CN104297401B - The HPLC-ELSD content assaying method of songorine in Radix Aconiti Kusnezoffii - Google Patents
The HPLC-ELSD content assaying method of songorine in Radix Aconiti Kusnezoffii Download PDFInfo
- Publication number
- CN104297401B CN104297401B CN201410348232.6A CN201410348232A CN104297401B CN 104297401 B CN104297401 B CN 104297401B CN 201410348232 A CN201410348232 A CN 201410348232A CN 104297401 B CN104297401 B CN 104297401B
- Authority
- CN
- China
- Prior art keywords
- songorine
- radix aconiti
- aconiti kusnezoffii
- compound
- acetonitrile
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)
- Steroid Compounds (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The present invention separates from Radix Aconiti Kusnezoffii first and obtains songorine, Aconitum soongaricum amine, karacoline, and on this basis, providing and a kind of utilize HPLC-ELSD method to measure the method for the content of songorine in Radix Aconiti Kusnezoffii, chromatographic condition and testing conditions are mobile phase: the volume ratio of acetonitrile-0.1% triethylamine solution is (40-50): (50-60); Column temperature: 20-30 DEG C; Flow velocity: 0.5-1.50mL min-1; Sample size: 10-30 �� L; The drift tube temperature of ELSD detector: 90-98 DEG C; Gas pressure: 2.00-3.00bar; Ram is closed mode, and the exploitation that the present invention is the quality standard and Semen Plantaginis that improve Radix Aconiti Kusnezoffii provide new scientific basis.
Description
Technical field
The present invention relates to and a kind of utilize HPLC-ELSD method to measure the method for the content of songorine in Radix Aconiti Kusnezoffii, belong to biomedicine field.
Background technology
The dried root that Radix Aconiti Kusnezoffii (AconitikusnezoffiiRadix) is ranunculaceae plant Aconitum kusnezoffii Reichb (AconitumkusnezoffiiReichb.), Medicinal is with a long history, is usually used in the symptoms such as treatment rheumatism, paralysis, colic of cold type, pain.
The primary bioactive components of Radix Aconiti Kusnezoffii is Diterpenoid Alkaloids, and they are active ingredient main in medical material, is again the composition that toxicity is very strong simultaneously, and inappropriate medication easily causes intoxicating phenomenon. Therefore, for ensureing drug safety, it is necessary to understand fully the kind of all alkaloids compositions in Radix Aconiti Kusnezoffii and strictly control the content of this type of alkaloid component in medical material by assay. Up to the present people establish content assaying method for the aconitine in Radix Aconiti Kusnezoffii, hypaconitine and mesaconitine. But without about the research of Aconitum soongaricum alkali content in Radix Aconiti Kusnezoffii. The invention provides and a kind of utilize HPLC-ELSD method to measure the method for the content of songorine in Radix Aconiti Kusnezoffii, Radix Aconiti Kusnezoffii quality standard can not only be improved, moreover it is possible to for the safe medication of Radix Aconiti Kusnezoffii and develop and provide scientific basis.
Summary of the invention
The technical problem to be solved in the present invention is to overcome existing defect, it is provided that a kind of utilize HPLC-ELSD method to measure the method for the content of songorine in Radix Aconiti Kusnezoffii.
In order to solve above-mentioned technical problem, the invention provides following technical scheme:
The HPLC-ELSD content assaying method of songorine in Radix Aconiti Kusnezoffii, preparing of its chromatographic condition and testing conditions, the preparation of need testing solution and reference substance solution is as follows:
Chromatographic condition and testing conditions:
Mobile phase: the volume ratio of acetonitrile-0.1% triethylamine solution is (40-50): (50-60)
Column temperature: 20-30 DEG C
Flow velocity: 0.5-1.50mL min-1
Sample size: 10-30 �� L
The drift tube temperature of ELSD detector: 90-98 DEG C
Gas pressure: 2.00-3.00bar
Ram is closed mode;
The preparation of need testing solution
It is appropriate that precision weighs Radix Aconiti Kusnezoffii powder, soaks with ammonia, chloroform heating and refluxing extraction 1-3 time, add ammonia solid-liquid ratio (g/ml) is 1:(0.5-2 every time), chloroform solid-liquid ratio (g/ml) be 1:(6-8), extraction time is 0.5-2.0h, filters, merging filtrate, decompression and solvent recovery, again it is dissolved in volumetric flask with acetonitrile again, constant volume, shakes up, filter, take subsequent filtrate as need testing solution;
The preparation of reference substance solution
Accurate Weigh Compound songorine, is dissolved in volumetric flask with acetonitrile, and constant volume shakes up, obtains 1.0-2.0mg mL-1Reference substance solution, cold preservation is standby.
Further, the HPLC-ELSD content assaying method of songorine, chromatographic condition and testing conditions in Radix Aconiti Kusnezoffii:
Mobile phase: the volume ratio of acetonitrile-0.1% triethylamine solution is 40:60
Column temperature: 25 DEG C
Flow velocity: 1.0mL min-1
Sample size: 20 �� L
The drift tube temperature of ELSD detector: 96 DEG C
Gas pressure: 2.50bar
Ram is closed mode.
Further, the HPLC-ELSD content assaying method of songorine in Radix Aconiti Kusnezoffii, the preparation of need testing solution:
Precision weighs Radix Aconiti Kusnezoffii powder 3.0g, soaks with ammonia, chloroform heating and refluxing extraction 2 times, add ammonia 3.0mL, chloroform 20mL every time, extraction time is 1.5h, 1.0h respectively, filters, merging filtrate, decompression and solvent recovery, again it is dissolved in 10mL volumetric flask with acetonitrile again, constant volume, shake up, cross 0.22 ��m of microporous filter membrane, take subsequent filtrate as need testing solution.
Further, the HPLC-ELSD content assaying method of songorine in Radix Aconiti Kusnezoffii, the preparation of reference substance solution:
Accurate Weigh Compound songorine 75mg, purity is 99.5%, is dissolved in 50mL volumetric flask with acetonitrile, and constant volume shakes up, obtains 1.5mg mL-1Reference substance solution, be placed in 4 DEG C of cold preservations standby.
