CN1975412A - Effective liquid phase chromatography for measuring alkali content of dita leaves - Google Patents
Effective liquid phase chromatography for measuring alkali content of dita leaves Download PDFInfo
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Abstract
The HPLC measures the picrinine content. The condition of HPLC is: the bulking agent of the chromatographic column is the carbon 18 alkyl bonding with the silica gel; the mobile phase is methanol or the acetonitrile mixed with the triethylamine or the diethylamine and the detection wave is 287nm. The invention can be used to detect the picrinine content not only in the Cornus controversa Hemsl (root, stem, skin and leaf), pharmaceutics (particle, sheet, capsule, soft extracts and peroral liquid) but also in other material. The method is quick, accurate and special. The all components in Cornus controversa Hemsl and pharmaceutics can flow out the chromatographic column within 40min.
Description
Technical field
The present invention relates to a kind of chromatogram analysis method, specifically is high performance liquid chromatography (HPLC) method of a kind of mensuration picrinine (Picrinine) content, belongs to the Pharmaceutical Analysis technical field.
Background technology
Cornus controversa Alstonia Scholaris (L.) R.Bro. also claims dita, common alstonia etc., is Apocynaceae alstonia yonnanensis platymiscium, and height greatly enhances green arbor, is born in hilly and mountainous land, the sparse woods of height above sea level below 850 meters.Domestic Yunnan, Guangxi, the Fujian etc. of being distributed in are distributed in India, Sri Lanka, Burma, Thailand, Vietnam, Philippine etc. abroad, aboundresources.
In Yunnan Province, Cornus controversa is the natural drug of multi-national use, its root, stem, skin, the Ye Junke application of being used as medicine.Han nationality claims " scandent hedyotis herb ", is the active drug of treatment respiratory disease; The Dai nationality claims " buying load not ", and it is many to cure mainly cough with lung heat's phlegm, and the parotid gland, submandibular lymph nodes swell and ache; The Lahu, Wa etc. all control bronchitis, cough, asthma etc. with leaf for tea-drinking.
Through long-term application, from the medicinal material of Cornus controversa for " Yunnan Province's drug standards ", " Chinese pharmacopoeia is recorded.Utilize the Cornus controversa medicinal material to be raw material, developed formulation products such as alstonia-leaf granule, tablet, capsule, soft extract, oral liquid.
Chemical research for Cornus controversa, Zhu Weiming is in its PhD dissertation " Primary Study of five kinds of resources of medicinal plant chemistry " (Kunming Inst. of Botany, Chinese Academy of Sciences, calendar year 2001) compares comprehensive literature review in, the result shows: 1. each position of Cornus controversa (root, stem, skin, leaf) all contains alkaloid, structurally indoles alkaloids that belong to more; 2. alkaloid is the important effective constituent of Cornus controversa; 3. picrinine (picrinine) is that each position of Cornus controversa is generally contained, and the highest alkaloid component of content.Fig. 1 seen in the chemical structural formula of picrinine.
Aspect the drug quality analysis, " Yunnan Province's drug standards " are only differentiated with simple alkaloid precipitation reaction the alstonia-leaf medicinal material, no assay project.In formulation products, health Chinese medicine standard promulgated by the ministries or commissions of the Central Government lamp stand blade (WS-10280) adopts acid base titration to survey alkaloid, and conversion is index components picrinine content, but the specificity of assay method is poor, can not adapt to the technical requirement of the modernization of Chinese medicine.
High performance liquid chromatography (HPLC) is applicable to analysis of complex ingredient, has become the main stream approach of quality analysis of traditional Chinese medicine at present, is used widely.Publication number is CN 1818636A, and the application people adopts the HPLC method to measure the quality that picrinine content is controlled medicinal material and preparation for the Chinese patent application of Zhu Guangrong.Its essential condition of determining is: with 2% ammoniacal liquor is to extract solvent, extract chloroform extraction; HPLC moving phase detects wavelength 232nm with aqueous hydrochloric acid solution (concentrated hydrochloric acid 1 → 100)-acetonitrile (80: 20).
Through inventor's systematic research, the result shows that there is major defect in the determined major technique condition of above-mentioned patented claim, is difficult to be applied to the quality analysis of alstonia-leaf medicinal material and preparation thereof.Content for main alkaloid composition picrinine (picrinine) in assay determination Cornus controversa medicinal material and the preparation thereof exactly, control drug quality better, the guarantee clinical drug safety is effective, need the efficient liquid-phase chromatography method of picrinine content in a kind of new mensuration Cornus controversa medicinal material and the preparation thereof
Summary of the invention
The objective of the invention is to overcome the shortcoming of prior art, a kind of efficient liquid-phase chromatography method of the picrinine of assay determination exactly content is provided, can be used for measuring Cornus controversa medicinal material (root, branch, skin, leaf), preparation (granule, tablet, capsule, soft extract, oral liquid) picrinine content, also can be used for measuring the picrinine content that contains in the other materials.
The feature of the inventive method is that liquid phase chromatogram condition is: chromatographic column is a filling agent with the carbon octadecyl silane, and the moving phase mixed solvent that to be methyl alcohol or acetonitrile form with rare triethylamine or diethylamine aqueous solution detects wavelength 287nm.Column temperature can be 30～40 ℃, and flow velocity can be 0.5～5ml/min, and sampling volume can be 10～20 μ l.Column temperature the best is 35 ℃, and flow velocity the best is 1ml/min.
When the moving phase mixed solvent that to be methyl alcohol form with rare triethylamine or rare diethylamine aqueous solution, the volume ratio of its composition can be methyl alcohol: rare triethylamine or rare diethylamine aqueous solution equal 40: 60～and 60: 40, the concentration that rare triethylamine or rare diethylamine aqueous solution contain triethylamine or diethylamine can be 0.01%～0.015%.As optimal condition, the volume ratio that mixed solvent is formed is a methyl alcohol: rare triethylamine or rare diethylamine aqueous solution equal 50: 50.
