CN102735789A

CN102735789A - Method for detecting quality of alkaloids in Tibetan medicine herba aconiti tangutici and preparations thereof

Info

Publication number: CN102735789A
Application number: CN2012102417462A
Authority: CN
Inventors: 邵成雷; 王海苹; 李丽; 任松鹏; 单玉刚; 刘玉芹; 郑亭亭
Original assignee: Shandong Arura Pharmaceutical Research & Development Co Ltd
Current assignee: Jinhe Tibetan Medicine (Shandong) Health Industry Co.,Ltd.
Priority date: 2012-07-12
Filing date: 2012-07-12
Publication date: 2012-10-17
Anticipated expiration: 2032-07-12
Also published as: CN102735789B

Abstract

The invention discloses a method for detecting quality of alkaloids in a Tibetan medicine herba aconiti tangutici and preparations thereof. The method comprises the steps of identification, check and content determination, wherein thin layer chromatography is adopted for identification and is characterized by taking a herba aconiti tangutici control medicine as the control and normal hexane-ethyl acetate-methanol-ammonia water as a developer; high performance liquid chromatography is adopted for check and is characterized by taking a mixed solution of an aconitine control substance, a hypaconitine control substance and a new aconitine control substance as the control, acetonitrile-tetrahydrofuran as a mobile phase A and an ammonium acetate water solution with glacial acetic acid as a mobile phase B to carry out gradient elution; high performance liquid chromatography is adopted for hordenine content determination and is characterized by taking a hordenine control substance as the control and an acetonitrile-potassium dihydrogen phosphate water solution as a mobile phase; and back titration is adopted for total alkaloid content determination. The method is stable and reliable, has strong specificity, can ensure the safety, effectiveness and quality controllability of the alkaloids in the Tibetan medicine herba aconiti tangutici and the preparations thereof, and has higher scientificity and application values.

Description

Alkaloidal quality determining method in Tibetan medicine Tangut aconite and the preparation thereof

Technical field

The present invention relates to a kind of quality determining method, be specifically related to alkaloidal quality determining method in a kind of Tibetan medicine Tangut aconite and the preparation thereof.This method comprises in the Tangut aconite in alkaloid and the preparation eight flavor Herba Swertiae bimaculatae balls thereof in the golden simply ball of alkaloid, ten that alkaloid, 13 flavor list loud, high-pitched sounds are loose, alkaloidal quality determining method in the peaceful particle of peace youngster.

Background technology

Tangut aconite (Herba Aconiti Tangutici) is the dry herb of ranunculaceae plant aconitum tanguticumStapf (Tangut aconite) Aconitum tanguticum (Maxim.) Stapf and ship helmet rhizome of Chinese monkshood Aconitum naviculare (Bruhl.) Stapf.Connect root excavate florescence at the beginning of autumn late summer, removes impurity, dries in the shade.These article are cool in nature, bitter, have clearing heat and detoxicating, promoting granulation and wound healing, the effect of eliminating dampness.Function cures mainly clearing heat and detoxicating, is mainly used in fever, food poisoning, the snakes and scorpions that liver heat, gallbladder-heat, lung heat, hepatitis, pneumonia, gastroenteritis, influenza, infectious disease causes and bites and grasserie.

The Aconitum medicinal plant is used as medicine with piece root, root mostly, have more dispel rheumatism, the effect of antalgic, wherein principal ingredient is a diester-type alkaloids, accounts for 7% ~ 10% of gross weight, has significant physiologically active, generally need concoct to reduce the property of medicine.Tangut aconite is with all herbal medicine, the high 10～30cm of draft, piece root 2; Little shape, its main pharmacodynamics composition alkaloid is mainly the lactone type diterpene alkaloid, contains a spot of diester-type alkaloids composition; Need not concoct and to be used as medicine, but overdose might bring healthy hidden danger.Tangut aconite records in " Tibetan medicine ministerial standard "; Tibetan medicine name list loud, high-pitched sound; Being medicinal herbs most in use in the Tibetan medicine prescribed preparation, is the medicinal material representative with Tibetan medicine characteristic simultaneously, and ministerial standard is only used its quality of medicinal material of microscopical identification control; Be difficult to guarantee that medicinal material is safe in utilization, effective, be necessary that therefore the quality determining method of setting up a kind of science estimates the quality of Tangut aconite and preparation thereof.

Summary of the invention

The objective of the invention is provides alkaloidal quality determining method in a kind of Tibetan medicine Tangut aconite and the preparation thereof for overcoming the deficiency of above-mentioned prior art.

For realizing above-mentioned purpose, the present invention adopts following technical proposals:

A kind of quality determining method of Tibetan medicine Tangut aconite; It comprises discriminating, inspection and assay project, and said discriminating is to be contrast, to be that developping agent launches on silica gel g thin-layer plate, is the thin-layered chromatography of developer with bismuth potassium iodide solution with volume parts than the normal hexane-ethyl acetate-methyl alcohol-ammoniacal liquor that is 5:4:1.5:0.2 with the Tangut aconite control medicinal material; Said inspection is the inspection to contained diester-type alkaloids in the medicinal material; It is that the mixed in equal amounts solution with aconitine reference substance, Hypaconitine reference substance, mesaconine reference substance is contrast; Is mobile phase A with volume parts than the acetonitrile-tetrahydrofuran that is 25:15, and adding the 0.5ml glacial acetic acid with every 1000ml0.1mol/l ammonium acetate aqueous solution is the high performance liquid chromatography that Mobile phase B is carried out gradient elution; Said assay comprises to be measured the assay of contained hordenine in the medicinal material and total alkaloid contents; Said hordenine assay is to be contrast with the hordenine reference substance, with volume parts than being acetonitrile-0.025mol/l potassium dihydrogen phosphate aqueous solution of 10:90 and using triethylamine to regulate the high performance liquid chromatography of PH to 7.2 as moving phase; Said Determination of Total Alkaloid is to adopt the mixed solution extraction of volume parts than 100:1 chloroform-ammoniacal liquor, merges washing lotion and filtrating, low-temperature reduced-pressure (below 50 ℃) evaporate to dryness; Add activated carbon decolorizing; Jolting, decolouring adds the sulfuric acid vs; Using the NaOH vs, is the back titration method of indicator solution with the methyl red.

The process of gradient elution is following in the said inspection:

Said discriminating is specially:

Get medicinal powder to be detected (crossing sieve No. three) 1g, add ammoniacal liquor 5ml, the 50ml that adds methylene chloride, sonicated (power 250W, frequency 40kHz) 30 minutes filters, the filtrating evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution.Get Tangut aconite control medicinal material 1g, shine medicinal material solution in pairs with legal system.Test according to thin-layered chromatography (an appendix VI of Pharmacopoeia of the People's Republic of China version in 2010 B); Drawing each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, is developping agent with volume parts than normal hexane-ethyl acetate-methyl alcohol-ammoniacal liquor of 5:4:1.5:0.2; Launch; Take out, dry, spray is with rare bismuth potassium iodide solution.In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show identical orange red spot.

Said inspection is specially:

Get medicinal powder to be detected (crossing sieve No. three) 2g, the accurate title, decide, and puts in the conical flask of tool plug, and the accurate mixed solution 50ml that adds volume parts than 1:100 hydrochloric acid-methyl alcohol claims to decide weight; Sonicated 40 minutes (temperature is no more than 25 ℃) is put coldly, claims to decide weight, supplies the weight that subtracts mistake with volume parts than the mixed solution of 1:100 hydrochloric acid-methyl alcohol, shakes up; Filter, precision is measured subsequent filtrate 25ml, and to doing, the residue precision adds 0.05mol/l sulfuric acid solution 25ml at decompression and solvent recovery below 40 ℃, and jolting makes dissolving; Centrifugal (rotating speed is 8000 rev/mins) 20 minutes, precision is measured supernatant 20ml, puts in the separating funnel, with washed with dichloromethane 4 times (20ml, 20ml, 15ml, 15ml); Discard dichloromethane solution, treat upper strata clarification after, transfer PH to 8-9 with ammonia solution, again with ether jolting extraction 5 times; Each 20ml merges ether solution, volatilizes, and residue is used the 0.05mol/l sulfuric acid dissolution; Be transferred in the 5ml measuring bottle, shake up, filter, get subsequent filtrate as need testing solution.It is an amount of to get aconitine reference substance, Hypaconitine reference substance, mesaconine reference substance, accurately claims surely, adds 0.05mol/l sulfuric acid and processes the mixed solution that every 1ml contains aconitine, Hypaconitine, each 50 μ g of mesaconine, compares article solution.Test according to high performance liquid chromatography (an appendix VI of Pharmacopoeia of the People's Republic of China version in 2010 D).With the octadecylsilane chemically bonded silica is filling agent; Is mobile phase A with volume parts than acetonitrile-tetrahydrofuran of 25:15, is Mobile phase B with 0.1mol/l ammonium acetate aqueous solution (every 1000ml adds glacial acetic acid 0.5ml), and the regulation in the according to the form below is carried out gradient elution, and the detection wavelength is 235nm.Number of theoretical plate calculates by the mesaconine peak should be not less than 2000.Accurate respectively reference substance solution and each 20 μ l of need testing solution of drawing inject liquid chromatograph, measure, and promptly get.

