CN102636589B - HPLC (High Performance Liquid Chromatography) quantitative method for cephaeline hydrochloride and ipecine hydrochloride in ipecacuanha medicinal material and preparation of ipecacuanha medicinal material - Google Patents
HPLC (High Performance Liquid Chromatography) quantitative method for cephaeline hydrochloride and ipecine hydrochloride in ipecacuanha medicinal material and preparation of ipecacuanha medicinal material Download PDFInfo
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- 244000284152 Carapichea ipecacuanha Species 0.000 title claims abstract description 75
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- 238000002360 preparation method Methods 0.000 title claims abstract description 45
- APCJEUAHYFIVKA-UHFFFAOYSA-N 5-trimethylsilylthiophene-2-carboxylic acid Chemical compound C[Si](C)(C)C1=CC=C(C(O)=O)S1 APCJEUAHYFIVKA-UHFFFAOYSA-N 0.000 title claims abstract description 44
- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 28
- 229960005208 ipecacuanha Drugs 0.000 title claims abstract description 12
- 229960002694 emetine Drugs 0.000 title abstract description 18
- AUVVAXYIELKVAI-CKBKHPSWSA-N emetine Chemical compound N1CCC2=CC(OC)=C(OC)C=C2[C@H]1C[C@H]1C[C@H]2C3=CC(OC)=C(OC)C=C3CCN2C[C@@H]1CC AUVVAXYIELKVAI-CKBKHPSWSA-N 0.000 title abstract description 18
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 title abstract description 8
- 238000004445 quantitative analysis Methods 0.000 title abstract 2
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 137
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims abstract description 48
- 238000000034 method Methods 0.000 claims abstract description 38
- 239000000284 extract Substances 0.000 claims abstract description 36
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 claims abstract description 31
- 239000000706 filtrate Substances 0.000 claims abstract description 27
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims abstract description 24
- 239000000843 powder Substances 0.000 claims abstract description 19
- 229940098465 tincture Drugs 0.000 claims abstract description 14
- 239000012530 fluid Substances 0.000 claims abstract description 11
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- HUEYSSLYFJVUIS-MRFSYGAJSA-N (2s,3r,11bs)-2-[[(1r)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolin-1-yl]methyl]-3-ethyl-9,10-dimethoxy-2,3,4,6,7,11b-hexahydro-1h-benzo[a]quinolizine;hydron;chloride Chemical compound Cl.N1CCC2=CC(OC)=C(OC)C=C2[C@H]1C[C@H]1C[C@H]2C3=CC(OC)=C(OC)C=C3CCN2C[C@@H]1CC HUEYSSLYFJVUIS-MRFSYGAJSA-N 0.000 claims description 55
- 229960001923 emetine hydrochloride Drugs 0.000 claims description 55
- 238000012360 testing method Methods 0.000 claims description 43
- 239000013558 reference substance Substances 0.000 claims description 32
- 230000007935 neutral effect Effects 0.000 claims description 21
- 239000007788 liquid Substances 0.000 claims description 20
- DTGZHCFJNDAHEN-YSFUMNCJSA-N Cephaeline Natural products O(C)c1c(OC)cc2c([C@H]3N(C[C@@H](CC)[C@@H](C[C@H]4NCCc5c4cc(OC)c(O)c5)C3)CC2)c1 DTGZHCFJNDAHEN-YSFUMNCJSA-N 0.000 claims description 18
- AUVVAXYIELKVAI-UHFFFAOYSA-N SJ000285215 Natural products N1CCC2=CC(OC)=C(OC)C=C2C1CC1CC2C3=CC(OC)=C(OC)C=C3CCN2CC1CC AUVVAXYIELKVAI-UHFFFAOYSA-N 0.000 claims description 18
- DTGZHCFJNDAHEN-OZEXIGSWSA-N cephaeline Chemical compound N1CCC2=CC(O)=C(OC)C=C2[C@H]1C[C@H]1C[C@H]2C3=CC(OC)=C(OC)C=C3CCN2C[C@@H]1CC DTGZHCFJNDAHEN-OZEXIGSWSA-N 0.000 claims description 18
- 238000013459 approach Methods 0.000 claims description 14
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- 235000008216 herbs Nutrition 0.000 claims 3
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- 238000002479 acid--base titration Methods 0.000 abstract description 10
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- 229930012976 ipecacuanha alkaloid Natural products 0.000 abstract 1
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 30
- 150000003797 alkaloid derivatives Chemical class 0.000 description 10
- 239000012071 phase Substances 0.000 description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 7
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonium chloride Substances [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 6
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 6
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 6
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- 241000123069 Ocyurus chrysurus Species 0.000 description 1
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Abstract
The invention relates to a HPLC (High Performance Liquid Chromatography) quantitative method for cephaeline hydrochloride and ipecine hydrochloride in an ipecacuanha medicinal material and a preparation of ipecacuanha medicinal material. The method is characterized by comprising the following steps: firstly, performing common isocratic elution according to PHLC method, and taking acetonitrile-carbinol-0.1% of phosphoric acid at a volume ratio of (8-9.5):(3-5):(86-88) as a flowing phase, wherein a detecting wavelength is 205nm; and simultaneously measuring the contents of the cephaeline hydrochloride and ipecine hydrochloride in the ipecacuanha medicinal material and a fluid extract, extract and tincture thereof so as to end the history of the measurement of total alkaloids of the ipecacuanha alkaloids in an acid base titration form according to the standard in multiple countries: after performing column chromatography separation for 4 times, respectively measuring the cephaeline hydrochloride and ipecine hydrochloride through delta A at 283nm and 350nm by adopting differential spectrophotometry. According to the method, only acidic aqueous carbinol or aqueous carbinol is used; after fine powder of the medicinal material is ultrasonically extracted or each preparation is diluted, a certain amount of subsequent filtrate is absorbed; and an alumina column is used for removing impurities so as to measure. The method is quick, convenient, accurate and capable of reappearing.