When the moving phase mixed solvent that to be acetonitrile form with rare triethylamine or rare diethylamine aqueous solution, the volume ratio of its composition is an acetonitrile: rare triethylamine or rare diethylamine aqueous solution equal 25: 75～and 30: 70, the concentration that rare triethylamine or rare diethylamine aqueous solution contain triethylamine or rare diethylamine is 0.01%～0.015%.
By following experimental study the present invention is further specified.
1, the preparation of picrinine (picrinine)
(1) extracts the separation alstonine
Use 95% alcohol reflux after the Cornus controversa pulverizing medicinal materials, reclaim solvent; Medicinal extract adds water and stirs the back of loosing to transfer pH with strong aqua be 9, and chloroform extraction reclaims solvent, total alkaloids.
The alkaloid part is through aluminum oxide column chromatography, with cyclohexane-ethyl acetate mixed solvent gradient elution (100: 5～100: 100), with merging the stream part that contains alstonine after the thin-layer chromatography inspection, recycle silicon glue column chromatography separating for several times, with chloroform-acetone mixed solvent gradient elution (100: 0～100: 30), in conjunction with the recrystallization preparation, obtain alstonine, HPLC checks purity about 95%.
(2) purifying alstonine
Above-mentioned alstonine purity does not reach the traditional Chinese medicine quality standard with reference substance requirement (more than 98%), further use sephadex lh-20 column chromatography purifying, with methyl alcohol is eluting solvent, merge stream part of only containing alstonine, reclaim solvent to small size, placement is spent the night, and separates out colourless needle, HPLC checks that its purity is 99.94%, is the alstonine reference substance.Fig. 2 is the HPLC collection of illustrative plates of picrinine reference substance.
(3) chemical constitution is identified
Colourless acicular crystal is soluble in methyl alcohol, chloroform, acetone, dissolves in ethyl acetate, ethanol, water insoluble and sherwood oil, but dissolve in watery hydrochloric acid and dilution heat of sulfuric acid.222～224 ℃ of fusing points.Optical activity is [a]
D-48 (c 0.21, CHCl
3, 25 ℃ of mensuration).
Infrared spectrum: IRv (KBr) cm
-1: 3430,3391 (vs, NH), (s, C-H), 1724 (s, C=O), 1610,1481,1465 (s, C=C), 1203-1167 (s, C-O and C-N) points out its structure to belong to alkaloid compound to 3009-2866.
Ultraviolet spectrum: UV λ (CH
3OH) nm: three main absorption peaks are arranged, be respectively 205,235.5,287.5, point out the phenyl ring that two keys and replacement are arranged in this compound structure.
Mass spectrum: positive ion FAB MS m/z:338[M]
+, 239
+Wherein the peak of m/z 338 is a molecular ion peak, and mass spectrometric data is analyzed in conjunction with the NMR spectroscopic data, and the molecular formula of its corresponding compound should be C
20H
22N
2O
3
Nuclear magnetic resoance spectrum: through test, its
1H NMR reaches
13C NMR (CDCl
3, ppm) data such as table one and table two.The reference literature report (Zhu Weiming: the Primary Study of five kinds of resources of medicinal plant chemistry. doctorate paper, Kunming plant institute of the Chinese Academy of Sciences, calendar year 2001), its structural identification is picrinine (picrinine).
The 1H NMR data and the ownership of table one picrinine
| H | 1H NMR | H | 1H NMR | H | 1H NMR | H | 1H NMR |
| 1 | 5.02，1H br.s | 9 | 7.15，1H br.d，J＝7.5Hz | 14 | 1.85，1H br.d， J＝14.0Hz 2.16，1H br.dd， J＝14.0， 4.2Hz | 17 | 3.10，3.79 each 1H br.d，J＝18Hz |
| 3 | 3.60，1H br.d，J＝4.2Hz | 10 | 6.80，1H br.t，J＝7.5Hz | 19 | 5.42，1H q，J＝6.9Hz | ||
| 5 | 4.83，1H br.s | 11 | 7.08，1H br.t，J＝7.5Hz | 15 | 3.28，1H br.s | 20 | 1.50，3H d，J＝6.9Hz |
| 6 | 3.45、2.27， each 1H br.d，J＝14Hz | 12 | 6.73，1H br.d，J＝7.5Hz | 16 | 2.46，1H br.s | 22 | 3.66，3H s |
Table two picrinine
13C NMR data and ownership
| C | 13C NMR | C | 13C NMR | C | 13C NMR | C | 13C NMR |
| 1 | ---- | 7 | 51.1(s) | 13 | 147.6(s) | 19 | 110.5(d) |
| 2 | 106.3(s) | 8 | 136.3(s) | 14 | 25.9(t) | 20 | 12.7(q) |
| 3 | 51.4(d) | 9 | 125.0(d) | 15 | 31.0(d) | 21 | 172.4(s) |
| 4 | ---- | 10 | 120.2(d) | 16 | 51.9(d) | 22 | 51.7(q) |
| 5 | 87.2(d) | 11 | 120.6(d) | 17 | 46.3(t) | ||
| 6 | 40.5(t) | 12 | 127.9(d) | 18 | 135.1(s) |
2, the preparation of Cornus controversa medicinal material and preparation test solution thereof
(1) preparation of Cornus controversa medicinal material test solution
The deliquescent universal law of alkaloid is: alkaloid is soluble in organic solvent and sour water, water insoluble and buck; Alkaloid salt is soluble in water, is insoluble to organic solvent.Therefore, extracting the unsuitable water of alkaloid from medicinal material is solvent, and should use organic solvent extraction.Different Extraction Method is measured and is relatively seen Table three, and wherein testing 1 is the method that Zhu Guangrong determines in its patented claim (publication number CN 1818636A).
The result shows that methyl alcohol has strong seepage force to plant tissue, target compound is had good dissolubility, so extraction effect is best; Water or 2% ammoniacal liquor have very strong seepage force to plant tissue, but the target compound indissoluble is separated, thereby extraction effect is bad; Acetone and chloroform have good dissolubility to target compound, but poor to the seepage force of plant tissue, so extract not exclusively, effect is also bad.Determine that thus the preparation method of Cornus controversa medicinal material test liquid is: the material of getting it filled, pulverize, add methanol eddy and extract, filter, promptly.