These article are pressed dry product and are calculated, and contain dibasic acid esters property alkaloid with aconitine (C ₃₄H ₄₇NO ₁₁), Hypaconitine (C ₃₃H ₄₅NO ₁₀) and mesaconine (C ₃₃H ₄₅NO ₁₁) the total amount meter, must not cross 0.1mg/g.

Said hordenine assay is specially:

Measure according to high performance liquid chromatography (" an appendix VI of Chinese pharmacopoeia version in 2010 D);

Chromatographic condition and system suitability test are filling agent with octyl silane group silica gel; Is moving phase with volume parts than acetonitrile-0.025mol/l potassium dihydrogen phosphate aqueous solution of 10:90 (triethylamine is regulated PH to 7.2); Detect wavelength 225nm; Number of theoretical plate calculates by the hordenine peak should be not less than 2000;

It is an amount of that the preparation precision of reference substance solution takes by weighing the hordenine reference substance, adds methyl alcohol and process the solution that contains hordenine 20 μ g among every 1ml, promptly gets;

Medicinal powder to be detected (crossing sieve No. three) 1g is got in the preparation of need testing solution, and accurate the title decides, and puts in the conical flask, adds the mixed solution 50ml of volume parts than 100:1 chloroform-ammoniacal liquor, claims to decide weight; Ultrasonic 30 minutes, placement was spent the night, and claimed to decide weight, supplied the weight that subtracts mistake with volume parts than the mixed solution of 100:1 chloroform-ammoniacal liquor, filtered; Get subsequent filtrate 25ml, decompression low temperature (below 50 ℃) evaporate to dryness, the accurate 0.05mol/l sulfuric acid solution 25ml that adds of residue makes dissolving, centrifugal (rotating speed is 8000 rev/mins) 20 minutes; Get supernatant, put in the separating funnel, transfer PH to 8-9, extract 5 times with the ethyl acetate jolting again, each 20ml with ammonia solution; Combining extraction liquid volatilizes, and residue is used dissolve with methanol, is transferred in the 5ml measuring bottle; Shake up, filter, get subsequent filtrate, promptly get.

Determination method is drawn reference substance solution and each 10 μ l of need testing solution respectively, injects liquid chromatograph, measures, and promptly gets.

These article are pressed dry product and are calculated, and contain hordenine (C ₁₀H ₁₅NO) must not be less than 0.2mg/g.

Said Determination of Total Alkaloid is specially:

Get medicinal powder to be detected (crossing sieve No. three) 5g, the accurate title, decide, and puts in the conical flask, adds the mixed solution 50ml of volume parts than 100:1 chloroform-ammoniacal liquor; Claim to decide weight, ultrasonic 30min, placement is spent the night; Filter, residue adds the mixed solution 50ml of volume parts than 100:1 chloroform-ammoniacal liquor, jolting 1 hour; Filter, residue use volume parts is washed 3 times than the mixed solution of 100:1 chloroform-ammoniacal liquor, each 15m; Merge washing lotion and filtrating, low-temperature reduced-pressure (below 50 ℃) evaporate to dryness, residue add ethanol 10ml makes dissolving; Add activated charcoal 1g; Jolting makes decolouring, and precision is measured subsequent filtrate 5ml again; Accurate sulfuric acid vs (0.01mol/l) 15ml, the water 15ml and 3 of methyl red indicator solutions of adding uses NaOH vs (0.02mol/l) titration to yellow.Every 1ml sulfuric acid vs (0.01mol/l) is equivalent to the hordenine (C of 1.65mg ₁₀H ₁₅NO).

These article are pressed dry product and are calculated, and contain alkaloid with hordenine (C ₁₀H ₁₅NO) meter must not be less than 3.6mg/g.

The present invention also provides the alkaloidal quality determining method of Tangut aconite in a kind of pharmaceutical preparation, adopts said method that Tangut aconite alkaloid in the preparation is carried out quality testing.

The alkaloidal quality determining method of Tangut aconite is specially in the pharmaceutical preparation:

Thin-layer chromatography is differentiated:

Get preparation powder to be detected (crossing sieve No. three) 5-8g, add ammoniacal liquor 5ml, the 50ml that adds methylene chloride, sonicated (power 250W, frequency 40kHz) 30 minutes filters, the filtrating evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution.Get Tangut aconite control medicinal material 1g, shine medicinal material solution in pairs with legal system.Test according to thin-layered chromatography (an appendix VI of Pharmacopoeia of the People's Republic of China version in 2010 B); Drawing each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, is developping agent with volume parts than normal hexane-ethyl acetate-methyl alcohol-ammoniacal liquor of 5:4:1.5:0.2; Launch; Take out, dry, spray is with rare bismuth potassium iodide solution.In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show identical orange red spot.

The diester-type alkaloids inspection:

Get preparation powder to be detected (crossing sieve No. three) 8-15g, the accurate title, decide, and puts in the conical flask of tool plug, and the accurate mixed solution 50ml that adds volume parts than 1:100 hydrochloric acid-methyl alcohol claims to decide weight; Sonicated 40 minutes (temperature is no more than 25 ℃) is put coldly, claims to decide weight, supplies the weight that subtracts mistake with volume parts than the mixed solution of hydrochloric acid-methyl alcohol of 1:100, shakes up; Filter, precision is measured subsequent filtrate 25ml, and to doing, the residue precision adds 0.05mol/l sulfuric acid solution 25ml at decompression and solvent recovery below 40 ℃, and jolting makes dissolving; Centrifugal (rotating speed is 8000 rev/mins) 20 minutes, precision is measured supernatant 20ml, puts in the separating funnel, with washed with dichloromethane 4 times (20ml, 20ml, 15ml, 15ml); Discard dichloromethane solution, treat upper strata clarification after, transfer PH to 8-9 with ammonia solution, again with ether jolting extraction 5 times; Each 20ml merges ether solution, volatilizes, and residue is used the 0.05mol/l sulfuric acid dissolution; Be transferred in the 5ml measuring bottle, shake up, filter, get subsequent filtrate as need testing solution.It is an amount of to get aconitine reference substance, Hypaconitine reference substance, mesaconine reference substance, accurately claims surely, adds 0.05mol/l sulfuric acid and processes the mixed solution that every 1ml contains aconitine, Hypaconitine, each 50 μ g of mesaconine, compares article solution.Test according to high performance liquid chromatography (an appendix VI of Pharmacopoeia of the People's Republic of China version in 2010 D).With the octadecylsilane chemically bonded silica is filling agent; Is mobile phase A with volume parts than acetonitrile-tetrahydrofuran of 25:15, is Mobile phase B with 0.1mol/l ammonium acetate aqueous solution (every 1000ml adds glacial acetic acid 0.5ml), and the regulation in the according to the form below is carried out gradient elution, and the detection wavelength is 235nm.Number of theoretical plate calculates by the mesaconine peak should be not less than 2000.Accurate respectively reference substance solution and each 20 μ l of need testing solution of drawing inject liquid chromatograph, measure, and promptly get.

The hordenine assay:

Preparation powder to be detected (crossing sieve No. three) 4-6g is got in the preparation of need testing solution, and accurate the title decides, and puts in the conical flask, adds the mixed solution 50ml of volume parts than 100:1 chloroform-ammoniacal liquor, claims to decide weight; Ultrasonic 30 minutes, placement was spent the night, and claimed to decide weight, supplied the weight that subtracts mistake with volume parts than the mixed solution of chloroform-ammoniacal liquor of 100:1, filtered; Get subsequent filtrate 25ml, decompression low temperature (below 50 ℃) evaporate to dryness, the accurate 0.05mol/l sulfuric acid solution 25ml that adds of residue makes dissolving, centrifugal (rotating speed is 8000 rev/mins) 20 minutes; Get supernatant, put in the separating funnel, transfer PH to 8-9, extract 5 times with the ethyl acetate jolting again, each 20ml with ammonia solution; Combining extraction liquid volatilizes, and residue is used dissolve with methanol, is transferred in the 5ml measuring bottle; Shake up, filter, get subsequent filtrate, promptly get.

Determination of Total Alkaloid:

Get preparation powder to be detected (crossing sieve No. three) 20-30g, the accurate title, decide, and puts in the conical flask, adds the mixed solution 50ml of volume parts than 100:1 chloroform-ammoniacal liquor; Claim to decide weight, ultrasonic 30min, placement is spent the night; Filter, residue adds the mixed solution 50ml of volume parts than 100:1 chloroform-ammoniacal liquor, jolting 1 hour; Filter, residue use volume parts is washed 3 times than the mixed solution of 100:1 chloroform-ammoniacal liquor, each 15m; Merge washing lotion and filtrating, low-temperature reduced-pressure (below 50 ℃) evaporate to dryness, residue add ethanol 10ml makes dissolving; Add activated charcoal 1g; Jolting makes decolouring, and precision is measured subsequent filtrate 5ml again; Accurate sulfuric acid vs (0.01mol/l) 15ml, the water 15ml and 3 of methyl red indicator solutions of adding uses NaOH vs (0.02mol/l) titration to yellow; Every 1ml sulfuric acid vs (0.01mol/l) is equivalent to the hordenine (C of 1.65mg ₁₀H ₁₅NO).

Said preparation is that eight flavor Herba Swertiae bimaculatae balls, ten golden simply balls or 13 flavor list loud, high-pitched sounds are loose, the peaceful particle of peace youngster.