Description
Technical field
The present invention relates to Cephaeline Hydrochloride and emetine hydrochloride HPLC quantivative approach in a kind of ipecac medicinal material and the preparation thereof.
Background technology
Ipecac is the dry rhizome of madder wort Cephaelis ipecacuanha (Brot.) A.Rich. or Cephaelis acuminata Karsten.It is the expelling phlegm and arresting coughing medicine.Be Brazil, Costa Rica or India's import medicinal material, record at American Pharmacopeia (24 editions) and European Pharmacopoeia (7.0).American Pharmacopeia records the quality standard of ipecac medicinal material, powdered ipecac and ipecac syrup, and its assay is: the content of measuring ipecac total alkaloids, ipecine and cephaeline respectively.After total alkaloids adopted chloroform to extract, soda acid back titration method was measured total alkaloid content.The assay of ipecine and cephaeline is then extremely loaded down with trivial details, time-consuming, need to use 4 root chromatogram columns, with different eluting solvents, through 4 column chromatographies, ipecine with after cephaeline separates, is adopted differential spectrophotometry, respectively at 283nm and 350nm wavelength place, measure the difference of absorbance log A, calculate content with Δ A.Flow process is more long, and the loss of composition to be measured is more many, causes the reappearance of method and accuracy more poor.So far, also do not find relevant ipecac alkaloids, with the report of efficient liquid phase quantitative measurement.
On the detection method basis of American Pharmacopeia (24 editions) and European Pharmacopoeia (7.0), producer drafts by import, Nat'l Pharmaceutical ﹠ Biological Products Control Institute checks, formulated the test stone of Chinese ipecac medicinal material, what assay still adopted is that soda acid back titration method was measured total alkaloid content after organic solvent extracted.Sample volume is big, and the organic solvent of cost is many.Moreover, former acid base titration method exists fatal defective in the preparation of need testing solution." get ipecac medicinal powder 7.5g, accurate claim surely, 100ml adds diethyl ether; jolting 5 minutes, adds ammonia solution 5ml again, jolting 1 hour; add water 5 ml, and ether layer is told in violent jolting; cotton is filtered; filtrate is put in the flask, and residue is with ether washing 2 times, each 25 ml; merge ether extracted liquid at first to see the sample pre-treatments program of titrimetry, evaporate to dryness, residue add 90% ethanol 2ml makes dissolving, evaporate to dryness, and 100 ℃ of heat dryings 5 minutes, residue adds 90% neutral alcohol 5ml, and the water-bath heating makes dissolving, precision add hydrochloric acid vs (0.1mol/L) 15ml, 1～2 of the red indicator solution of methylate is with NaOH vs (0.1mol/L) titration.Every 1ml hydrochloric acid vs (0.1mol/L) is equivalent to the ipecine (C of 24.03mg
29H
40N
2O
4).The unreasonable part of this handling procedure is:
1) the extraction solvent ether of Xuan Zeing, penetration power too a little less than, can't directly from the plant fine powder, alkaloid quantitatively be extracted.
2) purpose that adds the little ammonia test solution is in order to make the alkaloid in the vegetable cell free, to increase the penetration power of ether, alkaloid is easily come out by extracted by ether, should add ammonia solution earlier, adding diethyl ether.Can operate but is to add diethyl ether earlier, adds ammonia solution again, and ether has coated fine powder like this, and ammonia solution can not fully contact with fine powder, and the penetration power of ether can not get improving, and alkaloid is difficult to extract fully.
3) from the plant fine powder, extract alkaloid, except utilize to extract solvent similar mix with powerful penetration power, also will be by means of external force, i.e. heating or ultrasonic." jolting " operation in the former processing program had not both had the jolting frequency, did not have jolting intensity and jolting number of times yet, and like this, " jolting " operation can vary with each individual, and the reappearance of method is difficult to guarantee.