Reference substance songorine used in the present invention is self-control, and purity is 99.5%, and the preparation method of songorine is as follows:
(1) extract
Take dry Radix Aconiti Kusnezoffii, with chloroform-ammonia spirit (20:1) for mobile phase seepage pressure effects, extracting solution is concentrated, being acidified to pH=1��2 with the HCl of 0.5%, chloroform extracts 4 times, is merged by water layer, concentrates, alkalize to pH=11��12 with the NaOH of 0.5% again, chloroform extracts 4 times, takes chloroform layer and merges, concentrates, obtains crude extract.
(2) separate
Crude extract is carried out silica gel column chromatography, with eluant 1��5 for eluent gradient eluting, TCL checks, merge stream part that component is identical, recycling design, obtains three parts, contains songorine by detecting TCL wherein Part I, Part II contains Aconitum soongaricum amine, and Part III contains mesaconitine.
(3) purification
By step (2) gained three part difference purification repeatedly, final purification obtains 8 compounds, is songorine, Aconitum soongaricum amine, compound talastisamine, karacoline, 3-deoxyaconitine, aconitine, hypaconitine, mesaconitine respectively. The purification process of the employing in step (3) is silica gel column chromatography and/or recrystallization.
On the basis of the studies above, the present invention establishes HPLC-ELSD (high performance liquid chromatography-evaporation Laser Light Scattering detector) content assaying method of songorine in Radix Aconiti Kusnezoffii, and recording the average content of songorine in the Anguo product Radix Aconiti Kusnezoffii of Hebei is 0.9238mg g-1, it is 1.319mg g that Jilin huge rock produces the average content of songorine in Radix Aconiti Kusnezoffii-1, it is 1.768mg g that the average content of songorine in Radix Aconiti Kusnezoffii is produced in Dali-1, it is 1.635mg g that Hui nationality produces the average content of songorine in Radix Aconiti Kusnezoffii-1.The content of songorine difference to some extent in the Radix Aconiti Kusnezoffii of different sources, but except individual samples content is lower than 1.0mg g-1(would be likely to occur the unequal error component of sampling) outward, all the other are all at 1.0mg g-1(0.1%), more than, the higher alkaloids composition of content (total amount that 2010 editions pharmacopeia record Radix Aconiti Kusnezoffii mesaconitine, hypaconitine and mesaconitine is 0.10%��0.50%) is belonged to. Therefore, include the assay index of songorine in Radix Aconiti Kusnezoffii quality standard and be conducive to the quality control of Radix Aconiti Kusnezoffii.
Accompanying drawing explanation
Accompanying drawing is for providing a further understanding of the present invention, and constitutes a part for description, is used for together with embodiments of the present invention explaining the present invention, is not intended that limitation of the present invention. In the accompanying drawings:
Fig. 1 is the chromatogram of reference substance in embodiments of the invention mode;
Fig. 2 is the chromatogram of embodiments of the invention mode Chinese crude drug;
Fig. 3 is the extraction Comparative result of different solvents in embodiments of the invention mode;
Fig. 4 is the extraction Comparative result adding ammonia in embodiments of the invention mode with being not added with ammonia;
Fig. 5 is the contrast of the asynchronous extraction result of extracting method in embodiments of the invention mode;
Fig. 6 is the contrast of the asynchronous extraction result of extraction time in embodiments of the present invention;
Fig. 7 is the contrast of the asynchronous extraction result of Extraction solvent volume in embodiments of the present invention.
Fig. 8 is the linear relationship curve in embodiments of the invention 1.
Detailed description of the invention
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are illustrated, it will be appreciated that preferred embodiment described herein is merely to illustrate and explains the present invention, is not intended to limit the present invention.
One, the extraction of Radix Aconiti Kusnezoffii chemical composition and separation
1, experimental raw and reagent material
Experimental raw
This experiment is extracted and separation Radix Aconiti Kusnezoffii is purchased from Hebei Anguo.
Reagent material
This experiment agents useful for same is analytical pure, including petroleum ether, acetone, diethylamine, ammonia etc. Petroleum ether and diethylamine are purchased from Tianjin Tian Tai fine chemicals company limited; Acetone, ethyl acetate, chloroform and ammonia are purchased from Beijing Chemical Plant. TLCsilicagel60F254 and TLCsilicagel60RP-18F254S is purchased from Merck, and column chromatography silica gel (200-300 order) is purchased from Haiyang Chemical Plant, Qingdao.
2, the extraction of chemical composition and separation
(1) extract
Take dry Radix Aconiti Kusnezoffii 10kg, pulverize, with chloroform-ammonia spirit (20:1) for mobile phase seepage pressure effects, extracting solution is concentrated, is acidified to pH=1��2 with the HCl of 0.5%, chloroform extracts 4 times, being merged by water layer, concentrate, then alkalize to pH=11��12 with the NaOH of 0.5%, chloroform extracts 4 times, take chloroform layer to merge, concentrate, obtain crude extract 310g.
(2) separation and purification
Crude extract (310g) is carried out silica gel column chromatography, with eluant 1��5 for eluent gradient eluting, thin layer chromatography (TLC) detects, merge same composition, decompression and solvent recovery, obtains three parts, contains songorine by detecting TCL wherein Part I, Part II contains Aconitum soongaricum amine, and Part III contains mesaconitine.
