CN104422747A - Identification method for twenty aconitine compounds in traditional Chinese medicinal composition preparation - Google Patents
Identification method for twenty aconitine compounds in traditional Chinese medicinal composition preparation Download PDFInfo
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Abstract
The invention discloses an identification method for twenty aconitine compounds in a traditional Chinese medicinal composition preparation, belonging to the field of pharmaceutical analysis. The identification method comprises the following steps: preparation of a sample solution, chromatogram and mass spectrum analysis, and component identification. C19 diterpenoid alkaloids in the active part of Qiliqiangxin capsules are detected and identified by virtue of UPLC/Q-TOF-MS/MS, so that a strong evidence is provided for research on material base.
Description
Technical field
The invention belongs to Pharmaceutical Analysis field, be specifically related to the discrimination method of active ingredient of Chinese herbs.
Background technology
This Chinese medicine composition is by the Radix Astragali, monkshood, ginseng, the red sage root, lepidium seed, rhizoma alismatis, safflower, radix polygonati officinalis, dried orange peel, cassia twig, cortex periplocae etc. ten Chinese herbal medicine simply.Monkshood (Radix Aconiti Lateralis Preparata) is the dried side root of the Ranunculaceae aconitum plant rhizome of Chinese monkshood, poisonous, and has analgesia, anti-inflammatory, cardiac stimulant and the pharmacological action widely such as anticancer.Diterpenoid Alkaloids is the main chemical compositions of monkshood, be divided three classes by substituent structure difference: monoester alkaloid (monoester-diterpenoid alkaloids), diester-type alkaloids (diester-diterpenoid alkaloids) and ester-type alkaloids (1ipo-alkaloids), these are also likely one of cardiac stimulant compositions of this Chinese medicine composition.In the research of early stage for the active site composition of this Chinese medicine composition, the composition detected in monkshood is not reported to some extent.
The structure of Diterpenoid Alkaloids
R
1=CH
3or C
2H
5
R
2=H or OH
R
3=H or OH
R=CH
3COO(diester-diterpenoid alkaloids)
or OH(monoester-diterpenoid alkaloids)
Or formic acid tty acyl (lipo-alkaloids)
Summary of the invention
The object of this invention is to provide the discrimination method of 20 kinds of aconitine compounds in a kind of Chinese medicinal composition preparation.
The technical solution adopted in the present invention is:
The discrimination method of 20 kinds of aconitine compounds in a kind of Chinese medicinal composition preparation, described Chinese medicine composition is made up of the bulk drug of following weight portion: Radix Astragali 150-450 part, monkshood 40-120 part, ginseng or Radix Codonopsis 75-225 part, red sage root 75-225 part, lepidium seed 50-150 part, cortex periplocae or CORTEX ACANTHOPANACIS 60-180 part, rhizoma alismatis 75-225 part, radix polygonati officinalis 25-75 part, cassia twig 30-90 part, safflower 30-90 part, dried orange peel 25-75 part, the method adopts high performance liquid chromatography to be separated composition, with mass spectrum, compound is differentiated, described discrimination method comprises the following steps:
The preparation of A, sample solution: take Chinese medicinal composition preparation, with the ultrasonic extraction of methyl alcohol, centrifugal, get supernatant, add water, cross SPE post, then use methanol-eluted fractions, collect eluent, water-bath dries up, with methyl alcohol: water redissolves, and obtains sample solution;
B, liquid phase chromatogram condition: mobile phase is methyl alcohol and the H containing 0.1% formic acid
2o, flow velocity: 0.5ml/min; Sample size is 10 μ L; Gradient program: 0-2min, 90% containing the H of 0.1% formic acid
2o, 10% methyl alcohol; 2-13min, 90-60% contain the H of 0.1% formic acid the morning
2o, 10%-40% methyl alcohol, the H containing 0.1% formic acid of 13-19min, 60-30%
2o, 40%-70% methyl alcohol, 19-20min, 30-0% are containing the H of 0.1% formic acid
2o, 70%-100% methyl alcohol; Column temperature 40 DEG C;
Mass Spectrometry Conditions: ionization mode is ESI, positive and negative ion scans, capillary voltage is 3.5 kV, CE voltage is 45 V, and desolventizing gas is nitrogen, and desolventizing temperature is 550 DEG C, ion source temperature is 120 DEG C, MS/MS mode-10 MS/MS at 80 ms each 50-1300 m/z, IDA, dynamic background deduction is opened; Cycling time: 1 second; Data processing: PeakView
software
tMdata processing software;
C, Components identification: the molecular formula list of aconitine compound is imported in the XIC Manager in data processing software, the accurate mass number of molecular formula list and the Theoretical Mass number of calculating are contrasted the composition filtering out and meet, according to the cracking mode of the patch information analysis of compounds in second order ms, identify 20 aconitine compounds.
As optimal way, in described steps A, methyl alcohol is 50-90% methyl alcohol, and bath temperature is 40 DEG C, methyl alcohol: water is 1:1, SPE post is Waters Oasis HLB SPE pillar, 3cc 60mg.
As optimal way, in described step B, liquid phase post is Brownlee SPP liquid phase post, and specification is 2.7 μm, 2.1mm × 100mm, and nitrogen flow rate is 900 Lh
1.