4) in the emulsus mixed solution container that ipecac fine powder, water and ammonia solution mix mutually, how under the situation of not changing container, ether extracted liquid is separated with fine powder quantitatively and be to influence method accuracy and reproducible factor.
5) " residue adds 90% ethanol 2ml makes dissolving, evaporate to dryness ", whether this operation changes container, and literally the meaning is not change container, if so, where is the meaning of this step operation?
Not only running program is long, the ether consumption is big for reason to sum up, former acid base titration method, contaminated environment, and has numerous uncertain factors, can't quantitatively alkaloid be extracted, and also just can't carry out quantitative measurement exactly.
Fluid ipecac extract, ipecacuanha extract all are that ipecac tincture is to make by the ethanol diluent stream medicinal extract of stipulating under an appendix IN of Chinese Pharmacopoeia version in 2010 item by the preparation of the regulation under an appendix IO of Chinese Pharmacopoeia version in 2010 item.Fluid ipecac extract and ipecac tincture are raw material preparations subsidiary under the particles for eliminating phlegm and stopping cough for children agent quality standard item, and its method for quantitatively determining all is the acid dissolving, chloroform extraction removal of impurities, alkalization again, behind the chloroform extraction alkaloid, evaporate to dryness, acid base titration, complex operation, contaminated environment, accuracy is difficult to guarantee.
Under the above-mentioned background situation, for the quality of easy, quick, science, accurate, many indexs control ipecac medicinal material and its liquid extract, medicinal extract and tincture, Cephaeline Hydrochloride and ipecine HPLC quantivative approach in ipecac medicinal material and the preparation thereof have been invented.
Summary of the invention
Reported first the spectral scan figure (seeing Fig. 1,2) of Cephaeline Hydrochloride and emetine hydrochloride, on this basis, adopt HPLC method, common isocratic elution, reverse-phase chromatographic column, volume ratio is 8～9.5: 3～5: acetonitrile-methyl alcohol of 86～88-0.1% phosphoric acid is the phase that flows; 205nm is for detecting wavelength, measured the content of Cephaeline Hydrochloride and emetine hydrochloride in ipecac medicinal material and liquid extract, medicinal extract and the tincture simultaneously, and the ipecac alkaloids that is through with always is acid base titration mensuration total alkaloids in multinational statutory standards; After 4 column chromatography for separation, adopt differential spectrophotometry, respectively in the history of 283nm and 350nm place mensuration cephaeline and ipecine.Inventive method only need be with acidic aqueous methyl alcohol or aqueous methanol, ultrasonic extraction medicinal material fine powder or dilute its liquid extract, medicinal extract and tincture after, draw a certain amount of subsequent filtrate, the alumina column removal of impurities can be measured by sample introduction, method is easy, quick, accurate, reappear.
By methodological study, the Cephaeline Hydrochloride sample size is good linear relationship at 0.01456～0.2184 μ g with peak area, and regression equation is: Y=8184939.7X+1642, and, γ=0.99997 (see Table 1, Fig. 3); The emetine hydrochloride sample size is good linear relationship with peak area, regression equation at 0.0321～0.321 μ g: Y=6856886.5X-18354, γ=0.99997 (see Table 2, Fig. 4).Adopt application of sample to reclaim experiment, the result shows: the average recovery rate of Cephaeline Hydrochloride is 96.93% (n=9), and RSD is 1.31% (seeing Table 3); The average recovery rate of emetine hydrochloride is 99.47% (n=9), and RSD is 2.02% (seeing Table 4).Precision (seeing Table 5), stability (seeing Table 6), repeatability (seeing Table 7), post durability (seeing Table 8) experiment all meet the methodology requirement.Be applicable to the quantitative measurement of the middle Cephaeline Hydrochloride of ipecac medicinal material (seeing Fig. 5,6) and preparation (seeing Fig. 7,8) thereof and emetine hydrochloride.
