CN103308625A - Method for determining content of peimisine in sour pear lung-moistening paste - Google Patents
Method for determining content of peimisine in sour pear lung-moistening paste Download PDFInfo
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- CN103308625A CN103308625A CN2013102636426A CN201310263642A CN103308625A CN 103308625 A CN103308625 A CN 103308625A CN 2013102636426 A CN2013102636426 A CN 2013102636426A CN 201310263642 A CN201310263642 A CN 201310263642A CN 103308625 A CN103308625 A CN 103308625A
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Abstract
The invention discloses a method for determining content of peimisine in a sour pear lung-moistening paste, and aims to solve the technical problems of complicated operation, low accuracy, poor stability and poor result repeatability of the traditional method for controlling the quality of the sour pear lung-moistening paste. The adopted technical scheme is that the determination method comprises the following steps of: adding analytical pure methanol to a peimisine reference substance to prepare reference substance solution for future use; weighing the sour pear lung-moistening paste; agitating with kieselguhr; adding the analytical pure methanol to reflux and extract; filtering, evaporating filtrate, fixing volume, and filtering to obtain sample solution for future use; absorbing isometric reference substance solution and sample solution; respectively injecting into an efficient liquid-phase chromatographic instrument; and measuring and calculating according to an external standard method, namely obtaining the content of the peimisine in the sour pear lung-moistening paste. The method has the technical effects of high precision, stability, reproducibility and recovery rate. By using the method, the content of the peimisine in the sour pear lung-moistening paste can be conveniently, rapidly and accurately determined. Thus, the quality of the sour pear lung-moistening paste is well controlled.
Description
Technical field
The invention belongs to the control method technical field of drug quality, relate to a kind of assay method, particularly the assay method of Peimisine content in a kind of autumn pear lung-nourishing semifluid extract.
Background technology
The autumn pear lung-nourishing semifluid extract is comprised of pears, lily, tendril-leaved fritillary bulb, the tuber of dwarf lilyturf, tussilago, have moisten the lung and relieve the cough, the effect of secretion replenishing and throat moistening, be used for chronic cough, small amount of sputum sticky in nature, dryness of the mouth and throat.Wherein pears are monarch drug in a prescription, the bulb of fritillary, lily is ministerial drug, its active component is Peimisine and colchicin, wherein the content of Peimisine should be no less than 0.60mg/g, but act.std only has the thin-layered chromatography of a tuber of dwarf lilyturf to control the quality of medicine, do not have the assay item, the thin layer differential method is that fixing phase (such as silica gel) evenly is coated on the base plate (or rod) thinly, sample point is at thin layer one end, in developing tank, launch, because the displacement of each component on thin layer is different, forms spot disconnected from each other, measures the position of each spot and density thereof and just can finish qualitative to sample, the chromatography of quantitative test.But this assay method complicated operation, accuracy is low, poor stability, poor reproducibility as a result, thereby can not control well the quality of autumn pear lung-nourishing semifluid extract.
Summary of the invention
The objective of the invention is, poor stability low for the method complicated operation, the accuracy that solve existing control autumn pear lung-nourishing semifluid extract quality, the technical matters of poor reproducibility as a result, in order to address this problem, we have designed the assay method of Peimisine content in a kind of autumn pear lung-nourishing semifluid extract, utilize high performance liquid chromatography to measure the content of Peimisine in the autumn pear lung-nourishing semifluid extract, the mensuration process is convenient, fast, accurate, can control better the quality of autumn pear lung-nourishing semifluid extract.
The present invention for realizing the concrete technical scheme that goal of the invention adopts is: the assay method of Peimisine content in a kind of autumn pear lung-nourishing semifluid extract, and key is: described assay method may further comprise the steps:
A, in the Peimisine reference substance, add to analyze pure methyl alcohol and make the reference substance solution that Peimisine content is 55～65 μ g/ml, for subsequent use;
B, take by weighing the autumn pear lung-nourishing semifluid extract, admix zeyssatite, add and analyze pure methanol eddy extraction, filter evaporate to dryness filtrate, constant volume, refilter, get need testing solution, for subsequent use, wherein the massfraction ratio of autumn pear lung-nourishing semifluid extract, zeyssatite and the pure methyl alcohol of analysis is 1: (0.8～1.1): (499～501);
C, the reference substance solution of drawing isopyknic a step gained and the need testing solution of b step gained inject respectively high performance liquid chromatograph, measure, and calculate by external standard method, namely get Peimisine content in the autumn pear lung-nourishing semifluid extract.