As optimal way, described discrimination method comprises the following steps:
The preparation of A, sample solution: take Chinese medicinal composition preparation, with 50% methyl alcohol ultrasonic extraction 30min, extract evaporate to dryness, obtain dry sample, taking dry sample, add methyl alcohol ultrasonic dissolution, centrifugal, get supernatant, add water, Waters Oasis HLB SPE pillar, use 90% methanol-eluted fractions again, collect eluent, 40 DEG C of water-baths dry up, take ratio as the methyl alcohol of 1:1: water redissolves, and obtains sample solution;
B, liquid phase chromatogram condition: mobile phase is methyl alcohol and the H containing 0.1% formic acid
2o, flow velocity: 0.5ml/min; Sample size is 10 μ L; Gradient program: 0-2min, 90% containing the H of 0.1% formic acid
2o, 10% methyl alcohol; 2-13min, 90-60% are containing the H of 0.1% formic acid
2o, 10%-40% methyl alcohol, the H containing 0.1% formic acid of 13-19min, 60-30%
2o, 40%-70% methyl alcohol, 19-20min, 30-0% are containing the H of 0.1% formic acid
2o, 70%-100% methyl alcohol; Column temperature 40 DEG C;
Mass Spectrometry Conditions: ionization mode is ESI, positive and negative ion scans, capillary voltage is 3.5 kV, CE voltage is 45 V, and desolventizing gas is nitrogen, and desolventizing temperature is 550 DEG C, ion source temperature is 120 DEG C, MS/MS mode-10 MS/MS at 80 ms each 50-1300 m/z, IDA, dynamic background deduction is opened; Cycling time: 1 second; Data processing: PeakView
software
tMdata processing software;
C, Components identification: the molecular formula list of aconitine compound is imported in the XIC Manager in data processing software, the accurate mass number of molecular formula list and the Theoretical Mass number of calculating are contrasted the composition filtering out and meet, according to the cracking mode of the patch information analysis of compounds in second order ms, identify 20 aconitine compounds.
As optimal way, described Chinese medicine composition is made up of the bulk drug of following weight portion:
The Radix Astragali 450 parts, monkshood 112.5 parts, ginseng or Radix Codonopsis 225 parts, the red sage root 225 parts, lepidium seed 150 parts, cortex periplocae or CORTEX ACANTHOPANACIS 180 parts, rhizoma alismatis 225 parts, radix polygonati officinalis 75 parts, cassia twig 90 parts, 90 parts, safflower, dried orange peel 75 parts.
Or
The Radix Astragali 150 parts, monkshood 40 parts, ginseng or Radix Codonopsis 225 parts, the red sage root 225 parts, lepidium seed 50 parts, cortex periplocae or CORTEX ACANTHOPANACIS 180 parts, rhizoma alismatis 75 parts, radix polygonati officinalis 75 parts, cassia twig 30 parts, 90 parts, safflower, dried orange peel 25 parts.
Or
The Radix Astragali 250 parts, monkshood 112.5 parts, ginseng or Radix Codonopsis 200 parts, the red sage root 120 parts, lepidium seed 135 parts, cortex periplocae or CORTEX ACANTHOPANACIS 150 parts, rhizoma alismatis 200 parts, radix polygonati officinalis 60 parts, cassia twig 75 parts, 75 parts, safflower, dried orange peel 60 parts.
The active component of described Chinese medicine composition is made up of the following step:
(1) by the Radix Astragali, lepidium seed, rhizoma alismatis, ginseng or Radix Codonopsis, cortex periplocae or CORTEX ACANTHOPANACIS 7-9 times amount 70% alcohol extract, filter, concentrated extracting solution is the clear cream of 1.25-1.30 to the relative density when 60 DEG C of heat are surveyed;
(2) volatile oil of cassia twig, dried orange peel is extracted;
(3) monkshood, the red sage root, radix polygonati officinalis, safflower add 6-10 times amount soak by water 2 times, each 2 hours, merge extract, filter; Cassia twig, dried orange peel are put forward the aqueous solution after oil and are filtered, and collect aqueous solution filtrate, then add 6-10 times amount soak by water residue 1 hour, filter, and merge aqueous solution; The various aqueous solution of above-mentioned gained merged, being concentrated into relative density is the clear cream of 1.25-1.30, adds ethanol in stirring, to determining alcohol 70%, leaves standstill, filters, and it is the clear cream of 1.25-1.30 that filtrate is concentrated into the relative density when 60 DEG C of heat are surveyed.
In order to realize technique scheme, described Chinese medicine composition is made capsule, tablet, granule, powder or pill.
20 described aconitine compounds are respectively: middle aconine, Karma aconine, jesaconitine, aconine, secondary aconine, Fuziline, neoline, tower draws ground Sa Min, Cha Simanding, 14-acetyl tower draws ground Sa Min, 10-hydroxyl 14-Benzoylmesaconitine, 14-Benzoylmesaconitine, aconitine, 14-benzoyl aconine, 14-benzoyl time aconine, 14-benzoyl deoxidation aconine, mesaconitine, 10-hydroxyl aconitine, Hypaconitine, deoxyaconitine.
Owing to have employed technique scheme, the technical progress that the present invention obtains is:
The present invention uses UPLC/Q-TOF-MS/MS to the C in QiLi capsule active site
19diterpenoid Alkaloids detects and identifies, can provide strong evidence for the research of its material base.
Accompanying drawing explanation
Fig. 1 is the extraction chromatography of ions figure of 20 aconitine compounds, wherein 1 is middle aconine, 2 is Karma aconines, 3 is jesaconitines, 4 is aconines, 5 is time aconines, 6 is Fuzilines, 7 is neolines, 8 is that tower draws ground Sa Min, 9 is Cha Simanding, 10 is that 14-acetyl tower draws ground Sa Min, 11 is 10-hydroxyl 14-Benzoylmesaconitines, 12 is 14-Benzoylmesaconitines, 13 is aconitines, 14 is 14-benzoyl aconines, 15 is 14-benzoyl time aconines, 16 is 14-benzoyl deoxidation aconines, 17 is mesaconitine, 18 is 10-hydroxyl aconitines, 19 is Hypaconitines, 20 is deoxyaconitines,
Fig. 2 is the second order ms figure of aconitine;
Fig. 3 is the second order ms figure of mesaconitine;
Fig. 4 is the second order ms figure of Hypaconitine;
Fig. 5 is the second order ms figure of deoxyaconitine;
Fig. 6 is the second order ms figure of jesaconitine;
Fig. 7 is the second order ms figure of 10-hydroxyl aconitine;
Fig. 8 is the second order ms figure of middle aconine;
Fig. 9 is the second order ms figure of aconine;
Figure 10 is the second order ms figure of time aconine;
Figure 11 is the second order ms figure of 14-Benzoylmesaconitine;
Figure 12 is the second order ms figure of 10-hydroxyl-14-Benzoylmesaconitine;
Figure 13 is the second order ms figure of 14-benzoyl aconine;
Figure 14 is the second order ms figure of 14-benzoyl time aconine;
Figure 15 is the second order ms figure of 14-benzoyl deoxidation aconine;
Figure 16 is the second order ms figure of Karma aconine;
Figure 17 is the second order ms figure of Fuziline;
Figure 18 is the second order ms figure of neoline;
Figure 19 is the second order ms figure that tower draws ground Sa Min;
Figure 20 is the second order ms figure of Cha Simanding;
Figure 21 is the second order ms figure that 14-acetyl tower draws ground Sa Min.