The present invention measures the technical scheme that Cephaeline Hydrochloride and emetine hydrochloride content adopt in ipecac medicinal material and the preparation thereof:
(1) chromatographic condition is 8～9.5: 3～5 with the system suitability test with volume ratio: acetonitrile-methyl alcohol of 86～88-0.1% phosphoric acid is the phase that flows; Column temperature: 40 ℃; The detection wavelength is 205nm; Number of theoretical plate calculates by the emetine hydrochloride peak should be not less than 1500;
(2) preparation of reference substance solution is got Cephaeline Hydrochloride and the emetine hydrochloride reference substance is an amount of, accurately claims surely, puts in the brown measuring bottle, add methyl alcohol and make stoste, get stoste again, adding flows makes the solution of hydrochloric cephaeline 10～30 μ g of every 1ml, emetine hydrochloride 30～10 μ g mutually, namely;
(3) ipecac medicinal material fine powder 0.1g is got in the preparation of need testing solution, and accurate the title decides, and puts in the tool plug conical flask; Or get the preparation that is equivalent to 1g ipecac medicinal material, put in the measuring bottle of 25ml; Accurate hydrochloric acid-60% methyl alcohol mixed solution the 25ml that adds 1: 200 of medicinal material fine powder, close plug claims decide weight, and with power 250W, the ultrasonic processing of frequency 40kHz 30 minutes is put coldly, claims to decide weight again, supplies the weight that subtracts mistake with methyl alcohol, shakes up filtration; The wine of ipecac adds 60% methyl alcohol to scale, shakes up, and filters; Precision is measured medicinal material fine powder subsequent filtrate 2ml or preparation subsequent filtrate 1ml, put on the neutral alumina post that 100-120 order neutral alumina 1～1.5 g internal diameter 1cm is housed, and be eluted in the measuring bottle of 10 ml to about 9 ml, add 1 phosphoric acid, methyl alcohol with 60% is diluted to scale, shakes up, and filters with 0.45 μ m miillpore filter, get subsequent filtrate as need testing solution, namely;
(4) the accurate reference substance solution 5 μ l that draw of determination method difference, need testing solution 2-10 μ l injects liquid chromatograph, measures, namely.
Principle of the present invention is as follows:
Cephaeline and ipecine are organic base, can generate salt with acid, increase the solubleness in hydrophilic solvent, are quantitatively extracted.So directly with acidic aqueous methyl alcohol or aqueous methanol, ultrasonic extraction medicinal material fine powder or dilute its liquid extract, medicinal extract or tincture after, draw a certain amount of subsequent filtrate, the alumina column removal of impurities can sample introduction be measured.Method does not have extraction, no evaporate to dryness, easy, quick, practical.By adjusting the volume ratio of acetonitrile, methyl alcohol and 0.1% phosphoric acid, make cephaeline and ipecine on different chromatographic columns, crest separates well.The sample size of two kinds of alkaloid components of foundation presents good linear relationship with its peak area within the specific limits again, and is used for quantitative measurement.
Innovative point of the present invention and beneficial effect are as follows:
(1) reported first the spectral scan figure of Cephaeline Hydrochloride and emetine hydrochloride, the result shows: the two spectral scan figure basically identical, present three absorption peaks, and be respectively 203 ± 1nm, 225 ± 2nm, 282 ± 2nm, the sensitivity of its crest, successively decrease successively by the ascending of wavelength, three absorption peaks can both be considered but pollute, prolong its serviceable life from the minimizing chromatographic column as this alkaloidal detection wavelength, selection is bordering on the 205nm place, as detecting wavelength, sampling amount is few, and is highly sensitive.This result provides reference for the mensuration that the colleague carries out ipecac medicinal material and preparation.
(2) adopt the HPLC method first, with common isocratic elution, reverse-phase chromatographic column, volume ratio is 8～9.5: 3～5: acetonitrile-methyl alcohol of 86～88-0.1% phosphoric acid is the phase that flows; 205nm is for detecting wavelength, measured the content of Cephaeline Hydrochloride and emetine hydrochloride in ipecac medicinal material and liquid extract, medicinal extract and the tincture simultaneously.Method only need behind ultrasonic extraction medicinal material fine powder or the diluent stream medicinal extract, be drawn a certain amount of subsequent filtrate with acidic aqueous methyl alcohol or aqueous methanol, and the alumina column removal of impurities can be measured by sample introduction, concentrated, nothing extraction that method does not have, no evaporate to dryness, easy, quick, accurate, reproduction.
(3) appearance of this assay method, the ipecac alkaloids that is through with always are acid base titration mensuration total alkaloids in multinational statutory standards; After 4 column chromatography for separation, adopt differential spectrophotometry, respectively at 283nm and 350nm place, measure the history of cephaeline and ipecine with Δ A.Reduce sampling amount significantly, saved detection time, got rid of murder by poisoning reagent environmental pollution, improved accuracy and the reappearance of method.
(4) by adopting acid base titration method and HPLC method, to ipecac medicinal material Determination on content relatively, presentation of results: not only running program is long, the ether consumption is big for former acid base titration method, contaminated environment, and exist and extract solvent and select that addition sequence improper, ammonia solution is not proper, method is described numerous uncertain factors such as not rigorous, cause and quantitatively the ipecac alkaloids to be extracted from plant tissue, also just can't carry out quantitative measurement exactly.The total alkali of acid base titration has appearred less than the situation (seeing Table 9) of two alkali sums of HPLC method mensuration.