The extracting method of table three Cornus controversa medicinal material relatively
| Experiment | Solvent | Experimental phenomena and |
| 1 | 2% | 1. measure content 0.019%.2. poorly soluble to target substance, extract not exclusively. |
| 2 | | 1. measure content 0.022%.2. poorly soluble to target substance, extract not exclusively. |
| 3 | | 1. measure content 0.067%.2. extract fully. |
| 4 | | 1. measure content 0.066%.2. extract fully. |
| 5 | | 1. measure content 0.052%.2. poor to the seepage force of medicinal material, extract not exclusively. |
| 6 | | 1. measure content 0.041%.2. poor to the seepage force of medicinal material, extract not exclusively. |
According to alkaloid dissolubility universal law, show in conjunction with above-mentioned experimental studies results, there is major defect in Zhu Guangrong in the technical conditions of patented claim (publication number CN 1818636A) determined " extracting with pH=9 ammoniacal liquor ", can not extract the alkaloid component in the medicinal material fully, the later stage measurement result also just can not reflect the truth of medicinal material, is difficult to be applied to accurately the quality analysis work of alstonia-leaf medicinal material.
(2) preparation of Cornus controversa preparation test solution
The Cornus controversa formulation products is to add auxiliary material with medicinal extract to make, and according to universal experience, assay can be with regulating the pH value behind the water-soluble powder preparation, again with the chloroform extraction alkaloid.Screen suitable extraction conditions to guarantee that extraction is complete, operation feasible is main points, major influence factors is the control method and the degree of pH value, test findings such as table four.
The extracting method of table four Cornus controversa preparation relatively
| Experiment | Institute adds alkali and institute's adjust pH | Experimental phenomena and result |
| 1 | 1% NaOH; Transfer pH=9 | 1. accent is excessive easily, difficult operation.2. emulsification is serious, difficult extraction. |
| 2 | 0.5% NaOH; Transfer pH=8 | 1. accent is excessive easily, difficult operation.2. emulsification is serious, difficult extraction. |
| 3 | 0.5% NaOH; Transfer pH=7 | 1. accent is excessive easily, difficult operation.2. emulsion has improvement, still is difficult to extract fully. |
| 4 | 1% NaCO 3Transfer pH=9 | 1. emulsification is serious, difficult extraction. |
| 5 | 1% NaCO 3Transfer pH=8 | 1. emulsification is serious, difficult extraction. |
| 6 | 1% NaCO 3Transfer pH=7 | 1. emulsion makes moderate progress, and still is difficult to extraction fully. |
| 7 | 5% ammoniacal liquor; Transfer pH=8 | 1. accent is excessive easily, difficult operation.2. emulsion is arranged slightly, can extract, but length consuming time. |
| 8 | 5% ammoniacal liquor; Transfer pH=7 | 1. accent is excessive easily, difficult operation.2. do not have the emulsification phenomenon substantially, extraction easily. |
| 9 | 2% ammoniacal liquor; Transfer pH=8 | 1. emulsion is arranged, can extract, but length consuming time. |
| 10 | 2% ammoniacal liquor; Transfer pH=7 | 1. not emulsification, extraction is thorough, processing ease. |
Comparing result shows that formulation soln is transferred pH=7 with 2% ammoniacal liquor, not emulsification during extraction, and processing ease, extraction is thoroughly.Thus, determine that formulation products need testing solution preparation method is: get preparation, add that the water jolting is molten looses, transfer pH=7 with 2% ammoniacal liquor, put in the separating funnel with chloroform extraction, chloroform solution evaporate to dryness, residue are with dissolve with methanol promptly.
3, high-efficient liquid phase chromatogram condition
(1) chromatographic column is selected to determine
Consider the application ubiquity, select for use HPLC to measure the most frequently used carbon octadecyl silane post and test.Compared the Luna C-18 of Phenomenex company post, the STAR RP-18 of Merck company post, the XDB-C18 of Agilent company post etc., better separating effect is all arranged, therefore determine: selecting for use with the carbon octadecyl silane is the chromatographic column of filling agent.
(2) detection method and detection wavelength are determined
Picrinine tool uv absorption, available UV-detector detects.Picrinine is dissolved in methyl alcohol respectively, HPLC moving phase is measured ultraviolet spectrum, and data see Table five.The result shows three absorption peaks, and the peak of 209.0nm should not be elected to be the detection wavelength near terminal and big with solvent change; 235.5, the peak degree of separation of 287.0nm is all better, and, all can be used for detecting not with solvent change.Contrast the HPLC collection of illustrative plates that these two wavelength detect, less with the Interference Peaks in the 287nm wavelength collection of illustrative plates, therefore determine: use uv detection method, detect wavelength 287nm.
The ultraviolet spectrum data of table five picrinine
| Absorption peak in the methyl alcohol | 287.0nm | Absorption peak in the HPLC moving phase | 287.0nm |
| 235.5nm | 235.5nm | ||
| 205.0nm | 209.0nm |
(3) selection of moving phase is determined
The picrinine complex structure selects suitable moving phase to obtain the key that good separating effect is an analytical approach.Based on the general known fact and working experience, in analyzing, regulates HPLC the Acidity of Aikalinity of moving phase, and the acid of employing mainly is: phosphoric acid,diluted, dilute sulfuric acid, acetate, formic acid; The alkali that adopts mainly is: diethylamine, triethylamine, weak aqua ammonia.
Zhu Guangrong determines " HPLC moving phase aqueous hydrochloric acid solution (concentrated hydrochloric acid 1 → 100)-acetonitrile (80: 20) " in patented claim (publication number CN 1818636A).This moving phase is difficult to be applied to real work, and the one, the connecting tube of liquid chromatograph, chromatogram cylinder etc. is stainless steel material, can not tolerate the dilute hydrochloric acid solution corrosion; The 2nd, acidity is strong (pH is less than 1) too, causes the degraded of carbon octadecyl silane filler easily.
