025～1.708, No. 5 peaks: 0.709～1.182;

Non-total peak area must not surpass 5% of total peak area;

B. the assay of cinnamic acid

According to high effective liquid chromatography for measuring;

Chromatographic condition and system suitability test are the C of filling agent with octadecylsilane chemically bonded silica ₁₈Post; 0.1% phosphoric acid solution-20～24% acetonitrile of 20～21% methyl alcohol-55～60% is a moving phase; The detection wavelength is 278nm; Number of theoretical plate calculates by the cinnamic acid peak should be not less than 1500;

It is an amount of that the cinnamic acid reference substance is got in the preparation of reference substance solution, and accurate the title decides, and adds 50% methyl alcohol and makes the solution that contains 2.0 μ g among every 1ml;

The preparation precision of need testing solution is measured parenteral solution 1.0ml, puts in the 10ml measuring bottle, adds 50% methyl alcohol and is diluted to 10ml, shakes up, promptly;

Accurate respectively reference substance solution and each the 20 μ l of need testing solution of drawing of determination method inject liquid chromatograph, measure, promptly;

C. the assay of total chlorogenic acid

It is an amount of that the chlorogenic acid reference substance is got in the preparation of reference substance solution, and accurate the title decides, and adds water and makes the solution that contains 0.4mg among every 1ml, promptly;

Typical curve prepares precision and measures reference substance solution 0.5ml, 1.0ml, 1.5ml, 2.0ml, 2.5ml, puts respectively in the 50ml measuring bottle, adds water to scale, shakes up; With water is blank, according to spectrophotometric method, measures absorbance log at the wavelength place of 347nm; With the absorbance log is ordinate, is horizontal ordinate with concentration, the drawing standard curve;

The determination method precision is measured parenteral solution 1.0ml, puts in the 100ml measuring bottle, adds water to 100ml, shakes up, and measures absorbance log at 347nm wavelength place, reads chlorogenic acid weight the need testing solution from typical curve, calculates, promptly.

5, a kind of method of quality control for the treatment of the MAILUONING ZHUSHEYE of thrombosis as claimed in claim 4 is characterized in that the test sample finger-print is in this method:

The test sample finger-print has 10 total peaks;

No. 1 peak of relative retention time: 0.364, No. 2 peaks: 0.678, No. 3 peaks: 1.000, No. 4 peaks: 1.043, No. 5 peaks: 1.083, No. 6 peaks: 1.362, No. 7 peaks: 1.794, No. 8 peaks: 1.833, No. 9 peaks: 2.020, No. 10 peaks: 2.149;

No. 1 peak of relative peak area ratio: 6.780, No. 2 peaks: 1.363, No. 4 peaks: 1.220, No. 5 peaks: 0.932.

6, a kind of method of quality control for the treatment of the MAILUONING ZHUSHEYE of thrombosis as claimed in claim 4 is characterized in that the moving phase of cinnamic acid assay in this method is: 21: 55: 24 methyl alcohol-0.1% phosphoric acid solution-acetonitrile.

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