(5) the HPLC chromatogram of ipecac medicinal material and liquid extract thereof shows, the two is all after 60 minutes, also has a strong unknown crest that keeps, its area accounts for about 30% (seeing Fig. 9,11) of total peak area, must be by the alumina column removal of impurities, it is removed, shorten the working time (seeing Figure 10,12) of assay.Point out with this: principal ingredient is not cephaeline and the ipecine of bibliographical information in ipecac medicinal material and liquid extract thereof, also contains the not principal component that another accounts for total peak area 30%, and its chemical constitution and drug effect remain further to be studied.
Description of drawings
The spectral scan figure at Fig. 1 Cephaeline Hydrochloride peak
The spectral scan figure at Fig. 2 emetine hydrochloride peak
Fig. 3 Cephaeline Hydrochloride linear relationship chart
Fig. 4 emetine hydrochloride linear relationship chart
Fig. 5 Cephaeline Hydrochloride and emetine hydrochloride reference substance HPLC chromatogram
Fig. 6 ipecac medicinal material HPLC chromatogram
Fig. 7 Cephaeline Hydrochloride and emetine hydrochloride reference substance HPLC chromatogram
Fig. 8 fluid ipecac extract agent HPLC chromatogram
The unknown peak of Fig. 9 ipecac medicinal material retention time after 60 minutes
Figure 10 aluminium oxide is removed the chromatogram at unknown peak in the medicinal material
The unknown peak of Figure 11 fluid ipecac extract agent retention time after 60 minutes
Figure 12 aluminium oxide is removed the chromatogram at unknown peak in the liquid extract
Among Fig. 1, Fig. 2, ordinate is absorbance log; Horizontal ordinate is wavelength (nm)
Among Fig. 3, Fig. 4, ordinate is peak area; Horizontal ordinate is sample size (μ g)
Among Fig. 5～Figure 12,1 is the Cephaeline Hydrochloride peak, and 2 is the emetine hydrochloride peak, and 3 is unknown peak
The specific embodiment of the invention
Embodiment 1: Cephaeline Hydrochloride and ipecine HPLC quantivative approach in the ipecac medicinal material
(1) test of chromatographic condition and system suitability is that acetonitrile-methyl alcohol-0.1% phosphoric acid of 9: 3: 88 be mobile phase with volume ratio; Column temperature: 40 ℃; The detection wavelength is 205nm; Number of theoretical plate calculates by the emetine hydrochloride peak should be not less than 1500;
(2) preparation of reference substance solution is got Cephaeline Hydrochloride and the emetine hydrochloride reference substance is an amount of, accurately claims surely, puts in the brown measuring bottle, add methyl alcohol and make stoste, get stoste again, adding flows makes the solution of hydrochloric cephaeline 10～30 μ g of every 1ml, emetine hydrochloride 30～10 μ g mutually, namely;
(3) ipecac medicinal material fine powder 0.1g is got in the preparation of need testing solution, and accurate the title decides, and puts in the tool plug conical flask, accurate hydrochloric acid-60% methyl alcohol mixed solution the 25ml that adds 1: 200, close plug claims to decide weight, with power 250W, the ultrasonic processing of frequency 40kHz 30 minutes is put coldly, claims to decide weight again, supply the weight that subtracts mistake with methyl alcohol, shake up, filter, precision is measured subsequent filtrate 2ml, put on the neutral alumina post that 100-120 order neutral alumina 1.5 g internal diameter 1cm are housed, and be eluted in the measuring bottle of 10ml to about 9ml, adding 1 phosphoric acid, the methyl alcohol with 60% is diluted to scale, shake up, filter with 0.45 μ m miillpore filter, get subsequent filtrate as need testing solution, namely;
(4) the accurate reference substance solution 5 μ l that draw of determination method difference, need testing solution 5-10 μ l injects liquid chromatograph, measures calculating content.Measure the ipecac medicinal material content in 6 batches of different places of production, the results are shown in Table 9.
Embodiment 2: Cephaeline Hydrochloride and ipecine HPLC quantivative approach in the fluid ipecac extract
(1) test of chromatographic condition and system suitability is that acetonitrile-methyl alcohol-0.1% phosphoric acid of 8.5: 4: 87.5 be mobile phase with volume ratio; Column temperature: 40 ℃; The detection wavelength is 205nm; Number of theoretical plate calculates by the emetine hydrochloride peak should be not less than 1500;
(2) preparation of reference substance solution is got Cephaeline Hydrochloride and the emetine hydrochloride reference substance is an amount of, accurately claims surely, puts in the brown measuring bottle, add methyl alcohol and make stoste, get stoste again, adding flows makes the solution of hydrochloric cephaeline 10～30 μ g of every 1ml, emetine hydrochloride 30～10 μ g mutually, namely;
(3) the accurate fluid ipecac extract 1ml that draws of the preparation of need testing solution puts in the measuring bottle of 25ml, adds 60% methyl alcohol to scale, shake up, filter, precision is measured subsequent filtrate 1ml, put on the neutral alumina post that 100-120 order neutral alumina 1g internal diameter 1cm is housed, and be eluted in the measuring bottle of 10ml to about 9ml, adding 1 phosphoric acid, the methyl alcohol with 60% is diluted to scale, shake up, filter with 0.45 μ m miillpore filter, get subsequent filtrate as need testing solution, namely;
(4) the accurate reference substance solution 5 μ l that draw of determination method difference, need testing solution 2-3 μ l injects liquid chromatograph, measures calculating content.Measure 3 batches of fluid ipecac extract agent contents, the results are shown in Table 9.