According to physicochemical property and experience, we select methanol-water, acetonitrile-water (all scalable soda acids) solvent system contrast screening.Chromatographic column Luna C-18 post (using the pH scope is 1.5～10 for Φ 4.6 * 150mm, 5 μ m); Flow velocity 1.0ml/min; 35 ℃ of column temperatures; Detect wavelength 287nm.Comparative study data and analysis result thereof see Table six.
Table six moving phase contrast shaker test evaluation result
| No | Chromatographic condition | The absorption peak parameter | System synthesis is estimated |
| The picrinine peak | |||
| 1 | Methanol-water (50: 50) | Retention time: 20.0min number of theoretical plate: 1569 tailing factors: 1.559 | Retention time: moderate number of theoretical plate: too low absorption peak shape: asymmetric |
| 2 | Methanol-water (60: 40) | Retention time: 14.0min number of theoretical plate: 1582 tailing factors: 1.512 | Retention time: short slightly number of theoretical plate: too low absorption peak shape: asymmetric |
| 3 | Methyl alcohol-0.02% phosphoric acid (60: 40) | Retention time: 7.2min number of theoretical plate: 694 tailing factors: 2.456 | Retention time: too short number of theoretical plate: very low absorption peak shape: asymmetric |
| 4 | Acetonitrile-water (50: 50) | Retention time: 12.3min number of theoretical plate: 1519 tailing factors: 1.771 | Retention time: shorter number of theoretical plate: too low absorption peak shape: asymmetric |
| 5 | Acetonitrile-0.02% phosphoric acid (50: 50) | Retention time: 4.3min number of theoretical plate: 656 tailing factors: 2.417 | Retention time: too short number of theoretical plate: very low absorption peak shape: asymmetric |
| 6 | Methyl alcohol-0.01% triethylamine (60: 40) | Retention time: 12.6min number of theoretical plate: 7210 tailing factors: 1.084 | Retention time: shorter number of theoretical plate: higher absorption peak shape: symmetrical substantially |
| 7 | Methyl alcohol-0.01% triethylamine (50: 50) | Retention time: 21.6min number of theoretical plate: 9774 tailing factors: 1.066 | Retention time: moderate number of theoretical plate: higher absorption peak shape: symmetry |
| 8 | Methyl alcohol-0.012% triethylamine (50: 50) | Retention time: 20.8min number of theoretical plate: 9184 tailing factors: 1.049 | Retention time: moderate number of theoretical plate: higher absorption peak shape: symmetry |
| 9 | Methyl alcohol-0.015% triethylamine (50: 50) | Retention time: 20.1min number of theoretical plate: 9918 tailing factors: 1.061 | Retention time: moderate number of theoretical plate: higher absorption peak shape: symmetry |
| 10 | Methyl alcohol-0.012% triethylamine (40: 60) | Retention time: 35.6min number of theoretical plate: 7978 tailing factors: 1.086 | Retention time: long slightly number of theoretical plate: higher absorption peak shape: symmetrical substantially |
| 11 | Methyl alcohol-0.01% diethylamine (50: 50) | Retention time: 20.2min number of theoretical plate: 10344 tailing factors: 1.050 | Retention time: moderate number of theoretical plate: higher absorption peak shape: symmetry |
| 12 | Methyl alcohol-0.015% diethylamine (50: 50) | Retention time: 18.5min number of theoretical plate: 9342 tailing factors: 1.047 | Retention time: moderate number of theoretical plate: higher absorption peak shape: symmetry |
| 13 | Acetonitrile-0.01% triethylamine (30: 70) | Retention time: 12.9min number of theoretical plate: 12368 tailing factors: 1.105 | Retention time: shorter number of theoretical plate: very high absorption peak shape: symmetrical substantially |
| 14 | Acetonitrile-0.01% diethylamine (25: 75) | Retention time: 24.3min number of theoretical plate: 9271 tailing factors: 1.060 | Retention time: moderate number of theoretical plate: higher absorption peak shape: symmetry |
With preferred top condition experiment 8[methyl alcohol-0.012% triethylamine (50: 50)], experiment 14[acetonitrile-0.01% diethylamine (25: 75)] carry out analytical test, result such as table seven.
The result shows: the mixed solvent that moving phase is formed with methyl alcohol-rare machine amine (triethylamine, diethylamine) has best separating effect; The suitable concentration of rare machine amine aqueous solution is 0.01%～0.015%; The volume ratio of components of methyl alcohol and rare machine amine aqueous solution between 60: 40～40: 60 all can, the ratio of optimization is to see attached Figure 4 and 5 at 50: 50, is respectively Cornus controversa medicinal material, preparations by HPLC collection of illustrative plates.
Secondly, the mixed solvent of forming with acetonitrile-rare machine amine (triethylamine, diethylamine) also can be used for analyzing, and the suitable concentration of rare machine amine aqueous solution is 0.01%～0.015%; The volume proportion of composing of acetonitrile and rare machine amine aqueous solution all could between 25: 75～30: 70.