Embodiment
Below in conjunction with embodiment, the present invention is described in further details:
Mass spectrometer is AB SCIEX TripleTOF
tM5600, liquid phase post is Brownlee SPP liquid phase post, and specification is 2.7 μm, 2.1mm × 100mm.
The preparation of embodiment 1 medicine capsule of the present invention
Prescription: Radix Astragali 450g monkshood 112.5g ginseng 225g red sage root 225g lepidium seed 150g
Cortex periplocae 180g rhizoma alismatis 225g safflower 90g radix polygonati officinalis 75g dried orange peel 75g cassia twig 90g
Preparation method:
(1) Radix Astragali, lepidium seed, rhizoma alismatis, ginseng, cortex periplocae are added 8 times amount 70% alcohol refluxs according to above-mentioned prescription and extract 2 times, 3 hours first times, second time 2 hours, merge extract, filter, decompression filtrate recycling ethanol, is concentrated into the clear cream that relative density is 1.25 (60 DEG C of heat are surveyed), for subsequent use;
(2) cassia twig, dried orange peel are according to prescription distillating extracting oil, and put forward the aqueous solution after oil and filter, for subsequent use, residue adds 8 times amount soak by water 1 hour again, filters, and merges decocting liquid, for subsequent use;
(3) monkshood, the red sage root, radix polygonati officinalis, safflower 9 times amount that add water decoct 2 times, each 2 hours, merge extract, filter, merge with cassia twig, dried orange peel decocting liquid in step (2), being concentrated into relative density is 1.25 (60 DEG C of heat are surveyed) clear cream, ethanol is added in stirring, to determining alcohol 70%, less than 4 DEG C leave standstill 24 hours, filter, decompression filtrate recycling ethanol, being concentrated into relative density is 1.30 (60 DEG C of heat are surveyed) clear cream, mixes, 65 DEG C of oven dry with the clear cream of alcohol extracting of step (1);
(4) dry cream mixed powder is broken into 100 order powder, adds 70% appropriate amount of ethanol and granulates, spray into cassia twig, Pericarpium Citri Reticulatae volatile oil, mixing, encapsulated, makes 1000, to obtain final product.
The discrimination method of aconitine compound, the method adopts high performance liquid chromatography to be separated composition, carries out discriminating comprise the following steps with mass spectrum to compound:
The preparation of A, sample solution: take Chinese medicinal composition preparation 1.2g, with 50% methyl alcohol ultrasonic extraction 30min, extract evaporate to dryness, obtain dry sample, then take dry sample 10mg, add 5ml methyl alcohol ultrasonic dissolution, centrifugal, get supernatant, add water, Waters Oasis HLB SPE pillar, use 90% methanol-eluted fractions again, collect eluent, 40 DEG C of water-baths dry up, take ratio as the methyl alcohol of 1:1: water redissolves, and obtains sample solution;
B, liquid phase chromatogram condition: mobile phase is methyl alcohol and the H containing 0.1% formic acid
2o, flow velocity: 0.5ml/min; Sample size is 10 μ L; Gradient program: 0-2min, 90% containing the H of 0.1% formic acid
2o, 10% methyl alcohol; The H containing 0.1% formic acid of 2-13min, 90-60%
2o, 10%-40% methyl alcohol, the H containing 0.1% formic acid of 13-19min, 60-30%
2o, 40%-70% methyl alcohol, 19-20min, 30-0% are containing the H of 0.1% formic acid
2o, 70%-100% methyl alcohol; Column temperature 40 DEG C;
Mass Spectrometry Conditions: ionization mode is ESI, positive and negative ion scans, and capillary voltage is 3.5 kV, CE voltage is 45 V, and desolventizing gas is nitrogen, and flow velocity is 900 Lh
1,desolventizing temperature is 550 DEG C, and ion source temperature is 120 DEG C, MS/MS mode-10 MS/MS at 80 ms each 50-1300 m/z, IDA, and dynamic background deduction (DBS) is opened; Cycling time: 1 second; Data processing: PeakView
software
tMdata processing software;
C, Components identification: the molecular formula list of aconitine compound is imported in the XIC Manager in data processing software, the accurate mass number of molecular formula list and the Theoretical Mass number of calculating are contrasted the composition filtering out and meet, according to the cracking mode of the patch information analysis of compounds in second order ms, identify 20 aconitine compounds, in table 1, Fig. 1.