Embodiment 3: Cephaeline Hydrochloride and ipecine HPLC quantivative approach in the ipecac tincture
(1) test of chromatographic condition and system suitability is that acetonitrile-methyl alcohol-0.1% phosphoric acid of 9.5: 3.1: 87.4 be mobile phase with volume ratio; Column temperature: 40 ℃; The detection wavelength is 205nm; Number of theoretical plate calculates by the emetine hydrochloride peak should be not less than 1500;
(2) preparation of reference substance solution is got Cephaeline Hydrochloride and the emetine hydrochloride reference substance is an amount of, accurately claims surely, puts in the brown measuring bottle, add methyl alcohol and make stoste, get stoste again, adding flows makes the solution of hydrochloric cephaeline 10～30 μ g of every 1ml, emetine hydrochloride 30～10 μ g mutually, namely;
(3) the accurate ipecac tincture 20ml that draws of the preparation of need testing solution puts in the measuring bottle of 25ml, adds 60% methyl alcohol to scale, shake up, filter, precision is measured subsequent filtrate 1ml, put on the neutral alumina post that 100-120 order neutral alumina 1g internal diameter 1cm is housed, and be eluted in the measuring bottle of 10ml to about 9ml, adding 1 phosphoric acid, the methyl alcohol with 60% is diluted to scale, shake up, filter with 0.45 μ m miillpore filter, get subsequent filtrate as need testing solution, namely;
(4) the accurate reference substance solution 5 μ l that draw of determination method difference, need testing solution 2-3 μ l injects liquid chromatograph, measures calculating content.Measure 1 batch of ipecac tincture content, the results are shown in Table 9.
Embodiment 4: Cephaeline Hydrochloride and ipecine HPLC quantivative approach in the ipecacuanha extract agent
(1) test of chromatographic condition and system suitability is that acetonitrile-methyl alcohol-0.1% phosphoric acid of 9: 3.5: 87.5 be mobile phase with volume ratio; Column temperature: 40 ℃; The detection wavelength is 205nm; Number of theoretical plate calculates by the emetine hydrochloride peak should be not less than 1500;
(2) preparation of reference substance solution is got Cephaeline Hydrochloride and the emetine hydrochloride reference substance is an amount of, accurately claims surely, puts in the brown measuring bottle, add methyl alcohol and make stoste, get stoste again, adding flows makes the solution of hydrochloric cephaeline 10～30 μ g of every 1ml, emetine hydrochloride 30～10 μ g mutually, namely;
(3) ipecacuanha extract 0.5g is got in the preparation of need testing solution, puts in the measuring bottle of 25ml, and accurate the title decides, add 60% methyl alcohol to scale, shake up, filter, precision is measured subsequent filtrate 1ml, puts on the neutral alumina post that 100-120 order neutral alumina 1g internal diameter 1cm is housed, and is eluted in the measuring bottle of 10ml to about 9ml, add 1 phosphoric acid, methyl alcohol with 60% is diluted to scale, shakes up, and filters with 0.45 μ m miillpore filter, get subsequent filtrate as need testing solution, namely;
(4) the accurate reference substance solution 5 μ l that draw of determination method difference, need testing solution 2-3 μ l injects liquid chromatograph, measures calculating content.Measure 1 batch of ipecacuanha extract content, the results are shown in Table 9.