The invention has the beneficial effects as follows that adopt the alkaloid component that contains in thin-layered chromatography qualitative identification Tangut aconite and the preparation thereof, this method is easy, reliable, specificity is strong; Adopt the diester-type alkaloids composition that contains in high performance liquid chromatography inspection Tangut aconite and the preparation thereof; Adopt the hordenine content that contains in quantitative determination of high-performance liquid Tangut aconite and the preparation thereof; Hordenine has the similar ephedrine in a lot of aspects of pharmacologically active; Be Tangut aconite index property composition; Control its quality, can effectively guarantee the Tangut aconite quality, and then guaranteed the safety of patient's medication.Method of quality control of the present invention is reliable and stable, and selectivity is strong, can guarantee in Tibetan medicine Tangut aconite and the preparation thereof alkaloidal safely, effective, quality controllable, and bigger science and using value are arranged.

Description of drawings

Fig. 1 is the high-efficient liquid phase chromatogram of diester-type alkaloids inspection blank solvent in the Tangut aconite;

Fig. 2 is the high-efficient liquid phase chromatogram chromatogram of diester-type alkaloids inspection aconitine reference substance in the Tangut aconite;

Fig. 3 is the high-efficient liquid phase chromatogram chromatogram of diester-type alkaloids inspection Hypaconitine reference substance in the Tangut aconite;

Fig. 4 is the high-efficient liquid phase chromatogram chromatogram of diester-type alkaloids inspection mesaconine reference substance in the Tangut aconite;

Fig. 5 is the high-efficient liquid phase chromatogram chromatogram of diester-type alkaloids inspection test sample in the Tangut aconite;

Fig. 6 is an aconitine bioassay standard curve map;

Fig. 7 is a Hypaconitine bioassay standard curve map;

Fig. 8 is a mesaconine bioassay standard curve map;

Fig. 9 is the high-efficient liquid phase chromatogram chromatogram of hordenine assay hordenine reference substance in the Tangut aconite;

Figure 10 is the high-efficient liquid phase chromatogram chromatogram of hordenine assay test sample in the Tangut aconite;

Figure 11 is a hordenine bioassay standard curve map.

Wherein, the horizontal ordinate of Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 9, Figure 10 is the time, unit: Minutes; Ordinate is a voltage, unit: Volts; The horizontal ordinate of Fig. 6, Fig. 7, Fig. 8, Figure 11 is a concentration C, unit: μ g/ml; Ordinate is peak area A, unit: μ Vs.

Embodiment

Through instantiation the present invention is further set forth below, should be noted that following explanation only is in order to explain the present invention, its content not to be limited.

Experimental example 1: Tangut aconite thin-layer chromatography identification experiment

Tangut aconite, Qinghai gold are scolded Tibetan medicine medicine company incorporated company and are provided.

Thin-layer chromatography is differentiated:

Interpretation of result: investigated chloroform-methanol respectively, chloroform-methanol-formic acid, under the developping agent condition of different developping agents such as xylene-ethyl acetate-diethylamine, normal hexane-ethyl acetate-methyl alcohol and different volumes portion rate thereof, the expansion effect of Tangut aconite.The result finds; When being developping agent with volume parts than normal hexane-ethyl acetate-methyl alcohol of 5:4:1.5, the alkaloid component in the Tangut aconite has and launches effect preferably, but spot slightly trails; After adding ammoniacal liquor; Hangover disappears, and wherein, it is best that volume parts is launched effect than the developping agent for normal hexane-ethyl acetate of 5:4:1.5:0.2-methyl alcohol-ammoniacal liquor.

Experimental example 2: diester-type alkaloids inspection in the Tangut aconite

1. instrument, reagent and confession test agent

Instrument: high performance liquid chromatograph: the L-2100 of Hitachi pump, the L-2400 of Hitachi UV-detector;

Electronic balance: Tianjin, island AUW220D type.

Reference substance: aconitine reference substance (Nat'l Pharmaceutical & Biological Products Control Institute), lot number: 110720-200410; Hypaconitine (Nat'l Pharmaceutical & Biological Products Control Institute), lot number: 110798-200805; Mesaconine (Nat'l Pharmaceutical & Biological Products Control Institute), lot number: 110799-200505.

Sample: Tangut aconite (the Qinghai gold is scolded Tibetan medicine medicine company incorporated company and provided), lot number is respectively: 110701,110702,110703.

2. detect the selection of wavelength

Get aconitine reference substance, Hypaconitine reference substance, mesaconine reference substance mixed solution, in the 190-400nm wavelength coverage, scan, the result shows that mixing has absorption maximum to impinging upon the 235nm place, is the detection wavelength according to the selected 235nm of ultraviolet absorpting spectrum.

3. moving phase is selected

Having investigated methyl alcohol-volume parts ratio respectively is 0.1% triethylamine aqueous solution, methyl alcohol-0.04mol/l triethylamine (phosphorus acid for adjusting pH value to 6), acetonitrile-5mmol/l NaH ₂PO ₄Solution (phosphoric acid transfers to pH4.5); [volume parts is than acetonitrile-tetrahydrofuran of 25:15]-separating effect of need testing solution mesaconitine, Hypaconitine, mesaconine when different volumes portion rates such as [0.1mol/1 ammonium acetate aqueous solution (transferring to pH9.74 with glacial acetic acid)] is moving phase; Discover; Is mobile phase A with volume parts than acetonitrile-tetrahydrofuran of 25:15, is Mobile phase B with 0.1mol/l ammonium acetate aqueous solution (every 1000ml adds glacial acetic acid 0.5ml), when selecting for use appropriate condition to carry out gradient elution; Can reach separating effect preferably, and peak shape is better.Gradient is seen table 1.

Table 1 gradient table

4. specificity test

Get 0.05mol/l sulfuric acid as blank solvent.

It is an amount of to get aconitine reference substance, Hypaconitine reference substance, mesaconine reference substance, accurately claims surely, adds 0.05mol/l sulfuric acid respectively and processes the solution that every 1ml contains aconitine, Hypaconitine, each 50 μ g of mesaconine, respectively as reference substance solution.

Get medicinal powder to be detected (crossing sieve No. three) 2g, the accurate title, decide, and puts in the conical flask of tool plug, and the accurate mixed solution 50ml that adds volume parts than 1:100 hydrochloric acid-methyl alcohol claims to decide weight; Sonicated 40 minutes (temperature is no more than 25 ℃) is put coldly, claims to decide weight, supplies the weight that subtracts mistake with volume parts than the mixed solution of 1:100 hydrochloric acid-methyl alcohol, shakes up; Filter, precision is measured subsequent filtrate 25ml, and to doing, the residue precision adds 0.05mol/l sulfuric acid solution 25ml at decompression and solvent recovery below 40 ℃, and jolting makes dissolving; Centrifugal (rotating speed is 8000 rev/mins) 20 minutes, precision is measured supernatant 20ml, puts in the separating funnel, with washed with dichloromethane 4 times (20ml, 20ml, 15ml, 15ml); Discard dichloromethane solution, treat upper strata clarification after, transfer PH to 8-9 with ammonia solution, again with ether jolting extraction 5 times; Each 20ml merges ether solution, volatilizes, and residue is used the 0.05mol/l sulfuric acid dissolution; Be transferred in the 5ml measuring bottle, shake up, filter, get subsequent filtrate as need testing solution.

Under above-mentioned chromatographic condition, accurate respectively blank solvent, reference substance solution, each 20 μ l of need testing solution of drawing inject liquid chromatograph, the record chromatogram.The result shows that test sample chromatogram mesaconitine, Hypaconitine, mesaconine are adjacent the degree of separation at peak all greater than 1.5, and good separating effect can be used in the detection of diester-type alkaloids.The peak retention order of reference substance is followed successively by mesaconine, Hypaconitine, aconitine.The result sees accompanying drawing 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5.

5. the investigation of the preparation of typical curve and linear relationship

Precision takes by weighing aconitine reference substance (11.42mg), Hypaconitine reference substance (10.04mg), mesaconine (11.32mg); Put respectively in the volumetric flask of 10ml; With the sulfuric acid dissolution of 0.05mol/l and be diluted to scale, get each 5ml of above-mentioned reference substance solution to the volumetric flask of same 50ml, with the Dilution of sulphuric acid of 0.05mol/l to scale; Shake up, as the reference substance storing solution; Get reference substance storing solution 1ml, 3ml, 5ml, 7ml, 10ml, put respectively in the 10ml volumetric flask, the 0.05mol/l Dilution of sulphuric acid shakes up to scale, and each accurate sample introduction 20 μ l is a horizontal ordinate with concentration (C), and peak area (A) is an ordinate, carries out linear regression.The result sees table 2-1, table 2-2, table 2-3 and accompanying drawing 6, Fig. 7, Fig. 8.

Table 2-1 aconitine typical curve result

Regression equation: A=12284C+3132.7

Related coefficient: R=0.9999

The result shows: in 11.42 μ g/ml ~ 114.20 μ g/ml scopes, the peak area of aconitine (A) is good with concentration (C) linear relationship.

Table 2-2 Hypaconitine typical curve result

Regression equation: A=11756C+5608.5

Related coefficient: R=0.9999

The result shows: in 10.04 μ g/ml ~ 100.40 μ g/ml scopes, the peak area of Hypaconitine (A) is good with concentration (C) linear relationship.

