CN104931607A - Content measurement method for Lingqijia oral solution - Google Patents
Content measurement method for Lingqijia oral solution Download PDFInfo
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- CN104931607A CN104931607A CN201510275564.0A CN201510275564A CN104931607A CN 104931607 A CN104931607 A CN 104931607A CN 201510275564 A CN201510275564 A CN 201510275564A CN 104931607 A CN104931607 A CN 104931607A
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Abstract
The invention relates to a content measurement method for Lingqijia oral solution. The method comprises the following steps of creating a chromatographic condition, preparing a reference solution and preparing and detecting a test sample solution. The accuracy, the precision and the durability of the method for detecting content by adopting an evaporative light chromatograph are consistent with corresponding standards, so that the method is feasible.
Description
Technical field
The present invention relates to Chinese medicine compound prescription detection method, be specifically related to a kind of detection method of ganoderma astragalus oral liquid content.
Background technology
Ganoderma astragalus oral liquid, in State Food and Drug Administration's national drug standards, the assay method having write content in WS-11254 (ZD-1254)-2002 exactly is thin-layer chromatographic analysis method, wherein the preparation method of need testing solution is: precision measures this product 30ml, 2 times are extracted with chloroform jolting, each 25ml, discard chloroform solution, the water saturated normal butyl alcohol jolting of water liquid extracts 3 times, each 20ml, merge normal butyl alcohol liquid, 2 times are washed with ammonia solution, each 20ml, discard ammonia solution, normal butyl alcohol liquid evaporate to dryness, the residue 5ml that adds water makes dissolving, by D101 type large pore resin absorption column (internal diameter 1cm, long 12cm), with water 50ml wash-out, discard water liquid, use 30% ethanol 50ml wash-out again, discard eluent, continue with 70% ethanol 50ml wash-out, collect eluent, evaporate to dryness, residue adds methyl alcohol and dissolves and be transferred in 2ml measuring bottle, add methanol dilution to scale, shake up, as need testing solution.By the similar principle that mixes, chloroform is removing impurity; Normal butyl alcohol dissolves effective constituent; D101 type large pore resin absorption column is carrier of separating, plays suction-operated, removing impurity.
On the whole, prior art is prepared in test sample experimental procedure and is applied chloroform organic reagent, and toxicity is large, and costly, step is numerous and diverse, is unfavorable for experimental study and pharmaceutical production, inspection.Content assaying method is thin-layer chromatographic analysis method, and its precision, accuracy have much room for improvement.
Summary of the invention
The object of this invention is to provide a kind of method solving the mensuration ganoderma astragalus oral liquid of prior art.
The assay method of a kind of ganoderma astragalus oral liquid content provided by the invention, comprises the following steps: the preparation of chromatographic condition, reference substance solution, the preparation and determination methods of need testing solution.
In described assay method:
Chromatographic condition is: take octadecylsilane chemically bonded silica as filling agent; With acetonitrile-water (30:70) for mobile phase; Detect with evaporative light-scattering device, number of theoretical plate calculates should be not less than 3500 by Astragaloside IV peak;
The preparation of reference substance solution: get Astragaloside IV reference substance appropriate, accurately weighed, add methyl alcohol and make the solution of every 1ml containing 0.5mg, to obtain final product;
The preparation of need testing solution: precision measures ganoderma astragalus oral liquid 25-30ml, extracts 3 times with water-saturated n-butanol jolting, each 25-30ml, merge normal butyl alcohol liquid, wash with ammonia solution 20-25ml, discard ammoniacal liquor, wash with the water 20-25ml that normal butyl alcohol is saturated again, divide and get normal butyl alcohol liquid, evaporate to dryness, residue methyl alcohol dissolves, and is transferred in 5ml measuring bottle, adds methyl alcohol to scale, shake up, filter, get subsequent filtrate, to obtain final product;
Measure: accurate absorption reference substance solution 2 μ l, 5 μ l, need testing solution 10 μ l, injection liquid chromatography, measures, calculate with external standard two-point method logarithmic equation.