Table 1 Cephaeline Hydrochloride sample size and peak area
Table 2 emetine hydrochloride sample size and peak area
The recovery test result of Cephaeline Hydrochloride in table 3 sample
[0067]
The recovery test result of emetine hydrochloride in table 4 sample
Table 5 precision experimental result (peak area)
Table 6 stability experiment result (peak area)
Table 7 replica test result (mg/g)
Table 8 ipecac medicinal material acid base titration method and HPLC method measurement result are relatively
Cephaeline Hydrochloride and emetine hydrochloride assay result in table 9 medicinal material and the preparation
Annotate: brother Si is Costa Rican abbreviation
Claims (7)
1. the HPLC quantivative approach of Cephaeline Hydrochloride and emetine hydrochloride in an ipecac medicinal material and the preparation thereof is characterized in that:
(1) chromatographic condition is 8～9.5: 3～5 with the system suitability test with volume ratio: acetonitrile-methyl alcohol of 86～88-0.1% phosphoric acid is the phase that flows; Column temperature: 40 ℃; The detection wavelength is 205nm; Number of theoretical plate calculates by the emetine hydrochloride peak should be not less than 1500;
(2) preparation of reference substance solution is got Cephaeline Hydrochloride and the emetine hydrochloride reference substance is an amount of, accurately claims surely, puts in the brown measuring bottle, add methyl alcohol and make stoste, get stoste again, adding flows makes the solution of hydrochloric cephaeline 10～30 μ g of every 1ml, emetine hydrochloride 10～30 μ g mutually, namely;
(3) ipecac medicinal material fine powder 0.1g is got in the preparation of need testing solution, and accurate the title decides, and puts in the tool plug conical flask; Or get the preparation that is equivalent to 1g ipecac medicinal material, put in the measuring bottle of 25ml; Accurate hydrochloric acid-60% methyl alcohol mixed solution the 25ml that adds 1: 200 of medicinal material fine powder, close plug claims decide weight, and with power 250W, the ultrasonic processing of frequency 40kHz 30 minutes is put coldly, claims to decide weight again, supplies the weight that subtracts mistake with methyl alcohol, shakes up filtration; The wine of ipecac adds 60% methyl alcohol to scale, shakes up, and filters; Precision is measured medicinal material fine powder subsequent filtrate 2ml or preparation subsequent filtrate 1ml, put on the neutral alumina post that 100-120 order neutral alumina 1～1.5g internal diameter 1cm is housed, and be eluted in the measuring bottle of 10ml to about 9m1, add 1 phosphoric acid, methyl alcohol with 60% is diluted to scale, shakes up, and filters with 0.45 μ m miillpore filter, get subsequent filtrate as need testing solution, namely;
(4) the accurate reference substance solution 5 μ l that draw of determination method difference, need testing solution 2-10 μ l injects liquid chromatograph, measures, namely.
2. the HPLC quantivative approach of Cephaeline Hydrochloride and emetine hydrochloride in a kind of ipecac medicinal material according to claim 1 and the preparation thereof is further characterized in that its preparation means fluid ipecac extract, ipecacuanha extract and ipecac tincture.
3. the HPLC quantivative approach of Cephaeline Hydrochloride and emetine hydrochloride in a kind of ipecac medicinal material according to claim 2 and the preparation thereof is further characterized in that the every 1ml of fluid ipecac extract is equivalent to the 1g raw medicinal herbs; The every 1ml of ipecacuanha extract is equivalent to 2～5g raw medicinal herbs; The every 1ml of ipecac tincture is equivalent to the 0.05g raw medicinal herbs.
4. the HPLC quantivative approach of Cephaeline Hydrochloride and emetine hydrochloride in a kind of ipecac medicinal material according to claim 1 and the preparation thereof is further characterized in that:
(1) test of chromatographic condition and system suitability is that acetonitrile-methyl alcohol-0.1% phosphoric acid of 9: 3: 88 be mobile phase with volume ratio; Column temperature: 40 ℃; The detection wavelength is 205nm; Number of theoretical plate calculates by the emetine hydrochloride peak should be not less than 1500;
(2) preparation of reference substance solution is got Cephaeline Hydrochloride and the emetine hydrochloride reference substance is an amount of, accurately claims surely, puts in the brown measuring bottle, add methyl alcohol and make stoste, get stoste again, adding flows makes the solution of hydrochloric cephaeline 10～30 μ g of every 1ml, emetine hydrochloride 10～30 μ g mutually, namely;
(3) ipecac medicinal material fine powder 0.1g is got in the preparation of need testing solution, and accurate the title decides, and puts in the tool plug conical flask, accurate hydrochloric acid-60% methyl alcohol mixed solution the 25ml that adds 1: 200, close plug claims to decide weight, with power 250W, the ultrasonic processing of frequency 40kHz 30 minutes is put coldly, claims to decide weight again, supply the weight that subtracts mistake with methyl alcohol, shake up, filter, precision is measured subsequent filtrate 2ml, put on the neutral alumina post that 100-120 order neutral alumina 1.5g internal diameter 1cm is housed, and be eluted in the measuring bottle of 10ml to about 9ml, adding 1 phosphoric acid, the methyl alcohol with 60% is diluted to scale, shake up, filter with 0.45 μ m miillpore filter, get subsequent filtrate as need testing solution, namely;
(4) the accurate reference substance solution 5 μ l that draw of determination method difference, need testing solution 5-10 μ l injects liquid chromatograph, measures, namely.