Table 2-3 mesaconine typical curve result

Regression equation: A=13338C+274.47

Related coefficient: R=0.9999

The result shows: in 11.32 μ g/ml ~ 113.20 μ g/ml scopes, the peak area of mesaconine (A) is good with concentration (C) linear relationship.

6. precision test

Under above-mentioned chromatographic condition, the accurate absorption mixed reference substance solution 20 μ l, injects liquid chromatograph, each METHOD FOR CONTINUOUS DETERMINATION 6 times, and the record peak area also calculates relative standard deviation.The result shows that instrument precision is good.The result sees table 3-1, table 3-2, table 3-3.

Table 3-1 aconitine Precision test result

Table 3-2 Hypaconitine Precision test result

Table 3-3 mesaconine Precision test result

7. quantitative limit

Get respectively in aconitine contrast solution (57.10 μ g/ml), Hypaconitine contrast solution (50.20 μ g/ml), each 1ml to 25ml volumetric flask of mesaconine (56.60 μ g/ml); Respectively with the dilution of the sulfuric acid solution of 0.05mol/l and be settled to scale; Shake up sample introduction.Signal to noise ratio (S/N ratio) all is about 30 as a result.Get respectively again in the volumetric flask of above-mentioned solution 3ml to 10ml, with the dilution of the sulfuric acid solution of 0.05mol/l and be settled to scale, shake up sample introduction.Signal to noise ratio (S/N ratio) all is about 10 as a result.The result shows: when aconitine concentration was 0.685 μ g/ml, signal to noise ratio (S/N ratio) was about 10, and promptly aconitine quantitatively is limited to 13.70ng; When Hypaconitine concentration was 0.602 μ g/ml, signal to noise ratio (S/N ratio) was about 10, and promptly Hypaconitine quantitatively is limited to 12.05ng; When mesaconine concentration was 0.679 μ g/ml, signal to noise ratio (S/N ratio) was about 10, and promptly mesaconine quantitatively is limited to 13.58ng.

8. replica test

These article of getting (lot number: 110701), replication 6 times, the content and the relative standard deviation of record peak area and difference calculation sample mesaconitine, Hypaconitine, mesaconine.The result shows that this analytical approach repeatability is good.The result sees table 4-1, table 4-2, table 4-3.

Table 4-1 aconitine replica test result

Table 4-2 Hypaconitine replica test result

Table 4-3 mesaconine replica test result

9. recovery test

The reference substance storing solution is got aconitine reference substance, Hypaconitine reference substance, each 5mg of mesaconine reference substance; The accurate title, decide; Put respectively in the 10ml measuring bottle, add the 0.05mol/l sulfuric acid dissolution and be diluted to scale, respectively get in the measuring bottle that 1ml puts same 100ml; Hydrochloric acid-methyl alcohol (1:100) dilutes and is settled to scale, as mixing the reference substance storing solution.

The recovery test need testing solution is got medicinal powder to be detected (the cross No. three sieve) 1g (lot number: 110701), accurately claim surely, put in the conical flask of tool plug the accurate 2ml mixing reference substance storing solution that adds of known diester-type alkaloids content; Add the mixed solution 48ml of volume parts than 1:100 hydrochloric acid-methyl alcohol again, claim to decide weight, sonicated 40 minutes (temperature is no more than 25 ℃) is put cold; Claim to decide weight, supply the weight that subtracts mistake than the mixed solution of 1:100 hydrochloric acid-methyl alcohol, shake up, filter with volume parts; Precision is measured subsequent filtrate 25ml, and to doing, the residue precision adds 0.05mol/l sulfuric acid solution 25ml at decompression and solvent recovery below 40 ℃, and jolting makes dissolving; Centrifugal (rotating speed is 8000 rev/mins) 20 minutes, precision is measured supernatant 20ml, puts in the separating funnel, with washed with dichloromethane 4 times (20ml, 20ml, 15ml, 15ml); Discard dichloromethane solution, treat upper strata clarification after, transfer PH to 8-9 with ammonia solution, again with ether jolting extraction 5 times; Each 20ml merges ether solution, volatilizes, and residue is used the 0.05mol/l sulfuric acid dissolution; Be transferred in the 5ml measuring bottle, shake up, filter, get subsequent filtrate as need testing solution.Prepare 6 parts altogether.

Press content assaying method and measure calculate recovery rate and relative standard deviation thereof.The result shows: it is accurate that this assay method is measured the result.The result sees table 5.

Table 5 recovery test result

10. sample determination

Get 3 batches of Tangut aconite medicinal materials, the content of diester-type alkaloids is checked by above-mentioned content assaying method.The result sees table 6.

Diester-type alkaloids is measured the result in table 6 Tangut aconite

Experimental example 3: the assay of hordenine experiment in the Tangut aconite

1. instrument, reagent and confession test agent

Electronic balance: Tianjin, island AUW220D type.

Reference substance: hordenine reference substance (Nat'l Pharmaceutical & Biological Products Control Institute), lot number: 100762-200901.

2. detect the selection of wavelength

Get the hordenine reference substance solution, in the 190-400nm wavelength coverage, scan, according to ultraviolet absorpting spectrum, selected 225nm is for detecting wavelength.

3. moving phase and chromatographic column are selected

Having investigated acetonitrile-volume parts ratio respectively is 0.5% triethylamine aqueous solution (phosphoric acid is transferred pH3.0); Acetonitrile-0.025mol/l potassium dihydrogen phosphate aqueous solution (triethylamine is regulated PH to 7.2); Acetonitrile-0.1mol/l sodium dihydrogen phosphate-the separating effect of hordenine in need testing solution when different volumes portion rates such as 0.025mol/l sodium dodecyl sulfate solution are moving phase; Discover; When selecting acetonitrile-0.025mol/l potassium dihydrogen phosphate aqueous solution (triethylamine is regulated PH to 7.2) for use, can access separating effect preferably, when acetonitrile-0.025mol/l potassium dihydrogen phosphate aqueous solution (triethylamine is regulated PH to 7.2) volume parts ratio is 10:90 for moving phase; Hordenine main peak retention time is 7.9min, and separating effect is best.

Studied Waters C respectively ₁₈(250mm * 4.6mm, 5 μ m), Thermo C ₈(250mm * 4.6mm, 5 μ m), Welch

Phenyl-Ether different chromatographic column hordenines such as (250mm * 4.6mm, 5 μ m) separating effect, the result finds to use Thermo C ₈(250mm * 4.6mm, 5 μ m) chromatographic column, the peak shape of hordenine is better.

4. system suitability test

Under above-mentioned chromatographic condition, accurate respectively reference substance solution, each 10 μ l of need testing solution of drawing inject liquid chromatograph, the record chromatogram.The result shows that the degree of separation that hordenine is adjacent chromatographic peak in reference substance, the test sample chromatogram is all greater than 1.5.See accompanying drawing 9, Figure 10.

5. reference substance preparation

It is an amount of that precision takes by weighing the hordenine reference substance, adds methyl alcohol and process the solution that contains hordenine 20 μ g among every 1ml, promptly gets.

6. test sample preparation

Reported in the literature Hordenine with thermal instability, were investigated ultrasonic, vibration Oscillating two methods of extraction; Hordenine easily soluble in alcohol, chloroform, and the alkaline solution can make the alkaloids fully free, were investigated ethanol volume fraction ratio 100:1 - ammonia, methanol volume fraction ratio 100:1 - ammonia, volume fraction ratio 100:1 chloroform - ammonia three kinds of solvent extraction; also were investigated 20ml, 40ml, 60ml amount of different extraction solvent extraction.The result sees table 7, table 8 and table 9.

Table 7 method for distilling is investigated test findings

Table 8 extracts solvent and investigates test findings

Table 9 extracts quantity of solvent and investigates test findings

The result shows, adopts ultrasonic Extraction more abundant, extracts solvent preferred volume portion rate 100:1 chloroform-ammoniacal liquor, extracts quantity of solvent and selects for use 50ml to guarantee that hordenine extracts fully.Concrete grammar is following:

Get medicinal powder to be detected (crossing sieve No. three) 1g, the accurate title, decide, and puts in the conical flask, adds the mixed solution 50ml of volume parts than 100:1 chloroform-ammoniacal liquor, claims to decide weight; Ultrasonic 30 minutes, placement was spent the night, and claimed to decide weight, supplied the weight that subtracts mistake with volume parts than the mixed solution of 100:1 chloroform-ammoniacal liquor, filtered; Get subsequent filtrate 25ml, decompression low temperature (below 50 ℃) evaporate to dryness, the accurate 0.05mol/l sulfuric acid solution 25ml that adds of residue makes dissolving, centrifugal (rotating speed is 8000 rev/mins) 20 minutes; Get supernatant, put in the separating funnel, transfer PH to 8-9, extract 5 times with the ethyl acetate jolting again, each 20ml with ammonia solution; Combining extraction liquid volatilizes, and residue is used dissolve with methanol, is transferred in the 5ml measuring bottle; Shake up, filter, get subsequent filtrate, promptly get.

