CN103512998B - Chinese ephedra medicinal material, and content determination method of three alkaloids in preparation thereof - Google Patents
Chinese ephedra medicinal material, and content determination method of three alkaloids in preparation thereof Download PDFInfo
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Abstract
The invention relates to a Chinese ephedra medicinal material, and a content determination method of three alkaloids in a preparation thereof. The method has the characteristics that: a common reversed-phase column and a non-buffer salt which is a mobile phase of acetonitrile-methanol-0.1% phosphoric acid with a volume ratio of 1-1.5:4-4.2:94.5-95.0 are adopted; at 207+/-2nm, contents of ephedrine hydrochloride, pseudoephedrine hydrochloride, and ephedrine methyl hydrochloride in the Chinese ephedra medicinal material and the preparation thereof are simultaneously determined. A sample is only processed through water-containing methanol ultrasonic extraction and alumina impurity removing, such that a near-colorless transparent solution is obtained. The method is simple, fast, reproducible, and accurate. According to the quantitative chromatogram of the three alkaloids, a baseline is stable, peak separation is good, and the peak appearing is completed within 20min. Compared with a traditional method with a buffer-salt solution, a specific chromatographic column, and unconventional organic phase ratio and flow rate, with the method provided by the invention, instrument and chromatographic column service lives are prolonged, cost is reduced, and detection conditions are communized. The method is suitable for popularization and application of basic units.
Description
Technical field
The invention belongs to traditional Chinese medicine quality control field, relate to the quality determining method of a kind of Herba Ephedrae and preparation thereof, particularly, relate to three kinds of alkaloidal content assaying methods in a kind of Herba Ephedrae and preparation thereof.
Background technology
Chinese ephedra is the dry herbaceous stem stem of Ephedraceae plant plait Chinese ephedra Ephedra sinica Stapf, epheday intermedia Ephedra intermedia Schrenket C.A.Mey. or ephedra equisetina Ephedra equisetina Bge..Be the conventional Chinese medicine in a kind of cough-relieving compound preparation, tool Pulvis Diaphoreticus is trembled with fear, and freeings lung and relieving asthma, effect of inducing diuresis for removing edema.For anemofrigid cold, uncomfortable in chestly to breath with cough, geomantic omen edema.Herba ephedrae moistens the lung and relieve the cough.Preparation means Chinese ephedra and almond containing cough stopping sheet and golden Chinese ephedra and almond containing cough stopping sheet, is all the compound preparation of cough-relieving.The prescription of Chinese ephedra and almond containing cough stopping sheet, method for making and function cure mainly as follows:
Prescription: Chinese ephedra 600g semen armeniacae amarae 400g gypsum 1200g honey-fried licorice root 300g
Method for making: above four tastes, gets gypsum 60g, is ground into fine powder; Residue gypsum and Chinese ephedra, honey-fried licorice root are ground into meal, boiling secondary, each 2 hours, filter, merging filtrate; After semen armeniacae amarae deoils, boiling 2 hours, filters, filtrate and above-mentioned filtrate merge, and are evaporated to the clear cream that relative density is 1.10 ~ 1.15 (70 DEG C), are evaporated to dry cream, pulverize, appropriate with gypsum fine powder and starch, mixing, make particle, drying, adds Magnesium Stearate proper quantity, is pressed into 1000, sugar coating or film-coating, to obtain final product.
Function cures mainly: antibechic, eliminates the phlegm, and relievings asthma.For acute and chronic bronchitis and pant.
The prescription of gold Chinese ephedra and almond containing cough stopping sheet, method for making and function cure mainly as follows:
Prescription: Chinese ephedra 150-200 part processed by golden yellow a kind of reed mentioned in ancient books 150-190 part honeyed folium eriobotryae 280-350 part honey
Semen armeniacae amarae 150-200 part root of purple-flowered peucedanum 250-320 part root bark of white mulberry 250-320 part
Snakegourd Fruit 280-310 part balloonflower root 160-200 part honeysuckle 250-300 part
Caulis bambusae in taenian 250-320 part
Method for making: above ten tastes, Pi is leaf (honey is processed), Chinese ephedra (honey is processed), semen armeniacae amarae, the root of purple-flowered peucedanum 10 times amount 60% alcohol extract secondaries, and each 2 hours, merge extract, filter, filtrate recycling ethanol is to without alcohol taste, for subsequent use; The root bark of white mulberry, caulis bambusae in taenian, Snakegourd Fruit, balloonflower root, honeysuckle, adding 10 times amount soak by water to boiling, adding the root of large-flowered skullcap, decoct secondary, each 2 hours, merge extract, filter, filtrate is condensed into relative density 1.05-1.10 (60 DEG C of heat are surveyed) clear cream, adds 95% ethanol, limit edged stirs, to determining alcohol 70%, and refrigeration placement 24 hours, filter, filtrate recycling ethanol, to without alcohol taste, merges with alcohol extract, be concentrated into relative density 1.10-1.15 (60 DEG C of heat are surveyed) clear cream, spraying dry.Get spray powder and appropriate amount of starch, mixing, particle processed, is pressed into 1000, film coating, obtains final product.