5. the HPLC quantivative approach of Cephaeline Hydrochloride and emetine hydrochloride in a kind of ipecac medicinal material according to claim 1 and the preparation thereof is further characterized in that:
(1) test of chromatographic condition and system suitability is that acetonitrile-methyl alcohol-0.1% phosphoric acid of 8.5: 4: 87.5 be mobile phase with volume ratio; Column temperature: 40 ℃; The detection wavelength is 205nm; Number of theoretical plate calculates by the emetine hydrochloride peak should be not less than 1500;
(2) preparation of reference substance solution is got Cephaeline Hydrochloride and the emetine hydrochloride reference substance is an amount of, accurately claims surely, puts in the brown measuring bottle, add methyl alcohol and make stoste, get stoste again, adding flows makes the solution of hydrochloric cephaeline 10～30 μ g of every 1ml, emetine hydrochloride 10～30 μ g mutually, namely;
(3) the accurate fluid ipecac extract 1ml that draws of the preparation of need testing solution puts in the measuring bottle of 25ml, adds 60% methyl alcohol to scale, shake up, filter, precision is measured subsequent filtrate 1ml, put on the neutral alumina post that 100-120 order neutral alumina 1g internal diameter 1cm is housed, and be eluted in the measuring bottle of 10ml to about 9ml, adding 1 phosphoric acid, the methyl alcohol with 60% is diluted to scale, shake up, filter with 0.45 μ m miillpore filter, get subsequent filtrate as need testing solution, namely;
(4) accurate reference substance solution 5 μ 1 that draw of determination method difference, need testing solution 2-3 μ l injects liquid chromatograph, measures, namely.
6. the HPLC quantivative approach of Cephaeline Hydrochloride and emetine hydrochloride in a kind of ipecac medicinal material according to claim 1 and the preparation thereof is further characterized in that:
(1) test of chromatographic condition and system suitability is that acetonitrile-methyl alcohol-0.1% phosphoric acid of 9.5: 3.1: 87.4 be mobile phase with volume ratio; Column temperature: 40 ℃; The detection wavelength is 205nm; Number of theoretical plate calculates by the emetine hydrochloride peak should be not less than 1500;
(2) preparation of reference substance solution is got Cephaeline Hydrochloride and the emetine hydrochloride reference substance is an amount of, accurately claims surely, puts in the brown measuring bottle, add methyl alcohol and make stoste, get stoste again, adding flows makes the solution of hydrochloric cephaeline 10～30 μ g of every 1ml, emetine hydrochloride 10～30 μ g mutually, namely;
(3) the accurate ipecac tincture 20ml that draws of the preparation of need testing solution puts in the measuring bottle of 25ml, adds 60% methyl alcohol to scale, shake up, filter, precision is measured subsequent filtrate 1ml, put on the neutral alumina post that 100-120 order neutral alumina 1g internal diameter 1cm is housed, and be eluted in the measuring bottle of 10ml to about 9ml, adding 1 phosphoric acid, the methyl alcohol with 60% is diluted to scale, shake up, filter with 0.45 μ m miillpore filter, get subsequent filtrate as need testing solution, namely;
(4) the accurate reference substance solution 5 μ l that draw of determination method difference, need testing solution 2-3 μ l injects liquid chromatograph, measures, namely.
7. the HPLC quantivative approach of Cephaeline Hydrochloride and emetine hydrochloride in a kind of ipecac medicinal material according to claim 1 and the preparation thereof is further characterized in that:
(1) test of chromatographic condition and system suitability is that acetonitrile-methyl alcohol-0.1% phosphoric acid of 9: 3.5: 87.5 be mobile phase with volume ratio; Column temperature: 40 ℃; The detection wavelength is 205nm; Number of theoretical plate calculates by the emetine hydrochloride peak should be not less than 1500;
(2) preparation of reference substance solution is got Cephaeline Hydrochloride and the emetine hydrochloride reference substance is an amount of, accurately claims surely, puts in the brown measuring bottle, add methyl alcohol and make stoste, get stoste again, adding flows makes the solution of hydrochloric cephaeline 10～30 μ g of every 1ml, emetine hydrochloride 10～30 μ g mutually, namely;
(3) ipecacuanha extract 0.5g is got in the preparation of need testing solution, and accurate the title decides, and puts in the measuring bottle of 25ml, add 60% methyl alcohol to scale, shake up, filter, precision is measured subsequent filtrate 1ml, puts on the neutral alumina post that 100-120 order neutral alumina 1g internal diameter 1cm is housed, and is eluted in the measuring bottle of 10ml to about 9ml, add 1 phosphoric acid, methyl alcohol with 60% is diluted to scale, shakes up, and filters with 0.45 μ m miillpore filter, get subsequent filtrate as need testing solution, namely;
(4) the accurate reference substance solution 5 μ l that draw of determination method difference, need testing solution 2-3 μ l injects liquid chromatograph, measures, namely.
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Address after: 055550 Xingtai City, Cao Zhuang management area, Hebei Patentee after: Jing Jing Pharmaceutical Co., Ltd. Address before: 055550, Xingtai, Hebei province Cao Cao Zhuang management area, a branch of the East Patentee before: Jingjing Pharmaceutical Co., Ltd. |