CN104297401A - HPLC-ELSD content determination method for songorine in aconiti kusnezoffii radix - Google Patents
HPLC-ELSD content determination method for songorine in aconiti kusnezoffii radix Download PDFInfo
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Abstract
Songorine, songoramine and karacoline are firstly separated and obtained from aconiti kusnezoffii radix, and on the above basis, the invention provides a method of determining the content of songorine in aconiti kusnezoffii radix by utilizing a HPLC-ELSD process. The chromatographic conditions and the detection conditions comprise that the mobile phase is acetonitrile and a triethylamine solution with the concentration of 0.1% according to the volume ratio of (40-50):(50-60), the column temperature is 20-30 DEG C, the flow velocity is 0.5-1.50 mL/min, the sample size is 10-30 mu L, the drift tube temperature of an ELSD detector is 90-98 DEG C, the gas pressure is 2.00-3.00 bar, and a striker shows a closed state. The method provides new scientific basis for completing the quality standard of aconiti kusnezoffii radix and exploitation and utilization of aconiti kusnezoffii radix.
Description
Technical field
The present invention relates to a kind of method utilizing HPLC-ELSD method to measure the content of songorine in radix aconiti agrestis, belong to biomedicine field.
Background technology
Radix aconiti agrestis (Aconiti kusnezoffii Radix) is the dried root of the ranunculaceae plant north rhizome of Chinese monkshood (Aconitum kusnezoffii Reichb.), Medicinal is with a long history, is usually used in treatment rheumatism, paralysis, the cold symptom such as hernia, pain.
The primary bioactive components of radix aconiti agrestis is Diterpenoid Alkaloids, and they are effective component main in medicinal material, and be again the composition that toxicity is very strong, inappropriate medication easily causes intoxicating phenomenon simultaneously.Therefore, be ensure drug safety, be necessary to understand fully the kind of all alkaloids compositions in radix aconiti agrestis and strictly controlled the content of this type of alkaloid component in medicinal material by assay.Up to the present people establish content assaying method for the aconitine in radix aconiti agrestis, Hypaconitine and mesaconine.But also not about the research of aconitum soongoricum Stapf alkali content in radix aconiti agrestis.The invention provides a kind of method utilizing HPLC-ELSD method to measure the content of songorine in radix aconiti agrestis, Radix Aconiti Kusnezoffii quality standard can not only be improved, scientific basis can also be provided for the safe medication of radix aconiti agrestis and exploitation.
Summary of the invention
The technical problem to be solved in the present invention overcomes existing defect, provides a kind of method utilizing HPLC-ELSD method to measure the content of songorine in radix aconiti agrestis.
In order to solve the problems of the technologies described above, the invention provides following technical scheme:
The HPLC-ELSD content assaying method of songorine in radix aconiti agrestis, its chromatographic condition and testing conditions, the preparation of need testing solution and being prepared as follows of reference substance solution:
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: 0.5-1.50mLmin
-1
Sample size: 10-30 μ L
The drift tube temperature of ELSD detecting device: 90-98 DEG C
Gaseous tension: 2.00-3.00bar
Ram is closed condition;
The preparation of need testing solution
It is appropriate that precision takes radix aconiti agrestis powder, soaks, chloroform heating and refluxing extraction 1-3 time with ammoniacal liquor, add ammoniacal liquor solid-liquid ratio (g/ml) is 1:(0.5-2 at every turn), chloroform solid-liquid ratio (g/ml) is 1:(6-8), extraction time is 0.5-2.0h, filters, merging filtrate, decompression and solvent recovery, again be dissolved in volumetric flask with acetonitrile, constant volume, shakes up again, filter, get subsequent filtrate as need testing solution;
The preparation of reference substance solution
Accurate Weigh Compound songorine, is dissolved in volumetric flask with acetonitrile, constant volume, shakes up, obtain 1.0-2.0mgmL
-1reference substance solution, refrigerate for subsequent use.
Further, the HPLC-ELSD content assaying method of songorine in radix aconiti agrestis, 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: 1.0mLmin
-1
Sample size: 20 μ L
The drift tube temperature of ELSD detecting device: 96 DEG C
Gaseous tension: 2.50bar
Ram is closed condition.
Further, the HPLC-ELSD content assaying method of songorine in radix aconiti agrestis, the preparation of need testing solution:
Precision takes radix aconiti agrestis powder 3.0g, soaks with ammoniacal liquor, chloroform heating and refluxing extraction 2 times, add ammoniacal liquor 3.0mL, chloroform 20mL at every turn, extraction time is respectively 1.5h, 1.0h, filters, merging filtrate, decompression and solvent recovery, again be dissolved in 10mL volumetric flask with acetonitrile again, constant volume, shakes up, cross 0.22 μm of miillpore filter, get subsequent filtrate as need testing solution.