Function cures mainly: clearing lung and eliminating phlegm, relieving cough and asthma.For the phlegm acute tracheobronchitis that causes of heat and AECB, disease sees cough, expectoration, asthma, and phlegm is yellow or glue in vain, body heat, uncomfortable in chest, dry, and pharyngalgia, urine is yellow, just do, red tongue with yellowish and greasy fur, slippery and rapid pulse.
Bibliographical information, the principle active component of Chinese ephedra is alkaloids, substantially has six kinds of alkaloids, i.e. ephedrine, d-pseudo-ephedrine, demethyl ephedrine, Nor pseudo ephedrine, methylephedrine, methyl pseudoephedrine.What wherein content was higher is ephedrine, d-pseudo-ephedrine and methylephedrine, and its sequence is ephedrine > pseudoephedrine > methylephedrine, and three's sum accounts for more than 80% of six kinds of ephedrine total amounts.Remaining methyl pseudoephedrine, demethyl ephedrine and Nor pseudo ephedrine content are very low, generally extract dosage in preparation at compound humble, so at present in the statutory standards of compound preparation, be all control content limit with the content summation of ephedrine and pseudoephedrine.
In compound preparation, except ephedrine and pseudoephedrine, what content can be detected also has methylephedrine, and pharmacodynamic study shows: ephedrine has nervous centralis and sympathetic activation effect, and its sweating is relievingd asthma, cholagogic, the effect such as blood pressure, vasoconstriction that rises are all stronger; Pseudoephedrine has very strong anti-inflammatory and diuresis, and the snuffles aspect curative effect caused schneiderian membrane swelling is certain; Methylephedrine is suitable to the dilating effect of tracheal smooth muscle and antitussive effect and ephedrine, and has good anti-allergy action.So with the total amount of three kinds of Herba Ephedrae alkaloids, control the content of medicinal material and its compound preparation, the quality of medicinal material and preparation can be reacted more all sidedly.
By consulting literatures is learnt, the Herba Ephedrae that Chinese Pharmacopoeia version one in 2010 is recorded and infantile lung clearing phlegm transforming oral liquid, little Green Dragon particle, chill are only coughed ball etc., it is all the content summation controlling ephedrine and pseudoephedrine, the chromatographic column of Herba Ephedrae assay must be equipped with specially, and its filling agent is that the polarity ether that pole is of little use connects phenyl bonded silica.And the mobile phase of these medicinal materials and preparation is the lower phosphoric acid buffer salt of not advising using of Chinese Pharmacopoeia version in 2010 annex VID (3) item entirely, and organic phase ratio is many lower than 5%, bring harm not only to the serviceable life of instrument and chromatographic column, also to after mensuration, the flushing of chromatographic column adds operation steps.
Although also there is the report of five kinds of alkaloids (ephedrine, d-pseudo-ephedrine, methylephedrine, demethyl ephedrine, Nor pseudo ephedrine) in Simultaneously test Herba Ephedrae, four kinds of alkaloids (ephedrine, d-pseudo-ephedrine, methylephedrine, demethyl ephedrine) and three kinds of alkaloids (ephedrine, d-pseudo-ephedrine, methylephedrine), but its five kinds alkaloidal mensuration need special phenyl post, column length is 250mm, mobile phase is phosphoric acid buffer salt system, the ratio of organic phase and buffer salt water system is 4: 96, and flow velocity is 0.6ml/min; Though four kinds of alkaloidal mensuration are common Waters-ODS post, its column length is 250mm, and mobile phase is phosphoric acid buffer salt system, and the ratio of organic phase and buffer salt water system is 3: 97.25, and flow velocity is 0.8ml/min; And three kinds of alkaloidal Phenomenex synersi posts that are determined as, its length is 250mm, and mobile phase is phosphoric acid buffer salt system, and the ratio of organic phase and buffer salt water system is 2: 98, and flow velocity is 1ml/min.In view of this, the Simultaneously test of multiple ephedrines composition, the setting of its various parameter is all outside pharmacopeia annex VID high performance liquid chromatography regulation parameter, all needs brokenly conventional independent setting.
In above-mentioned background situation, for the quality of easy, quick, science, specification, multi-objective control medicinal material and its preparation, invent the common chromatographic column of employing, non-buffered salt has been mobile phase, in pharmacopeia annex VID high performance liquid chromatography defined parameter area, three kinds of alkaloidal content in Simultaneously test Herba Ephedrae and preparation thereof.
Summary of the invention
First with common reverse-phase chromatographic column, non-buffered salt mobile phase, its volume ratio is acetonitrile-methanol-0.1% phosphoric acid of 1 ~ 1.5: 4 ~ 4.2: 94.5 ~ 95.0, at 207 ± 2nm place, the content of ephedrine hydrochloride, pseudoephedrine hydrochloride and mephedrine in Simultaneously test Herba Ephedrae and preparation thereof.According to three kinds of alkaloidal quantitative chromatographic figure, crest is separated good, and baseline is steady, goes out peak complete (Fig. 1,2,3,4,5) in 20 minutes.