The result of preferred optimal flow facies analysis medicinal material of table seven and preparation
| No | Chromatographic condition | The absorption peak parameter | System synthesis is estimated |
| Cornus controversa medicinal material sample: picrinine peak | |||
| Methyl alcohol-0.012% triethylamine (50: 50) (optimum condition 8) | Retention time: 20.8min number of theoretical plate: 7562 tailing factors: 1.052 degree of separation: 1.67 | Retention time: moderate number of theoretical plate: higher absorption peak shape: symmetrical separation case: complete baseline separation | |
| Acetonitrile-0.01% diethylamine (25: 75) (condition 14) | Retention time: 25.1min number of theoretical plate: 8732 tailing factors: 1.068 degree of separation: 1.87 | Retention time: moderate number of theoretical plate: higher absorption peak shape: symmetrical separation case: complete baseline separation | |
| Alstonia-leaf formulation samples: picrinine peak | |||
| Methyl alcohol-0.012% triethylamine (50: 50) (optimum condition 8) | Retention time: 21.0min number of theoretical plate: 6637 tailing factors: 1.056 degree of separation: 1.75 | Retention time: moderate number of theoretical plate: higher absorption peak shape: symmetrical separation case: complete baseline separation | |
| Acetonitrile-0.01% diethylamine (25: 75) (condition 14) | Retention time: 25.4min number of theoretical plate: 9121 tailing factors: 1.062 degree of separation: 1.86 | Retention time: moderate number of theoretical plate: higher absorption peak shape: symmetrical separation case: complete baseline separation | |
(4) the specificity research of Ce Dinging
1. reference substance adds: in the test liquid of Cornus controversa and preparation thereof, sneak into reference substance solution sample introduction mensuration and the contrast of test liquid chromatogram, no new absorption peak occurs, and picrinine absorption peak relative area increases.2. 3-D scanning: with diode array detector (DAD), in the scanning samples chromatogram and the absorption peak of reference substance peak same position, peak purity is more than 950, and ultraviolet spectrum is consistent with reference substance.Above evidence: the material of the absorption peak correspondence of mensuration is a picrinine, and this content assaying method has the specificity of height.
4, assay method is investigated and is determined
(1) stability test
Reference substance, need testing solution are measured in placing the back different time, the result shows: reference substance solution stable in 72h at least (peak area RSD=0.98%, n=7), need testing solution is stable (peak area RSD=1.02% in 48h at least, n=5), can satisfy the assay demand.
Table eight stability test data
| Reference substance solution | |||||||||||
| Standing time | 0h | 2h | 4h | 8h | 24h | 48h | 72h | ||||
| Peak area | 627885 | 622908 | 621910 | 623704 | 639773 | 628574 | 630802 | ||||
| The RSD=0.98% of peak area | |||||||||||
| For trial target solution | |||||||||||
| Standing time | 0h | 4h | 16h | 24h | 48h | ||||||
| Peak area | 391100 | 383250 | 386952 | 392926 | 391419 | ||||||
| The RSD=1.02% of peak area | |||||||||||
(2) detectability and linear relationship are determined
It is an amount of to get dita alkali, makes the 1mg/ml storing solution with moving phase, and constantly the dilution back is measured lowest detection and is limited to 30ng (signal/noise=3), assay method sensitivity.Getting the storing solution dilution respectively is variable concentrations, measures, and the relation of sample size and peak area sees Table nine.
In sample size 0.1 μ g～14 μ g scopes, picrinine peak area and sample size are the good linear relation.Regression equation intercept (1202) is 0.53% of an absorption coefficient (227514), and the tropic is almost crossed initial point, and for simplifying computing method, available one point external standard method is quantitative, sees Fig. 3, picrinine bioassay standard curve.
Table nine detectability and linear relationship data and regression equation
| Lowest detectable limit: 30ng, S/N=3 | ||||||
| | 14 | 10 | 4 | 2 | 1 | 0.1 |
| Peak area | 3177771 | 2281612 | 912780 | 453521 | 221877 | 20932 |
| Regression equation: Y (peak area)=227514X (sample size)-1202, r=0.99999 | ||||||
(3) reappearance test
Get same lot number Cornus controversa medicinal material (20051001), alstonia-leaf particle (20060204) takes by weighing 6 parts simultaneously, according to the parallel preparation test solution of test liquid preparation method, measures the content of picrinine, and data see Table ten.Medicinal material is measured the RSD=1.88% of content for six times, and preparation alstonia-leaf particle is measured the RSD=1.32% of content for six times, and the assay method reappearance is good.
The test of table ten methodological study----reappearance
| Cornus controversa medicinal material (20051001) | ||||||
| Content (mg/g) | 0.161 | 0.168 | 0.159 | 0.164 | 0.164 | 0.163 |
| Mean value: 0.163mg/g, RSD=1.88% | ||||||
| Alstonia-leaf particle (20060204) | ||||||
| Content (mg/g) | 0.106 | 0.107 | 0.108 | 0.105 | 0.109 | 0.107 |
| Mean value: 0.107mg/g, RSD=1.32% | ||||||
(4) application of sample recovery test
(20060204,0.107mg/g), totally 9 parts, the accurate title, decide, and is divided into three groups respectively to get known content alstonia-leaf particle.Get an amount of picrinine reference substance, make the solution of 0.22mg/ml, according to being equivalent to 80%, 100%, 120% of picrinine content in the sample, join in three groups of samples respectively with methyl alcohol.Extract according to the test liquid preparation method, measure content, data see Table 11.The result shows that the assay method average recovery rate is 98.6%, RSD=1.43% (n=9).The reference substance energy that adds is accurate, the recovery of reproduction, and assay method accurately and reliably.
Table ten methodological study----application of sample recovery test data
| Numbering | Sampling amount (g) | Sample size (mg) | Addition (mg) | The amount of measuring (mg) | Yield (mg) | The recovery (%) | Average recovery rate | |
| 80 | 1 | 4.9231 | 0.5268 | 0.44 | 0.9634 | 0.4366 | 99.2 | On average: 98.6% RSD:1.43% |
| 2 | 5.0006 | 0.5351 | 0.9656 | 0.4305 | 97.8 | |||
| 3 | 4.8715 | 0.5212 | 0.9482 | 0.4270 | 97.0 | |||
| 100 | 1 | 4.7654 | 0.5099 | 0.55 | 1.0442 | 0.5343 | 97.1 | |
| 2 | 4.7879 | 0.5123 | 1.0474 | 0.5351 | 97.3 | |||
| 3 | 5.0008 | 0.5351 | 1.0760 | 0.5409 | 98.3 | |||
| 120 | 1 | 5.0561 | 0.5410 | 0.66 | 1.2026 | 0.6616 | 100.2 | |
| 2 | 5.1045 | 0.5462 | 1.2052 | 0.6590 | 99.8 | |||
| 3 | 5.1813 | 0.5544 | 1.2191 | 0.6647 | 100.7 | |||
5, analytical approach serviceability test
(1) change in flow is to the influence of measurement result
Keep chromatographic column, column temperature, detection wavelength, moving phase constant.Flow velocity is set at 0.8ml/min, 1.0ml/min, 1.2ml/min, measures respectively, and data see Table 12.The result shows: flow velocity change reach ± 20% o'clock, the accurate content of lamp stand leaf alkali in the working sample still.