7. the investigation of the preparation of typical curve and linear relationship

Precision is measured hordenine reference substance stock solution solution (41.60 μ g/ml) 1ml, 3ml, 5ml, 8ml, 10ml; Put respectively in the 10ml volumetric flask, methyl alcohol is diluted to scale, shakes up; Each accurate sample introduction 10 μ l; With peak area (A) reference substance concentration (C) is carried out linear regression, get hordenine regression equation: A=9280.2C+2913.0, related coefficient: R=0.9997.The result shows: hordenine is in 4.16 μ g/ml ~ 41.60 μ g/ml scopes, and the peak area of hordenine (A) is good with concentration (C) linear relationship.The result sees table 10 and accompanying drawing 11.

Table 10 hordenine linear relationship is investigated the result

8. precision test

The accurate hordenine reference substance solution 10 μ l that draw inject liquid chromatograph, each METHOD FOR CONTINUOUS DETERMINATION 6 times, and the record peak area also calculates relative standard deviation, relative standard deviation RSD=0.72%.The result shows that instrument precision is good.The result sees table 11.

Table 11 Precision test result

9. stability test

After the need testing solution preparation was accomplished, the accurate 10 μ l that draw injected liquid chromatograph, and the record peak area was whenever measured once at a distance from 2 hours later on, investigated 6 hours, calculated the relative standard deviation of peak area, RSD=0.66%.The result shows: in 6 hours, to measure the result stable for hordenine in the test sample.The result sees table 12.

Table 12 sample stability test findings

10. replica test

These article of getting (lot number: 110701), replication 6 times, the content of hordenine in the calculation sample, relative standard deviation RSD=1.17%.Show that analytical approach repeatability is good.The result sees table 13.

Table 13 replica test result

11. recovery test

Precision takes by weighing 6 parts of same lot sample article, and (lot number: 110701), the accurate hordenine reference substance that adds is measured its content, and calculate recovery rate, hordenine average recovery rate are 97.92%, relative standard deviation RSD=1.32%.It is accurate to show that this assay method is measured the result.The result sees table 14.

Table 14 recovery experimental result

12. sample determination

Get 3 batches of Tangut aconite medicinal materials, hordenine is measured content by above-mentioned content assaying method.The result sees table 15.

Table 15 hordenine assay result

Experimental example 4: total alkaloid contents determination experiment in the Tangut aconite

1. instrument, reagent and confession test agent

Instrument: electronic balance: Tianjin, island AUW220D type; Buret (Beijing Glass Implement Factory, 25ml).

2. blank test

Precision is measured ethanol 5ml, puts in the 250ml conical flask, the accurate 15ml sulfuric acid vs (0.0116mol/l) that adds, and 3 of 15ml water and methyl red index liquid drip to yellow with NaOH vs (0.0205mol/l) titration.The result consumes alkali lye 16.95ml, shows that this solvent system does not produce interference to measuring the result.

3. reappearance test

Get 6 parts in 110701 lot number samples, the method for pressing in the sample under the Determination of Total Alkaloid item is measured, and calculates total alkaloid content and relative standard deviation thereof.The result shows: this method repeatability is good.The result sees table 16.

Table 16 Determination of Total Alkaloid reproducible test results

4. recovery test

Precision takes by weighing 6 parts of the same lot sample article of known content, and (lot number: 110701), the accurate respectively hordenine reference substance that adds press in the sample method mensuration under the Determination of Total Alkaloid item, calculate recovery rate and relative standard deviation thereof.The result shows: it is accurate that this assay method is measured the result.The result sees table 17.

Table 17 recovery experimental result

5. Determination of Total Alkaloid in the sample

Get medicinal powder to be detected (crossing sieve No. three) 5g, the accurate title, decide, and puts in the conical flask, adds the mixed solution 50ml of volume parts than 100:1 chloroform-ammoniacal liquor; Claim to decide weight, ultrasonic 30min, placement is spent the night; Filter, residue adds the mixed solution 50ml of volume parts than 100:1 chloroform-ammoniacal liquor, jolting 1 hour; Filter, residue use volume parts is washed 3 times than the mixed solution of 100:1 chloroform-ammoniacal liquor, each 15ml; Merge washing lotion and filtrating, low-temperature reduced-pressure (below 50 ℃) evaporate to dryness, residue add ethanol 10ml makes dissolving; Add activated charcoal 1g; Jolting makes decolouring, and precision is measured subsequent filtrate 5ml again; Accurate sulfuric acid vs (0.01mol/l) 15ml, the water 15ml and 3 of methyl red indicator solutions of adding uses NaOH vs (0.02mol/l) titration to yellow.Every 1ml sulfuric acid vs (0.01mol/l) is equivalent to the hordenine (C of 1.65mg ₁₀H ₁₅NO).The result sees table 18.

Table 18 Determination of Total Alkaloid result

Following embodiment all can realize the described effect of above-mentioned experimental example.

Find through consulting the domestic and foreign literature data, the Tangut aconite chemical constitution study seldom, wherein compound structure is quite similar; When thin layer is differentiated, be difficult to its exclusive compound and other impurity are separated, interference component is many; Therefore its thin layer is differentiated and brought very big difficulty; We grope through a large amount of experiments to this, finally adopt the Tangut aconite control medicinal material to be contrast, filter out to launch developping agent effective, that repeatability is good; It is with respect to original discrimination method, and is easier, reliable and specificity is strong.For inspection item and assay; Equal avirulence introduction of Tangut aconite and content assaying method in bibliographical information and the quality standard; In order to guarantee the safe and effective of correct use of Tangut aconite and preparation thereof; So be necessary aconitine, Hypaconitine, mesaconine etc. to set up main alkaloid composition hordenine and total alkaloid contents assay method in the Tangut aconite simultaneously for the diester-type alkaloids toxic component of representative carries out limit examine.Therefore we grope through experiment; The diester-type alkaloids inspection is at first carried out pre-treatment to test sample; Remove most of interference component; Select for use different mobile phase, different mobile phase volume portion rate isochromatic spectrum condition to be optimized then, filter out the chromatographic condition of good separating effect, and the inspection method of being set up has been carried out methodological study; The hordenine assay respectively to different chromatographic column fillers, different moving phase and volume parts thereof than isochromatic spectrum condition; Different Extraction Method, the different test sample disposal routes such as solvent, different extraction quantity of solvent of extracting are optimized; Preferred top condition, and the content assaying method of being set up carried out methodological study; Determination of Total Alkaloid adopts the back titration method, and has investigated methodological studies such as blank, reappearance, average recovery.The result shows, inspection of being set up and content assaying method, and precision is high, good reproducibility, the recovery is high, and the result has stronger specificity accurately and reliably.

Embodiment 1: alkaloidal quality determining method in the Tangut aconite

Thin-layer chromatography is differentiated:

The diester-type alkaloids inspection:

The hordenine assay:

Determination method is drawn reference substance solution and each 10 μ l of need testing solution respectively, injects liquid chromatograph, measures, and promptly gets;

Determination of Total Alkaloid:

Differentiate in the detection that following standard application is accordinged in said Tangut aconite control medicinal material source:

Tangut aconite is the dry herb of ranunculaceae plant Tangut aconite Aconitum tanguicum (Mazxim.) Stapf and ship helmet rhizome of Chinese monkshood Aco-nitum naviculare (Bruhl.) Stapf.Main product in Qinghai, ground such as Gansu, Tibet, Sichuan, the Tangut aconite control medicinal material should meet standard (being drafted by the institute for drug control, Qinghai Province) under 85 pages of items of Tibetan medicine ministerial standard.Standard is following:

Tangut aconite Tibetan medicine name: list loud, high-pitched sound

Phonetic name: Tanggute Wutou

English name: HERBA ACONITI TANGUTICI

Book page number: C1-85

Standard numbering: WS3-BC-0085-95

These article are the dry herb of ranunculaceae plant Tangut aconite Aconitum tanguticum (Maxim.) Stapf and ship helmet rhizome of Chinese monkshood Aco-nitum naviculare (Bruhl.) Stapf.Connect root excavate florescence at the beginning of autumn late summer, removes impurity, dries in the shade.

[proterties] these article piece root is tiny, and spindle-like or taper shape, female root head have sub-root 2～7 of living vestiges; Sub-roots and tops has the scar of a deflection, and is not of uniform size, long 2～3cm, and diameter 3～4mm, the epidermis tawny is to pitchy, the section off-white color.Stem is cylindric, shrinkage, and varying length, diameter 2～3mm, the epidermis celadon is to dirty-green, and matter is crisp, frangible, section hollow.Leaf-shrinkage curls, and the intact leaf kidney is circular, oval, palmate drastic crack, sliver 2～3 shallow splitting.Raceme stalk and pedicel are by pubescence; Calyx 5, purple or bluish violet, last sepal helmet shape; Petal 2, minimum; Stamen is most; Filigree is extremely short, flattens tool hair, flower pesticide black; Ovary does not have hair.Gas is little, bitter.

[discriminating] these article powder sepia.Conduit is rare, and scalariform and screw thread are main with scalariform duct, diameter 18～32 μ m.Fiber is often cataclasm, diameter 18～28 μ m, and end wall point, heavy wall are crooked slightly, and minority inclination simple pit is arranged.

[process of preparing Chinese medicine] removed impurity.

[nature and flavor] hardship, cold.