The invention has the beneficial effects as follows: the accuracy of high performance liquid chromatography, stability, reappearance and the recovery are all higher, utilize this method can determine convenient, fast, exactly the content of Peimisine in the autumn pear lung-nourishing semifluid extract, thereby control better the quality of autumn pear lung-nourishing semifluid extract.
Embodiment
The assay method of Peimisine content in a kind of autumn pear lung-nourishing semifluid extract, key is: described assay method may further comprise the steps:
A, in the Peimisine reference substance, add to analyze pure methyl alcohol and make the reference substance solution that Peimisine content is 55～65 μ g/ml, for subsequent use;
B, take by weighing the autumn pear lung-nourishing semifluid extract, admix zeyssatite, add and analyze pure methanol eddy extraction, filter evaporate to dryness filtrate, constant volume, refilter, get need testing solution, for subsequent use, wherein the massfraction ratio of autumn pear lung-nourishing semifluid extract, zeyssatite and the pure methyl alcohol of analysis is 1: (0.8～1.1): (499～501);
C, the reference substance solution of drawing isopyknic a step gained and the need testing solution of b step gained inject respectively high performance liquid chromatograph, measure, and calculate by external standard method, namely get Peimisine content in the autumn pear lung-nourishing semifluid extract.
Described step b is refluxing extraction 25～35min, is settled to 0.1 part before the refluxing extraction, and the diameter of used miillpore filter is 0.45 μ m when refiltering.
The chromatographic condition of described high performance liquid chromatograph is: take octadecylsilane chemically bonded silica as fixing phase, mixed liquor take volume ratio as 37: 63: 1 trifluoroacetic acid aqueous solution, high purity water and diethylamine is as mobile phase, the flow velocity of mobile phase is 1ml/min, detect with evaporative light-scattering detector, the drift tube temperature of evaporative light-scattering detector is 33 ℃, atomization gas is nitrogen, and nebulizer gas pressure is 2.5 * 10
-5Pa utilizes external standard method to calculate the content of measuring Peimisine in the autumn pear lung-nourishing semifluid extract.
Described theoretical cam curve is calculated with the Peimisine peak should be not less than 2000.
This assay method is that the inventor draws through a large amount of experimental study and method validations, and is specific as follows:
One, reagent and instrument
Sample: effluent north is driven Zhi Lin pharmaceutcal corporation, Ltd and is provided, and lot number is 20130411,20130412,20130413.
Reference substance: Peimisine provides (for assay) by Nat'l Pharmaceutical ﹠ Biological Products Control Institute, and purity is 100%, and lot number is 715-9003.
Reagent: acetonitrile is chromatographically pure, and phosphoric acid is pure for analyzing, and methyl alcohol is pure for analyzing, and water is high purity water.
Instrument: produce 510 type infusion pumps with U.S. Waters company, the U6K injector, U.S. Beckman company produces 163 type Variable wavelength UV detectors and 427 type integration registering instruments are measured.
Two, experimental technique and result:
1, chromatographiccondition
System suitability, the chromatographic condition of described high performance liquid chromatograph is: take octadecylsilane chemically bonded silica as fixing phase, take the trifluoroacetic acid aqueous solution of volume ratio as 37: 63: 1: high purity water: diethylamine detects with the evaporative light-scattering device as mobile phase.
2, the selection of experiment condition
2.1, the selection of chromatogram flow phase: compared methanol-water-phosphoric acid (47: 53: 0.2), acetonitrile-0.025M phosphoric acid (28: 72), acetonitrile: water: diethylamine (37: 63: 1), find that the latter is high to Peimisine peak degree of separation in this product test liquid, its theoretical cam curve can reach more than 2000.