Part I (40g) is carried out silica gel column chromatography, and with eluant 7��10 for eluent gradient eluting, TLC detects, and merges same composition, decompression and solvent recovery, obtains A (10g), two parts of B (5g). Again part A being carried out silica gel column chromatography, with 8,9,10 for eluent gradient eluting, TLC detects, and merges same composition, decompression and solvent recovery, precipitates out white powder and is compound cw-1 (2.0g);By mother liquor concentrations, carry out silica gel column chromatography, obtain white powder and be compound cw-6 (860mg). Part B is carried out silica gel column chromatography, and with 8,9,10 for eluent gradient eluting, TLC detects, merge same composition, decompression and solvent recovery, the mixed solution crystallization of white precipitate petroleum ether and acetone, recrystallization, obtain colourless lump shaped crystalline and be compound cw-3 (1.0g). Part II (35g) is carried out silica gel column chromatography, and with eluant 6��9 for eluent gradient eluting, TLC detects, and merges same composition, decompression and solvent recovery, obtains C (9.4g), two parts of D (3.0g). Again C portion being carried out silica gel column chromatography, with 6,8,9 for eluent gradient eluting, TLC detects, and merges same composition, decompression and solvent recovery, obtains compound cw-7 (white powder, 560mg) and C-1 (5.2g) part. Again C-1 part is carried out silica gel column chromatography, with eluant 6,8,9 for eluent gradient eluting, TLC detects, merge same composition, decompression and solvent recovery, the mixed solution crystallization of white precipitate petroleum ether and acetone, recrystallization, obtain colourless granular crystal and be compound cw-5 (200mg). D part is carried out silica gel column chromatography, with eluant 6,8,9,10 for eluent gradient eluting, TLC detects, merge same composition, decompression and solvent recovery, the mixed solution crystallization of white precipitate petroleum ether and acetone, recrystallization, obtain colourless lump shaped crystalline and be compound cw-2 (550mg). Part III (42g) is carried out silica gel column chromatography, and with eluant 6,8,9,11 for eluent gradient eluting, TLC detects, and merges same composition, decompression and solvent recovery, obtains E (17g) part. Again E part being carried out silica gel column chromatography, with eluant 7��10 for eluent gradient eluting, TLC detects, and merges same composition, decompression and solvent recovery, obtains compound cw-8 (white powder, 4.0g) and E-1 (5.0g) part. Again E-1 part is carried out silica gel column chromatography, with eluant 7��10 for eluent gradient eluting, TLC detects, merge same composition, decompression and solvent recovery, the mixed solution crystallization of white precipitate petroleum ether and acetone, recrystallization, obtain colourless granular crystal and be compound cw-4 (70mg).
Two, the Structural Identification of chemical composition
The Structural Identification of compound cw-1
Compound cw-1 is white powder, and m.p.195.5��196.5 DEG C are soluble in chloroform, acetone, are slightly soluble in acetonitrile, and bismuth potassium iodide reaction is positive.
IR (KBr) spectrum: 3500cm-1The appearance locating strong absworption peak illustrates hydroxyl in this chemical constitution, 1700cm-1The appearance of the strong absworption peak in place illustrates in this structure containing carbonyl, 1650cm-1The appearance of place's absworption peak illustrates in this structure containing double bond.
ESI-MS:m/z358.3[M+H]+, the molecular weight pointing out this compound is 357.3.
13C-NMR(CDCl3, 125MHz) spectrum provide 22 carbon signals altogether, in conjunction with DEPT compose it can be seen that compound cw-1 has 2 methyl signals (��c, ppm:26.09,13.62), 8 methylene signals (��c, ppm:111.45,57.42,50.95,38.16,37.34,32.19,31.66,23.21), 7 methine signals (��c, ppm:77.32,70.37,66.03,53.80,49.19,43.52,35.22), 5 quaternary carbon signal (��c, ppm:210.03,151.01,52.41,50.00,34.11), wherein ��c210.03ppm place's quaternary carbon signal is carbonyl carbon signals, ��c151.01,111.45ppm place quaternary carbon, methylene signals respectively hold two carbon signals of alkene.
1H-NMR(CDCl3, 500MHz) and spectrum: ��H(ppm) 0.771 (3H, s) for the proton signal of methyl, ��H1.072 (3H, t, J=7.5Hz) are the proton signal of methyl, �� on nitrogen ethylH2.491 (2H, m) for the proton signal of methylene on nitrogen ethyl, ��H3.849 (1H, s), 4.365 (1H, d, J=8.0Hz) respectively two the even proton signal on hydroxyl methine carbons, ��H5.201,5.293 (each 1H, s) for two proton signals of end alkene.
According to more than13C-NMR and1H-NMR signal characteristic and the regularity of distribution, preliminary deduction compound cw-1 is C20Diterpene alkaloid compounds, through compareing with document, it is determined that compound cw-1 is that songorine (has another name called songorine, songorine, C22H31NO3). This compound known obtains for separating from Radix Aconiti Kusnezoffii first by literature search. Its chemical structural formula is as follows:
The Structural Identification of compound cw-2
Compound cw-2 is colourless lump shaped crystalline (petroleum ether-acetone), and m.p.212��213 DEG C are soluble in chloroform, acetonitrile, and bismuth potassium iodide reaction is positive.
IR (KBr) spectrum: 3400cm-1The appearance locating strong absworption peak illustrates hydroxyl in this chemical constitution, 1700cm-1The appearance locating strong absworption peak illustrates in this structure containing carbonyl, 1650cm-1The appearance locating strong absworption peak illustrates in this structure containing double bond.
ESI-MS:m/z356.2[M+H]+, the molecular weight pointing out this compound is 355.2.
13C-NMR(CDCl3, 125MHz) spectrum provide 22 carbon signals altogether, in conjunction with DEPT compose it can be seen that compound cw-2 has 2 methyl signals (��c, ppm:18.89,14.25), 7 methylene signals (��c, ppm:111.94,48.32,37.39,31.21,29.71,24.27,23.99), 8 methine signals (��c, ppm:92.94,76.94,67.75,66.14,53.05,48.47,45.93,31.35), 5 quaternary carbon signal (��c, ppm:208.81,149.61,51.71,50.11,37.81). Wherein ��c208.81ppm place's quaternary carbon signal is carbonyl carbon signals, ��c149.61,111.94ppm place quaternary carbon, methylene signals respectively hold two carbon signals of alkene.
1H-NMR(CDCl3, 500MHz) and spectrum: ��H(ppm) 0.849 (3H, s) for the proton signal of methyl, ��H1.036 (3H, t, J=6.0Hz) are the proton signal of methyl, �� on nitrogen ethylH2.671 (2H, m) for the proton signal of methylene on nitrogen ethyl, ��H3.715 (1H, s), 3.992 (1H, d, J=4.0Hz) be the even proton signal on oxygen methine carbon, ��H4.404 (1H, d, J=5.5Hz) connect the proton signal on hydroxyl methine carbon, ��H5.207,5.311 (each 1H, s) for two proton signals of end alkene.