[function with cure mainly] is clearing heat and detoxicating, promoting granulation and wound healing, eliminating dampness.Be used for the heating that infectious disease causes, the liver and gall pyreticosis, mass formed by blood stasis, stomach energy, the ulcer sore, snakes and scorpions is bitten and grasserie.

[usage and consumption] 0.6～1.2g.

[storage] put aeration-drying place.

The Tangut aconite control medicinal material that this patent uses is originated through Qinghai Province Tibetan medicine hospital's fame president, the Tibetan medicine principal expert of Qinghai Province Nima according to above standard evaluation.

Alkaloidal quality determining method in 2: eight flavors of embodiment Herba Swertiae bimaculatae ball

Eight flavor Herba Swertiae bimaculatae balls are from the Tibetan medicine ministerial standard, and standard is numbered: WS3-BC-0241-95.Form by Herba Swertiae bimaculatae 300g, Lagotis glauca 200g, Semen Herpetospermi 80g, angle fennel 200g, list loud, high-pitched sound 200g, barberry skin 160g, rock ginseng 240g, banksia rose 200g.

Thin-layer chromatography is differentiated:

Get preparation powder to be detected (crossing sieve No. three) 5g, add ammoniacal liquor 5ml, the 50ml that adds methylene chloride, sonicated (power 250W, frequency 40kHz) 30 minutes filters, the filtrating evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution.Get Tangut aconite control medicinal material 1g, shine medicinal material solution in pairs with legal system.Test according to thin-layered chromatography (an appendix VI of Pharmacopoeia of the People's Republic of China version in 2010 B); Drawing each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, is developping agent with volume parts than normal hexane-ethyl acetate-methyl alcohol-ammoniacal liquor of 5:4:1.5:0.2; Launch; Take out, dry, spray is with rare bismuth potassium iodide solution.In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show identical orange red spot.

The diester-type alkaloids inspection:

Get preparation powder to be detected (crossing sieve No. three) 8g, the accurate title, decide, and puts in the conical flask of tool plug, and the accurate mixed solution 50ml that adds volume parts than 1:100 hydrochloric acid-methyl alcohol claims to decide weight; Sonicated 40 minutes (temperature is no more than 25 ℃) is put coldly, claims to decide weight, supplies the weight that subtracts mistake with volume parts than the mixed solution of 1:100 hydrochloric acid-methyl alcohol, shakes up; Filter, precision is measured subsequent filtrate 25ml, and to doing, the residue precision adds 0.05mol/l sulfuric acid solution 25ml at decompression and solvent recovery below 40 ℃, and jolting makes dissolving; Centrifugal (rotating speed is 8000 rev/mins) 20 minutes, precision is measured supernatant 20ml, puts in the separating funnel, with washed with dichloromethane 4 times (20ml, 20ml, 15ml, 15ml); Discard dichloromethane solution, treat upper strata clarification after, transfer PH to 8-9 with ammonia solution, again with ether jolting extraction 5 times; Each 20ml merges ether solution, volatilizes, and residue is used the 0.05mol/l sulfuric acid dissolution; Be transferred in the 5ml measuring bottle, shake up, filter, get subsequent filtrate as need testing solution.It is an amount of to get aconitine reference substance, Hypaconitine reference substance, mesaconine reference substance, accurately claims surely, adds 0.05mol/l sulfuric acid and processes the mixed solution that every 1ml contains aconitine, Hypaconitine, each 50 μ g of mesaconine, compares article solution.Test according to high performance liquid chromatography (an appendix VI of Pharmacopoeia of the People's Republic of China version in 2010 D).With the octadecylsilane chemically bonded silica is filling agent; Is mobile phase A with volume parts than acetonitrile-tetrahydrofuran of 25:15, is Mobile phase B with 0.1mol/l ammonium acetate aqueous solution (every 1000ml adds glacial acetic acid 0.5ml), and the regulation in the according to the form below is carried out gradient elution, and the detection wavelength is 235nm.Number of theoretical plate calculates by the mesaconine peak should be not less than 2000.Accurate respectively reference substance solution and each 20 μ l of need testing solution of drawing inject liquid chromatograph, measure, and promptly get.

These article contain dibasic acid esters property alkaloid with aconitine (C ₃₄H ₄₇NO ₁₁), Hypaconitine (C ₃₃H ₄₅NO ₁₀) and mesaconine (C ₃₃H ₄₅NO ₁₁) the total amount meter, must not cross 0.012mg/g.

The hordenine assay:

Preparation powder to be detected (crossing sieve No. three) 4g is got in the preparation of need testing solution, and accurate the title decides, and puts in the conical flask, adds the mixed solution 50ml of volume parts than 100:1 chloroform-ammoniacal liquor, claims to decide weight; Ultrasonic 30 minutes, placement was spent the night, and claimed to decide weight, supplied the weight that subtracts mistake with volume parts than the mixed solution of 100:1 chloroform-ammoniacal liquor, filtered; Get subsequent filtrate 25ml, decompression low temperature (below 50 ℃) evaporate to dryness, the accurate 0.05mol/l sulfuric acid solution 25ml that adds of residue makes dissolving, centrifugal (rotating speed is 8000 rev/mins) 20 minutes; Get supernatant, put in the separating funnel, transfer PH to 8-9, extract 5 times with the ethyl acetate jolting again, each 20ml with ammonia solution; Combining extraction liquid volatilizes, and residue is used dissolve with methanol, is transferred in the 5ml measuring bottle; Shake up, filter, get subsequent filtrate, promptly get.

These article contain hordenine (C ₁₀H ₁₅NO) must not be less than 0.025mg/g.

Determination of Total Alkaloid:

Get preparation powder to be detected (crossing sieve No. three) 20g, the accurate title, decide, and puts in the conical flask, adds the mixed solution 50ml of volume parts than 100:1 chloroform-ammoniacal liquor; Claim to decide weight, ultrasonic 30min, placement is spent the night; Filter, residue adds the mixed solution 50ml of volume parts than 100:1 chloroform-ammoniacal liquor, jolting 1 hour; Filter, residue use volume parts is washed 3 times than the mixed solution of 100:1 chloroform-ammoniacal liquor, each 15m; Merge washing lotion and filtrating, low-temperature reduced-pressure (below 50 ℃) evaporate to dryness, residue add ethanol 10ml makes dissolving; Add activated charcoal 1g; Jolting makes decolouring, and precision is measured subsequent filtrate 5ml again; Accurate sulfuric acid vs (0.01mol/l) 15ml, the water 15ml and 3 of methyl red indicator solutions of adding uses NaOH vs (0.02mol/l) titration to yellow; Every 1ml sulfuric acid vs (0.01mol/l) is equivalent to the hordenine (C of 1.65mg ₁₀H ₁₅NO).

These article contain alkaloid with hordenine (C ₁₀H ₁₅NO) meter must not be less than 0.45mg/g.

Alkaloidal quality determining method in 3: ten golden simply balls of embodiment

Ten golden simply balls are from the Tibetan medicine ministerial standard, and standard is numbered: WS3-BC-0168-95.Form by myrobalan's (stoning) 75g, dried feces of Sus scrofa L 50g, seed of pomegranate 40g, the tame and docile cream 25g of slag, Semen Herpetospermi 20g, list loud, high-pitched sound 26g, angle fennel 40g, common embelia fruit 35g, rose 100g, iron staff hammer 20g, Moschus 2.5g.

Thin-layer chromatography is differentiated:

Get preparation powder to be detected (crossing sieve No. three) 8g, add ammoniacal liquor 5ml, the 50ml that adds methylene chloride, sonicated (power 250W, frequency 40kHz) 30 minutes filters, the filtrating evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution.Get Tangut aconite control medicinal material 1g, shine medicinal material solution in pairs with legal system.Test according to thin-layered chromatography (an appendix VI of Pharmacopoeia of the People's Republic of China version in 2010 B); Drawing each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, is developping agent with volume parts than normal hexane-ethyl acetate-methyl alcohol-ammoniacal liquor of 5:4:1.5:0.2; Launch; Take out, dry, spray is with rare bismuth potassium iodide solution.In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show identical orange red spot.

The diester-type alkaloids inspection:

Get preparation powder to be detected (crossing sieve No. three) 15g, the accurate title, decide, and puts in the conical flask of tool plug, and the accurate mixed solution 50ml that adds volume parts than 1:100 hydrochloric acid-methyl alcohol claims to decide weight; Sonicated 40 minutes (temperature is no more than 25 ℃) is put coldly, claims to decide weight, supplies the weight that subtracts mistake with volume parts than the mixed solution of 1:100 hydrochloric acid-methyl alcohol, shakes up; Filter, precision is measured subsequent filtrate 25ml, and to doing, the residue precision adds 0.05mol/l sulfuric acid solution 25ml at decompression and solvent recovery below 40 ℃, and jolting makes dissolving; Centrifugal (rotating speed is 8000 rev/mins) 20 minutes, precision is measured supernatant 20ml, puts in the separating funnel, with washed with dichloromethane 4 times (20ml, 20ml, 15ml, 15ml); Discard dichloromethane solution, treat upper strata clarification after, transfer PH to 8-9 with ammonia solution, again with ether jolting extraction 5 times; Each 20ml merges ether solution, volatilizes, and residue is used the 0.05mol/l sulfuric acid dissolution; Be transferred in the 5ml measuring bottle, shake up, filter, get subsequent filtrate as need testing solution.It is an amount of to get aconitine reference substance, Hypaconitine reference substance, mesaconine reference substance, accurately claims surely, adds 0.05mol/l sulfuric acid and processes the mixed solution that every 1ml contains aconitine, Hypaconitine, each 50 μ g of mesaconine, compares article solution.Test according to high performance liquid chromatography (an appendix VI of Pharmacopoeia of the People's Republic of China version in 2010 D).With the octadecylsilane chemically bonded silica is filling agent; Is mobile phase A with volume parts than acetonitrile-tetrahydrofuran of 25:15, is Mobile phase B with 0.1mol/l ammonium acetate aqueous solution (every 1000ml adds glacial acetic acid 0.5ml), and the regulation in the according to the form below is carried out gradient elution, and the detection wavelength is 235nm.Number of theoretical plate calculates by the mesaconine peak should be not less than 2000.Accurate respectively reference substance solution and each 20 μ l of need testing solution of drawing inject liquid chromatograph, measure, and promptly get.