By methodological study, the sample size of ephedrine hydrochloride is at 0.1029-0.686 μ g, and be good linear relationship (see Fig. 6) with peak area, regression equation is: Y=2303175.3X-1526, r=0.9999; Pseudoephedrine hydrochloride sample size is at 0.02511-0.1674 μ g, and be good linear relationship (see Fig. 7) with peak area, regression equation is Y=2307131X-4611, r=0.9996; Mephedrine sample size is at 0.02223 ~ 0.1482 μ g, and be good linear relationship (see Fig. 8) with peak area, regression equation is: Y=2054332.3X-1016, r=0.9999.Adopt application of sample recovery experiment, result shows: the average recovery rate of ephedrine hydrochloride is 100.56% (n=6), RSD is 0.20% (see table 1); The average recovery rate of pseudoephedrine hydrochloride is 97.84% (n=6), RSD is 0.94% (see table 2).The average recovery rate of mephedrine is 98.45% (n=6), RSD is 1.05% (see table 3).Precision (see table 4), stability (see table 5), repeatability (see table 6), specificity (see Fig. 3,4,5) etc., empirical tests all meets methodology requirement.Be applicable to three kinds of alkaloidal quantitative measurements in Herba Ephedrae and preparation thereof.
The technical solution adopted for the present invention to solve the technical problems is:
(1) chromatographic condition and system suitability take octadecylsilane chemically bonded silica as filling agent; Be that acetonitrile-methanol-0.1% phosphoric acid of 1 ~ 1.5: 4 ~ 4.2: 94.5 ~ 95.0 is for mobile phase with volume ratio; Column temperature: 40 DEG C; Determined wavelength is 207nm; Number of theoretical plate calculates should be not less than 3000 by ephedrine hydrochloride peak;
(2) to get ephedrine hydrochloride, pseudoephedrine hydrochloride and mephedrine appropriate in the preparation of reference substance solution, accurately weighed, add hydrochloric acid-70% methanol solution that volume ratio is 1: 200, make the solution of every 1ml hydrochloric ephedrine 30 ~ 15 μ g, pseudoephedrine hydrochloride 10 ~ 5 μ g, mephedrine 6 ~ 3 μ g, product solution in contrast;
(3) Herba Ephedrae fine powder 0.2 ~ 0.3g is got in the preparation of need testing solution, accurately weighed, or Chinese ephedra preparation 0.5 ~ 0.8g, accurately weighed, put in tool plug conical flask respectively, precision adds 70% methyl alcohol 25ml, close plug, weighed weight, with power 250W, the ultrasonic process of frequency 33kHz 10 ~ 30 minutes, let cool, weighed weight again, the weight of less loss is supplied with 70% methyl alcohol, shake up, , filter, precision measures subsequent filtrate 2ml, put in the chromatographic column of the internal diameter 1cm that 100-120 order neutral alumina 1 ~ 2g is housed, with in 70% methanol-eluted fractions to the measuring bottle of 10ml to about 9ml, add 1 phosphoric acid, methanol dilution with 70% is to scale, shake up, filter with the miillpore filter of 0.45 μm, get subsequent filtrate, as need testing solution,
(4) determination method is accurate respectively draws above-mentioned reference substance solution and each 10 μ l of need testing solution, injection liquid chromatography, three kinds of alkaloidal content in working sample.
For the preferred determination techniques scheme of Herba Ephedrae be:
(1) chromatographic condition and system suitability take octadecylsilane chemically bonded silica as filling agent; Be that acetonitrile-methanol-0.1% phosphoric acid of 1.5: 4: 94.5 is for mobile phase with volume ratio; Column temperature: 40 DEG C; Determined wavelength is 207nm; Number of theoretical plate calculates should be not less than 3000 by ephedrine hydrochloride peak;
(2) to get ephedrine hydrochloride, pseudoephedrine hydrochloride and mephedrine respectively appropriate in the preparation of reference substance solution, accurately weighed, add hydrochloric acid-70% methanol solution that volume ratio is 1: 200, make the solution of every 1ml hydrochloric ephedrine 20 μ g, pseudoephedrine hydrochloride 10 μ g, mephedrine 3 μ g, to obtain final product;
(3) Herba Ephedrae fine powder 0.2g is got in the preparation of need testing solution, accurately weighed, put in tool plug conical flask, precision adds 70% methyl alcohol 25ml, close plug, weighed weight, with power 250W, the ultrasonic process of frequency 33kHz 30 minutes, let cool, weighed weight again, the weight of less loss is supplied with 70% methyl alcohol, shake up, filter paper filters, precision measures subsequent filtrate 2ml, put in the chromatographic column of the internal diameter 1cm that 100-120 order neutral alumina 1.5g is housed, with in 70% methanol-eluted fractions to the measuring bottle of 10ml to about 9ml, add 1 phosphoric acid, methanol dilution with 70% is to scale, shake up, filter with 0.45 μm of miillpore filter, get subsequent filtrate, as need testing solution,
(4) determination method is accurate respectively draws above-mentioned reference substance solution and each 10 μ l of need testing solution, injection liquid chromatography, measures three kinds of alkaloidal content in Herba Ephedrae.