Further, the HPLC-ELSD content assaying method of songorine in radix aconiti agrestis, the preparation of reference substance solution:
Accurate Weigh Compound songorine 75mg, purity is 99.5%, and be dissolved in 50mL volumetric flask with acetonitrile, constant volume, shakes up, and obtains 1.5mgmL
-1reference substance solution, it is for subsequent use to be placed in 4 DEG C of refrigerations.
Reference substance songorine used by the present invention is self-control, and purity is 99.5%, and the preparation method of songorine is as follows:
(1) extract
Get dry radix aconiti agrestis, with chloroform-ammonia spirit (20:1) for mobile phase seepage pressure effects, extract is concentrated, be acidified to pH=1 ~ 2 with the HCl of 0.5%, chloroform extraction 4 times, water layer is merged, concentrate, alkalize to pH=11 ~ 12 with the NaOH of 0.5% again, chloroform extraction 4 times, gets chloroform layer and merges, concentrates, obtain crude extract.
(2) be separated
Crude extract is carried out silica gel column chromatography, be eluent gradient wash-out with eluant, eluent 1 ~ 5, TCL checks, merge component phase homogeneous turbulence part, recycling design, obtains three parts, by detect TCL wherein Part I contain songorine, Part II contains aconitum soongoricum Stapf amine, and Part III contains mesaconine.
(3) purifying
By step (2) gained three part difference purifying repeatedly, final purification obtains 8 compounds, is songorine, aconitum soongoricum Stapf amine, compound talastisamine, karacoline, 3-deoxyaconitine, aconitine, Hypaconitine, mesaconine respectively.The purification process of the employing in step (3) is silica gel column chromatography and/or recrystallization.
On the basis of above-mentioned research, the present invention establishes HPLC-ELSD (high performance liquid chromatography-evaporation Laser Light Scattering detector) content assaying method of songorine in radix aconiti agrestis, and the average content recording songorine in the Anguo product radix aconiti agrestis of Hebei is 0.9238mgg
-1, the average content that Jilin huge rock produces songorine in radix aconiti agrestis is 1.319mgg
-1, the average content that songorine in radix aconiti agrestis is produced in Dali is 1.768mgg
-1, the average content that Hui nationality produces songorine in radix aconiti agrestis is 1.635mgg
-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 1.0mgg
-1(may there is the unequal error component of sampling) outward, all the other are all at 1.0mgg
-1(0.1%), more than, the higher alkaloids composition of content (total amount that 2010 editions pharmacopeia record radix aconiti agrestis mesaconitine, Hypaconitine and mesaconine is 0.10% ~ 0.50%) is belonged to.Therefore, the assay index of songorine is included in the quality control that radix aconiti agrestis quality standard is conducive to Radix Aconiti Kusnezoffii.
Accompanying drawing explanation
Accompanying drawing is used to provide a further understanding of the present invention, and forms a part for instructions, together with embodiments of the present invention for explaining the present invention, is not construed as limiting the 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 ammoniacal liquor in embodiments of the invention mode Yu do not add ammoniacal liquor;
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.
Embodiment
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described, should be appreciated that preferred embodiment described herein is only for instruction and explanation of the present invention, is not intended to limit the present invention.
One, the extraction of radix aconiti agrestis chemical composition and separation
1, experimental raw and reagent material
Experimental raw
This experiment extraction and separation Radix Aconiti Kusnezoffii are purchased from Hebei Anguo.
Reagent material
It is pure that this experiment agents useful for same is analysis, comprises sherwood oil, acetone, diethylamine, ammoniacal liquor etc.Sherwood oil and diethylamine are purchased from Tianjin Tian Tai fine chemicals company limited; Acetone, ethyl acetate, chloroform and ammoniacal liquor are purchased from Beijing Chemical Plant.TLC silica gel60F254 and TLC silica gel60RP-18F254S is purchased from Merck, and column chromatography silica gel (200-300 order) is purchased from Haiyang Chemical Plant, Qingdao.
2, the Extraction and separation of chemical composition
(1) extract
Get dry radix aconiti agrestis 10kg, pulverize, with chloroform-ammonia spirit (20:1) for mobile phase seepage pressure effects, extract is concentrated, be acidified to pH=1 ~ 2 with the HCl of 0.5%, chloroform extraction 4 times, water layer is merged, concentrates, then alkalize to pH=11 ~ 12 with the NaOH of 0.5%, chloroform extraction 4 times, get chloroform layer to merge, concentrate, obtain crude extract 310g.