These article contain dibasic acid esters property alkaloid with aconitine (C ₃₄H ₄₇NO ₁₁), Hypaconitine (C ₃₃H ₄₅NO ₁₀) and mesaconine (C ₃₃H ₄₅NO ₁₁) the total amount meter, must not cross 0.005mg/g.

The hordenine assay:

Preparation powder to be detected (crossing sieve No. three) 6g is got in the preparation of need testing solution, and accurate the title decides, and puts in the conical flask, adds the mixed solution 50ml of volume parts than 100:1 chloroform-ammoniacal liquor, claims to decide weight; Ultrasonic 30 minutes, placement was spent the night, and claimed to decide weight, supplied the weight that subtracts mistake with volume parts than the mixed solution of 100:1 chloroform-ammoniacal liquor, filtered; Get subsequent filtrate 25ml, decompression low temperature (below 50 ℃) evaporate to dryness, the accurate 0.05mol/l sulfuric acid solution 25ml that adds of residue makes dissolving, centrifugal (rotating speed is 8000 rev/mins) 20 minutes; Get supernatant, put in the separating funnel, transfer PH to 8-9, extract 5 times with the ethyl acetate jolting again, each 20ml with ammonia solution; Combining extraction liquid volatilizes, and residue is used dissolve with methanol, is transferred in the 5ml measuring bottle; Shake up, filter, get subsequent filtrate, promptly get.

These article contain hordenine (C ₁₀H ₁₅NO) must not be less than 0.011mg/g.

Determination of Total Alkaloid:

Get preparation powder to be detected (crossing sieve No. three) 30g, the accurate title, decide, and puts in the conical flask, adds the mixed solution 50ml of volume parts than 100:1 chloroform-ammoniacal liquor; Claim to decide weight, ultrasonic 30min, placement is spent the night; Filter, residue adds the mixed solution 50ml of volume parts than 100:1 chloroform-ammoniacal liquor, jolting 1 hour; Filter, residue use volume parts is washed 3 times than the mixed solution of 100:1 chloroform-ammoniacal liquor, each 15m; Merge washing lotion and filtrating, low-temperature reduced-pressure (below 50 ℃) evaporate to dryness, residue add ethanol 10ml makes dissolving; Add activated charcoal 1g; Jolting makes decolouring, and precision is measured subsequent filtrate 5ml again; Accurate sulfuric acid vs (0.01mol/l) 15ml, the water 15ml and 3 of methyl red indicator solutions of adding uses NaOH vs (0.02mol/l) titration to yellow; Every 1ml sulfuric acid vs (0.01mol/l) is equivalent to the hordenine (C of 1.65mg ₁₀H ₁₅NO).

These article contain alkaloid with hordenine (C ₁₀H ₁₅NO) meter must not be less than 0.21mg/g.

Alkaloidal quality determining method during 4: ten three flavors of embodiment list loud, high-pitched sound is loose

It is Tibetan's proved recipe that 13 flavor list loud, high-pitched sounds are loose, and is made up of list loud, high-pitched sound 60g, Semen Herpetospermi 30g, Radix Gentianae Macrophyllae flower 40g, Herba Swertiae bimaculatae 40g, BAXIAGA 40g, denticulate ixeris herb 40g, flood even 40g, barberry skin 40g, strobilization angle fennel 40g, Chrysosplenium nudicaule Bunge 30g, cow-bezoar 3g, safflower 20g, seed of Cortex Holarrhenae Antidysentericae 30g.

Thin-layer chromatography is differentiated:

The diester-type alkaloids inspection:

These article contain dibasic acid esters property alkaloid with aconitine (C ₃₄H ₄₇NO ₁₁), Hypaconitine (C ₃₃H ₄₅NO ₁₀) and mesaconine (C ₃₃H ₄₅NO ₁₁) the total amount meter, must not cross 0.013mg/g.

The hordenine assay:

These article contain hordenine (C ₁₀H ₁₅NO) must not be less than 0.026mg/g.

Determination of Total Alkaloid:

These article contain alkaloid with hordenine (C ₁₀H ₁₅NO) meter must not be less than 0.47mg/g.

Embodiment 5: alkaloidal quality determining method in the peaceful particle of peace youngster

The peaceful particle of peace youngster is Tibetan's proved recipe, is made up of Tabasheer 66.7g, safflower 53.3g, calculus bovis factitius 5.3g, purple bergenia herb 53.3g, Radix Glycyrrhizae 53.3g, high mountain Tian jin cabbage pickled in sweet and sour 53.3g, short tube rabbit grass 66.7g, Santalum album 66.7g, Tangut aconite 66.7g.

Thin-layer chromatography is differentiated:

The diester-type alkaloids inspection:

The hordenine assay:

These article contain hordenine (C ₁₀H ₁₅NO) must not be less than 0.009mg/g.

Determination of Total Alkaloid:

These article contain alkaloid with hordenine (C ₁₀H ₁₅NO) meter must not be less than 0.16mg/g.

Claims

1. the quality determining method of a Tibetan medicine Tangut aconite; It is characterized in that; It comprises discriminating, inspection and assay project, and said discriminating is to be contrast, to be that developping agent launches on silica gel g thin-layer plate, is the thin-layered chromatography of developer with bismuth potassium iodide solution with volume parts than the normal hexane-ethyl acetate-methyl alcohol-ammoniacal liquor that is 5:4:1.5:0.2 with the Tangut aconite control medicinal material; Said inspection is the inspection to contained diester-type alkaloids in the medicinal material; It is that the mixed in equal amounts solution with aconitine reference substance, Hypaconitine reference substance, mesaconine reference substance is contrast; Is mobile phase A with volume parts than the acetonitrile-tetrahydrofuran that is 25:15, and adding the 0.5ml glacial acetic acid with every 1000ml 0.1mol/l ammonium acetate aqueous solution is the high performance liquid chromatography that Mobile phase B is carried out gradient elution; Said assay comprises to be measured the assay of contained hordenine in the medicinal material and total alkaloid contents; Said hordenine assay is to be contrast with the hordenine reference substance, with volume parts than being acetonitrile-0.025mol/l potassium dihydrogen phosphate aqueous solution of 10:90 and using triethylamine to regulate the high performance liquid chromatography of PH to 7.2 as moving phase; Said Determination of Total Alkaloid is to adopt the mixed solution extraction of volume parts than 100:1 chloroform-ammoniacal liquor, merges washing lotion and filtrating, low-temperature reduced-pressure evaporate to dryness below 50 ℃; Add activated carbon decolorizing; Jolting, decolouring adds the sulfuric acid vs; Using the NaOH vs, is the back titration method of indicator solution with the methyl red.

2. quality determining method as claimed in claim 1 is characterized in that, the process of gradient elution is following in the said inspection:

3. quality determining method as claimed in claim 1 is characterized in that, said discriminating is specially:

Get medicinal powder 1g to be detected, add ammoniacal liquor 5ml, the 50ml that adds methylene chloride, sonicated 30 minutes filters, the filtrating evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution; Get Tangut aconite control medicinal material 1g, shine medicinal material solution in pairs with legal system; According to the thin-layered chromatography test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate; Is developping agent with volume parts than the normal hexane-ethyl acetate-methyl alcohol-ammoniacal liquor that is 5:4:1.5:0.2, launches, and takes out; Dry, spray is with rare bismuth potassium iodide solution; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show identical orange red spot.