For the preferred determination techniques scheme of Chinese ephedra and almond containing cough stopping sheet be:
(1) chromatographic condition and system suitability take octadecylsilane chemically bonded silica as filling agent; Be that acetonitrile-methanol-0.1% phosphoric acid of 1: 4.2: 94.8 is for mobile phase with volume ratio; Column temperature: 40 DEG C; Determined wavelength is 207nm; Number of theoretical plate calculates should be not less than 3000 by ephedrine hydrochloride peak;
(2) to get ephedrine hydrochloride, pseudoephedrine hydrochloride and mephedrine respectively appropriate in the preparation of reference substance solution, accurately weighed, add hydrochloric acid-70% methanol solution that volume ratio is 1: 200, make the solution of every 1ml hydrochloric ephedrine 30 μ g, pseudoephedrine hydrochloride 10 μ g, mephedrine 5 μ g, to obtain final product;
(3) Chinese ephedra and almond containing cough stopping sheet 20 is got in the preparation of need testing solution, removing dressing, accurately weighed, porphyrize, cross 50 mesh sieves, get 0.8g, accurately weighed, put in tool plug conical flask, precision adds 70% methyl alcohol 25ml, close plug, weighed weight, with power 250W, the ultrasonic process of frequency 33kHz 10 minutes, let cool, weighed weight again, the weight of less loss is supplied with methyl alcohol, shake up, filter, precision measures subsequent filtrate 2ml, put in the chromatographic column of the internal diameter 1cm that 100-120 order neutral alumina 1.5g is housed, with in 70% methanol-eluted fractions to the measuring bottle of 10ml to about 9ml, add 1 phosphoric acid, methanol dilution with 70% is to scale, shake up, filter with the miillpore filter of 0.45 μm, get subsequent filtrate, as need testing solution,
(4) determination method is accurate respectively draws above-mentioned reference substance solution and each 10 μ l of need testing solution, injection liquid chromatography, measures three kinds of alkaloidal content in Chinese ephedra and almond containing cough stopping sheet.
For the preferred determination techniques scheme of golden Chinese ephedra and almond containing cough stopping sheet be:
(1) chromatographic condition and system suitability take octadecylsilane chemically bonded silica as filling agent; Be that acetonitrile-methanol-0.1% phosphoric acid of 1: 4: 95 is for mobile phase with volume ratio; Column temperature: 40 DEG C; Determined wavelength is 207nm; Number of theoretical plate calculates should be not less than 3000 by ephedrine hydrochloride peak;
(2) to get ephedrine hydrochloride, pseudoephedrine hydrochloride and mephedrine respectively appropriate in the preparation of reference substance solution, accurately weighed, add hydrochloric acid-70% methanol solution that volume ratio is 1: 200, make the solution of every 1ml hydrochloric ephedrine 15 μ g, pseudoephedrine hydrochloride 5 μ g, mephedrine 3 μ g, to obtain final product;
(3) golden Chinese ephedra and almond containing cough stopping sheet 20 is got in the preparation of need testing solution, removing dressing, accurately weighed, porphyrize, cross 50 mesh sieves, get 0.8g, accurately weighed, put in tool plug conical flask, precision adds 70% methyl alcohol 25ml, close plug, weighed weight, with power 250W, the ultrasonic process of frequency 33kHz 10 minutes, let cool, weighed weight again, the weight of less loss is supplied with methyl alcohol, shake up, filter, precision measures subsequent filtrate 2ml, put in the chromatographic column of the internal diameter 1cm that 100-120 order neutral alumina 1.8g is housed, with in 70% methanol-eluted fractions to the measuring bottle of 10ml to about 9ml, add 1 phosphoric acid, methanol dilution with 70% is to scale, shake up, filter with the miillpore filter of 0.45 μm, get subsequent filtrate, as need testing solution,
(4) determination method is accurate respectively draws above-mentioned reference substance solution and each 10 μ l of need testing solution, and injection liquid chromatography, measures three kinds of alkaloidal content in golden Chinese ephedra and almond containing cough stopping sheet.
Principle of the present invention is as follows:
Utilize ephedrine hydrochloride, pseudoephedrine hydrochloride and mephedrine, maximum absorption band (see Fig. 9,10,11) is all presented at 205 ± 2nm, take 207nm as determined wavelength, these three kinds of alkaloidal sample sizes within the specific limits, good linear relationship is presented respectively with its peak area, and for quantitative measurement.
Innovative point of the present invention and beneficial effect as follows:
(1) first with common reverse-phase chromatographic column, non-buffered salt mobile phase, namely volume ratio is acetonitrile-methanol-0.1% phosphoric acid of 1 ~ 1.5: 4 ~ 4.2: 94.5 ~ 95.0, at 207 ± 2nm place, the content of ephedrine hydrochloride, pseudoephedrine hydrochloride and mephedrine in Simultaneously test Herba Ephedrae and preparation thereof.Measurement result is pointed out, these three kinds of alkaloidss, and in Herba Ephedrae and preparation, content belongs to higher, and the mensuration with evaluation quality is worth.