(2) separation and purifying
Crude extract (310g) is carried out silica gel column chromatography, be eluent gradient wash-out with eluant, eluent 1 ~ 5, thin-layer chromatography (TLC) detects, merge same composition, decompression and solvent recovery, obtains three parts, by detect TCL wherein Part I contain songorine, Part II contains aconitum soongoricum Stapf amine, and Part III contains mesaconine.
Part I (40g) is carried out silica gel column chromatography, is eluent gradient wash-out with eluant, eluent 7 ~ 10, and TLC detects, and merge same composition, decompression and solvent recovery, obtains A (10g), B (5g) two parts.Again part A being carried out silica gel column chromatography, is eluent gradient wash-out with 8,9,10, and TLC detects, and merges same composition, decompression and solvent recovery, separates 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, is eluent gradient wash-out with 8,9,10, and TLC detects, merge same composition, decompression and solvent recovery, the mixed solution crystallization of white precipitate sherwood oil 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, is eluent gradient wash-out with eluant, eluent 6 ~ 9, and TLC detects, and merge same composition, decompression and solvent recovery, obtains C (9.4g), D (3.0g) two parts.Again C part being carried out silica gel column chromatography, is eluent gradient wash-out with 6,8,9, and TLC detects, and merge 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, be eluent gradient wash-out with eluant, eluent 6,8,9, TLC detects, merge same composition, decompression and solvent recovery, the mixed solution crystallization of white precipitate sherwood oil and acetone, recrystallization, obtain colourless granular crystal and be compound cw-5 (200mg).D part is carried out silica gel column chromatography, be eluent gradient wash-out with eluant, eluent 6,8,9,10, TLC detects, merge same composition, decompression and solvent recovery, the mixed solution crystallization of white precipitate sherwood oil and acetone, recrystallization, obtain colourless lump shaped crystalline and be compound cw-2 (550mg).Part III (42g) is carried out silica gel column chromatography, is eluent gradient wash-out with eluant, eluent 6,8,9,11, and TLC detects, and merge same composition, decompression and solvent recovery, obtains E (17g) part.Again E part being carried out silica gel column chromatography, is eluent gradient wash-out with eluant, eluent 7 ~ 10, and TLC detects, and merge 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, be eluent gradient wash-out with eluant, eluent 7 ~ 10, TLC detects, merge same composition, decompression and solvent recovery, the mixed solution crystallization of white precipitate sherwood oil 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 absorption peak illustrates hydroxyl in this chemical constitution, 1700cm
-1the appearance of the strong absorption peak in place illustrates containing carbonyl in this structure, 1650cm
-1the appearance of place's absorption peak illustrates in this structure containing double bond.
ESI-MS:m/z358.3 [M+H]
+, point out the molecular weight of this compound to be 357.3.
13c-NMR (CDCl
3, 125MHz) spectrum provide 22 carbon signals altogether, in conjunction with DEPT spectrum known, compound cw-1 has 2 methyl signals (δ
c, ppm:26.09,13.62), 8 methylene signals (δ
c, ppm:111.45,57.42,50.95,38.16,37.34,32.19,31.66,23.21), 7 methine signals (δ
c, ppm:77.32,70.37,66.03,53.80,49.19,43.52,35.22), 5 quaternary carbon signal (δ
c, ppm:210.03,151.01,52.41,50.00,34.11), wherein δ
c210.03ppm place quaternary carbon signal is carbonyl carbon signals, δ
c151.01,111.45ppm place quaternary carbon, methylene signals be respectively end alkene two carbon signals.
1h-NMR (CDCl
3, 500MHz) and spectrum: δ
h(ppm) 0.771 (3H, s) be methyl proton signal, δ
h1.072 (3H, t, J=7.5Hz) are the proton signal of methyl on nitrogen ethyl, δ
h2.491 (2H, m) are the proton signal of methylene on nitrogen ethyl, δ
h3.849 (1H, s), 4.365 (1H, d, J=8.0Hz) are respectively the proton signal on two company's hydroxyl methine carbons, δ
h5.201,5.293 (each 1H, s) are two proton signals of end alkene.
According to more than
13c-NMR and
1h-NMR signal characteristic and the regularity of distribution, preliminary deduction compound cw-1 is C
20diterpene alkaloid compounds, through contrasting with document, deterministic compound cw-1 is that songorine (has another name called songorine, songorine, C
22h
31nO
3).This compound known obtains for being separated from radix aconiti agrestis 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 (sherwood oil-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 absorption peak illustrates hydroxyl in this chemical constitution, 1700cm
-1the appearance locating strong absorption peak illustrates containing carbonyl in this structure, 1650cm
-1the appearance locating strong absorption peak illustrates in this structure containing double bond.