Table ten two method serviceability test----change in flow are to the influence of measurement result
| Batch number | 20060302 | |||||
| Change in flow | 0.8ml/min | 1.0ml/min | 1.2ml/min | |||
| Tailing factor | 1.049 | 1.028 | 1.029 | |||
| Number of theoretical plate | 11019 | 8523 | 8209 | |||
| Content mg/g | 0.106 | 0.108 | 0.108 | 0.106 | 0.109 | 0.106 |
| Data analysis | Mean value=0.107; RSD=1.24% (n=6) | |||||
(2) detect the influence of wavelength shift to measurement result
Keep chromatographic column, column temperature, moving phase, flow velocity constant.The detection wavelength set is 284nm, 287nm, 290nm, measures respectively, and data see Table 13.The result shows: detecting wavelength shift and reach ± during 3nm, still the accurate content of lamp stand leaf alkali in the working sample.
Table ten three serviceability tests----detect the influence of wavelength shift to measurement result
| Batch number | 20060302 | |||||
| Wavelength variations | 284nm | 287nm | 290nm | |||
| Tailing factor | 1.039 | 1.028 | 1.048 | |||
| Number of theoretical plate | 9968 | 8523 | 9856 | |||
| Content mg/g | 0.105 | 0.108 | 0.108 | 0.106 | 0.107 | 0.107 |
| Data analysis | Mean value=0.107; RSD=1.09% (n=6) | |||||
(3) the moving phase ratio changes the influence to measurement result
Keep chromatographic column, column temperature, detection wavelength, flow velocity constant.The proportion of composing of appropriate change moving phase is tested, and data see 14.The result shows: the moving phase ratio change reach ± 3% situation under, the accurate content of lamp stand leaf alkali in the working sample still.
Table ten four serviceability tests----moving phase ratio changes the influence to measurement result
| Batch number | 20060302 | |||||
| Moving phase | Methyl alcohol-0.01% triethylamine | |||||
| Ratio changes | 47∶53 | 50∶50 | 53∶47 | |||
| Tailing factor | 1.023 | 1.028 | 1.067 | |||
| Number of theoretical plate | 9397 | 8523 | 9931 | |||
| Content mg/g | 0.106 | 0.109 | 0.108 | 0.106 | 0.106 | 0.107 |
| Data analysis | Mean value=0.107; RSD=1.18% (n=6) | |||||
(4) comparison of different chromatographic column measurement results
Keep column temperature, detect wavelength, moving phase, flow velocity is constant.Use that different chromatographic columns are measured and the system evaluation analysis instead, data see Table 15.The result shows: this analytical approach all has peak shape and separating effect preferably on different chromatographic columns, with different chromatographic columns, and equal accurate content of lamp stand leaf alkali in the working sample.Chromatographic column relatively has following three kinds:
Luna C-18 post: Phenomenex company, specification Φ 4.6mm * 150mm uses pH scope 1.5～10, pillar sequence number 339618-40.
Purospher Star C-18 post: Merck company, specification Φ 4.6mm * 150mm uses pH scope 1.5～11.5, pillar sequence number 147565.
Zorbax C-18 post: Agilent company, specification Φ 4.6mm * 150mm uses pH scope 1～12, specification Φ 4.6mm * 150mm, pillar sequence number USKH009350.
The comparison of the different chromatographic column measurement results of table ten five serviceability tests----
| Batch number | 20060302 | |||||
| Chromatographic column | The C-18 filler requires the pH tolerance value should reach (moving phase pH=9.3) more than 9.5 | |||||
| Factory plate model | Purospher Star C-18 | Luna C-18 | Zorbax C-18 | |||
| Tailing factor | 1.038 | 1.028 | 1.062 | |||
| Number of theoretical plate | 8908 | 8523 | 10309 | |||
| Content mg/g | 0.109 | 0.107 | 0.108 | 0.106 | 0.107 | 0.106 |
| Data analysis | Mean value=0.107; RSD=1.09% (n=6) | |||||
The above results shows that this analytical approach has good durability: 1. can be applicable to the C-18 chromatographic column of different labels, peak shape and separating effect are preferably all arranged.2. can adapt to the change of moving phase proportion of composing in ± 3% scope.3. can adapt to the change of flow velocity in ± 20% scope.4. can tolerant detect the skew of wavelength in ± 3nm scope.
(5) checking of analytical approach on different manufacturers liquid chromatography instrument
Above-mentioned research is finished on the LC-10AvP high performance liquid chromatograph of Tianjin, island, for further verifying the durability of this analytical approach, select again now to use more Agilent1100, Waters1525 liquid chromatograph, carried out the checking of this analytical approach on different manufacturers liquid chromatography instrument and measured.Instrument condition is respectively: 1. Tianjin, island LC-10AvP; 2. Agilent1100; 3. Waters1525.Determination data sees Table 16.
The result shows that this method measurement result on the high performance liquid chromatograph of different labels has consistance, and analytical instrument is had adaptability more widely, and durability is good.
Table ten six serviceability tests----different manufacturers PLC instrument measurement result relatively
| Batch number | 20060302 | |||||
| The instrument model | Tianjin, island LC-10AvP | Waters1525 | Agilent1100 | |||
| Chromatographic column | Luna C-18 | Purospher Star C-18 | Zorbax C-18 | |||
| Content mg/g | 0.109 | 0.106 | 0.108 | 0.106 | 0.106 | 0.106 |
| Data analysis | RSD＝1.09％(n＝6) | |||||
Assay method: it is an amount of to get picrinine, and accurate the title decides, and makes the reference substance solution that every 1ml contains 10 μ g～100 μ g with the dissolving of HPLC moving phase.According to the above-mentioned method of determining, preparation Cornus controversa medicinal material, and the test solution of preparation.Accurate reference substance solution, the test solution drawn injected liquid chromatograph (HPLC), measures, promptly.