2.2, the test sample extraction conditions selects: compared and prepared test liquid with the organic solvent wash-out again after test sample is directly admixed the dispersion of equivalent zeyssatite with organic solvent lixiviate and test sample, the latter obviously is better than the former.
Thereby extraction process is defined as: precision takes by weighing autumn pear lung-nourishing semifluid extract 200mg, admixes zeyssatite 200mg, adds methyl alcohol 100ml refluxing extraction 30min, filter, filtrate is steamed near and is done, and is settled to 10ml, miillpore filter (0.45 μ m) filters, and gets filtrate as need testing solution.Accurate reference substance solution and each 10 μ l of need testing solution of drawing inject respectively high performance liquid chromatograph, measure the peak area integrated value, calculate with external standard method, namely get Peimisine content in the autumn pear lung-nourishing semifluid extract.
3, the investigation of linear relationship
Precision is drawn reference substance solution (63.2 μ g/ml) 5 μ l, 10 μ l, 15 μ l, 20 μ l and 25 μ l respectively, inject high performance liquid chromatograph, measure peak area by above-mentioned chromatographic condition, take the micrograms of Peimisine as horizontal ordinate, take the peak area integrated value as ordinate drawing standard curve.Calculate regression equation Y=-1.434+131.77X by above typical curve, correlation coefficient r=0.9999, Peimisine is good linear relationship with peak area in 0.316-1.58 μ g scope.
4, blank test
Autumn pear lung-nourishing semifluid extract (not containing the bulb of fritillary) extracts according to preparation technology's flow process and obtains medicinal extract.Obtain the blank test test liquid by high performance liquid chromatography test liquid preparation method again, sample introduction 10 μ L detect by above-mentioned chromatographic condition, without the Peimisine peak.
5, accuracy test
Take by weighing the Peimisine reference substance, add the solution that methyl alcohol is made 63.2 μ g/ml.The above-mentioned reference substance solution 10 μ L of accurate absorption repeat sample introduction 5 times, the record chromatogram, and the result is as described in Table 1, and the relative standard deviation of Peimisine peak area integrated value is less than 2%.
Table 1
6, stability test
The accurate need testing solution 10 μ l that draw above-mentioned new preparation, sample introduction (totally 6 times) during respectively at 0h, 2h, 4h, 8h, 12h and 24h records Peimisine peak area integrated value, calculates relative standard deviation RSD, and is as shown in table 2.The result shows, the data stabilization that Peimisine records in 24 hours under this condition.
Table 2
7, reappearance test
Precision takes by weighing 5 parts of test sample autumn pear lung-nourishing semifluid extracts (lot number 20130411), and every part of 200mg prepares test liquid according to said method, with high effective liquid chromatography for measuring Peimisine content, calculate relative standard deviation RSD＜2%, as shown in table 3, show that the repeatability of high performance liquid chromatography is good.
Table 3
8, recovery test
Adopt application of sample absorption method precision to take by weighing the autumn pear lung-nourishing semifluid extract (lot number 20130411) of 5 parts of known Peimisine content, every part of 100mg, precision is added the Peimisine reference substance respectively, use high effective liquid chromatography for measuring, the result is as shown in table 4, RSD＜2% shows that the recovery of high performance liquid chromatography is higher.
Table 4
9, sample determination
Get three batches of test samples, prepare need testing solution according to the preparation method of need testing solution, configure simultaneously reference substance solution, sample introduction is 3 times respectively, records chromatogram with high performance liquid chromatography, measures peak area, calculates Peimisine content, and the result is as shown in table 5.
Table 5
According to the test figure shown in the table 5, contain the bulb of fritillary in this product and be no less than 0.60mg/g in Peimisine, meet the quality standard of autumn pear lung-nourishing semifluid extract.
In sum, the present invention can determine the content of Peimisine in the autumn pear lung-nourishing semifluid extract convenient, fast, exactly, for the quality control of autumn pear lung-nourishing semifluid extract provides good examination criteria.