ESI-MS:m/z356.2 [M+H]
+, point out the molecular weight of this compound to be 355.2.
13c-NMR (CDCl
3, 125MHz) spectrum provide 22 carbon signals altogether, in conjunction with DEPT spectrum known, compound cw-2 has 2 methyl signals (δ
c, ppm:18.89,14.25), 7 methylene signals (δ
c, ppm:111.94,48.32,37.39,31.21,29.71,24.27,23.99), 8 methine signals (δ
c, ppm:92.94,76.94,67.75,66.14,53.05,48.47,45.93,31.35), 5 quaternary carbon signal (δ
c, ppm:208.81,149.61,51.71,50.11,37.81).Wherein δ
c208.81ppm place quaternary carbon signal is carbonyl carbon signals, δ
c149.61,111.94ppm place quaternary carbon, methylene signals be respectively end alkene two carbon signals.
1h-NMR (CDCl
3, 500MHz) and spectrum: δ
h(ppm) 0.849 (3H, s) be methyl proton signal, δ
h1.036 (3H, t, J=6.0Hz) are the proton signal of methyl on nitrogen ethyl, δ
h2.671 (2H, m) are the proton signal of methylene on nitrogen ethyl, δ
h3.715 (1H, s), 3.992 (1H, d, J=4.0Hz) connect the proton signal on oxygen methine carbon, δ
h4.404 (1H, d, J=5.5Hz) connect the proton signal on hydroxyl methine carbon, δ
h5.207,5.311 (each 1H, s) are two proton signals of end alkene.
According to more than
13c-NMR and
1h-NMR signal characteristic and the regularity of distribution, preliminary deduction compound cw-2 is C
20diterpene alkaloid compounds, through contrasting with document, deterministic compound cw-2 is that Zhunger Basin aconine (has another name called dehydrogenation songorine, songoramine, C
22h
29nO
3).This compound known obtains for being separated from radix aconiti agrestis 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 (sherwood oil-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 absorption peak illustrates hydroxyl in this chemical constitution.
ESI-MS:m/z422.3 [M+H]
+, point out the molecular weight of this compound to be 421.3.
13c-NMR (CDCl
3, 125MHz) spectrum provide 24 carbon signals altogether, in conjunction with DEPT spectrum known, compound cw-3 has 4 methyl signals (δ
c, ppm:59.49,56.49,56.32,13.71), 8 methylene signals (δ
c, ppm:79.49,53.17,49.52,38.35,32.79,27.69,25.84,24.75), 9 methine signals (δ
c, ppm:86.37,82.25,75.60,62.96,46.96,46.06,45.90,45.80,37.56), 3 quaternary carbon signal (δ
c, ppm:72.81,48.72,38.67).Wherein δ
c59.49,56.49,56.32ppm place methyl signals is three methoxyl carbon signals.
1h-NMR (CDCl
3, 500MHz) and spectrum: δ
h(ppm) 1.057 (3H, t, J=7.0Hz) are the proton signal of methyl on nitrogen ethyl, δ
h2.529 (2H, m) are the proton signal of methylene on nitrogen ethyl, δ
h3.267,3.299,3.346 (each 3H, s) are the proton signal of three methoxyls.
According to more than
13c-NMR and
1h-NMR signal characteristic and the regularity of distribution, preliminary deduction compound cw-3 is C
19diterpene alkaloid compounds, through contrasting with document, deterministic compound cw-3 is that talastisamine (has another name called tower and draws ground Sa Min, talatizamine, C
24h
39nO
5).Its chemical structural formula is as follows:
The Structural Identification of compound cw-4
Compound cw-4 is colourless granular crystal (sherwood oil-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]
+, point out the molecular weight of this compound to be 377.2.
13c-NMR (CDCl
3, 125MHz) spectrum provide 22 carbon signals altogether, in conjunction with DEPT spectrum known, compound cw-4 has 3 methyl signals (δ
c, ppm:56.33,27.57,13.05), 7 methylene signals (δ
c, ppm:60.24,48.41,42.26,31.31,29.68,28.46,25.14), 9 methine signals (δ
c, ppm:81.98,75.84,72.50,63.38,46.71,46.59,45.09,44.05,39.95), 3 quaternary carbon signal (δ
c, ppm:74.23,48.82,32.92).Wherein δ
c56.33ppm place methyl signals is a methoxyl carbon signal.
1h-NMR (CDCl
3, 500MHz) and spectrum: δ
h(ppm) 0.882 (3H, s) be methyl proton signal, δ
h1.118 (3H, t, J=7.5Hz) are the proton signal of methyl on nitrogen ethyl, δ
h2.531 (2H, m) are the proton signal of methylene on nitrogen ethyl, δ
hthe proton signal that (3.346 3H, s) is methoxyl.