According to more than13C-NMR and1H-NMR signal characteristic and the regularity of distribution, preliminary deduction compound cw-2 is C20Diterpene alkaloid compounds, through compareing with document, it is determined that compound cw-2 is that Zhunger Basin aconine (has another name called dehydrogenation songorine, songoramine, C22H29NO3). This compound known obtains for separating from Radix Aconiti Kusnezoffii first by literature search. Its chemical structural formula is as follows:
The Structural Identification of compound cw-3
Compound cw-3 is colourless lump shaped crystalline (petroleum ether-acetone), and m.p.134.5��135.5 DEG C are soluble in chloroform, acetonitrile, and bismuth potassium iodide reaction is positive.
IR (KBr) spectrum: 3400cm-1The appearance locating strong absworption peak illustrates hydroxyl in this chemical constitution.
ESI-MS:m/z422.3[M+H]+, the molecular weight pointing out this compound is 421.3.
13C-NMR(CDCl3, 125MHz) spectrum provide 24 carbon signals altogether, in conjunction with DEPT compose it can be seen that compound cw-3 has 4 methyl signals (��c, ppm:59.49,56.49,56.32,13.71), 8 methylene signals (��c, ppm:79.49,53.17,49.52,38.35,32.79,27.69,25.84,24.75), 9 methine signals (��c, ppm:86.37,82.25,75.60,62.96,46.96,46.06,45.90,45.80,37.56), 3 quaternary carbon signal (��c, ppm:72.81,48.72,38.67).Wherein ��c59.49,56.49,56.32ppm place methyl signals be three methoxyl group carbon signals.
1H-NMR(CDCl3, 500MHz) and spectrum: ��H(ppm) 1.057 (3H, t, J=7.0Hz) are the proton signal of methyl, �� on nitrogen ethylH2.529 (2H, m) for the proton signal of methylene on nitrogen ethyl, ��H3.267,3.299,3.346 (each 3H s) is the proton signal of three methoxyl groups.
According to more than13C-NMR and1H-NMR signal characteristic and the regularity of distribution, preliminary deduction compound cw-3 is C19Diterpene alkaloid compounds, through compareing with document, it is determined that compound cw-3 is that talastisamine (has another name called tower and draws ground Sa Min, talatizamine, C24H39NO5). Its chemical structural formula is as follows:
The Structural Identification of compound cw-4
Compound cw-4 is colourless granular crystal (petroleum ether-acetone), and m.p.187.5��188.0 DEG C are soluble in chloroform, acetone, are slightly soluble in acetonitrile, and bismuth potassium iodide reaction is positive.
ESI-MS:m/z378.2[M+H]+, the molecular weight pointing out this compound is 377.2.
13C-NMR(CDCl3, 125MHz) spectrum provide 22 carbon signals altogether, in conjunction with DEPT compose it can be seen that compound cw-4 has 3 methyl signals (��c, ppm:56.33,27.57,13.05), 7 methylene signals (��c, ppm:60.24,48.41,42.26,31.31,29.68,28.46,25.14), 9 methine signals (��c, ppm:81.98,75.84,72.50,63.38,46.71,46.59,45.09,44.05,39.95), 3 quaternary carbon signal (��c, ppm:74.23,48.82,32.92). Wherein ��c56.33ppm place's methyl signals is a methoxyl group carbon signal.
1H-NMR(CDCl3, 500MHz) and spectrum: ��H(ppm) 0.882 (3H, s) for the proton signal of methyl, ��H1.118 (3H, t, J=7.5Hz) are the proton signal of methyl, �� on nitrogen ethylH2.531 (2H, m) for the proton signal of methylene on nitrogen ethyl, ��H3.346 (3H, s) for the proton signal of methoxyl group.
According to more than13C-NMR and1H-NMR signal characteristic and the regularity of distribution, preliminary deduction compound cw-4 is C19Diterpene alkaloid compounds, warp and document] compare, it is determined that compound cw-4 is karacoline (karakoline, C22H35NO4). This compound known obtains for separating from Radix Aconiti Kusnezoffii first by literature search. Its chemical structural formula is as follows:
The Structural Identification of compound cw-5
Compound cw-5 is colourless granular crystal (petroleum ether-acetone), and m.p.176.5��177.0 DEG C are soluble in chloroform, acetone, are slightly soluble in acetonitrile, and bismuth potassium iodide reaction is positive.
IR (KBr) spectrum: 3500cm-1The appearance locating strong absworption peak illustrates hydroxyl in this chemical constitution, 1600��1450cm-1Aromatic ring frame vibration performance peak, 1700cm occur-1The appearance locating strong absworption peak illustrates in this structure containing carbonyl.
ESI-MS:m/z630.4[M+H]+, the molecular weight pointing out this compound is 629.4.
13C-NMR(CDCl3, 125MHz) spectrum provide 32 carbon signals altogether, in conjunction with DEPT compose it can be seen that compound cw-5 has 6 methyl signals (��c, ppm:61.02,59.07,58.00,56.27,21.45,13.47), 6 methylene signals (��c, ppm:80.31,53.17,49.06,36.67,35.27,26.39), 13 methine signals (��c, ppm:133.22,129.63,128.63,90.18,85.26,83.28,79.01,78.86,61.39,49.22,45.15,44.62,41.01), 7 quaternary carbon signal (��c, ppm:172.40,166.15,129.90,92.13,74.14,49.95,39.08). Wherein ��c61.02,59.07,58.00,56.27ppm place methyl signals be four methoxyl group carbon signals, ��c172.40,166.15ppm place quaternary carbon signal be two carbonyl carbon signals, ��c129.90 (quaternary carbon), 133.22 (methines), 129.63 (methines), 128.63 (methine) ppm place signal are the carbon signal on monosubstituted phenyl ring.
1H-NMR(CDCl3, 500MHz) and spectrum: ��H(ppm) 1.077 (3H, t, J=7.5Hz) are the proton signal of methyl, �� on nitrogen ethylH1.375 (3H, s) for the proton signal of methyl on acetyl group, ��H3.158,3.266,3.283,3.738 (each 3H s) is the proton signal of four methoxyl groups, ��H7.438��8.042 (5H) three groups signal is the proton signal on phenyl ring.