embodiment 2: the preparation of medicinal tablet of the present invention
Prescription: Radix Astragali 150g monkshood 40g Radix Codonopsis 225g red sage root 225g lepidium seed 50g
CORTEX ACANTHOPANACIS 180g rhizoma alismatis 75g radix polygonati officinalis 75g cassia twig 30g safflower 90g dried orange peel 25g
Preparation method:
(1) Radix Astragali, lepidium seed, rhizoma alismatis, Radix Codonopsis, CORTEX ACANTHOPANACIS are proportionally measured take and add 8 times amount 70% alcohol refluxs and extract 2 times, 3 hours first times, second time 2 hours, merge extract, filter, decompression filtrate recycling ethanol, is concentrated into the clear cream that relative density is 1.25 (60 DEG C of heat are surveyed), for subsequent use;
(2) cassia twig, dried orange peel are according to recipe quantity distillating extracting oil, and put forward the aqueous solution after oil and filter, for subsequent use, residue adds 8 times amount soak by water 1 hour again, filters, and merges decocting liquid, for subsequent use;
(3) monkshood, the red sage root, radix polygonati officinalis, safflower 9 times amount that add water decoct 2 times, each 2 hours, merge extract, filter, merge with cassia twig, dried orange peel decocting liquid in step (2), being concentrated into relative density is 1.30 (60 DEG C of heat are surveyed) clear cream, ethanol is added in stirring, to determining alcohol 70%, less than 4 DEG C leave standstill 24 hours, filter, decompression filtrate recycling ethanol, being concentrated into relative density is 1.25 (60 DEG C of heat are surveyed) clear cream, mixes, 70 DEG C of oven dry with the clear cream of alcohol extracting of step (1);
(4) formulation method makes tablet routinely.
The discrimination method of aconitine compound, the method adopts high performance liquid chromatography to be separated composition, carries out discriminating comprise the following steps with mass spectrum to compound:
The preparation of A, sample solution: take Chinese medicinal composition preparation 1.2g, with 50% methyl alcohol ultrasonic extraction 30min, extract evaporate to dryness, obtain dry sample, then take dry sample 10mg, add 5ml methyl alcohol ultrasonic dissolution, centrifugal, get supernatant, add water, Waters Oasis HLB SPE pillar, use 90% methanol-eluted fractions again, collect eluent, 40 DEG C of water-baths dry up, take ratio as the methyl alcohol of 1:1: water redissolves, and obtains sample solution;
B, liquid phase chromatogram condition: mobile phase is methyl alcohol and the H containing 0.1% formic acid
2o, flow velocity: 0.5ml/min; Applied sample amount is 10 μ L; Gradient program: 0-2min, 90% containing the H of 0.1% formic acid
2o, 10% methyl alcohol; The H containing 0.1% formic acid of 2-13min, 90-60%
2o, 10%-40% methyl alcohol, the H containing 0.1% formic acid of 13-19min, 60-30%
2o, 40%-70% methyl alcohol, 19-20min, 30-0% are containing the H of 0.1% formic acid
2o, 70%-100% methyl alcohol; Column temperature 40 DEG C;
Mass Spectrometry Conditions: ionization mode is ESI, positive and negative ion scans, and capillary voltage is 3.5 kV, CE voltage is 45 V, and desolventizing gas is nitrogen, and flow velocity is 900 Lh
1,desolventizing temperature is 550 DEG C, and ion source temperature is 120 DEG C, MS/MS mode-10 MS/MS at 80 ms each 50-1300 m/z, IDA, and dynamic background deduction (DBS) is opened; Cycling time: 1 second; Data processing: PeakView
software
tMdata processing software;
C, Components identification: the molecular formula list of aconitine compound is imported in the XIC Manager in data processing software, the accurate mass number of molecular formula list and the Theoretical Mass number of calculating are contrasted the composition filtering out and meet, according to the cracking mode of the patch information analysis of compounds in second order ms, identify 20 aconitine compounds.
embodiment 3: medicine of the present inventiongranule
preparation
Prescription: Radix Astragali 250g monkshood 112.5g Radix Codonopsis 200g red sage root 120g lepidium seed 135g
Cortex periplocae 150g rhizoma alismatis 200g radix polygonati officinalis 60g cassia twig 75g safflower 75g dried orange peel 60g
Preparation method:
(1) Radix Astragali, lepidium seed, rhizoma alismatis, Radix Codonopsis, cortex periplocae are added 8 times amount 70% alcohol refluxs according to above-mentioned prescription and extract 2 times, 3 hours first times, second time 2 hours, merge extract, filter, decompression filtrate recycling ethanol, is concentrated into the clear cream that relative density is 1.28 (60 DEG C of heat are surveyed), for subsequent use;
(2) cassia twig, dried orange peel are according to prescription distillating extracting oil, and put forward the aqueous solution after oil and filter, for subsequent use, residue adds 6 times amount soak by water 1 hour again, filters, and merges decocting liquid, for subsequent use;
(3) monkshood, the red sage root, radix polygonati officinalis, safflower 9 times amount that add water decoct 2 times, each 2 hours, merge extract, filter, merge with cassia twig, dried orange peel decocting liquid in step (2), being concentrated into relative density is 1.27 (60 DEG C of heat are surveyed) clear cream, adds ethanol in stirring, to determining alcohol 70%, less than 4 DEG C leave standstill 24 hours, filter, decompression filtrate recycling ethanol, being concentrated into relative density is 1.30 (60 DEG C of heat are surveyed) clear cream, mixes with the clear cream of alcohol extracting of step (1);
(4) formulation method makes granule routinely.