(2) By consulting literatures is learnt, current Herba Ephedrae has the alkaloidal content report of mensuration more than three kinds, in the preparation of compound composition, due to increasing of flavour of a drug, the interference of composition is all measure ephedrine at present, or the report of ephedrine and pseudoephedrine total amount, its mobile phase is also is mostly buffering salts, the optics of perishable instrument and damage chromatographic column.
(3) non-buffered salt mobile phase of the present invention, available protecting instrument performance, extends the chromatographic column life-span, simplifies after detection terminates, the cleaning procedure of chromatographic column.
(4) common reverse-phase chromatographic column of the present invention is compared with the specific chromatographic column of report, first the length range of chromatographic column expands, the chromatographic column of 150 ~ 250nm length can be applied, and crest separation all meets the requirements, no longer by the restriction of chromatographic column brand, make testing conditions generalization, cost reduces, and is beneficial to the universal of grass-roots unit and applies.
(5) in the Herba Ephedrae reported, five kinds of alkaloidal mensuration, the ratio of organic phase is 4, and flow velocity is 0.6ml/min; Four kinds of alkaloidal mensuration, the ratio 3 of organic phase, flow velocity is 0.8ml/min; Three kinds of alkaloidal mensuration, the ratio 2 of organic phase, flow velocity is 1ml/min.Not only its organic phase ratio is less than 5, and flow velocity is less than 1ml/min more, like this, will extend the working time of mensuration, and the column length time containing buffer salt water system, not only there will be C a high proportion of
18the component retention that the random coils of chain causes changes, and the chromatographic system caused is unstable, and the theoretical cam curve straight line that also there will be chromatographic column declines, and crest alteration of form, do not meet mensuration requirement, chromatographic column is scrapped.Therefore, mobile phase of the present invention, the organic phase ratio specified under the ratio of organic phase being fall into Chinese Pharmacopoeia version in 2010 annex VID item should be not less than 5% regulation usually, avoids C
18the random coils of chain and cause adverse consequences, maintains the normal service life of chromatographic column.Mobile phase of the present invention effectively can also be separated six kinds of alkaloids (see Fig. 2) in Herba Ephedrae, but is limited to the consistance with preparation, and its evaluation index just has quality control meaning, selects to measure three kinds of higher alkaloids of content.
(6) appearance of the inventive method, solve the difficult problem that measures of easy, quick, low cost simultaneously of multiple alkaloid in Herba Ephedrae and preparation Chinese ephedra and almond containing cough stopping sheet thereof and golden Chinese ephedra and almond containing cough stopping sheet, achieve the object of multicomponent, multi objective, comprehensive descision drug quality.Be also other preparations containing Herba Ephedrae, provide multi objective quantitative measurement new way, tool example and exemplary role.
Accompanying drawing explanation
Fig. 1 tri-kinds of alkaloid reference substance HPLC chromatograms
Fig. 2 Herba Ephedrae HPLC chromatogram
Fig. 3 gold Chinese ephedra and almond containing cough stopping sheet HPLC chromatogram
Fig. 4 Chinese ephedra and almond containing cough stopping sheet HPLC chromatogram
The blank HPLC chromatogram of Fig. 5 Chinese ephedra and almond containing cough stopping sheet
The linear relationship chart of Fig. 6 ephedrine hydrochloride
The linear relationship chart of Fig. 7 pseudoephedrine hydrochloride
The linear relationship chart of Fig. 8 mephedrine
Fig. 9 ephedrine hydrochloride spectral scan figure
Figure 10 pseudoephedrine hydrochloride spectral scan figure
Figure 11 mephedrine spectral scan figure
In Fig. 6, Fig. 7, Fig. 8, ordinate is peak area; Horizontal ordinate is sample size (μ g)
Fig. 9, Figure 10, Tu11Zhong, ordinate is absorbance unit (mAU); Horizontal ordinate is wavelength (nm)
In Fig. 1, Fig. 2, Fig. 3, Fig. 4, being 1. ephedrine hydrochloride peak, is 2. pseudoephedrine hydrochloride peak, is 3. mephedrine peak, 4. hydrochloride methyl pseudoephedrine 5. hydrochloric acid demethyl ephedrine 6. hydrochloric acid Nor pseudo ephedrine
The specific embodiment of the invention
Embodiment 1:
(1) chromatographic condition and system suitability take octadecylsilane chemically bonded silica as filling agent; Be that acetonitrile-methanol-0.1% phosphoric acid of 1 ~ 1.5: 4 ~ 4.2: 94.5 ~ 95.0 is for mobile phase with volume ratio; Column temperature: 40 DEG C; Determined wavelength is 207nm; Number of theoretical plate calculates should be not less than 3000 by ephedrine hydrochloride peak;
(2) to get ephedrine hydrochloride, pseudoephedrine hydrochloride and mephedrine appropriate in the preparation of reference substance solution, accurately weighed, add hydrochloric acid-70% methanol solution that volume ratio is 1: 200, make the solution of every 1ml hydrochloric ephedrine 30 ~ 15 μ g, pseudoephedrine hydrochloride 10 ~ 5 μ g, mephedrine 6 ~ 3 μ g, product solution in contrast;
(3) Herba Ephedrae fine powder 0.2 ~ 0.3g is got in the preparation of need testing solution, accurately weighed, or Chinese ephedra preparation 0.5 ~ 0.8g, accurately weighed, put in tool plug conical flask respectively, precision adds 70% methyl alcohol 25ml, close plug, weighed weight, with power 250W, the ultrasonic process of frequency 33kHz 10 ~ 30 minutes, let cool, weighed weight again, the weight of less loss is supplied with 70% methyl alcohol, shake up, , filter, precision measures subsequent filtrate 2ml, put in the chromatographic column of the internal diameter 1cm that 100-120 order neutral alumina 1 ~ 2g is housed, with in 70% methanol-eluted fractions to the measuring bottle of 10ml to about 9ml, add 1 phosphoric acid, methanol dilution with 70% is to scale, shake up, filter with the miillpore filter of 0.45 μm, get subsequent filtrate, as need testing solution,
(4) determination method is accurate respectively draws above-mentioned reference substance solution and each 5 ~ 10 μ l of need testing solution, injection liquid chromatography, the content of three kinds of ephedrines in working sample.Determine commercially available 3 batches of Chinese ephedra content, the results are shown in Table 7.