Preferably, described assay method comprises the following steps:
Chromatographic condition is: take octadecylsilane chemically bonded silica as filling agent; Take volume ratio as the acetonitrile-water of 30:70 be mobile phase; Detect with evaporative light-scattering device, number of theoretical plate calculates should be not less than 3500 by Astragaloside IV peak;
The preparation of reference substance solution: get Astragaloside IV reference substance appropriate, accurately weighed, add methyl alcohol and make the solution of every 1ml containing 0.5mg, to obtain final product;
The preparation of need testing solution: precision measures ganoderma astragalus oral liquid 25ml, extracts 3 times with water-saturated n-butanol jolting, each 30ml, merge normal butyl alcohol liquid, wash with ammonia solution 20ml, discard ammoniacal liquor, wash with the water 20ml that normal butyl alcohol is saturated again, divide and get normal butyl alcohol liquid, evaporate to dryness, residue methyl alcohol dissolves, and is transferred in 5ml measuring bottle, adds methyl alcohol to scale, shake up, filter, get subsequent filtrate, to obtain final product;
Measure: accurate absorption reference substance solution 2 μ l, 5 μ l, need testing solution 10 μ l, injection liquid chromatography, measures, calculate with external standard two-point method logarithmic equation.
Detection method provided by the invention finally detects Astragaloside content in every 1ml ganoderma astragalus oral liquid and is not less than 32 μ g.
Detection method provided by the invention has the following advantages:
1, adopt normal butyl alcohol effective component extracting when prepared by test sample, do not reach the object of low toxicity, Green experiment with chloroform; Though Astragaloside IV molecular weight is comparatively large, have glycoside character after all, must be noted that to reduce the loss, extraction and wash volumes should be unsuitable excessive.
2, also report is had, the detection method of Astragaloside IV in Chinese angelica tonifying blood oral liquid, with the present invention closely, as CN200910064944.4 (publication number is CN101551365A), but the present invention uses, and acetonitrile-water (30:70) is high as the testing result accuracy of mobile phase, relative deviation is little;
The number of times extracted in the preparation of need testing solution is few, wash time is short, save time, fast, by experiment result prove to reach identical result, obtain the effective constituent with amount.
2, employing evaporative light chromatograph provided by the invention detects the method for this content, and accuracy, precision, durability all meet respective standard, show that method is feasible.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.
Embodiment 1
1, instrument and reagent
SEDEX75 (France), Astragaloside IV reference substance, source: Nat'l Pharmaceutical & Biological Products Control Institute, acetonitrile: be chromatographically pure, all the other are pure for analyzing.
2, chromatographic condition
Take octadecylsilane chemically bonded silica as filling agent (5 μ, 150 × 4.6mm); With acetonitrile-water (30:70) for mobile phase; Detect with evaporative light-scattering device, number of theoretical plate is calculated should be not less than 3500 by Astragaloside IV peak.
The preparation of 2.1 reference substance solution: get Astragaloside IV reference substance appropriate, accurately weighed, add methyl alcohol and make the solution of every 1ml containing 0.5mg, to obtain final product.
2.2 the preparation of need testing solution:
Precision measures ganoderma astragalus oral liquid 25ml, extracts 3 times with water-saturated n-butanol jolting, each 30ml, merge normal butyl alcohol liquid, wash with ammonia solution 20ml, discard ammoniacal liquor, wash with the water 20ml that normal butyl alcohol is saturated again, divide and get normal butyl alcohol liquid, evaporate to dryness, residue methyl alcohol dissolves, and is transferred in 5ml measuring bottle, adds methyl alcohol to scale, shake up, filter, get subsequent filtrate, to obtain final product.