The discrimination method of aconitine compound, the method adopts high performance liquid chromatography to be separated composition, carries out discriminating comprise the following steps with mass spectrum to compound:
The preparation of A, sample solution: take Chinese medicinal composition preparation 1.5g, with 90% methyl alcohol ultrasonic extraction 30min, extract evaporate to dryness, obtain dry sample, then take dry sample 10mg, add 5ml methyl alcohol ultrasonic dissolution, centrifugal, get supernatant, add water, Waters Oasis HLB SPE pillar, use 50% methanol-eluted fractions again, collect eluent, 40 DEG C of water-baths dry up, take ratio as the methyl alcohol of 1:1: water redissolves, and obtains sample solution;
B, liquid phase chromatogram condition: mobile phase is methyl alcohol and the H containing 0.1% formic acid
2o, flow velocity: 0.5ml/min; Applied sample amount is 10 μ L; Gradient program: 0-2min, 90% containing the H of 0.1% formic acid
2o, 10% methyl alcohol; The H containing 0.1% formic acid of 2-13min, 90-60%
2o, 10%-40% methyl alcohol, the H containing 0.1% formic acid of 13-19min, 60-30%
2o, 40%-70% methyl alcohol, 19-20min, 30-0% are containing the H of 0.1% formic acid
2o, 70%-100% methyl alcohol; Column temperature 40 DEG C;
Mass Spectrometry Conditions: ionization mode is ESI, positive and negative ion scans, and capillary voltage is 3.5 kV, CE voltage is 45 V, and desolventizing gas is nitrogen, and flow velocity is 900 Lh
1,desolventizing temperature is 550 DEG C, and ion source temperature is 120 DEG C, MS/MS mode-10 MS/MS at 80 ms each 50-1300 m/z, IDA, and dynamic background deduction (DBS) is opened; Cycling time: 1 second; Data processing: PeakView
software
tMdata processing software;
C, Components identification: the molecular formula list of aconitine compound is imported in the XIC Manager in data processing software, the accurate mass number of molecular formula list and the Theoretical Mass number of calculating are contrasted the composition filtering out and meet, according to the cracking mode of the patch information analysis of compounds in second order ms, identify 20 aconitine compounds.
Embodiment 4
Prescription: Radix Astragali 450g monkshood 40g ginseng 225g red sage root 225g lepidium seed 150g
Cortex periplocae 180g rhizoma alismatis 225g safflower 90g radix polygonati officinalis 75g dried orange peel 75g cassia twig 90g
Preparation method:
(1) Radix Astragali, lepidium seed, rhizoma alismatis, ginseng, cortex periplocae are added 9 times amount 70% alcohol refluxs according to above-mentioned prescription and extract 2 times, 3 hours first times, second time 2 hours, merge extract, filter, decompression filtrate recycling ethanol, is concentrated into the clear cream that relative density is 1.30 (60 DEG C of heat are surveyed), for subsequent use;
(2) cassia twig, dried orange peel are according to prescription distillating extracting oil, and put forward the aqueous solution after oil and filter, for subsequent use, residue adds 10 times amount soak by water 1 hour again, filters, and merges decocting liquid, for subsequent use;
(3) monkshood, the red sage root, radix polygonati officinalis, safflower 10 times amount that add water decoct 2 times, each 2 hours, merge extract, filter, merge with cassia twig, dried orange peel decocting liquid in step (2), being concentrated into relative density is 1.25 (60 DEG C of heat are surveyed) clear cream, ethanol is added in stirring, to determining alcohol 70%, less than 4 DEG C leave standstill 24 hours, filter, decompression filtrate recycling ethanol, being concentrated into relative density is 1.25 (60 DEG C of heat are surveyed) clear cream, mixes, 65 DEG C of oven dry with the clear cream of alcohol extracting of step (1);
(4) pill is made according to a conventional method.
The discrimination method of aconitine compound, the method adopts high performance liquid chromatography to be separated composition, carries out discriminating comprise the following steps with mass spectrum to compound:
The preparation of A, sample solution: take Chinese medicinal composition preparation 1g, with 70% methyl alcohol ultrasonic extraction 30min, extract evaporate to dryness, obtain dry sample, then take dry sample 10mg, add 5ml methyl alcohol ultrasonic dissolution, centrifugal, get supernatant, add water, Waters Oasis HLB SPE pillar, use 80% methanol-eluted fractions again, collect eluent, 40 DEG C of water-baths dry up, take ratio as the methyl alcohol of 1:1: water redissolves, and obtains sample solution;
B, liquid phase chromatogram condition: mobile phase is methyl alcohol and the H containing 0.1% formic acid
2o, flow velocity: 0.5ml/min; Applied sample amount is 10 μ L; Gradient program: 0-2min, 90% containing the H of 0.1% formic acid
2o, 10% methyl alcohol; The H containing 0.1% formic acid of 2-13min, 90-60%
2o, 10%-40% methyl alcohol, the H containing 0.1% formic acid of 13-19min, 60-30%
2o, 40%-70% methyl alcohol, 19-20min, 30-0% are containing the H of 0.1% formic acid
2o, 70%-100% methyl alcohol; Column temperature 40 DEG C;
Mass Spectrometry Conditions: ionization mode is ESI, positive and negative ion scans, and capillary voltage is 3.5 kV, CE voltage is 45 V, and desolventizing gas is nitrogen, and flow velocity is 900 Lh
1,desolventizing temperature is 550 DEG C, and ion source temperature is 120 DEG C, MS/MS mode-10 MS/MS at 80 ms each 50-1300 m/z, IDA, and dynamic background deduction (DBS) is opened; Cycling time: 1 second; Data processing: PeakView
software
tMdata processing software;
C, Components identification: the molecular formula list of aconitine compound is imported in the XIC Manager in data processing software, the accurate mass number of molecular formula list and the Theoretical Mass number of calculating are contrasted the composition filtering out and meet, according to the cracking mode of the patch information analysis of compounds in second order ms, identify 20 aconitine compounds.
Embodiment 5
Prescription: Radix Astragali 150g monkshood 120g Radix Codonopsis 75g red sage root 75g lepidium seed 50g
CORTEX ACANTHOPANACIS 60g rhizoma alismatis 75g safflower 30g radix polygonati officinalis 25g dried orange peel 25g cassia twig 30g
Preparation method:
(1) Radix Astragali, lepidium seed, rhizoma alismatis, Radix Codonopsis, CORTEX ACANTHOPANACIS are added 8 times amount 70% alcohol refluxs according to above-mentioned prescription and extract 2 times, 3 hours first times, second time 2 hours, merge extract, filter, decompression filtrate recycling ethanol, is concentrated into the clear cream that relative density is 1.28 (60 DEG C of heat are surveyed), for subsequent use;
(2) cassia twig, dried orange peel are according to prescription distillating extracting oil, and put forward the aqueous solution after oil and filter, for subsequent use, residue adds 6 times amount soak by water 1 hour again, filters, and merges decocting liquid, for subsequent use;
(3) monkshood, the red sage root, radix polygonati officinalis, safflower 9 times amount that add water decoct 2 times, each 2 hours, merge extract, filter, merge with cassia twig, dried orange peel decocting liquid in step (2), being concentrated into relative density is 1.27 (60 DEG C of heat are surveyed) clear cream, ethanol is added in stirring, to determining alcohol 70%, less than 4 DEG C leave standstill 24 hours, filter, decompression filtrate recycling ethanol, being concentrated into relative density is 1.29 (60 DEG C of heat are surveyed) clear cream, mixes, 67 DEG C of oven dry with the clear cream of alcohol extracting of step (1);
(4) powder is made according to a conventional method.