According to more than13C-NMR and1H-NMR signal characteristic and the regularity of distribution, preliminary deduction compound cw-5 is C19Aconitine type compound, through compareing with document, it is determined that compound cw-5 is 3-deoxyaconitine (3-deoxyaconitine, C34H47NO10). Its chemical structural formula is as follows:
The Structural Identification of compound cw-6
Compound cw-6 is white powder, and m.p.190.5��191.5 DEG C are soluble in chloroform, acetone, and bismuth potassium iodide reaction is positive. Thin layer chromatography (TLC) is utilized to compare with aconitine standard substance, developing solvent is petroleum ether-acetone-diethylamine (silica gel), normal hexane-acetone-diethylamine (silica gel), acetone-water-diethylamine (ODS), developer is bismuth potassium iodide test solution and sulphuric acid-ethanol test solution, both spot colors and Rf value are all consistent, and mixed melting point does not decline, therefore can determine that compound cw-6 is aconitine (aconitine, C34H47NO11). Its chemical structural formula is as follows:
The Structural Identification of compound cw-7
Compound cw-7 is white powder, and m.p.177.5��178.0 DEG C are soluble in chloroform, acetone, and bismuth potassium iodide reaction is positive. Thin layer chromatography (TLC) is utilized to compare with hypaconitine standard substance, developing solvent is petroleum ether-acetone-diethylamine (silica gel), normal hexane-acetone-diethylamine (silica gel), acetone-water-diethylamine (ODS), developer is bismuth potassium iodide test solution and sulphuric acid-ethanol test solution, both spot colors and Rf value are all consistent, and mixed melting point does not decline, therefore can determine that compound cw-7 is hypaconitine (hypaconitine, C33H45NO10). Its chemical structural formula is as follows:
The Structural Identification of compound cw-8
Compound cw-8 is white powder, and m.p.194.5��196.0 DEG C are soluble in chloroform, acetone, and bismuth potassium iodide reaction is positive. Thin layer chromatography (TLC) is utilized to compare with mesaconitine standard substance, developing solvent is petroleum ether-acetone-diethylamine (silica gel), normal hexane-acetone-diethylamine (silica gel), acetone-water-diethylamine (ODS), developer is bismuth potassium iodide test solution and sulphuric acid-ethanol test solution, both spot colors and Rf value are all consistent, and mixed melting point does not decline, therefore can determine that compound cw-8 is mesaconitine (mesaconitine, C33H45NO11). Its chemical structural formula is as follows:
Three, HPLC-ELSD method measures the selection of the chromatographic condition of the content of songorine in Radix Aconiti Kusnezoffii
1, the selection of mobile phase
First, this experiment adopts DAD detector, during with methanol-water, acetonitrile-water for mobile phase isocratic elution, find that the peak shape hangover of target compound cw-1 is more serious, and after adding the triethylamine of 0.1% in aqueous phase, peak shape has had and has been obviously improved, and therefore selects methanol-0.1% triethylamine solution or acetonitrile-0.1% triethylamine solution as mobile phase. Due to mobile phase meta-alkalescence, therefore select and be applicable to the higher Extend-C of PH18Chromatographic column. Then, this effects separating effect being mobile phase with methanol-0.1% triethylamine solution, it has been found that this mobile phase condition cannot make target compound cw-1 reach to efficiently separate with other compositions in medical material.The separating effect that it is mobile phase with acetonitrile-0.1% triethylamine solution that this experiment has been investigated again, it has been found that although cw-1 can efficiently separate with other compositions, but baseline fluctuation is relatively big, causes that detection limit is unable to reach mensuration requirement. And find after using Alltech2000ELSD detector to compare, baseline, peak shape are substantially better than DAD detector, and detect limit, precision and all can reach mensuration requirement. Therefore for making all the components in target compound and medical material reach baseline separation, and obtaining good peak shape, this experiment finally selects use ELSD detector, and using acetonitrile-0.1% triethylamine solution as mobile phase. Finally determine that mobile phase is acetonitrile-0.1% triethylamine solution (40:60) through repetition test.
2, the setting of ELSD parameter
Finally determine that through repetition test the parameter of ELSD is as follows:
Drift tube temperature is 96 DEG C, and gas (air) pressure is 2.50bar, and ram is closed mode. The chromatogram of reference substance and medical material is as shown in Figure 1, 2.
3, the selection of extraction conditions
For ensureing that target compound extracts completely, now the influence factors such as Extraction solvent, extracting method, extraction time, solvent load are investigated, so that it is determined that optimum extraction conditions.
3.1, the selection of Extraction solvent
Precision weighs 3.0g Radix Aconiti Kusnezoffii powder, totally 5 parts, all soaks with 3.0mL ammonia, then is separately added into hexamethylene, petroleum ether, chloroform, ethyl acetate, acetone 20mL, being heated to reflux 1.5h, filter, filtrate decompression is evaporated, again it is dissolved in 10mL volumetric flask with acetonitrile again, constant volume, shake up. Cross 0.22 ��m of microporous filter membrane, take filtrate 20 �� L and inject HPLC, measure the peak area of cw-1. Result (Fig. 3) shows, in chloroform extracted solution, the peak area of compound cw-1 is maximum, illustrates that extraction effect is best, and acetone takes second place, and both of which, higher than hexamethylene, petroleum ether and ethyl acetate, therefore selects chloroform as Extraction solvent.
3.2, the comparison adding ammonia and be not added with ammonia
Precision weighs 3.0g Radix Aconiti Kusnezoffii powder, totally 2 parts, and portion soaks with 3.0mL ammonia, adds chloroform 20mL, and another part is directly added into chloroform 20mL, is heated to reflux 1.5h, filters, and filtrate decompression is evaporated, is more again dissolved in 10mL volumetric flask with acetonitrile, and constant volume shakes up. Cross 0.22 ��m of microporous filter membrane, take filtrate 20 �� L and inject HPLC, measure the peak area of cw-1. Result (Fig. 4) shows, with ammonia soak extracting solution in the peak area of compound cw-1 apparently higher than the extracting solution being not added with ammonia, illustrate that the extraction effect adding ammonia is good, therefore select ammonification water-soaked.