Description of drawings
Fig. 1 is the chemical structural formula of picrinine.
Fig. 2 is the HPLC collection of illustrative plates of picrinine reference substance.
Fig. 3 is a picrinine bioassay standard curve.
Fig. 4 is the HPLC collection of illustrative plates of Cornus controversa medicinal material.
Fig. 5 is a Cornus controversa preparations by HPLC collection of illustrative plates.
Beneficial effect of the present invention and technological progress are:
1. pass through the comparative studies of system, set up a kind of efficient liquid-phase chromatography method of measuring the dita leaves alkali content, both can be used for measuring the content of picrinine in Cornus controversa medicinal material (root, stem, skin, leaf), the preparation (granule, tablet, capsule, soft extract, oral liquid), also can be used for measuring the dita leaves alkali content that contains in the other materials.
2. analytical method has good separating effect, and it is accurate, sensitive to measure, and specificity is strong, measures and waits fast technical characterstic. Each composition absworption peak can all flow out chromatographic column in Cornus controversa medicinal material, the preparation in 40 minutes, finished analytical work.
The present invention can directly apply to the quality analysis of actual production process and product.
Embodiment
Following specific embodiment can further specify the present invention and application thereof, but does not constitute the restriction of the scope that claim of the present invention is protected.
Principle of work and process: test liquid injects liquid chromatograph, and moving phase carries composition such as picrinine and enters chromatographic column, separates each composition, makes them arrive detecting device successively and is identified; The response at picrinine peak in the detecting device working sample, and according to the size of response and the content that relatively comes picrinine in the calculation sample of picrinine standard specimen.
Embodiment 1: the assay of picrinine in the lamp stand leaf
Get the lamp stand leaf, pulverize, cross 60 eye mesh screens, precision takes by weighing 2g, puts in the 100ml round-bottomed flask, accurately adds methyl alcohol 50ml, weighs, and water-bath refluxing extraction 1 hour is put coldly, supplies the weight that subtracts mistake with methyl alcohol, filters, and promptly gets test solution.It is an amount of to get picrinine, and accurate the title decides, and makes the reference substance solution that every 1ml contains 50 μ g with the dissolving of HPLC moving phase.
According to the above-mentioned method of determining (table six-experiment 8), accurate reference substance solution, the test solution drawn injected liquid chromatograph, measures, promptly.The picrinine assay the results are shown in Table 17 in the lamp stand leaf medicinal material in the different places of production.
Picrinine assay result in the table ten seven different places of production lamp stand leaves
| The sample lot number | The place of production | Content mg/g | The sample lot number | The place of production | Content mg/g |
| The S-20051101 leaf | Simao City | 0.72 | X-20060601 | Xishuangbanna | 0.63 |
| The S-20060301 leaf | 0.57 | X-20060901 | 0.68 | ||
| The S-20060601 leaf | 0.62 | D-20060601 | The Dehong state | 0.47 | |
| The S-20060901 leaf | 0.65 | D-20060901 | 0.53 |
Embodiment 2: the assay of picrinine in the lamp stand tree root
Method is with embodiment 1, and the medicinal part of mensuration is the root of Cornus controversa, and the picrinine assay the results are shown in Table 18 in the lamp stand tree root in the different places of production.
Picrinine assay result in the table ten eight different places of production Cornus controversa root herbs
| The sample lot number | The place of production | Content mg/g | The sample lot number | The place of production | Content mg/g |
| The S-20051101 root | Simao City | 1.38 | X-20060601 | Xishuangbanna | 1.02 |
| The S-20060601 root | 1.13 | X-20060901 | 1.24 | ||
| The S-20060901 root | 1.47 | D-20060901 | The Dehong state | 1.39 |
Embodiment 3: the assay of picrinine in the lamp stand branch
Method is with embodiment 1, and the medicinal part of mensuration is the branch of Cornus controversa, and the picrinine assay the results are shown in Table 19 in the Cornus controversa stem in the different places of production.
Picrinine assay result in the table ten nine different places of production lamp stand branches
| The sample lot number | The place of production | Content mg/g | The sample lot number | The place of production | Content mg/g |
| The S-20051101 stem | Simao City | 0.87 | X-20060601 | Xishuangbanna | 0.74 |
| The S-20060601 stem | 0.79 | X-20060901 | 0.80 | ||
| The S-20060901 stem | 0.81 | D-20060901 | The Dehong state | 0.71 |
Embodiment 4: the assay of picrinine in the lamp stand bark
Method is with embodiment 1, and the medicinal part of mensuration is the skin of Cornus controversa, and the picrinine assay the results are shown in Table 20 in the lamp stand bark in the different places of production.
Picrinine assay result in the table two ten different places of production lamp stand barks
| The sample lot number | The place of production | Content mg/g | The sample lot number | The place of production | Content mg/g |
| The S-20051101 skin | Simao City | 1.76 | X-20060601 | Xishuangbanna | 1.53 |
| The S-20060601 skin | 1.63 | X-20060901 | 1.74 | ||
| The S-20060901 skin | 1.70 | D-20060901 | The Dehong state | 1.69 |
Embodiment 5: the content of measuring picrinine in the lamp stand leaf with different moving phases
Method is with embodiment 1, selects for use in the above-mentioned table six experiment-6～7,9～14 moving phases to form respectively and measures, and the results are shown in Table 21 (sample lot number S-20051101 leaves).