Embodiment 2:
(1) chromatographic condition and system suitability take octadecylsilane chemically bonded silica as filling agent; Be that acetonitrile-methanol-0.1% phosphoric acid of 1: 4.2: 94.8 is for mobile phase with volume ratio; Column temperature: 40 DEG C; Determined wavelength is 207nm; Number of theoretical plate calculates should be not less than 3000 by ephedrine hydrochloride peak;
(2) to get ephedrine hydrochloride, pseudoephedrine hydrochloride and mephedrine respectively appropriate in the preparation of reference substance solution, accurately weighed, add hydrochloric acid-70% methanol solution that volume ratio is 1: 200, make the solution of every 1ml hydrochloric ephedrine 30 μ g, pseudoephedrine hydrochloride 10 μ g, mephedrine 5 μ g, to obtain final product;
(3) Chinese ephedra and almond containing cough stopping sheet 20 is got in the preparation of need testing solution, removing dressing, accurately weighed, porphyrize, cross 50 mesh sieves, get 0.8g, accurately weighed, put in tool plug conical flask, precision adds 70% methyl alcohol 25ml, close plug, weighed weight, with power 250W, the ultrasonic process of frequency 33kHz 10 minutes, let cool, weighed weight again, the weight of less loss is supplied with methyl alcohol, shake up, filter, precision measures subsequent filtrate 2ml, put in the chromatographic column of the internal diameter 1cm that 100-120 order neutral alumina 1.5g is housed, with in 70% methanol-eluted fractions to the measuring bottle of 10ml to about 9ml, add 1 phosphoric acid, methanol dilution with 70% is to scale, shake up, filter with the miillpore filter of 0.45 μm, get subsequent filtrate, as need testing solution,
(4) determination method is accurate respectively draws above-mentioned reference substance solution and each 10 μ l of need testing solution, injection liquid chromatography, measures the content of three kinds of ephedrines in test sample.Determine commercially available 3 batches of Chinese ephedra and almond containing cough stopping sheet content, the results are shown in Table 7.
Embodiment 3:
(1) chromatographic condition and system suitability take octadecylsilane chemically bonded silica as filling agent; Be that acetonitrile-methanol-0.1% phosphoric acid of 1: 4: 95 is for mobile phase with volume ratio; Column temperature: 40 DEG C; Determined wavelength is 207nm; Number of theoretical plate calculates should be not less than 3000 by ephedrine hydrochloride peak;
(2) to get ephedrine hydrochloride, pseudoephedrine hydrochloride and mephedrine respectively appropriate in the preparation of reference substance solution, accurately weighed, add hydrochloric acid-70% methanol solution that volume ratio is 1: 200, make the solution of every 1ml hydrochloric ephedrine 15 μ g, pseudoephedrine hydrochloride 5 μ g, mephedrine 3 μ g, to obtain final product;
(3) golden Chinese ephedra and almond containing cough stopping sheet 20 is got in the preparation of need testing solution, removing dressing, accurately weighed, porphyrize, cross 50 mesh sieves, get 0.8g, accurately weighed, put in tool plug conical flask, precision adds 70% methyl alcohol 25ml, close plug, weighed weight, with power 250W, the ultrasonic process of frequency 33kHz 10 minutes, let cool, weighed weight again, the weight of less loss is supplied with methyl alcohol, shake up, filter, precision measures subsequent filtrate 2ml, put in the chromatographic column of the internal diameter 1cm that 100-120 order neutral alumina 1.8g is housed, with in 70% methanol-eluted fractions to the measuring bottle of 10ml to about 9ml, add 1 phosphoric acid, methanol dilution with 70% is to scale, shake up, filter with the miillpore filter of 0.45 μm, get subsequent filtrate, as need testing solution,
(4) determination method is accurate respectively draws above-mentioned reference substance solution and each 10 μ l of need testing solution, injection liquid chromatography, measures the content of three kinds of ephedrines in test sample.Determine 3 batches of golden Chinese ephedra and almond containing cough stopping sheet content, the results are shown in Table 7.