The discrimination method of aconitine compound, the method adopts high performance liquid chromatography to be separated composition, carries out discriminating comprise the following steps with mass spectrum to compound:
The preparation of A, sample solution: take Chinese medicinal composition preparation 1.2g, with 50% methyl alcohol ultrasonic extraction 30min, extract evaporate to dryness, obtain dry sample, then take dry sample 10mg, add 5ml methyl alcohol ultrasonic dissolution, centrifugal, get supernatant, add water, Waters Oasis HLB SPE pillar, use 90% methanol-eluted fractions again, collect eluent, 40 DEG C of water-baths dry up, take ratio as the methyl alcohol of 1:1: water redissolves, and obtains sample solution;
B, liquid phase chromatogram condition: mobile phase is methyl alcohol and the H containing 0.1% formic acid
2o, flow velocity: 0.5ml/min; Applied sample amount is 10 μ L; Gradient program: 0-2min, 90% containing the H of 0.1% formic acid
2o, 10% methyl alcohol; The H containing 0.1% formic acid of 2-13min, 90-60%
2o, 10%-40% methyl alcohol, the H containing 0.1% formic acid of 13-19min, 60-30%
2o, 40%-70% methyl alcohol, 19-20min, 30-0% are containing the H of 0.1% formic acid
2o, 70%-100% methyl alcohol; Column temperature 40 DEG C;
Mass Spectrometry Conditions: ionization mode is ESI, positive and negative ion scans, and capillary voltage is 3.5 kV, CE voltage is 45 V, and desolventizing gas is nitrogen, and flow velocity is 900 Lh
1,desolventizing temperature is 550 DEG C, and ion source temperature is 120 DEG C, MS/MS mode-10 MS/MS at 80 ms each 50-1300 m/z, IDA, and dynamic background deduction (DBS) is opened; Cycling time: 1 second; Data processing: PeakView
software
tMdata processing software;
C, Components identification: the molecular formula list of aconitine compound is imported in the XIC Manager in data processing software, the accurate mass number of molecular formula list and the Theoretical Mass number of calculating are contrasted the composition filtering out and meet, according to the cracking mode of the patch information analysis of compounds in second order ms, identify 20 aconitine compounds.
Concrete aconitine compound is in table 1
The mass spectrometric data of 20 compounds that table 1. adopts UPLC/Q-TOF-MS/MS to identify from QiLi capsule
1.1 diester-type alkaloids
Diester-type alkaloids be without concoct monkshood in principal ingredient, be also toxic component, but the process of preparing Chinese medicine after major part can be hydrolyzed generation monoester alkaloid, toxicity reduce to before 1/2000.
Composition
13mass spectrogram provide 646.3186 quasi-molecular ion peak, use Fomular Finder, the general structure according to diterpene alkaloid limits C number≤50, H≤200, N, O≤20, infers that its possible chemical composition is C
34h
47nO
11.MS
2m/z 554.2709 [M+H-AcOH-CH is had in spectrum
3oH]
+, 105.0332 [C
7h
5o]
+, warp and aconitine standard control
t r, accurate molecular quality is all consistent, be accredited as aconitine, see Fig. 2.
Composition
17mass spectrogram provide the quasi-molecular ion peak of m/z 632.3026, use Fomular Finder to infer that its possible chemical composition is C
33h
45nO
11, MS
2m/z 572.2832 [M+H-AcOH] is had in spectrum
+, m/z 540.2532 [M+H-AcOH-CH
3oH]
+, m/z 512.2636 [M+H-AcOH-CH
3oH-CO]
+, 105.0336 [C
7h
5o]
+fragmention, through with document to being accredited as mesaconitine, see Fig. 3.
Composition
19mass spectrogram provide the quasi-molecular ion peak of m/z 616.3090, use Fomular Finder to infer that its possible chemical composition is C
33h
45nO
10, MS
2m/z 584.2851 [M+H-CH is had in spectrum
3oH]
ten, m/z 556.2891 [M+H-AcOH]
+, m/z 524.2636 [M+H-AcOH-CH
3oH]
+, m/z 496.2669 [M+H-AcOH-CH
3oH-CO]
+, 105.0337 [C
7h
5o]
+fragmention, be accredited as Hypaconitine through to contrast with document, see Fig. 4.
Composition
20mass spectrogram provide the quasi-molecular ion peak of m/z 630.3238, use Fomular Finder to infer that its possible chemical composition is C
34h
47nO
10, MS
2m/z 570.3037 [M+H-AcOH] is had in spectrum
+, m/z 538.2769 [M+H-AcOH-CH
3oH]
+, m/z 510.2809 [M+H-AcOH-CH
3oH-CO]
+with m/z 506.2556 [M+ H-AcOH-2CH
3oH]
+fragmention, be inferred as deoxyaconitine through to contrast with document, see Fig. 5.
Same method also identifies composition in conjunction with document
3(m/z 638.2712),
18(m/z 662.3136) is respectively jesaconitine and 10-hydroxyl aconitine, sees Fig. 6, Fig. 7.
1.2 monoester alkaloid
Diester-type alkaloids can produce monoester alkaloid, C after hydrolysis or degraded
8the R base of position becomes hydroxyl from acetyl group or forms 8,9 double bonds.