According to more than
13c-NMR and
1h-NMR signal characteristic and the regularity of distribution, preliminary deduction compound cw-4 is C
19diterpene alkaloid compounds, warp and document] contrast, deterministic compound cw-4 is karacoline (karakoline, C
22h
35nO
4).This compound known obtains for being separated from radix aconiti agrestis 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 (sherwood oil-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 absorption peak illustrates hydroxyl in this chemical constitution, 1600 ~ 1450cm
-1there is aromatic ring frame vibration performance peak, 1700cm
-1the appearance locating strong absorption peak illustrates in this structure containing carbonyl.
ESI-MS:m/z630.4 [M+H]
+, point out the molecular weight of this compound to be 629.4.
13c-NMR (CDCl
3, 125MHz) spectrum provide 32 carbon signals altogether, in conjunction with DEPT spectrum known, compound cw-5 has 6 methyl signals (δ
c, ppm:61.02,59.07,58.00,56.27,21.45,13.47), 6 methylene signals (δ
c, ppm:80.31,53.17,49.06,36.67,35.27,26.39), 13 methine signals (δ
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 (δ
c, ppm:172.40,166.15,129.90,92.13,74.14,49.95,39.08).Wherein δ
c61.02,59.07,58.00,56.27ppm place methyl signals is four methoxyl carbon signals, δ
c172.40,166.15ppm place quaternary carbon signal is two carbonyl carbon signals, δ
c129.90 (quaternary carbons), 133.22 (methines), 129.63 (methines), 128.63 (methine) ppm place signal are the carbon signal on monosubstituted phenyl ring.
1h-NMR (CDCl
3, 500MHz) and spectrum: δ
h(ppm) 1.077 (3H, t, J=7.5Hz) are the proton signal of methyl on nitrogen ethyl, δ
h1.375 (3H, s) is the proton signal of methyl on acetyl group, δ
h3.158,3.266,3.283,3.738 (each 3H, s) are the proton signal of four methoxyls, δ
h7.438 ~ 8.042 (5H) three groups signal is the proton signal on phenyl ring.
According to more than
13c-NMR and
1h-NMR signal characteristic and the regularity of distribution, preliminary deduction compound cw-5 is C
19aconitine type compound, through contrasting with document, deterministic compound cw-5 is 3-deoxyaconitine (3-deoxyaconitine, C
34h
47nO
10).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 contrast with aconitine standard items, developping agent is sherwood oil-acetone-diethylamine (silica gel), normal hexane-acetone-diethylamine (silica gel), acetone-water-diethylamine (ODS), developer is bismuth potassium iodide test solution and sulfuric acid-ethanol test solution, both spot colors and Rf value all consistent, and mixed melting point does not decline, therefore can deterministic compound cw-6 be aconitine (aconitine, C
34h
47nO
11).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 contrast with Hypaconitine standard items, developping agent is sherwood oil-acetone-diethylamine (silica gel), normal hexane-acetone-diethylamine (silica gel), acetone-water-diethylamine (ODS), developer is bismuth potassium iodide test solution and sulfuric acid-ethanol test solution, both spot colors and Rf value all consistent, and mixed melting point does not decline, therefore can deterministic compound cw-7 be Hypaconitine (hypaconitine, C
33h
45nO
10).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 contrast with mesaconine standard items, developping agent is sherwood oil-acetone-diethylamine (silica gel), normal hexane-acetone-diethylamine (silica gel), acetone-water-diethylamine (ODS), developer is bismuth potassium iodide test solution and sulfuric acid-ethanol test solution, both spot colors and Rf value all consistent, and mixed melting point does not decline, therefore can deterministic compound cw-8 be mesaconine (mesaconitine, C
33h
45nO
11).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 agrestis
1, the selection of mobile phase
First, this experiment adopts DAD detecting device, 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 add the triethylamine of 0.1% in aqueous phase after, peak shape has had obvious improvement, therefore selects methyl alcohol-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 PH
18chromatographic column.Then, the separating effect that it is mobile phase that this experiment has been investigated with methyl alcohol-0.1% triethylamine solution, finds that this mobile phase condition cannot make target compound cw-1 reach with other compositions in medicinal material and effectively be separated.The separating effect that it is mobile phase that this experiment has been investigated again with acetonitrile-0.1% triethylamine solution, although find that cw-1 effectively can be separated with other compositions, baseline fluctuation is comparatively large, causes detectability cannot reach mensuration requirement.And using Alltech2000ELSD detecting device to compare rear discovery, baseline, peak shape are obviously better than DAD detecting device, and detectability, precision all can reach mensuration requirement.Therefore for making all the components in target compound and medicinal material reach baseline separation, and obtain good peak shape, this tests final choice for use ELSD detecting device, 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 the parameter of ELSD is as follows through repetition test:
Drift tube temperature is 96 DEG C, and gas (air) pressure is 2.50bar, and ram is closed condition.The chromatogram of reference substance and medicinal material 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 being investigated, thus determining optimum extraction conditions.