The recovery test result of ephedrine hydrochloride in table 1 sample
The recovery test result of pseudoephedrine hydrochloride in table 2 sample
The recovery test result of mephedrine in table 3 sample
Note: for saving reference substance, the cumulative volume of sample is not 25ml, is 15ml, in the consistent situation of concentration, sampling amount reduces.
Table 4 reference substance Precision Experiment result (peak area)
Table 5 reference substance stability experiment result (peak area)
Table 6 replica test result (mg/g)
The assay result (mg/g) of table 7 Herba Ephedrae and preparation
Claims (5)
1. three kinds of alkaloidal content assaying methods in Herba Ephedrae and preparation thereof, is characterized in that:
(1) chromatographic condition and system suitability take octadecylsilane chemically bonded silica as filling agent; Be that acetonitrile-methanol-0.1% phosphoric acid of 1 ~ 1.5: 4 ~ 4.2: 94.5 ~ 95.0 is for mobile phase with volume ratio; Column temperature: 40 DEG C; Determined wavelength is 207nm; Number of theoretical plate calculates should be not less than 3000 by ephedrine hydrochloride peak;
(2) to get ephedrine hydrochloride, pseudoephedrine hydrochloride and mephedrine appropriate in the preparation of reference substance solution, accurately weighed, add hydrochloric acid-70% methanol solution that volume ratio is 1: 200, make the solution of every 1ml hydrochloric ephedrine 30 ~ 15 μ g, pseudoephedrine hydrochloride 10 ~ 5 μ g, mephedrine 6 ~ 3 μ g, product solution in contrast;
(3) Herba Ephedrae fine powder 0.2 ~ 0.3g is got in the preparation of need testing solution, accurately weighed, or Chinese ephedra preparation 0.5 ~ 0.8g, accurately weighed, put in tool plug conical flask respectively, precision adds 70% methyl alcohol 25ml, close plug, weighed weight, with power 250W, the ultrasonic process of frequency 33kHz 10 ~ 30 minutes, let cool, weighed weight again, the weight of less loss is supplied with 70% methyl alcohol, shake up, filter, precision measures subsequent filtrate 2ml, put in the chromatographic column of the internal diameter 1cm that 100-120 order neutral alumina 1 ~ 2g is housed, with in 70% methanol-eluted fractions to the measuring bottle of 10ml to 9ml, add 1 phosphoric acid, methanol dilution with 70% is to scale, shake up, filter with the miillpore filter of 0.45 μm, get subsequent filtrate, as need testing solution,
(4) determination method is accurate respectively draws above-mentioned reference substance solution and each 5 ~ 10 μ l of need testing solution, injection liquid chromatography, three kinds of alkaloidal content in working sample.
2. three kinds of alkaloidal content assaying methods in a kind of Herba Ephedrae according to claim 1 and preparation thereof, is characterized in that described preparation refers to Chinese ephedra and almond containing cough stopping sheet and golden Chinese ephedra and almond containing cough stopping sheet.
3. three kinds of alkaloidal content assaying methods in a kind of Herba Ephedrae according to claim 1 and preparation thereof, are further characterized in that:
(1) chromatographic condition and system suitability take octadecylsilane chemically bonded silica as filling agent; Be that acetonitrile-methanol-0.1% phosphoric acid of 1.5: 4: 94.5 is for mobile phase with volume ratio; Column temperature: 40 DEG C; Determined wavelength is 207nm; Number of theoretical plate calculates should be not less than 3000 by ephedrine hydrochloride peak;
(2) to get ephedrine hydrochloride, pseudoephedrine hydrochloride and mephedrine respectively appropriate in the preparation of reference substance solution, accurately weighed, add hydrochloric acid-70% methanol solution that volume ratio is 1: 200, make the solution of every 1ml hydrochloric ephedrine 20 μ g, pseudoephedrine hydrochloride 10 μ g, mephedrine 3 μ g, to obtain final product;
(3) Herba Ephedrae fine powder 0.2g is got in the preparation of need testing solution, accurately weighed, put in tool plug conical flask, precision adds 70% methyl alcohol 25ml, close plug, weighed weight, with power 250W, the ultrasonic process of frequency 33kHz 30 minutes, let cool, weighed weight again, the weight of less loss is supplied with 70% methyl alcohol, shake up, filter paper filters, precision measures subsequent filtrate 2ml, put in the chromatographic column of the internal diameter 1cm that 100-120 order neutral alumina 1.5g is housed, with in 70% methanol-eluted fractions to the measuring bottle of 10ml to 9ml, add 1 phosphoric acid, methanol dilution with 70% is to scale, shake up, filter with 0.45 μm of miillpore filter, get subsequent filtrate, as need testing solution,
(4) determination method is accurate respectively draws above-mentioned reference substance solution and each 10 μ l of need testing solution, injection liquid chromatography, measures three kinds of alkaloidal content in Herba Ephedrae.