Composition
1provide the quasi-molecular ion peak of m/z 486.2689, utilize Fomular Finder in conjunction with the general structure of aconite alkaloids, infer that its possible chemical composition is C
24h
40nO
9.MS
2m/z 468.2605 [M+H-H is had in spectrum
2o]
ten, m/z 454.2447 [M+H-CH
3oH]
ten, m/z 436.2331 [M+H-H
2o-CH
3oH]
ten, m/z 422.2190 [M+H-2CH
3oH]
ten, and m/z 404.2073 [M+H-H
2o-2CH
3oH]
tenfragmention, the Fracture in conjunction with document and aconitine compound is inferred as middle aconine, sees Fig. 8.
Composition
4provide the quasi-molecular ion peak of m/z 500.2844, its possible chemical composition is C to utilize Fomular Finder to infer
25h
42nO
9.MS
2m/z 468.2605 [M+H-H is had in spectrum
2o]
ten, m/z 454.2447 [M+H-CH
3oH]
ten, m/z 436.2331 [M+H-H
2o-CH
3oH]
ten, m/z 422.2190 [M+H-2CH
3oH]
ten, and m/z 404.2073 [M+H-H
2o-2CH
3oH]
tenfragmention, the Fracture in conjunction with document and aconitine compound is inferred as aconine, sees Fig. 9.Same method identifies composition
5(m/z 470.2740) is time aconine, sees Figure 10.
Composition
12provide the quasi-molecular ion peak of m/z 590.2941, Fomular Finder, in conjunction with the general structure of aconite alkaloids, infers that its possible chemical composition is C
31h
43nO
10.MS
2m/z 572.2851 [M+H-H is had in spectrum
2o]
ten, m/z 558.2685 [M+H-CH
3oH]
ten, m/z 540.2573 [M+H-H
2o-CH
3oH]
ten, m/z 526.2429 [M+H-2CH
3oH]
ten, and m/z 508.2321 [M+H-H
2o-2CH
3oH]
ten, 105.0331 [C
7h
5o]
+, 77.0392 [C
6h
5]
+fragmention, be inferred as 14-Benzoylmesaconitine in conjunction with document.What have similar fragment also has composition
11(m/z 606.2864),
14(m/z 604.3097),
15(m/z 574.2978),
16(m/z 588.3130), is accredited as 10-hydroxyl 14-Benzoylmesaconitine, 14-benzoyl aconine, 14-benzoyl time aconine and 14-benzoyl deoxidation aconine respectively.See Figure 11-15 respectively.
2.3 other alkaloids
Composition
2provide the quasi-molecular ion peak of m/z 408.2727, its possible chemical composition is C to utilize Fomular Finder to infer
23h
37nO
5.MS
2m/z 390.2626 [M+H-H is had in spectrum
2o]
ten, m/z 372.2541 [M+H-2H
2o]
ten, m/z 358.2373 [M+H-H
2o-CH
3oH]
ten, be accredited as Karma aconine in conjunction with document.Equally in conjunction with corresponding fragment information and document qualification composition
6(m/z 454.2808),
7(m/z 438.2839),
8(m/z 422.2877),
9(m/z 452.2984),
10(m/z 604.3097) is respectively Fuziline, neoline, tower draw ground Sa Min, Cha Simanding and 14-acetyl tower to draw ground Sa Min.See Figure 16-21.
Claims (10)
1. the discrimination method of 20 kinds of aconitine compounds in a Chinese medicinal composition preparation, described Chinese medicine composition is made up of the bulk drug of following weight portion: Radix Astragali 150-450 part, monkshood 40-120 part, ginseng or Radix Codonopsis 75-225 part, red sage root 75-225 part, lepidium seed 50-150 part, cortex periplocae or CORTEX ACANTHOPANACIS 60-180 part, rhizoma alismatis 75-225 part, radix polygonati officinalis 25-75 part, cassia twig 30-90 part, safflower 30-90 part, dried orange peel 25-75 part, the method adopts high performance liquid chromatography to be separated composition, with mass spectrum, compound is differentiated, it is characterized in that described discrimination method comprises the following steps:
The preparation of A, sample solution: take Chinese medicinal composition preparation, with the ultrasonic extraction of methyl alcohol, centrifugal, get supernatant, add water, cross SPE post, then use methanol-eluted fractions, collect eluent, water-bath dries up, with methyl alcohol: water redissolves, and obtains sample solution;
B, liquid phase chromatogram condition: mobile phase is methyl alcohol and the H containing 0.1% formic acid
2o, flow velocity: 0.5ml/min; Sample size is 10 μ L; Gradient program: 0-2min, 90% containing the H of 0.1% formic acid
2o, 10% methyl alcohol; 2-13min, 90-60% contain the H of 0.1% formic acid the morning
2o, 10%-40% methyl alcohol, the H containing 0.1% formic acid of 13-19min, 60-30%
2o, 40%-70% methyl alcohol, 19-20min, 30-0% are containing the H of 0.1% formic acid
2o, 70%-100% methyl alcohol; Column temperature 40 DEG C;
Mass Spectrometry Conditions: ionization mode is ESI, positive and negative ion scans, capillary voltage is 3.5 kV, CE voltage is 45 V, and desolventizing gas is nitrogen, and desolventizing temperature is 550 DEG C, ion source temperature is 120 DEG C, MS/MS mode-10 MS/MS at 80 ms each 50-1300 m/z, IDA, dynamic background deduction is opened; Cycling time: 1 second; Data processing: PeakView
software
tMdata processing software;
C, Components identification: the molecular formula list of aconitine compound is imported in the XIC Manager in data processing software, the accurate mass number of molecular formula list and the Theoretical Mass number of calculating are contrasted the composition filtering out and meet, according to the cracking mode of the patch information analysis of compounds in second order ms, identify 20 aconitine compounds.
2. discrimination method according to claim 1, it is characterized in that in described steps A, methyl alcohol is 50-90% methyl alcohol, bath temperature is 40 DEG C, methyl alcohol: water is 1:1, SPE post is Waters Oasis HLB SPE pillar.