4. quality determining method as claimed in claim 2 is characterized in that, said inspection is specially:

Get medicinal powder 2g to be detected, the accurate title, decide, and puts in the conical flask of tool plug, and accurate adding volume parts claims to decide weight than being the mixed solution 50ml of 1:100 hydrochloric acid-methyl alcohol; Sonicated 40 minutes, temperature is no more than 25 ℃, puts coldly, claims to decide weight, uses volume parts to supply the weight that subtracts mistake than as the mixed solution of 1:100 hydrochloric acid-methyl alcohol; Shake up, filter, precision is measured subsequent filtrate 25ml, and to doing, the residue precision adds 0.05mol/l sulfuric acid solution 25ml at decompression and solvent recovery below 40 ℃; Jolting makes dissolving, rotating speed be 8000 rev/mins centrifugal 20 minutes, precision is measured supernatant 20ml, puts in the separating funnel, with washed with dichloromethane 4 times; Discard dichloromethane solution, treat upper strata clarification after, transfer PH to 8-9 with ammonia solution, again with ether jolting extraction 5 times; Each 20ml merges ether solution, volatilizes, and residue is used the 0.05mol/l sulfuric acid dissolution; Be transferred in the 5ml measuring bottle, shake up, filter, get subsequent filtrate as need testing solution; It is an amount of to get aconitine reference substance, Hypaconitine reference substance, mesaconine reference substance; The accurate title, decide; Add 0.05mol/l sulfuric acid and process the mixed solution that every 1ml contains aconitine, Hypaconitine, each 50 μ g of mesaconine, compare article solution, according to the high performance liquid chromatography test; With the octadecylsilane chemically bonded silica is filling agent; Is mobile phase A with volume parts than the acetonitrile-tetrahydrofuran that is 25:15, and adding glacial acetic acid 0.5ml with 0.1mol/l ammonium acetate aqueous solution and every 1000ml is Mobile phase B, carries out gradient elution, and the detection wavelength is 235nm; Number of theoretical plate calculates by the mesaconine peak should be not less than 2000; Accurate respectively reference substance solution and each 20 μ l of need testing solution of drawing inject liquid chromatograph, measure, and promptly get.

5. quality determining method as claimed in claim 1 is characterized in that, said hordenine assay is specially:

According to high effective liquid chromatography for measuring;

Chromatographic condition and system suitability test are filling agent with octyl silane group silica gel; With volume parts than be 10:90 acetonitrile-0.025mol/l potassium dihydrogen phosphate aqueous solution and use triethylamine to regulate PH to 7.2 to be moving phase; Detect wavelength 225nm; Number of theoretical plate calculates by the hordenine peak should be not less than 2000;

Medicinal powder 1g to be detected is got in the preparation of need testing solution, and accurate the title decides, and puts in the conical flask, adds volume parts than being the mixed solution 50ml of 100:1 chloroform-ammoniacal liquor, claims to decide weight; Ultrasonic 30 minutes, placement was spent the night, and claimed to decide weight, used volume parts to supply the weight that subtracts mistake than as the mixed solution of 100:1 chloroform-ammoniacal liquor, filtered; Get subsequent filtrate 25ml, decompression low temperature evaporate to dryness, the accurate 0.05mol/l sulfuric acid solution 25ml that adds of residue makes dissolving, rotating speed be 8000 rev/mins centrifugal 20 minutes; Get supernatant, put in the separating funnel, transfer PH to 8-9, extract 5 times with the ethyl acetate jolting again, each 20ml with ammonia solution; Combining extraction liquid volatilizes, and residue is used dissolve with methanol, is transferred in the 5ml measuring bottle; Shake up, filter, get subsequent filtrate, promptly get.

6. quality determining method as claimed in claim 1 is characterized in that, said Determination of Total Alkaloid is specially:

Get medicinal powder 5g to be detected, the accurate title, decide, and puts in the conical flask, adds the mixed solution 50ml of volume parts than 100:1 chloroform-ammoniacal liquor; Claim to decide weight, ultrasonic 30min, placement is spent the night; Filter, residue adds the mixed solution 50ml of volume parts than 100:1 chloroform-ammoniacal liquor, jolting 1 hour; Filter, residue use volume parts is washed 3 times than the mixed solution of 100:1 chloroform-ammoniacal liquor, each 15m; Merge washing lotion and filtrating, low-temperature reduced-pressure evaporate to dryness, residue add ethanol 10ml makes dissolving; Add activated charcoal 1g; Jolting makes decolouring, and precision is measured subsequent filtrate 5ml again; Accurate 0.01mol/l sulfuric acid vs 15ml, the water 15ml and 3 of methyl red indicator solutions of adding uses the titration of 0.02mol/l NaOH vs to yellow; Every 1ml sulfuric acid vs is equivalent to the hordenine of 1.65mg.

7. the alkaloidal quality determining method of Tangut aconite in the pharmaceutical preparation is characterized in that, adopts claim 1 or 2 described methods that Tangut aconite alkaloid in the preparation is carried out quality testing.

8. quality determining method as claimed in claim 7 is characterized in that, said quality determining method is specially:

Thin-layer chromatography is differentiated:

Get preparation powder 5-8g to be detected, add ammoniacal liquor 5ml, the 50ml that adds methylene chloride, power 250W, frequency 40kHz sonicated 30 minutes filter, the filtrating evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution; Get Tangut aconite control medicinal material 1g, shine medicinal material solution in pairs with legal system; According to the thin-layered chromatography test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate; Is developping agent with volume parts than the normal hexane-ethyl acetate-methyl alcohol-ammoniacal liquor that is 5:4:1.5:0.2, launches, and takes out; Dry, spray is with rare bismuth potassium iodide solution; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show identical orange red spot;

The diester-type alkaloids inspection:

Get preparation powder 8-15g to be detected, the accurate title, decide, and puts in the conical flask of tool plug, and accurate adding volume parts claims to decide weight than being the mixed solution 50ml of 1:100 hydrochloric acid-methyl alcohol; Sonicated 40 minutes, temperature is no more than 25 ℃, puts coldly, claims to decide weight, uses volume parts to supply the weight that subtracts mistake than as the mixed solution of 1:100 hydrochloric acid-methyl alcohol; Shake up, filter, precision is measured subsequent filtrate 25ml, and to doing, the residue precision adds 0.05mol/l sulfuric acid solution 25ml at decompression and solvent recovery below 40 ℃; Jolting makes dissolving, rotating speed be 8000 rev/mins centrifugal 20 minutes, precision is measured supernatant 20ml, puts in the separating funnel, with washed with dichloromethane 4 times; Discard dichloromethane solution, treat upper strata clarification after, transfer PH to 8-9 with ammonia solution, again with ether jolting extraction 5 times; Each 20ml merges ether solution, volatilizes, and residue is used the 0.05mol/l sulfuric acid dissolution; Be transferred in the 5ml measuring bottle, shake up, filter, get subsequent filtrate as need testing solution; It is an amount of to get aconitine reference substance, Hypaconitine reference substance, mesaconine reference substance, accurately claims surely, adds 0.05mol/l sulfuric acid and processes the mixed solution that every 1ml contains aconitine, Hypaconitine, each 50 μ g of mesaconine, compares article solution; Test according to high performance liquid chromatography; With the octadecylsilane chemically bonded silica is filling agent; Is mobile phase A with volume parts than the acetonitrile-tetrahydrofuran that is 25:15, and adding glacial acetic acid 0.5ml with 0.1mol/l ammonium acetate aqueous solution and every 1000ml is Mobile phase B, and the regulation in the according to the form below is carried out gradient elution, and the detection wavelength is 235nm; Number of theoretical plate calculates by the mesaconine peak should be not less than 2000; Accurate respectively reference substance solution and each 20 μ l of need testing solution of drawing inject liquid chromatograph, measure, and promptly get;

The hordenine assay:

According to high effective liquid chromatography for measuring;

Preparation powder 4-6g to be detected is got in the preparation of need testing solution, and accurate the title decides, and puts in the conical flask, adds volume parts than being the mixed solution 50ml of 100:1 chloroform-ammoniacal liquor, claims to decide weight; Ultrasonic 30 minutes, placement was spent the night, and claimed to decide weight, used volume parts to supply the weight that subtracts mistake than as the mixed solution of 100:1 chloroform-ammoniacal liquor, filtered; Get subsequent filtrate 25ml, decompression low temperature evaporate to dryness, the accurate 0.05mol/l sulfuric acid solution 25ml that adds of residue makes dissolving, rotating speed be 8000 rev/mins centrifugal 20 minutes; Get supernatant, put in the separating funnel, transfer PH to 8-9, extract 5 times with the ethyl acetate jolting again, each 20ml with ammonia solution; Combining extraction liquid volatilizes, and residue is used dissolve with methanol, is transferred in the 5ml measuring bottle; Shake up, filter, get subsequent filtrate, promptly get;

Determination of Total Alkaloid:

Get preparation powder 20-30g to be detected, the accurate title, decide, and puts in the conical flask, adds the mixed solution 50ml of volume parts than 100:1 chloroform-ammoniacal liquor; Claim to decide weight, ultrasonic 30min, placement is spent the night; Filter, residue adds the mixed solution 50ml of volume parts than 100:1 chloroform-ammoniacal liquor, jolting 1 hour; Filter, residue use volume parts is washed 3 times than the mixed solution of 100:1 chloroform-ammoniacal liquor, each 15m; Merge washing lotion and filtrating, low-temperature reduced-pressure evaporate to dryness, residue add ethanol 10ml makes dissolving; Add activated charcoal 1g; Jolting makes decolouring, and precision is measured subsequent filtrate 5ml again; Accurate 0.01mol/l sulfuric acid vs 15ml, the water 15ml and 3 of methyl red indicator solutions of adding uses the titration of 0.02mol/l NaOH vs to yellow; Every 1ml sulfuric acid vs is equivalent to the hordenine of 1.65mg.

9. like claim 7 or 8 described quality determining methods, it is characterized in that said preparation is eight flavor Herba Swertiae bimaculatae balls, ten golden simply balls, 13 flavor list loud, high-pitched sounds are loose or pacify the peaceful particle of youngster.