2.3 determination methods: accurate absorption reference substance solution 2 μ l, 5 μ l, need testing solution 10 μ l, injection liquid chromatography, measures, calculate with external standard two-point method logarithmic equation.
3, method validation
3.1 negative control experiments
Get glossy ganoderma 15.0g, wilsonii 40.0g, by making without Astragali Solution 100ml under method for making item, precision gets 25ml, according to need testing solution, preparation method makes negative controls, and by above-mentioned chromatography determination, result shows that in ganoderma astragalus oral liquid, the mensuration of other compositions to Astragaloside IV is noiseless.
3.2 linear relationships are investigated: precision measures reference substance solution (0.864mg/ml) 1 μ l, 2 μ l, 3 μ l, 4 μ l, 6 μ l, 8 μ l, 10 μ l, 15 μ l, inject hplc determination peak area, with the logarithm of sample size for ordinate, with the logarithm of peak area for horizontal ordinate drawing standard curve, obtain regression equation lgM=0.6859lgA-3.8713, r=0.99993, concrete outcome is in table 1.
Table 1 Astragaloside IV linear relationship test findings
| Sampling volume (μ l) | Sample size (μ g) | Peak area mean value |
| 1 | 0.864 | 365153 |
| 2 | 1.728 | 992763.5 |
| 3 | 2.592 | 1758528.5 |
| 4 | 3.456 | 2702404.5 |
| 6 | 5.184 | 4917206.5 |
| 8 | 6.912 | 7420212.5 |
| 10 | 8.64 | 10369118.6 |
| 15 | 12.96 | 17986224 |
Table 1 result shows: Astragaloside IV has good linear relationship in sample size 0.864 ~ 12.96 μ g.
3.3 instrument precision tests: precision measures reference substance solution 10 μ l (0.432mg/ml), repeat sample introduction and measure peak area 5 times, the results are shown in Table 2.
Table 2 Astragaloside IV Precision test result
Table 2 result shows: RSD=0.3% (n=5), result shows, the precision of instrument is good.
3.4 replica tests: get same sample lots 6 parts, press sample size determination method respectively and measure, the results are shown in Table 3.
Table 3 Astragaloside IV replica test result
Table 3 result shows: RSD=1.3% (n=6), result shows, detection method provided by the invention is reproducible.
3.5 stability tests: get need testing solution, every certain hour sample introduction 1 time, continuous sample introduction 6 times, measure peak area, the results are shown in Table 4.
Table 4 Astragaloside IV stability test result
Table 4 result shows: RSD=1.7% (n=6), and display Astragaloside IV is stable at least 14h.
Result shows, detection method good stability provided by the invention.
3.6 recovery tests: precision measures the ganoderma astragalus oral liquid 6 parts of known content sample (0.0630mg/ml), it is appropriate that precision adds Astragaloside IV reference substance, measures, and calculate content, the results are shown in Table 5 by content determination.
Table 5 ganoderma astragalus oral liquid sample Astragaloside IV application of sample recovery test
Table 5 result shows: average recovery rate is 99.2%, RSD=1.7% (n=6), and result shows, the method recovery provided by the invention is good.
The selection of 3.7 mobile phases
Use C
18acetonitrile-water (30:70) solvent system that post uses under adopting Chinese Pharmacopoeia Milkvetch Root item reaches satisfied separating effect.
3.8 detectability
The signal that the signal measure Astragaloside IV chromatographic peak and blank solvent are measured compares, and the amount injecting instrument when taking signal to noise ratio (S/N ratio) as 3:1 determines detectability, records detection and is limited to 0.0864 μ g, confirms: the method for inspection is feasible, meets standard.
3.9 quantitative limit: repeat sample introduction 4 times with 10 times of Astragaloside IV reference substance solution to noise, sample size is 0.1728 μ g, in table 6.
Table 6 quantitative limit measurement result
Table 6 result shows: record and be quantitatively limited to 0.1728 μ g, repeats the RSD=1.1% of sample introduction peak area value.