4. three kinds of alkaloidal content assaying methods in a kind of Herba Ephedrae according to claim 1 and preparation thereof, are further characterized in that:.
(1) chromatographic condition and system suitability take octadecylsilane chemically bonded silica as filling agent; Be that acetonitrile-methanol-0.1% phosphoric acid of 1: 4.2: 94.8 is for mobile phase with volume ratio; Column temperature: 40 DEG C; Determined wavelength is 207nm; Number of theoretical plate calculates should be not less than 3000 by ephedrine hydrochloride peak;
(2) to get ephedrine hydrochloride, pseudoephedrine hydrochloride and mephedrine respectively appropriate in the preparation of reference substance solution, accurately weighed, add hydrochloric acid-70% methanol solution that volume ratio is 1: 200, make the solution of every 1ml hydrochloric ephedrine 30 μ g, pseudoephedrine hydrochloride 10 μ g, mephedrine 5 μ g, to obtain final product;
(3) Chinese ephedra and almond containing cough stopping sheet 20 is got in the preparation of need testing solution, removing dressing, accurately weighed, porphyrize, cross 50 mesh sieves, get 0.8g, accurately weighed, put in tool plug conical flask, precision adds 70% methyl alcohol 25ml, close plug, weighed weight, with power 250W, the ultrasonic process of frequency 33kHz 10 minutes, let cool, weighed weight again, the weight of less loss is supplied with methyl alcohol, shake up, filter, precision measures subsequent filtrate 2ml, put in the chromatographic column of the internal diameter 1cm that 100-120 order neutral alumina 1.5g is housed, with in 70% methanol-eluted fractions to the measuring bottle of 10ml to 9ml, add 1 phosphoric acid, methanol dilution with 70% is to scale, shake up, filter with the miillpore filter of 0.45 μm, get subsequent filtrate, as need testing solution,
(4) determination method is accurate respectively draws above-mentioned reference substance solution and each 10 μ l of need testing solution, injection liquid chromatography, measures three kinds of alkaloidal content in Chinese ephedra and almond containing cough stopping sheet.
5. three kinds of alkaloidal content assaying methods in a kind of Herba Ephedrae according to claim 1 and preparation thereof, are further characterized in that:
(1) chromatographic condition and system suitability take octadecylsilane chemically bonded silica as filling agent; Be that acetonitrile-methanol-0.1% phosphoric acid of 1: 4: 95 is for mobile phase with volume ratio; Column temperature: 40 DEG C; Determined wavelength is 207nm; Number of theoretical plate calculates should be not less than 3000 by ephedrine hydrochloride peak;
(2) to get ephedrine hydrochloride, pseudoephedrine hydrochloride and mephedrine respectively appropriate in the preparation of reference substance solution, accurately weighed, add hydrochloric acid-70% methanol solution that volume ratio is 1: 200, make the solution of every 1ml hydrochloric ephedrine 15 μ g, pseudoephedrine hydrochloride 5 μ g, mephedrine 3 μ g, to obtain final product;
(3) golden Chinese ephedra and almond containing cough stopping sheet 20 is got in the preparation of need testing solution, removing dressing, accurately weighed, porphyrize, cross 50 mesh sieves, get 0.8g, accurately weighed, put in tool plug conical flask, precision adds 70% methyl alcohol 25ml, close plug, weighed weight, with power 250W, the ultrasonic process of frequency 33kHz 10 minutes, let cool, weighed weight again, the weight of less loss is supplied with methyl alcohol, shake up, filter, precision measures subsequent filtrate 2ml, put in the chromatographic column of the internal diameter 1cm that 100-120 order neutral alumina 1.8g is housed, with in 70% methanol-eluted fractions to the measuring bottle of 10ml to 9ml, add 1 phosphoric acid, methanol dilution with 70% is to scale, shake up, filter with the miillpore filter of 0.45 μm, get subsequent filtrate, as need testing solution,
(4) determination method is accurate respectively draws above-mentioned reference substance solution and each 10 μ l of need testing solution, and injection liquid chromatography, measures three kinds of alkaloidal content in golden Chinese ephedra and almond containing cough stopping sheet.
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| CN105954379A (en) * | 2016-04-22 | 2016-09-21 | 广西壮族自治区梧州食品药品检验所 | Method for extracting ephedrine hydrochloride in lung ventilating and regulating pill |
| CN106950292A (en) * | 2017-01-17 | 2017-07-14 | 天津中新药业集团股份有限公司达仁堂制药厂 | A kind of lung-clearing anti-inflammatory ball quality standard detecting method |
| CN110297043A (en) * | 2018-03-21 | 2019-10-01 | 上海医药工业研究院 | A kind of analysis method of 2- methylamino -1- phenyl-1-propanol |
| CN114544786A (en) * | 2020-11-25 | 2022-05-27 | 中国科学院大连化学物理研究所 | Chromatographic separation method of organic amine alkaloid in relative ephedra by utilizing fluorine-containing fixation |
| CN113288922B (en) * | 2021-05-13 | 2022-04-08 | 海南葫芦娃药业集团股份有限公司 | Ephedra extract and preparation method thereof |
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