3.3, the selection of extracting method
Precision weighs 3.0g Radix Aconiti Kusnezoffii powder, totally 3 parts, all soaks with 3.0mL ammonia, and adds chloroform 20mL, being respectively adopted and be heated to reflux 1.5h, ultrasonic 1.5h, tri-kinds of methods extractions of merceration 24h, filter, filtrate decompression is evaporated, again it is dissolved in 10mL volumetric flask with acetonitrile again, constant volume, shake up. Cross 0.22 ��m of microporous filter membrane, take filtrate 20 �� L and inject HPLC, measure the peak area of cw-1. Result (Fig. 5) shows, in the extract being heated to reflux, the peak area of compound cw-1 is the highest, illustrates that extraction effect is best, and therefore selective extraction method is for being heated to reflux.
3.4, the selection of extraction time
Precision weighs 1 part of 3.0g Radix Aconiti Kusnezoffii powder, soak with ammonia, chloroform heating and refluxing extraction 3 times, adds ammonia 3.0mL, chloroform 20mL every time, and the time is 1.5h, 1.0h, 1.0h respectively, after extracting every time, filtering, filtrate decompression is evaporated, is more again dissolved in 10mL volumetric flask with acetonitrile, constant volume, shakes up.Cross 0.22 ��m of microporous filter membrane, take filtrate 20 �� L and inject HPLC, measure the peak area of cw-1. Result (Fig. 6) shows, the extraction ratio that the 1st time is heated to reflux is the highest, is not detected by the existence of compound cw-1 in the 3rd extracting solution, illustrates twice to be extracted completely by cw-1, and therefore selective extraction number of times is 2 times.
3.5, the selection of Extraction solvent consumption
Precision weighs 3.0g Radix Aconiti Kusnezoffii powder, totally 3 parts, all soaks with 3.0mL ammonia, then be separately added into chloroform 15,20,25,30mL, be heated to reflux 1.5h, filter, filtrate decompression is evaporated, is more again dissolved in 10mL volumetric flask with acetonitrile, and constant volume shakes up. Cross 0.22 ��m of microporous filter membrane, take filtrate 20 �� L and inject HPLC, measure the peak area of cw-1. Result (Fig. 7) shows, 20mL and 25,30mL chloroform extracting solution in cw-1 peak area maximum, but three difference less, therefore selective extraction solvent load is 20mL.
Embodiment 1
In Radix Aconiti Kusnezoffii, the HPLC-ELSD content assaying method of songorine, specifically comprises the following steps that
1, experimental apparatus, chromatographic condition and testing conditions, material and reference substance
Chromatograph: Agilent1200HPLC
Detector: Alltech2000ELSD
Chromatographic column: AgilentExtend-C18(4.6��250mm,5��m)
Chromatographic condition and testing conditions:
Mobile phase: the volume ratio of acetonitrile-0.1% triethylamine solution is 40:60
Column temperature: 25 DEG C
Flow velocity: 1.0mL min-1
Sample size: 20 �� L
The drift tube temperature of ELSD detector: 96 DEG C
Gas pressure: 2.50bar
Ram is closed mode.
Medical material: medical material used by content analysis is respectively purchased from Hebei Anguo, Jilin huge rock, Dali and Hui nationality.
Reference substance: songorine (purity 99.5%, self-control).
2, the preparation of need testing solution
Precision weighs Radix Aconiti Kusnezoffii powder 3.0g, soaks with ammonia, chloroform heating and refluxing extraction 2 times, adds ammonia 3.0mL, chloroform 20mL every time, extraction time is 1.5h, 1.0h respectively, filters, merging filtrate, evaporated under reduced pressure, again it is dissolved in 10mL volumetric flask with acetonitrile again, constant volume, shake up. Cross 0.22 ��m of microporous filter membrane, take subsequent filtrate as need testing solution.
3, the preparation of reference substance solution
Precision weighs songorine (purity 99.5%) 75mg, is dissolved in 50mL volumetric flask with acetonitrile, and constant volume shakes up, obtains 1.5mg mL-1Reference substance solution, it is standby to be placed in 4 DEG C of refrigerators cold preservation.
4, methodological study
For ensureing feasibility and the accuracy of assay method, now the factors such as linear relationship, precision, repeatability, stability, average recovery are investigated.
4.1, the investigation of linear relationship
Pipette 0.5 respectively, 1.0,2.0,4.0,6.0,8.0,10.0mL songorine reference substance solution (1.5mg mL-1) to 10mL volumetric flask, by dilution in acetonitrile to groove, shake up, obtain concentration be 0.075,0.15,0.30,0.60,0.90,1.20,1.50mg mL-1Reference substance solution. Cross 0.22 ��m of microporous filter membrane, take filtrate 20 �� L and inject HPLC, parallel assay 3 times, calculate the meansigma methods of peak area. With the common logarithm of reference substance sample introduction quality (m) for abscissa, average peak area (SAll) common logarithm be vertical coordinate, investigate lgSAllLinear relationship with lgm. Experimental data is in Table one, and standard curve is as shown in Figure 8. By result it will be seen that sample size lgS in 1.5 �� g��30 �� g rangeAllHaving good linear relationship with lgm, linear equation is: lgSAll=1.6311lgm+0.1061, R2=0.9991.
The linear relationship of table one compound songorine
4.2, Precision Experiment
4.2.1, withinday precision
Accurately pipette 2.0mL reference substance solution (1.5mg mL-1) to 10mL volumetric flask, by dilution in acetonitrile to groove, shake up, obtaining concentration is 0.3mg mL-1Reference substance solution. With 0.22 ��m of filtering with microporous membrane, take filtrate 20 �� L and inject HPLC, continuous sample introduction 6 times in a day, measure peak area, calculate RSD value. Experimental data is in Table two. It is shown that the RSD value of compound cw-1 peak area is 0.26%, less than 2%, it is seen that withinday precision is good.