Result shows: the method for inspection is feasible, meets standard.
3.10 scopes: by normal 60% and 140% sampling and measuring measuring sampling amount, investigate the precision measured at height sampling amount.Get same batch sample measures Astragaloside IV content at high and low sampling amount, the results are shown in Table 7.
Table 7 range finding result
Table 7 result shows: same batch sample measures the content of Astragaloside IV at high and low sampling amount, and result no significant difference, shows that method is feasible, meet standard.
3.11 durability
Adopt different brands octadecylsilane chemically bonded silica chromatographic column, enter need testing solution 10 μ l respectively, Astragaloside IV chromatographic peak is separated good.
4, sample size measures: carry out assay by content determination respectively to 10 batch samples, measurement result is in table 8.
Table 8 ganoderma astragalus oral liquid sample Astragaloside IV measurement result
Table 8 result shows: the assay average of this product 10 batch sample is 0.0592 (mg/ml), and the content of medicinal material used is 0.102%, and its rate of transform is 58.04%; Consider the difference of medicinal material content, with pharmacopeia Chinese crude drug content limit 0.040%, the rate of transform 58.04% calculates, then this product content is limited to 0.023mg/ml, and primary standard content is limited to 0.03mg/ml; Consider that high-efficient liquid phase method is sensitive in thin layer chromatography scanning, under the prerequisite not reducing quality standard, temporarily the content limit of this product is defined as 0.032mg/ml, namely the every 1ml of this product contains the Radix Astragali with Astragaloside IV (C
41h
68o
14) meter, 32 μ g must not be less than.
Result shows: shown by above-mentioned quality standards in Chinese drugs analytical approach confirmatory experiment result, the method is highly sensitive, accuracy good, can reach efficient, low toxicity, green inspection, thus improve the quality requirements of examination and test of products environment.
Comparative example 1: the preparation method of application reference CN200910064944.4 test sample, processes ganoderma astragalus oral liquid, adopts method of the present invention to detect.
Experimental example 1: comparison test
Adopt embodiment 1 and comparative example 1 to carry out content detection to ganoderma astragalus oral liquid respectively, the results are shown in Table 9.
Table 9: embodiment 1 and comparative example 1 content detection data
Table 9 result shows: compared with the prior art, detection method provided by the invention more saves time, low toxicity, green test result more accurately, high efficiency extraction effective constituent wherein.
Although above with general explanation, embodiment and test, the present invention is described in detail, and on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (8)
1. an assay method for ganoderma astragalus oral liquid content, comprises the following steps: the preparation of chromatographic condition, reference substance solution, the preparation and determination methods of need testing solution.
2. assay method according to claim 1, is characterized in that, described chromatographic condition is: take octadecylsilane chemically bonded silica as filling agent; Take volume ratio as 30:70 acetonitrile-water be mobile phase; Detect with evaporative light-scattering device, number of theoretical plate calculates should be not less than 3500 by Astragaloside IV peak.
3. detection method according to claim 1, is characterized in that, the preparation of described need testing solution: precision measures ganoderma astragalus oral liquid 25-30ml, extract 3 times with water-saturated n-butanol jolting, each 25-30ml, merge normal butyl alcohol liquid, wash with ammonia solution 20-25ml, discard ammoniacal liquor, then wash with the water 20-25ml that normal butyl alcohol is saturated, divide and get normal butyl alcohol liquid, evaporate to dryness, residue methyl alcohol dissolves, be transferred in 5ml measuring bottle, add methyl alcohol to scale, shake up, filter, get subsequent filtrate, to obtain final product.
4. detection method according to claim 1, is characterized in that, the preparation of described reference substance solution: get Astragaloside IV reference substance appropriate, accurately weighed, adds methyl alcohol and makes the solution of every 1ml containing 0.5mg, to obtain final product.