3.1, the selection of Extraction solvent
Precision takes 3.0g radix aconiti agrestis powder, totally 5 parts, all soaks with 3.0mL ammoniacal liquor, then adds cyclohexane, sherwood oil, chloroform, ethyl acetate, acetone 20mL respectively, add hot reflux 1.5h, filter, filtrate decompression evaporate to dryness, again be dissolved in 10mL volumetric flask with acetonitrile again, constant volume, shakes up.Cross 0.22 μm of miillpore filter, get filtrate 20 μ 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, and illustrate that extraction effect is best, acetone takes second place, and both are all higher than cyclohexane, sherwood oil and ethyl acetate, therefore selects chloroform as Extraction solvent.
3.2, add ammoniacal liquor and do not add comparing of ammoniacal liquor
Precision takes 3.0g radix aconiti agrestis powder, totally 2 parts, and portion soaks with 3.0mL ammoniacal liquor, then adds chloroform 20mL, and another part directly adds chloroform 20mL, adds hot reflux 1.5h, and filter, filtrate decompression evaporate to dryness, be more again dissolved in 10mL volumetric flask with acetonitrile, constant volume, shakes up.Cross 0.22 μm of miillpore filter, get filtrate 20 μ L and inject HPLC, measure the peak area of cw-1.Result (Fig. 4) shows, with ammoniacal liquor soak extract in the peak area of compound cw-1 apparently higher than the extract not adding ammoniacal liquor, illustrate that the extraction effect adding ammoniacal liquor is good, therefore select ammonification water-soaked.
3.3, the selection of extracting method
Precision takes 3.0g radix aconiti agrestis powder, totally 3 parts, all soaks with 3.0mL ammoniacal liquor, and adds chloroform 20mL, adopt respectively and add hot reflux 1.5h, ultrasonic 1.5h, cold soaking 24h tri-kinds of methods extractions, filter, filtrate decompression evaporate to dryness, again be dissolved in 10mL volumetric flask with acetonitrile again, constant volume, shakes up.Cross 0.22 μm of miillpore filter, get filtrate 20 μ L and inject HPLC, measure the peak area of cw-1.Result (Fig. 5) shows, the peak area adding compound cw-1 in the extract of hot reflux is the highest, illustrates that extraction effect is best, and therefore selective extraction method is for adding hot reflux.
3.4, the selection of extraction time
Precision takes 1 part, 3.0g radix aconiti agrestis powder, soak with ammoniacal liquor, chloroform heating and refluxing extraction 3 times, add ammoniacal liquor 3.0mL, chloroform 20mL, the time is respectively 1.5h, 1.0h, 1.0h at every turn, after each extraction, filter, filtrate decompression evaporate to dryness, be more again dissolved in 10mL volumetric flask with acetonitrile, constant volume, shakes up.Cross 0.22 μm of miillpore filter, get filtrate 20 μ L and inject HPLC, measure the peak area of cw-1.Result (Fig. 6) shows, the extraction ratio adding hot reflux for the 1st time is the highest, the existence of compound cw-1 do not detected in No. the 3rd extract, and illustrate and extracted completely by cw-1 for twice, therefore selective extraction number of times is 2 times.
3.5, the selection of Extraction solvent consumption
Precision takes 3.0g radix aconiti agrestis powder, totally 3 parts, all soaks with 3.0mL ammoniacal liquor, then add respectively chloroform 15,20,25,30mL, add hot reflux 1.5h, filter, filtrate decompression evaporate to dryness, be more again dissolved in 10mL volumetric flask with acetonitrile, constant volume, shakes up.Cross 0.22 μm of miillpore filter, get filtrate 20 μ L and inject HPLC, measure the peak area of cw-1.Result (Fig. 7) shows, in the extract of 20mL and 25,30mL chloroform, cw-1 peak area is maximum, but three's difference is less, and therefore selective extraction solvent load is 20mL.
Embodiment 1
The HPLC-ELSD content assaying method of songorine in radix aconiti agrestis, concrete steps are as follows:
1, experimental apparatus, chromatographic condition and testing conditions, material and reference substance
Chromatograph: Agilent1200HPLC
Detecting device: Alltech2000ELSD
Chromatographic column: Agilent Extend-C
18(4.6 × 250mm, 5 μm)
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: 1.0mLmin
-1
Sample size: 20 μ L
The drift tube temperature of ELSD detecting device: 96 DEG C
Gaseous tension: 2.50bar
Ram is closed condition.