The different moving phases of table two 11 usefulness are measured the content of picrinine in the lamp stand leaf
| Moving phase is formed | Content mg/g | Moving phase is formed | Content mg/g |
| Methyl alcohol-0.01% triethylamine (60: 40) | 0.71 | Methyl alcohol-0.01% diethylamine (50: 50) | 0.73 |
| Methyl alcohol-0.01% triethylamine (50: 50) | 0.72 | Methyl alcohol-0.015% diethylamine (50: 50) | 0.72 |
| Methyl alcohol-0.015% triethylamine (50: 50) | 0.72 | Acetonitrile-0.01% triethylamine (30: 70) | 0.71 |
| Methyl alcohol-0.012% triethylamine (40: 60) | 0.72 | Acetonitrile-0.01% diethylamine (25: 75) | 0.71 |
Embodiment 6: the assay of picrinine in the lamp stand blade
Get 20 in lamp stand blade, remove dressing, the label porphyrize is crossed 60 mesh sieves, and precision takes by weighing 2g, puts in the 100ml round-bottomed flask, accurately adds methyl alcohol 50ml, weighs, and water-bath refluxing extraction 1 hour is put coldly, supplies the weight that subtracts mistake with methyl alcohol.Filter, get subsequent filtrate 25ml evaporate to dryness, it is molten diffusing that residue adds the jolting of 25ml water, transfers pH=7 with 2% ammoniacal liquor, put in the separating funnel and divide three extractions, combined chloroform liquid, evaporate to dryness with the 50ml chloroform, residue dissolves with moving phase and is settled in the 5ml measuring bottle, shakes up, and promptly gets test solution.
According to the above-mentioned method of determining (table six-experiment 8), accurate reference substance solution, the test solution drawn injected liquid chromatograph, measures.The result of picrinine assay is in the lamp stand blade: 0.128mg/g.
Embodiment 7: the assay of picrinine in the alstonia-leaf particle
Get alstonia-leaf particle 5g, the accurate title, decide, and adds the jolting of 25ml water and dissolve, and adds 2% ammoniacal liquor and transfer pH=7, put in the separating funnel and divide three extractions with the 50ml chloroform, combined chloroform liquid, evaporate to dryness, residue dissolves with moving phase and is transferred in the 10ml measuring bottle, be diluted to scale, shake up, promptly.
Accurate reference substance solution, the test solution drawn injected liquid chromatograph, measures.The result of picrinine assay is in the lamp stand blade: 0.149mg/g.
Embodiment 8: the assay of picrinine in the alstonia-leaf capsule
Get alstonia-leaf capsule 's content 5g, following measurement operation is with embodiment 7.The result of picrinine assay is in the alstonia-leaf capsule: 0.117mg/g.
Embodiment 9: the assay of picrinine in the alstonia-leaf soft extracts
Get alstonia-leaf soft extracts 5g, add water-solubleization of 25ml, following measurement operation is with embodiment 7.The result of picrinine assay is in the alstonia-leaf soft extracts: 0.153mg/g.
Embodiment 10: the assay of picrinine in the alstonia-leaf oral liquid
Get alstonia-leaf oral liquid 10ml, add the 15ml dilution, following measurement operation is with embodiment 7.The result of picrinine assay is in the alstonia-leaf oral liquid: 0.161mg/ml.
Claims (6)
1, a kind of efficient liquid-phase chromatography method of measuring picrinine content, it is characterized in that liquid phase chromatogram condition is: chromatographic column is a filling agent with the carbon octadecyl silane, the moving phase mixed solvent that to be methyl alcohol or acetonitrile form with rare triethylamine or diethylamine aqueous solution detects wavelength 287nm.
2, as the said efficient liquid-phase chromatography method of claim 1, it is characterized in that column temperature is 30～40 ℃, flow velocity is 0.5～5ml/min, sampling volume is 10～20 μ l.
3, as the said efficient liquid-phase chromatography method of claim 2, it is characterized in that column temperature is 35 ℃, flow velocity is 1ml/min.
4, as the said efficient liquid-phase chromatography method of claim 1, it is characterized in that the moving phase mixed solvent that to be methyl alcohol form with rare triethylamine or rare diethylamine aqueous solution, the volume ratio of forming is a methyl alcohol: rare triethylamine or rare diethylamine aqueous solution equal 40: 60～and 60: 40, the concentration that rare triethylamine or rare diethylamine aqueous solution contain triethylamine or diethylamine is 0.01%～0.015%.
5, as the said efficient liquid-phase chromatography method of claim 4, it is characterized in that the volume ratio that mixed solvent is formed is a methyl alcohol: rare triethylamine or rare diethylamine aqueous solution equal 50: 50.
6, as the said efficient liquid-phase chromatography method of claim 1, it is characterized in that the moving phase mixed solvent that to be acetonitrile form with rare triethylamine or rare diethylamine aqueous solution, the volume ratio of forming is an acetonitrile: rare triethylamine or rare diethylamine aqueous solution equal 25: 75～and 30: 70, the concentration that rare triethylamine or rare diethylamine aqueous solution contain triethylamine or rare diethylamine is 0.01%～0.015%.
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| CN101983574A (en) * | 2010-11-18 | 2011-03-09 | 云南省农业科学院茶叶研究所 | Alstonia-leaf tea bag and processing method thereof |
| CN114177146A (en) * | 2022-01-17 | 2022-03-15 | 南宁市第一人民医院 | Preparation and content determination method of schefflera octophylla formula particles |
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| CN100356172C (en) * | 2006-03-16 | 2007-12-19 | 朱光荣 | Use of picrinine in quality control of Dengtaiye medicine and its preparation |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101983574A (en) * | 2010-11-18 | 2011-03-09 | 云南省农业科学院茶叶研究所 | Alstonia-leaf tea bag and processing method thereof |
| CN114177146A (en) * | 2022-01-17 | 2022-03-15 | 南宁市第一人民医院 | Preparation and content determination method of schefflera octophylla formula particles |
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Effective date of registration: 20170927 Address after: High tech Zone Jinpu Ma Cheng Road 650503 Yunnan city of Kunming province No. 2899 Patentee after: Yunnan Plant Pharmaceutical Industry Co., Ltd. Address before: 650034 Wang Jia dam 22, Guandu District, Yunnan, Kunming Patentee before: Kunming Zhenhua Pharmacy Co., Ltd. |