5. detection method according to claim 1, is characterized in that, described mensuration: accurate absorption reference substance solution 2 μ l, 5 μ l, need testing solution 10 μ l, injection liquid chromatography, measures, calculate with external standard two-point method logarithmic equation.
6. the detection method according to any one of claim 1-5, is characterized in that, described assay method comprises the following steps:
Chromatographic condition is: take octadecylsilane chemically bonded silica as filling agent; With acetonitrile-water (30:70) for mobile phase; Detect with evaporative light-scattering device, number of theoretical plate calculates should be not less than 3500 by Astragaloside IV peak;
The preparation of reference substance solution: get Astragaloside IV reference substance appropriate, accurately weighed, add methyl alcohol and make the solution of every 1ml containing 0.5mg, to obtain final product;
The preparation of need testing solution: precision measures ganoderma astragalus oral liquid 25-30ml, extracts 3 times with water-saturated n-butanol jolting, each 25-30ml, merge normal butyl alcohol liquid, wash with ammonia solution 20-25ml, discard ammoniacal liquor, wash with the water 20-25ml that normal butyl alcohol is saturated again, divide and get normal butyl alcohol liquid, evaporate to dryness, residue methyl alcohol dissolves, and is transferred in 5ml measuring bottle, adds methyl alcohol to scale, shake up, filter, get subsequent filtrate, to obtain final product;
Measure: accurate absorption reference substance solution 2 μ l, 5 μ l, need testing solution 10 μ l, injection liquid chromatography, measures, calculate with external standard two-point method logarithmic equation.
7. detection method according to claim 6, is characterized in that, described assay method comprises the following steps:
Chromatographic condition is: take octadecylsilane chemically bonded silica as filling agent; Take volume ratio as the acetonitrile-water of 30:70 be mobile phase; Detect with evaporative light-scattering device, number of theoretical plate calculates should be not less than 3500 by Astragaloside IV peak;
The preparation of reference substance solution: get Astragaloside IV reference substance appropriate, accurately weighed, add methyl alcohol and make the solution of every 1ml containing 0.5mg, to obtain final product;
The preparation of need testing solution: precision measures ganoderma astragalus oral liquid 25ml, extracts 3 times with water-saturated n-butanol jolting, each 30ml, merge normal butyl alcohol liquid, wash with ammonia solution 20ml, discard ammoniacal liquor, wash with the water 20ml that normal butyl alcohol is saturated again, divide and get normal butyl alcohol liquid, evaporate to dryness, residue methyl alcohol dissolves, and is transferred in 5ml measuring bottle, adds methyl alcohol to scale, shake up, filter, get subsequent filtrate, to obtain final product;
Measure: accurate absorption reference substance solution 2 μ l, 5 μ l, need testing solution 10 μ l, injection liquid chromatography, measures, calculate with external standard two-point method logarithmic equation.
8. the detection method of the ganoderma astragalus oral liquid according to any one of claim 1-7, is characterized in that, in every 1ml ganoderma astragalus oral liquid, Astragaloside content is not less than 32 μ g.
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| CN108434152A (en) * | 2018-03-26 | 2018-08-24 | 黑龙江珍宝岛药业股份有限公司 | A kind of pharmaceutical composition and its preparation method and application for improving sleep |
| CN108956811A (en) * | 2018-06-12 | 2018-12-07 | 江苏颐海药业有限责任公司 | One kind being used for the method for quality control of " invigorating heart dredging collateral oral solution " preparation |
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| CN108434152A (en) * | 2018-03-26 | 2018-08-24 | 黑龙江珍宝岛药业股份有限公司 | A kind of pharmaceutical composition and its preparation method and application for improving sleep |
| CN108956811A (en) * | 2018-06-12 | 2018-12-07 | 江苏颐海药业有限责任公司 | One kind being used for the method for quality control of " invigorating heart dredging collateral oral solution " preparation |
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Application publication date: 20150923 |