Table is in two days and day to day precision experimental result
4.2.2, day to day precision
Accurately pipette 2.0mL reference substance solution (1.5mg mL-1) to 10mL volumetric flask, by dilution in acetonitrile to groove, shake up, obtaining concentration is 0.3mg mL-1Reference substance solution. With 0.22 ��m of filtering with microporous membrane, taking filtrate 20 �� L and inject HPLC, continuous sample introduction 3 days, every day, sample introduction 2 times, measured peak area, calculated RSD value. Experimental data is in Table two. It is shown that the RSD value of songorine peak area is 1.54%, less than 2%, it is seen that day to day precision is good.
4.3, repeated experiment
According to " method of 2 prepares need testing solution 6 parts, takes 20 �� L and injects HPLC, every part of sample introduction 2 times, measures the average peak area of songorine, according to one point external standard method, calculates the content of cw-1 in need testing solution, and obtains RSD value. Experimental data is in Table three. Result shows, the average content of songorine is 1.563mg g-1, RSD value is 1.41%, less than 2%, it is seen that repeatability is good.
Table three repeatability experimental result
(note: songorine reference substance solution concentration is 0.45mg mL-1, average peak area is 4185237. )
4.4, stability experiment
Prepare need testing solution 1 part according to the method for " 2 ", take 20 �� L and inject HPLC, every 2h sample introduction 1 time in 0��10h, measure the peak area of cw-1 in need testing solution, and obtain RSD value. Experimental data is in Table four. It is shown that the RSD value of songorine peak area is 1.68%, less than 2%, it is seen that need testing solution is good at 10h internal stability.
Table four stability experiment result
4.5, average recovery experiment
Precision weighs 3.0g Radix Aconiti Kusnezoffii powder, and (Aconitum soongaricum alkali content is it is known that be 1.563mg g-1), totally 6 parts, the 1st, 2 parts add reference substance solution (1.5mg mL-1) 2.7mL, 3rd, 4 parts add reference substance solution 3.3mL, 5th, 6 parts add reference substance solution 4.0mL, make Standard entertion amount be Aconitum soongaricum alkali content in sample respectively 0.8 times, 1.0 times, 1.2 times, make mark-on need testing solution according to the method for " 2 ". Take 20 �� L and inject HPLC, measure the peak area of compound cw-1, according to one point external standard method, calculate the total content of songorine in need testing solution, then obtained average recovery and the RSD value of cw-1 by formula below. Experimental data is in Table five. It is shown that the mean sample recovery rate of compound songorine is 96.86%, RSD value is 1.67%, it is seen that the average recovery of the method is good.
Table five average recovery experimental result
(note: songorine reference substance solution concentration is 0.90mg mL-1, average peak area is 12285773. )
5, assay
5.1, the preparation of reference substance solution
Accurately pipette 3.0mL reference substance solution (1.5mg mL-1) to 10mL volumetric flask, by dilution in acetonitrile to groove, shake up, obtaining concentration is 0.45mg mL-1Reference substance solution.With 0.22 ��m of filtering with microporous membrane, filtrate is as the External standards solutions of assay.
5.2, the preparation of need testing solution
Take purchased from Hebei Anguo, Jilin huge rock, the Radix Aconiti Kusnezoffii of 2 batches of 3 batches of Dali and Hui nationality respectively, dry, pulverize, make need testing solution according to the method for " 2 ", carry out assay.
5.3, assay
According to the chromatographic condition of " 1 ", take 20 �� L reference substance solution and inject HPLC, parallel assay 3 times, calculate meansigma methods and the lgS of cw-1 (songorine) peak areaAll. Take 20 �� L need testing solutions and inject HPLC, every part of parallel assay 2 times, according to one point external standard method, calculate the content of cw-1 in need testing solution. Experimental result is in Table six. It is shown that cw-1 songorine in the Radix Aconiti Kusnezoffii of different sources different batches) content slightly difference.
The assay of compound songorine in table six different sources sample
(note: Gaer aconitine reference substance solution concentration is 0.45mg mL-1, average peak area is 4069804. )
The content of songorine in Radix Aconiti Kusnezoffii has been measured by this chapter invention HPLC-ELSD method.
Assay result illustrates, the content of songorine difference to some extent in the Radix Aconiti Kusnezoffii of different sources, but except individual samples content is lower than 1.0mg g-1Outward, all the other are all at 1.0mg g-1(0.1%), more than, the higher alkaloids composition of content (total amount that 2010 editions pharmacopeia record Radix Aconiti Kusnezoffii mesaconitine, hypaconitine and mesaconitine is 0.10%��0.50%) is belonged to. Therefore, include the assay index of songorine in Radix Aconiti Kusnezoffii quality standard and be conducive to the quality control of Radix Aconiti Kusnezoffii.
Last it is noted that the foregoing is only the preferred embodiments of the present invention, it is not limited to the present invention, although the present invention being described in detail with reference to previous embodiment, for a person skilled in the art, technical scheme described in foregoing embodiments still can be modified by it, or wherein portion of techniques feature carries out equivalent replacement. All within the spirit and principles in the present invention, any amendment of making, equivalent replacement, improvement etc., should be included within protection scope of the present invention.