Medicinal material: content analysis medicinal material used 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 takes radix aconiti agrestis powder 3.0g, soaks with ammoniacal liquor, chloroform heating and refluxing extraction 2 times, adds ammoniacal liquor 3.0mL, chloroform 20mL at every turn, extraction time is respectively 1.5h, 1.0h, filters, merging filtrate, evaporated under reduced pressure, again be dissolved in 10mL volumetric flask with acetonitrile again, constant volume, shakes up.Cross 0.22 μm of miillpore filter, get subsequent filtrate as need testing solution.
3, the preparation of reference substance solution
Precision takes songorine (purity 99.5%) 75mg, and be dissolved in 50mL volumetric flask with acetonitrile, constant volume, shakes up, and obtains 1.5mgmL
-1reference substance solution, be placed in 4 DEG C of refrigerator and cooled and hide for subsequent use.
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 (1.5mgmL
-1) in 10mL volumetric flask, by dilution in acetonitrile to groove, shake up, obtain concentration be 0.075,0.15,0.30,0.60,0.90,1.20,1.50mgmL
-1reference substance solution.Cross 0.22 μm of miillpore filter, get filtrate 20 μ L and inject HPLC, replicate determination 3 times, calculate the mean value of peak area.With the common logarithm of reference substance sample introduction quality (m) for horizontal ordinate, average peak area (S
all) common logarithm be ordinate, investigate lgS
allwith the linear relationship of lgm.Experimental data is in table one, and typical curve as shown in Figure 8.From result, sample size is lgS within the scope of 1.5 μ g ~ 30 μ g
allhave good linear relationship with lgm, linear equation is: lgS
all=1.6311lgm+0.1061, R
2=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 (1.5mgmL
-1) in 10mL volumetric flask, by dilution in acetonitrile to groove, shake up, obtaining concentration is 0.3mgmL
-1reference substance solution.With 0.22 μm of filtering with microporous membrane, get filtrate 20 μ L and inject HPLC, continuous sample introduction 6 times in a day, measure peak area, calculate RSD value.Experimental data is in table two.Result shows, the RSD value of compound cw-1 peak area is 0.26%, is less than 2%, and visible withinday precision is good.
In table two day and day to day precision experimental result
4.2.2, day to day precision
Accurately pipette 2.0mL reference substance solution (1.5mgmL
-1) in 10mL volumetric flask, by dilution in acetonitrile to groove, shake up, obtaining concentration is 0.3mgmL
-1reference substance solution.With 0.22 μm of filtering with microporous membrane, get filtrate 20 μ 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.Result shows, the RSD value of songorine peak area is 1.54%, is less than 2%, and visible day to day precision is good.
4.3, repeated experiment
According to " method of 2 prepares need testing solution 6 parts, gets 20 μ L and injects HPLC, every part of sample introduction 2 times, measure 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 1.563mgg
-1, RSD value is 1.41%, is less than 2%, and visible repeatability is good.
The repeated experimental result of table three
(note: songorine reference substance solution concentration is 0.45mgmL
-1, average peak area is 4185237.)
4.4, stability experiment
Prepare need testing solution 1 part according to the method for " 2 ", get 20 μ 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.Result shows, the RSD value of songorine peak area is 1.68%, is less than 2%, and visible need testing solution is good at 10h internal stability.
Table four stability experiment result
4.5, average recovery experiment
Precision takes 3.0g radix aconiti agrestis powder, and (aconitum soongoricum Stapf alkali content is known, is 1.563mgg
-1), totally 6 parts, the 1st, 2 part adds reference substance solution (1.5mgmL
-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 0.8 times, 1.0 times, 1.2 times of aconitum soongoricum Stapf alkali content in sample respectively, make mark-on need testing solution according to the method for " 2 ".Get 20 μ 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 obtain average recovery and the RSD value of cw-1 by formula below.Experimental data is in table five.Result shows, the mean sample recovery rate of compound songorine is 96.86%, RSD value is 1.67%, and the average recovery of visible the method is good.
Table five average recovery experimental result
(note: songorine reference substance solution concentration is 0.90mgmL
-1, average peak area is 12285773.)
5, assay
5.1, the preparation of reference substance solution
Accurately pipette 3.0mL reference substance solution (1.5mgmL
-1) in 10mL volumetric flask, by dilution in acetonitrile to groove, shake up, obtaining concentration is 0.45mgmL
-1reference substance solution.With 0.22 μm of filtering with microporous membrane, filtrate is as the External standards solutions of assay.