3. discrimination method according to claim 1, it is characterized in that in described step B, liquid phase post is Brownlee SPP liquid phase post, specification is 2.7 μm, 2.1mm × 100mm, and nitrogen flow rate is 900 Lh
1.
4. discrimination method according to claim 1, is characterized in that described discrimination method is made up of following steps:
The preparation of A, sample solution: take Chinese medicinal composition preparation, with 50% methyl alcohol ultrasonic extraction 30min, extract evaporate to dryness, obtain dry sample, taking dry sample, add methyl alcohol ultrasonic dissolution, centrifugal, get supernatant, add water, Waters Oasis HLB SPE pillar, use 90% methanol-eluted fractions again, collect eluent, 40 DEG C of water-baths dry up, take ratio as the methyl alcohol of 1:1: water redissolves, and obtains sample solution;
B, liquid phase chromatogram condition: mobile phase is methyl alcohol and the H containing 0.1% formic acid
2o, flow velocity: 0.5ml/min; Sample size is 10 μ L; Gradient program: 0-2min, 90% containing the H of 0.1% formic acid
2o, 10% methyl alcohol; 2-13min, 90-60% are containing the H of 0.1% formic acid
2o, 10%-40% methyl alcohol, the H containing 0.1% formic acid of 13-19min, 60-30%
2o, 40%-70% methyl alcohol, 19-20min, 30-0% are containing the H of 0.1% formic acid
2o, 70%-100% methyl alcohol; Column temperature 40 DEG C;
Mass Spectrometry Conditions: ionization mode is ESI, positive and negative ion scans, capillary voltage is 3.5 kV, CE voltage is 45 V, and desolventizing gas is nitrogen, and desolventizing temperature is 550 DEG C, ion source temperature is 120 DEG C, MS/MS mode-10 MS/MS at 80 ms each 50-1300 m/z, IDA, dynamic background deduction is opened; Cycling time: 1 second; Data processing: PeakView
software
tMdata processing software;
C, Components identification: the molecular formula list of aconitine compound is imported in the XIC Manager in data processing software, the accurate mass number of molecular formula list and the Theoretical Mass number of calculating are contrasted the composition filtering out and meet, according to the cracking mode of the patch information analysis of compounds in second order ms, identify 20 aconitine compounds.
5. discrimination method according to claim 1, is characterized in that described Chinese medicine composition is made up of the bulk drug of following weight portion:
The Radix Astragali 450 parts, monkshood 112.5 parts, ginseng or Radix Codonopsis 225 parts, the red sage root 225 parts, lepidium seed 150 parts, cortex periplocae or CORTEX ACANTHOPANACIS 180 parts, rhizoma alismatis 225 parts, radix polygonati officinalis 75 parts, cassia twig 90 parts, 90 parts, safflower, dried orange peel 75 parts.
6. discrimination method according to claim 1, is characterized in that described Chinese medicine composition is made up of the bulk drug of following weight portion:
The Radix Astragali 150 parts, monkshood 40 parts, ginseng or Radix Codonopsis 225 parts, the red sage root 225 parts, lepidium seed 50 parts, cortex periplocae or CORTEX ACANTHOPANACIS 180 parts, rhizoma alismatis 75 parts, radix polygonati officinalis 75 parts, cassia twig 30 parts, 90 parts, safflower, dried orange peel 25 parts.
7. discrimination method according to claim 1, is characterized in that described Chinese medicine composition is made up of the bulk drug of following weight portion:
The Radix Astragali 250 parts, monkshood 112.5 parts, ginseng or Radix Codonopsis 200 parts, the red sage root 120 parts, lepidium seed 135 parts, cortex periplocae or CORTEX ACANTHOPANACIS 150 parts, rhizoma alismatis 200 parts, radix polygonati officinalis 60 parts, cassia twig 75 parts, 75 parts, safflower, dried orange peel 60 parts.
8. the discrimination method according to any one of claim 1-7, is characterized in that, the active component of described Chinese medicine composition is made up of the following step:
(1) by the Radix Astragali, lepidium seed, rhizoma alismatis, ginseng or Radix Codonopsis, cortex periplocae or CORTEX ACANTHOPANACIS 7-9 times amount 70% alcohol extract, filter, concentrated extracting solution is the clear cream of 1.25-1.30 to the relative density when 60 DEG C of heat are surveyed;
(2) volatile oil of cassia twig, dried orange peel is extracted;
(3) monkshood, the red sage root, radix polygonati officinalis, safflower add 6-10 times amount soak by water 2 times, each 2 hours, merge extract, filter; Cassia twig, dried orange peel are put forward the aqueous solution after oil and are filtered, and collect aqueous solution filtrate, then add 6-10 times amount soak by water residue 1 hour, filter, and merge aqueous solution; The various aqueous solution of above-mentioned gained merged, being concentrated into relative density is the clear cream of 1.25-1.30, adds ethanol in stirring, to determining alcohol 70%, leaves standstill, filters, and it is the clear cream of 1.25-1.30 that filtrate is concentrated into the relative density when 60 DEG C of heat are surveyed.
9. the discrimination method according to any one of claim 1-7, is characterized in that, described Chinese medicine composition is capsule, tablet, granule, powder or pill as the pharmaceutical preparation of active component.
10. the discrimination method according to any one of claim 1-7, it is characterized in that 20 described aconitine compounds are respectively: middle aconine, Karma aconine, jesaconitine, aconine, secondary aconine, Fuziline, neoline, tower draws ground Sa Min, Cha Simanding, 14-acetyl tower draws ground Sa Min, 10-hydroxyl 14-Benzoylmesaconitine, 14-Benzoylmesaconitine, aconitine, 14-benzoyl aconine, 14-benzoyl time aconine, 14-benzoyl deoxidation aconine, mesaconitine, 10-hydroxyl aconitine, Hypaconitine, deoxyaconitine.
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