Claims (4)
1. the assay method of Peimisine content in the autumn pear lung-nourishing semifluid extract, it is characterized in that: described assay method may further comprise the steps:
A, in the Peimisine reference substance, add to analyze pure methyl alcohol and make the reference substance solution that Peimisine content is 55～65 μ g/ml, for subsequent use;
B, take by weighing the autumn pear lung-nourishing semifluid extract, admix zeyssatite, add and analyze pure methanol eddy extraction, filter evaporate to dryness filtrate, constant volume, refilter, get need testing solution, for subsequent use, wherein the massfraction ratio of autumn pear lung-nourishing semifluid extract, zeyssatite and the pure methyl alcohol of analysis is 1: (0.8～1.1): (499～501);
C, the reference substance solution of drawing isopyknic a step gained and the need testing solution of b step gained inject respectively high performance liquid chromatograph, measure, and calculate by external standard method, namely get Peimisine content in the autumn pear lung-nourishing semifluid extract.
2. the assay method of Peimisine content in a kind of autumn pear lung-nourishing semifluid extract according to claim 1, it is characterized in that: described step b is refluxing extraction 25～35min, be settled to 0.1 part before the refluxing extraction, the diameter of used miillpore filter is 0.45 μ m when refiltering.
3. the assay method of Peimisine content in each described a kind of autumn pear lung-nourishing semifluid extract in 2 according to claim 1, it is characterized in that: the chromatographic condition of described high performance liquid chromatograph is: take octadecylsilane chemically bonded silica as fixing phase, mixed liquor take volume ratio as 37: 63: 1 trifluoroacetic acid aqueous solution, high purity water and diethylamine is as mobile phase, the flow velocity of mobile phase is 1ml/min, detect with evaporative light-scattering detector, the drift tube temperature of evaporative light-scattering detector is 33 ℃, atomization gas is nitrogen, and nebulizer gas pressure is 2.5 * 10
-5Pa utilizes external standard method to calculate the content of measuring Peimisine in the autumn pear lung-nourishing semifluid extract.
4. the assay method of Peimisine content in a kind of autumn pear lung-nourishing semifluid extract according to claim 1 is characterized in that: described theoretical cam curve is calculated with the Peimisine peak should be not less than 2000.
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Citations (3)
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| CN100998286A (en) * | 2006-05-19 | 2007-07-18 | 四川阿坝天贝生物有限责任公司 | Sichuan fritillary bulb, and method for culture of the same |
| CN102359942A (en) * | 2011-09-27 | 2012-02-22 | 山东阿如拉药物研究开发有限公司 | Qingpeng ointment and quality control method of Qingpeng ointment preparation |
| CN103175938A (en) * | 2013-04-01 | 2013-06-26 | 山西振东开元制药有限公司 | Quality control method of drug for treating cough |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100998286A (en) * | 2006-05-19 | 2007-07-18 | 四川阿坝天贝生物有限责任公司 | Sichuan fritillary bulb, and method for culture of the same |
| CN102359942A (en) * | 2011-09-27 | 2012-02-22 | 山东阿如拉药物研究开发有限公司 | Qingpeng ointment and quality control method of Qingpeng ointment preparation |
| CN103175938A (en) * | 2013-04-01 | 2013-06-26 | 山西振东开元制药有限公司 | Quality control method of drug for treating cough |
Non-Patent Citations (2)
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| 余华等: "中药川贝母定量分析方法研究", 《中药川贝母定量分析方法研究》 * |
| 黄林芳等: "HPLC -ELSD法测定不同加工方法川贝母中贝母辛的含量", 《全国第八届天然药物资源学术研讨会论文集》 * |
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Denomination of invention: Method for determining content of peimisine in sour pear lung-moistening paste Effective date of registration: 20180417 Granted publication date: 20150204 Pledgee: Baoding Qingyuan District Rural Credit Union Limited by Share Ltd southern section Chuang Credit Union Pledgor: Hebei Yuzhilin Pharmaceutical Co., Ltd. Registration number: 2018130000004 |
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Date of cancellation: 20210524 Granted publication date: 20150204 Pledgee: Baoding Qingyuan District Rural Credit Union Limited by Share Ltd. southern section Chuang Credit Union Pledgor: HEBEI YUZHILIN PHARMACEUTICAL Co.,Ltd. Registration number: 2018130000004 |
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