5.2, the preparation of need testing solution
Get the Radix Aconiti Kusnezoffii purchased from Hebei Anguo, Jilin huge rock, 3 batches of Dali and 2 batches of 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 ", get 20 μ L reference substance solution and inject HPLC, replicate determination 3 times, calculate mean value and the lgS of cw-1 (songorine) peak area
all.Get 20 μ L need testing solutions and inject HPLC, every part of replicate determination 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.Result shows, 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 0.45mgmL
-1, average peak area is 4069804.)
This chapter invents and measures the content of songorine in radix aconiti agrestis by 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 1.0mgg
-1outward, all the other are all at 1.0mgg
-1(0.1%), more than, the higher alkaloids composition of content (total amount that 2010 editions pharmacopeia record radix aconiti agrestis mesaconitine, Hypaconitine and mesaconine is 0.10% ~ 0.50%) is belonged to.Therefore, the assay index of songorine is included in the quality control that radix aconiti agrestis quality standard is conducive to Radix Aconiti Kusnezoffii.
Last it is noted that the foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, although with reference to previous embodiment to invention has been detailed description, for a person skilled in the art, it still can be modified to the technical scheme described in foregoing embodiments, or carries out equivalent replacement to wherein portion of techniques feature.Within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (4)
1. the HPLC-ELSD content assaying method of songorine in radix aconiti agrestis, is characterized in that,
Chromatographic condition and testing conditions:
Mobile phase: the volume ratio of acetonitrile-0.1% triethylamine aqueous solution is (40-50): (50-60)
Column temperature: 20-30 DEG C
Flow velocity: 0.5-1.50mLmin
-1
Sample size: 10-30 μ L
The drift tube temperature of ELSD detecting device: 90-98 DEG C
Gaseous tension: 2.00-3.00bar
Ram is closed condition;
The preparation of need testing solution:
It is appropriate that precision takes radix aconiti agrestis powder, soaks, chloroform heating and refluxing extraction 1-3 time with ammoniacal liquor, add ammoniacal liquor solid-liquid ratio (g/ml) is 1:(0.5-2 at every turn), chloroform solid-liquid ratio (g/ml) is 1:(6-8), extraction time is 0.5-2.0h, filters, merging filtrate, decompression and solvent recovery, again be dissolved in volumetric flask with acetonitrile, constant volume, shake up, filter, get subsequent filtrate as need testing solution;
The preparation of reference substance solution:
Precision takes songorine, is dissolved in volumetric flask, constant volume, shakes up, obtain 1.0-2.0mgmL with acetonitrile
-1reference substance solution.
2. the HPLC-ELSD content assaying method of songorine in radix aconiti agrestis as claimed in claim 1, is characterized 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: 1.0mLmin
-1
Sample size: 20 μ L
The drift tube temperature of ELSD detecting device: 96 DEG C
Gaseous tension: 2.50bar
Ram is closed condition.
3. the HPLC-ELSD content assaying method of songorine in radix aconiti agrestis as claimed in claim 1 or 2, is characterized in that, the preparation of need testing solution:
Precision takes radix aconiti agrestis powder 3.0g, soaks with ammoniacal liquor, chloroform heating and refluxing extraction 2 times, add ammoniacal liquor 3.0mL, chloroform 20mL at every turn, extraction time is respectively 1.5h, 1.0h, filters, merging filtrate, decompression and solvent recovery, again be dissolved in 10mL volumetric flask with acetonitrile, constant volume, shakes up, cross 0.22 μm of miillpore filter, get subsequent filtrate as need testing solution.
4. the HPLC-ELSD content assaying method of songorine in radix aconiti agrestis as claimed in claim 1 or 2, is characterized in that, the preparation of reference substance solution:
Accurate Weigh Compound songorine 75mg, purity is 99.5%, and be dissolved in 50mL volumetric flask with acetonitrile, constant volume, shakes up, and obtains 1.5mgmL
-1reference substance solution, it is for subsequent use to be placed in 4 DEG C of refrigerations.
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| CN108375642A (en) * | 2018-01-25 | 2018-08-07 | 中国农业科学院蜜蜂研究所 | Biomarker of the diester-type aconitine as toxic honey and its application in differentiating toxic honey |
| CN108548885A (en) * | 2018-06-21 | 2018-09-18 | 江苏康缘药业股份有限公司 | The method that two-dimensional liquid chromatography detects compound Nanxing pain paste |
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| 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 |
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| 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 |
| CN108548885A (en) * | 2018-06-21 | 2018-09-18 | 江苏康缘药业股份有限公司 | The method that two-dimensional liquid chromatography detects compound Nanxing pain paste |
| 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 |
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