Claims (1)
1. the HPLC-ELSD content assaying method of songorine in Radix Aconiti Kusnezoffii, it is characterised in that
Chromatographic condition and testing conditions:
Mobile phase: the volume ratio of acetonitrile-0.1% triethylamine solution is 40:60
Column temperature: 25 DEG C
Flow velocity: 1.0mL min-1
Sample size: 20 �� L
The drift tube temperature of ELSD detector: 96 DEG C
Gas pressure: 2.50bar
Ram is closed mode;
The preparation of need testing solution:
Precision weighs Radix Aconiti Kusnezoffii powder 3.0g, soaks with ammonia, chloroform heating and refluxing extraction 2 times, add ammonia 3.0mL, chloroform 20mL every time, extraction time is 1.5h, 1.0h respectively, filters, merging filtrate, decompression and solvent recovery, again it is dissolved in 10mL volumetric flask with acetonitrile, constant volume, shake up, cross 0.22 ��m of microporous filter membrane, take subsequent filtrate as need testing solution;
The preparation of reference substance solution:
Accurate Weigh Compound songorine 75mg, purity is 99.5%, is dissolved in 50mL volumetric flask with acetonitrile, and constant volume shakes up, obtains 1.5mg mL-1Reference substance solution, be placed in 4 DEG C of cold preservations standby.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410348232.6A CN104297401B (en) | 2014-07-22 | 2014-07-22 | The HPLC-ELSD content assaying method of songorine in Radix Aconiti Kusnezoffii |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410348232.6A CN104297401B (en) | 2014-07-22 | 2014-07-22 | The HPLC-ELSD content assaying method of songorine in Radix Aconiti Kusnezoffii |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104297401A CN104297401A (en) | 2015-01-21 |
| CN104297401B true CN104297401B (en) | 2016-06-08 |
Family
ID=52317222
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410348232.6A Expired - Fee Related CN104297401B (en) | 2014-07-22 | 2014-07-22 | The HPLC-ELSD content assaying method of songorine in Radix Aconiti Kusnezoffii |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104297401B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106358654A (en) * | 2016-08-25 | 2017-02-01 | 北京华邈中药工程技术开发中心 | Method for improving stability of content of drug effective ingredients in aconitum kusnezoff radices |
| CN108375642B (en) * | 2018-01-25 | 2021-04-20 | 中国农业科学院蜜蜂研究所 | Diester-type aconitine as biomarker of toxic honey and application of diester-type aconitine in identifying toxic honey |
| CN108548885B (en) * | 2018-06-21 | 2021-04-30 | 江苏康缘药业股份有限公司 | Method for detecting compound arisaema analgesic plaster by two-dimensional liquid chromatography |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| SU425106A1 (en) * | 1972-07-31 | 1974-04-25 | METHOD FOR DETECTION OF ALKALOIDS OPI IN BIOLOGICAL LIQUIDS | |
| JPS5962312A (en) * | 1982-09-30 | 1984-04-09 | Sanki Eng Kk | Method and device for refining physiologically active natural organic compound |
| IT1309450B1 (en) * | 1999-07-20 | 2002-01-23 | Azienda Provinciale Per I Serv | PROCEDURE FOR THE QUANTITATIVE DETERMINATION OF COCAINE AND OTHER SIMILAR SUBSTANCES PRESENT IN A SAMPLE OBTAINED FROM A |
-
2014
- 2014-07-22 CN CN201410348232.6A patent/CN104297401B/en not_active Expired - Fee Related
Non-Patent Citations (2)
| Title |
|---|
| 草乌中生物碱类化学成分的研究;彭劭;《吉林大学硕士学位论文》;20140401;摘要，第2、35-46页 * |
| 草乌及其炮制品中6种生物碱类成分的含量测定;邓广海等;《中国实验方剂学杂志》;20130430;第19卷(第7期);35-38 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104297401A (en) | 2015-01-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102579612B (en) | Method for extracting total alkaloid of aconitum soongaricum | |
| CN103145677B (en) | Method for separating active ingredients from aquilaria sinensis lamina by utilizing high-speed countercurrent chromatography | |
| CN106706768B (en) | Method for separating and measuring empagliflozin and related substances thereof | |
| CN104297401B (en) | The HPLC-ELSD content assaying method of songorine in Radix Aconiti Kusnezoffii | |
| CN105131077A (en) | Method for extracting peroxyergosterol from wall-broken ganoderma lucidum spore powder | |
| CN112526051B (en) | Fmoc-lysine high performance liquid chromatography determination method | |
| CN106770737A (en) | HPLC analysis method of nicorandil related substances | |
| CN103926332B (en) | A kind of Ultra Performance Liquid Chromatography method of uridine, guanosine and adenosine content in Simultaneously test pinellia tuber extract | |
| CN105467021B (en) | A kind of method in relation to substance in HPLC method separation determination paricalcitol bulk pharmaceutical chemicals and preparation | |
| Feng et al. | An efficient and target-oriented sample enrichment method for preparative separation of minor alkaloids by pH-zone-refining counter-current chromatography | |
| CN107505409B (en) | A method of measuring isomer impurities ginsenoside RK1 in ginsenoside RZ1 raw materials or preparation | |
| CN106442842B (en) | A kind of high-efficiency liquid chromatography method for detecting of sirolimus | |
| CN110297061B (en) | Method for determining contents of chlorogenic acid, caffeic acid and luteolin in Ixeris denticulata by one-test-multiple-evaluation method | |
| Ranher et al. | A validated HPLC method for determination of artesunate in bulk and tablet formulation | |
| Dong et al. | Effect of inorganic salt on partition of high‐polarity parishins in two‐phase solvent systems and separation by high‐speed counter‐current chromatography from Gastrodia elata Blume | |
| Qin et al. | Ultrasonic-assisted liquid–liquid extraction and HILIC–ELSD analysis of ginsenoside Rb1, astragaloside IV and dulcitol in sugar-free “Fufangfufangteng Heji” | |
| CN104297402B (en) | The HPLC content assaying method of adenosine in plantain seed | |
| CN103145729B (en) | Bilobalide B compound and preparation method thereof | |
| Ning et al. | Separation and purification of quinolyridine alkaloids from seeds of Thermopsis lanceolata R. Br. by conventional and pH‐zone‐refining counter‐current chromatography | |
| CN114965748A (en) | Method for simultaneously detecting barbaloin, aloe-emodin and indirubin in new compound aloe capsule | |
| Wang et al. | Qualitative and quantitative analyses of bioactive secolignans from folk medicinal plant Peperomia dindygulensis using UHPLC-UV/Q-TOF-MS | |
| QIU et al. | Simultaneous determination of six quaternary ammonium alkaloids in Coptidis rhizoma by UPLC | |
| CN106483226A (en) | A kind of method of high performance liquid chromatography separation LIUSHEN WAN uv absorption composition | |
| CN102617656A (en) | Method for separating and preparing high purity flavonoid glycoside compounds from actinidia valvata dunn leaves | |
| CN102659549B (en) | Method for extracting brevifolin from Relinqing granula raw material and method for preparation quality control |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20160608 Termination date: 20190722 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |