CN104422747B - The discrimination method of 20 kinds of aconitine compounds containings in a kind of Chinese medicinal composition preparation - Google Patents
The discrimination method of 20 kinds of aconitine compounds containings in a kind of Chinese medicinal composition preparation Download PDFInfo
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Abstract
The invention discloses a kind of discrimination method of 20 kinds of aconitine compounds containings in Chinese medicinal composition preparation, belong to Pharmaceutical Analysis field, discrimination method comprises the following steps the preparation of sample solution, chromatograph mass spectrum analysis, Components identification, using UPLC/Q TOF MS/MS to the C in QiLi capsule active site19Diterpenoid Alkaloids are detected and identified, can provide strong evidence for the research of its material base.
Description
Technical field
The invention belongs to Pharmaceutical Analysis field, and in particular to the discrimination method of active ingredient of Chinese herbs.
Background technology
The 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, perfume (or spice)
Add Pi Dengshi Chinese herbal medicines simply.Monkshood (Radix Aconiti Lateralis Preparata) is Ranunculaceae aconitum plant
The drying lateral root of the rhizome of Chinese monkshood, it is poisonous, and with extensive pharmacological actions such as analgesia, anti-inflammatory, cardiac stimulant and anticancers.Diterpenoid Alkaloids are
The main chemical compositions of monkshood, it is divided into three classes by substituent structure difference：Monoester alkaloid (monoester-
Diterpenoid alkaloids), diester-type alkaloids (diester-diterpenoid alkaloids) and epoxy-type biology
Alkali (1ipo-alkaloids), these are it could also be possible that one of cardiac stimulant composition of the Chinese medicine composition.Early stage is for the Chinese medicine group
In the research of the active site composition of compound, the composition in detection monkshood has not been reported.
The structure of Diterpenoid Alkaloids
R1=CH3 or C2H5
R2=H or OH
R3=H or OH
R=CH3COO(diester-diterpenoid alkaloids)
or OH(monoester-diterpenoid alkaloids)
Or formic acid tty acyl (lipo-alkaloids)
The content of the invention
It is an object of the invention to provide a kind of discrimination method of 20 kinds of aconitine compounds containings in Chinese medicinal composition preparation.
The technical solution adopted in the present invention is：
The discrimination method of 20 kinds of aconitine compounds containings in a kind of Chinese medicinal composition preparation, the Chinese medicine composition be by with
The bulk drug of lower parts by weight is made：Radix Astragali 150-450 parts, monkshood 40-120 parts, ginseng or Radix Codonopsis 75-225 parts, red sage root 75-
225 parts, lepidium seed 50-150 parts, cortex periplocae or CORTEX ACANTHOPANACIS 60-180 parts, rhizoma alismatis 75-225 parts, radix polygonati officinalis 25-75 parts, cassia twig
30-90 parts, safflower 30-90 parts, dried orange peel 25-75 parts, this method is separated using high performance liquid chromatography to composition, with mass spectrum pair
Compound is differentiated that the discrimination method comprises the following steps：
A, the preparation of sample solution：Chinese medicinal composition preparation is weighed, with methanol ultrasonic extraction, centrifugation, supernatant is taken, adds
Water, SPE posts are crossed, again with methanol elution, collect eluent, water-bath drying, with methanol：Water redissolves, and obtains sample solution；
B, liquid phase chromatogram condition：Mobile phase is methanol and the H containing 0.1% formic acid2O, flow velocity：0.5ml/min；Sample size is
10μL；Gradient program：0-2min, 90% contains the H of 0.1% formic acid2O, 10% methanol；2-13min, the 90-60% morning contain 0.1% formic acid
H2O, 10%-40% methanol, 13-19min, the 60-30% H containing 0.1% formic acid2O, 40%-70% methanol, 19-20min, 30-0%
H containing 0.1% formic acid2O, 70%-100% methanol；40 DEG C of column temperature；
Mass Spectrometry Conditions：Ionization mode is ESI, and positive and negative ion scanning, capillary voltage is 3.5 kV, and CE voltages are
45 V, except solvent gas are nitrogen, except solvent 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, which deducts, to be opened；Circulation time：1 second；Data processing：
PeakViewSoftwareTMData processing software；
C, Components identification：The XIC that the molecular formula list of aconitine compounds containing is imported in data processing software
In Manager, the accurate mass number of molecular formula list is filtered out into the composition met with the Theoretical Mass number contrast calculated, according to
The cracking mode of patch information analysis of compounds in second order mses, identify 20 aconitine compounds containings.
It is preferred that methanol is 50-90% methanol in the step A, bath temperature is 40 DEG C, methanol：Water is 1:1,
SPE posts are Waters Oasis HLB SPE pillars, 3cc 60mg.
It is preferred that liquid phase post is Brownlee SPP liquid phase posts in the step B, specification is 2.7 μm, 2.1mm
× 100mm, nitrogen flow rate are 900 Lh−1。
It is preferred that the discrimination method comprises the following steps：
A, the preparation of sample solution：Chinese medicinal composition preparation is weighed, with 50% methanol ultrasonic extraction 30min, extract solution steams
It is dry, drying sample is obtained, is weighing drying sample, adds methanol ultrasonic dissolution, is centrifuged, is taken supernatant, add water, Waters Oasis
HLB SPE pillars, then eluted with 90% methanol, collect eluent, 40 DEG C of water-bath dryings, using ratio as 1:1 methanol：Water redissolves,
Obtain sample solution；
B, liquid phase chromatogram condition：Mobile phase is methanol and the H containing 0.1% formic acid2O, flow velocity：0.5ml/min；Sample size is
10μL；Gradient program：0-2min, 90% contains the H of 0.1% formic acid2O, 10% methanol；2-13min, 90-60% contain the H of 0.1% formic acid2O,
10%-40% methanol, 13-19min, the 60-30% H containing 0.1% formic acid2O, 40%-70% methanol, 19-20min, 30-0% contain 0.1%
The H of formic acid2O, 70%-100% methanol；40 DEG C of column temperature；
Mass Spectrometry Conditions：Ionization mode is ESI, and positive and negative ion scanning, capillary voltage is 3.5 kV, and CE voltages are
45 V, except solvent gas are nitrogen, except solvent 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, which deducts, to be opened；Circulation time：1 second；Data processing：
PeakViewSoftwareTMData processing software；
C, Components identification：The XIC that the molecular formula list of aconitine compounds containing is imported in data processing software
In Manager, the accurate mass number of molecular formula list is filtered out into the composition met with the Theoretical Mass number contrast calculated, according to
The cracking mode of patch information analysis of compounds in second order mses, identify 20 aconitine compounds containings.
It is preferred that the Chinese medicine composition is made up of the bulk drug of following parts by weight：
225 parts of 450 parts of the Radix Astragali, 112.5 parts of monkshood, ginseng or Radix Codonopsis, 225 parts of the red sage root, 150 parts of lepidium seed, cortex periplocae or
180 parts of CORTEX ACANTHOPANACIS, 225 parts of rhizoma alismatis, 75 parts of radix polygonati officinalis, 90 parts of cassia twig, 90 parts of safflower, 75 parts of dried orange peel.
Or
225 parts of 150 parts of the Radix Astragali, 40 parts of monkshood, ginseng or Radix Codonopsis, 225 parts of the red sage root, 50 parts of lepidium seed, cortex periplocae or south five
Add 180 parts of skin, 75 parts of rhizoma alismatis, 75 parts of radix polygonati officinalis, 30 parts of cassia twig, 90 parts of safflower, 25 parts of dried orange peel.
Or
200 parts of 250 parts of the Radix Astragali, 112.5 parts of monkshood, ginseng or Radix Codonopsis, 120 parts of the red sage root, 135 parts of lepidium seed, cortex periplocae or
150 parts of CORTEX ACANTHOPANACIS, 200 parts of rhizoma alismatis, 60 parts of radix polygonati officinalis, 75 parts of cassia twig, 75 parts of safflower, 60 parts of dried orange peel.
The active component of the Chinese medicine composition is made up of the following steps：
（1）The Radix Astragali, lepidium seed, rhizoma alismatis, ginseng or Radix Codonopsis, cortex periplocae or CORTEX ACANTHOPANACIS are measured into 70% ethanol with 7-9 times to carry
Take, filter, concentrated extracting solution to the clear cream that relative density is 1.25-1.30 when 60 DEG C of heat are surveyed；
（2）Extract cassia twig, the volatile oil of dried orange peel；
（3）Monkshood, the red sage root, radix polygonati officinalis, safflower add 6-10 times to measure decocting to boil 2 times, 2 hours every time, merge extract solution, filtration；
Cassia twig, dried orange peel propose the aqueous solution filtration after oil, collect aqueous solution filtrate, then add 6-10 times to measure decocting and boil residue 1 hour, filter,
Merge the aqueous solution；The various aqueous solution of above-mentioned gained are merged, it is 1.25-1.30 clear creams to be concentrated into relative density, is added in stirring
Enter ethanol, to determining alcohol 70%, stand, filtration, it is 1.25-1.30 clear creams that filtrate, which is concentrated into the relative density when 60 DEG C of heat are surveyed,.
In order to realize above-mentioned technical proposal, capsule, tablet, granule, powder or ball is made in the Chinese medicine composition
Agent.
20 described aconitine compounds containings are respectively：Middle aconine, Karma aconine, jesaconitine, crow
Former alkali, secondary aconine, Fuziline, neoline, tower draw ground Sa Min, Cha Simanding, 14- acetyl tower to draw ground Sa Min, 10- hydroxyl
14- Benzoylmesaconitines, 14- Benzoylmesaconitines, aconitine, 14- benzoyl aconines, 14- benzoyls
Aconine, 14- benzoyls deoxidation aconine, mesaconitine, 10- hydroxyls aconitine, Hypaconitine, deoxyaconitine.
By adopting the above-described technical solution, the technological progress that the present invention obtains is：
The present invention is using UPLC/Q-TOF-MS/MS to the C in QiLi capsule active site19Diterpenoid Alkaloids enter
Go and detected and identify, strong evidence can be provided for the research of its material base.
Brief description of the drawings
Fig. 1 is the extraction chromatography of ions figure of 20 aconitine compounds containings, wherein 1 is middle aconine, 2 be the Karma rhizome of Chinese monkshood
Former alkali, 3 be jesaconitine, and 4 be aconine, and 5 be time aconine, and 6 be Fuziline, and 7 be neoline, and 8 be that tower draws ground Sa
Quick, 9 be Cha Simanding, and 10 be that 14- acetyl tower draws ground Sa Min, and 11 be 10- hydroxyl 14- Benzoylmesaconitines, and 12 be 14- benzene
Aconine in formyl, 13 be aconitine, and 14 be 14- benzoyl aconines, and 15 be 14- benzoyl time aconines, and 16 are
14- benzoyls deoxygenate aconine, and 17 be mesaconitine, and 18 be 10- hydroxyl aconitines, and 19 be Hypaconitine, and 20 be deoxidation crow
Head alkali；
Fig. 2 is the second order mses figure of aconitine；
Fig. 3 is the second order mses figure of mesaconitine；
Fig. 4 is the second order mses figure of Hypaconitine；
Fig. 5 is the second order mses figure of deoxyaconitine；
Fig. 6 is the second order mses figure of jesaconitine；
Fig. 7 is the second order mses figure of 10- hydroxyl aconitines；
Fig. 8 is the second order mses figure of middle aconine；
Fig. 9 is the second order mses figure of aconine；
Figure 10 is the second order mses figure of time aconine；
Figure 11 is the second order mses figure of 14- Benzoylmesaconitines；
Figure 12 is the second order mses figure of 10- hydroxyl -14- Benzoylmesaconitines；
Figure 13 is the second order mses figure of 14- benzoyl aconines；
Figure 14 is the second order mses figure of 14- benzoyl time aconines；
Figure 15 is the second order mses figure of 14- benzoyls deoxidation aconine；
Figure 16 is the second order mses figure of Karma aconine；
Figure 17 is the second order mses figure of Fuziline；
Figure 18 is the second order mses figure of neoline；
Figure 19 is the second order mses figure that tower draws ground Sa Min；
Figure 20 is Cha Simanding second order mses figure；
Figure 21 is the second order mses figure that 14- acetyl tower draws ground Sa Min.
Embodiment
The present invention is described in further details with reference to embodiment：
Mass spectrograph is AB SCIEX TripleTOFTM 5600, liquid phase post is Brownlee SPP liquid phase posts, specification 2.7
μm, 2.1mm × 100mm.
The preparation of 1 medicine capsule of the present invention of embodiment
Prescription：Radix Astragali 450g monkshood 112.5g ginseng 225g red sage root 225g lepidium seeds 150g
Cortex periplocae 180g rhizoma alismatis 225g safflower 90g radix polygonati officinalis 75g dried orange peel 75g cassia twig 90g
Preparation method：
（1）The Radix Astragali, lepidium seed, rhizoma alismatis, ginseng, cortex periplocae are added into 8 times of 70% alcohol reflux of amount extractions 2 according to above-mentioned prescription
It is secondary, 3 hours for the first time, second 2 hours, merge extract solution, filtration, decompression filtrate recycling ethanol, being concentrated into relative density is
The clear cream of 1.25 (60 DEG C of heat are surveyed), it is standby；
（2）Cassia twig, dried orange peel propose the filtration of the aqueous solution after oil, standby, residue adds 8 again according to prescription distillating extracting oil
Amount decocting again is boiled 1 hour, is filtered, and merges decocting liquid, standby；
（3）Monkshood, the red sage root, radix polygonati officinalis, safflower add 9 times of amounts of water to decoct 2 times, 2 hours every time, merge extract solution, filtration, with step
Suddenly（2）Middle cassia twig, dried orange peel decocting liquid merge, and are concentrated into relative density as 1.25 (60 DEG C of heat are surveyed) clear creams, second is added in stirring
Alcohol, to determining alcohol 70%, less than 4 DEG C stand 24 hours, filtration, decompression filtrate recycling ethanol, are concentrated into relative density as 1.30
(60 DEG C of heat are surveyed) clear cream, with step（1）Alcohol extracting clear cream mixing, 65 DEG C drying；
（4）Dry cream co-grinding adds 70% appropriate amount of ethanol to pelletize into 100 mesh powder, sprays into cassia twig, Pericarpium Citri Reticulatae volatile oil, mixes,
It is encapsulated, 1000 are made, is produced.
The discrimination method of rhizome of Chinese monkshood alkali cpd, this method is separated using high performance liquid chromatography to composition, with mass spectrum pair
Compound carries out discriminating and comprised the following steps：
A, the preparation of sample solution：Chinese medicinal composition preparation 1.2g is weighed, with 50% methanol ultrasonic extraction 30min, extract solution
It is evaporated, obtains drying sample, then weigh drying sample 10mg, add 5ml methanol ultrasonic dissolutions, centrifuges, take supernatant, add water,
Waters Oasis HLB SPE pillars, then eluted with 90% methanol, collect eluent, 40 DEG C of water-bath dryings, using ratio as 1:1
Methanol：Water redissolves, and obtains sample solution；
B, liquid phase chromatogram condition：Mobile phase is methanol and the H containing 0.1% formic acid2O, flow velocity：0.5ml/min；Sample size is
10μL；Gradient program：0-2min, 90% contains the H of 0.1% formic acid2O, 10% methanol；2-13min, 90-60% containing 0.1% formic acid
H2O, 10%-40% methanol, 13-19min, the 60-30% H containing 0.1% formic acid2O, 40%-70% methanol, 19-20min, 30-0% contain
The H of 0.1% formic acid2O, 70%-100% methanol；40 DEG C of column temperature；
Mass Spectrometry Conditions：Ionization mode is ESI, and positive and negative ion scanning, capillary voltage is 3.5 kV, and CE voltages are
45 V, except solvent gas are nitrogen, flow velocity is 900 Lh1,Except solvent temperature is 550 DEG C, ion source temperature 120
DEG C, MS/MS mode-10 MS/MS at 80 ms each 50-1300 m/z, IDA, dynamic background deduct（DBS）Open；Follow
The ring time：1 second；Data processing：PeakViewSoftwareTMData processing software；
C, Components identification：The XIC that the molecular formula list of aconitine compounds containing is imported in data processing software
In Manager, the accurate mass number of molecular formula list is filtered out into the composition met with the Theoretical Mass number contrast calculated, according to
The cracking mode of patch information analysis of compounds in second order mses, 20 aconitine compounds containings are identified, are shown 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 seeds 50g
CORTEX ACANTHOPANACIS 180g rhizoma alismatis 75g radix polygonati officinalis 75g cassia twig 30g safflower 90g dried orange peels 25g
Preparation method：
（1）The Radix Astragali, lepidium seed, rhizoma alismatis, Radix Codonopsis, CORTEX ACANTHOPANACIS, which are proportionally measured, to weigh adds 8 times of 70% alcohol refluxs of amount to carry
Take 2 times, 3 hours for the first time, second 2 hours, merge extract solution, filtration, decompression filtrate recycling ethanol, be concentrated into relative density
It is standby for the clear cream of 1.25 (60 DEG C of heat are surveyed)；
（2）Cassia twig, dried orange peel propose the filtration of the aqueous solution after oil, standby, residue adds again according to recipe quantity distillating extracting oil
8 times of amount decoctings are boiled 1 hour, are filtered, and merge decocting liquid, standby；
（3）Monkshood, the red sage root, radix polygonati officinalis, safflower add 9 times of amounts of water to decoct 2 times, 2 hours every time, merge extract solution, filtration, with step
Suddenly（2）Middle cassia twig, dried orange peel decocting liquid merge, and are concentrated into relative density as 1.30 (60 DEG C of heat are surveyed) clear creams, second is added in stirring
Alcohol, to determining alcohol 70%, less than 4 DEG C stand 24 hours, filtration, decompression filtrate recycling ethanol, are concentrated into relative density as 1.25
(60 DEG C of heat are surveyed) clear cream, with step（1）Alcohol extracting clear cream mixing, 70 DEG C drying；
（4）Routinely tablet is made in formulation method.
The discrimination method of rhizome of Chinese monkshood alkali cpd, this method is separated using high performance liquid chromatography to composition, with mass spectrum pair
Compound carries out discriminating and comprised the following steps：
A, the preparation of sample solution：Chinese medicinal composition preparation 1.2g is weighed, with 50% methanol ultrasonic extraction 30min, extract solution
It is evaporated, obtains drying sample, then weigh drying sample 10mg, add 5ml methanol ultrasonic dissolutions, centrifuges, take supernatant, add water,
Waters Oasis HLB SPE pillars, then eluted with 90% methanol, collect eluent, 40 DEG C of water-bath dryings, using ratio as 1:1
Methanol：Water redissolves, and obtains sample solution；
B, liquid phase chromatogram condition：Mobile phase is methanol and the H containing 0.1% formic acid2O, flow velocity：0.5ml/min；Applied sample amount is
10μL；Gradient program：0-2min, 90% contains the H of 0.1% formic acid2O, 10% methanol；2-13min, 90-60% containing 0.1% formic acid
H2O, 10%-40% methanol, 13-19min, the 60-30% H containing 0.1% formic acid2O, 40%-70% methanol, 19-20min, 30-0% contain
The H of 0.1% formic acid2O, 70%-100% methanol；40 DEG C of column temperature；
Mass Spectrometry Conditions：Ionization mode is ESI, and positive and negative ion scanning, capillary voltage is 3.5 kV, and CE voltages are
45 V, except solvent gas are nitrogen, flow velocity is 900 Lh1,Except solvent temperature is 550 DEG C, ion source temperature 120
DEG C, MS/MS mode-10 MS/MS at 80 ms each 50-1300 m/z, IDA, dynamic background deduct（DBS）Open；Follow
The ring time：1 second；Data processing：PeakViewSoftwareTMData processing software；
C, Components identification：The XIC that the molecular formula list of aconitine compounds containing is imported in data processing software
In Manager, the accurate mass number of molecular formula list is filtered out into the composition met with the Theoretical Mass number contrast calculated, according to
The cracking mode of patch information analysis of compounds in second order mses, identify 20 aconitine compounds containings.
Embodiment 3：The preparation of medicinal granule of the present invention
Prescription：Radix Astragali 250g monkshood 112.5g Radix Codonopsis 200g red sage root 120g lepidium seeds 135g
Cortex periplocae 150g rhizoma alismatis 200g radix polygonati officinalis 60g cassia twig 75g safflower 75g dried orange peels 60g
Preparation method：
（1）The Radix Astragali, lepidium seed, rhizoma alismatis, Radix Codonopsis, cortex periplocae are added into 8 times of 70% alcohol reflux of amount extractions 2 according to above-mentioned prescription
It is secondary, 3 hours for the first time, second 2 hours, merge extract solution, filtration, decompression filtrate recycling ethanol, being concentrated into relative density is
The clear cream of 1.28 (60 DEG C of heat are surveyed), it is standby；
（2）Cassia twig, dried orange peel propose the filtration of the aqueous solution after oil, standby, residue adds 6 again according to prescription distillating extracting oil
Amount decocting again is boiled 1 hour, is filtered, and merges decocting liquid, standby；
（3）Monkshood, the red sage root, radix polygonati officinalis, safflower add 9 times of amounts of water to decoct 2 times, 2 hours every time, merge extract solution, filtration, with step
Suddenly（2）Middle cassia twig, dried orange peel decocting liquid merge, and are concentrated into relative density as 1.27 (60 DEG C of heat are surveyed) clear creams, second is added in stirring
Alcohol, to determining alcohol 70%, less than 4 DEG C stand 24 hours, filtration, decompression filtrate recycling ethanol, are concentrated into relative density as 1.30
(60 DEG C of heat are surveyed) clear cream, with step（1）Alcohol extracting clear cream mixing；
（4）Routinely granule is made in formulation method.
The discrimination method of rhizome of Chinese monkshood alkali cpd, this method is separated using high performance liquid chromatography to composition, with mass spectrum pair
Compound carries out discriminating and comprised the following steps：
A, the preparation of sample solution：Chinese medicinal composition preparation 1.5g is weighed, with 90% methanol ultrasonic extraction 30min, extract solution
It is evaporated, obtains drying sample, then weigh drying sample 10mg, add 5ml methanol ultrasonic dissolutions, centrifuges, take supernatant, add water,
Waters Oasis HLB SPE pillars, then eluted with 50% methanol, collect eluent, 40 DEG C of water-bath dryings, using ratio as 1:1
Methanol：Water redissolves, and obtains sample solution；
B, liquid phase chromatogram condition：Mobile phase is methanol and the H containing 0.1% formic acid2O, flow velocity：0.5ml/min；Applied sample amount is
10μL；Gradient program：0-2min, 90% contains the H of 0.1% formic acid2O, 10% methanol；2-13min, 90-60% containing 0.1% formic acid
H2O, 10%-40% methanol, 13-19min, the 60-30% H containing 0.1% formic acid2O, 40%-70% methanol, 19-20min, 30-0% contain
The H of 0.1% formic acid2O, 70%-100% methanol；40 DEG C of column temperature；
Mass Spectrometry Conditions：Ionization mode is ESI, and positive and negative ion scanning, capillary voltage is 3.5 kV, and CE voltages are
45 V, except solvent gas are nitrogen, flow velocity is 900 Lh1,Except solvent temperature is 550 DEG C, ion source temperature 120
DEG C, MS/MS mode-10 MS/MS at 80 ms each 50-1300 m/z, IDA, dynamic background deduct（DBS）Open；Follow
The ring time：1 second；Data processing：PeakViewSoftwareTMData processing software；
C, Components identification：The XIC that the molecular formula list of aconitine compounds containing is imported in data processing software
In Manager, the accurate mass number of molecular formula list is filtered out into the composition met with the Theoretical Mass number contrast calculated, according to
The cracking mode of patch information analysis of compounds in second order mses, identify 20 aconitine compounds containings.
Embodiment 4
Prescription：Radix Astragali 450g monkshood 40g ginseng 225g red sage root 225g lepidium seeds 150g
Cortex periplocae 180g rhizoma alismatis 225g safflower 90g radix polygonati officinalis 75g dried orange peel 75g cassia twig 90g
Preparation method：
（1）The Radix Astragali, lepidium seed, rhizoma alismatis, ginseng, cortex periplocae are added into 9 times of 70% alcohol reflux of amount extractions 2 according to above-mentioned prescription
It is secondary, 3 hours for the first time, second 2 hours, merge extract solution, filtration, decompression filtrate recycling ethanol, being concentrated into relative density is
The clear cream of 1.30 (60 DEG C of heat are surveyed), it is standby；
（2）Cassia twig, dried orange peel propose the filtration of the aqueous solution after oil, standby, residue adds 10 again according to prescription distillating extracting oil
Amount decocting again is boiled 1 hour, is filtered, and merges decocting liquid, standby；
（3）Monkshood, the red sage root, radix polygonati officinalis, safflower add 10 times of amounts of water to decoct 2 times, 2 hours every time, merge extract solution, filter, with
Step（2）Middle cassia twig, dried orange peel decocting liquid merge, and are concentrated into relative density as 1.25 (60 DEG C of heat are surveyed) clear creams, second is added in stirring
Alcohol, to determining alcohol 70%, less than 4 DEG C stand 24 hours, filtration, decompression filtrate recycling ethanol, are concentrated into relative density as 1.25
(60 DEG C of heat are surveyed) clear cream, with step（1）Alcohol extracting clear cream mixing, 65 DEG C drying；
（4）Pill is made according to a conventional method.
The discrimination method of rhizome of Chinese monkshood alkali cpd, this method is separated using high performance liquid chromatography to composition, with mass spectrum pair
Compound carries out discriminating and comprised the following steps：
A, the preparation of sample solution：Chinese medicinal composition preparation 1g is weighed, with 70% methanol ultrasonic extraction 30min, extract solution steams
It is dry, drying sample is obtained, then drying sample 10mg is weighed, add 5ml methanol ultrasonic dissolutions, centrifuge, take supernatant, add water,
Waters Oasis HLB SPE pillars, then eluted with 80% methanol, collect eluent, 40 DEG C of water-bath dryings, using ratio as 1:1
Methanol：Water redissolves, and obtains sample solution；
B, liquid phase chromatogram condition：Mobile phase is methanol and the H containing 0.1% formic acid2O, flow velocity：0.5ml/min；Applied sample amount is
10μL；Gradient program：0-2min, 90% contains the H of 0.1% formic acid2O, 10% methanol；2-13min, 90-60% containing 0.1% formic acid
H2O, 10%-40% methanol, 13-19min, the 60-30% H containing 0.1% formic acid2O, 40%-70% methanol, 19-20min, 30-0% contain
The H of 0.1% formic acid2O, 70%-100% methanol；40 DEG C of column temperature；
Mass Spectrometry Conditions：Ionization mode is ESI, and positive and negative ion scanning, capillary voltage is 3.5 kV, and CE voltages are
45 V, except solvent gas are nitrogen, flow velocity is 900 Lh1,Except solvent temperature is 550 DEG C, ion source temperature 120
DEG C, MS/MS mode-10 MS/MS at 80 ms each 50-1300 m/z, IDA, dynamic background deduct（DBS）Open；Follow
The ring time：1 second；Data processing：PeakViewSoftwareTMData processing software；
C, Components identification：The XIC that the molecular formula list of aconitine compounds containing is imported in data processing software
In Manager, the accurate mass number of molecular formula list is filtered out into the composition met with the Theoretical Mass number contrast calculated, according to
The cracking mode of patch information analysis of compounds in second order mses, identify 20 aconitine compounds containings.
Embodiment 5
Prescription：Radix Astragali 150g monkshood 120g Radix Codonopsis 75g red sage root 75g lepidium seeds 50g
CORTEX ACANTHOPANACIS 60g rhizoma alismatis 75g safflower 30g radix polygonati officinalis 25g dried orange peel 25g cassia twig 30g
Preparation method：
（1）The Radix Astragali, lepidium seed, rhizoma alismatis, Radix Codonopsis, CORTEX ACANTHOPANACIS are added into 8 times of amount 70% alcohol reflux extractions according to above-mentioned prescription
2 times, 3 hours for the first time, second 2 hours, merge extract solution, filtration, decompression filtrate recycling ethanol, being concentrated into relative density is
The clear cream of 1.28 (60 DEG C of heat are surveyed), it is standby；
（2）Cassia twig, dried orange peel propose the filtration of the aqueous solution after oil, standby, residue adds 6 again according to prescription distillating extracting oil
Amount decocting again is boiled 1 hour, is filtered, and merges decocting liquid, standby；
（3）Monkshood, the red sage root, radix polygonati officinalis, safflower add 9 times of amounts of water to decoct 2 times, 2 hours every time, merge extract solution, filtration, with step
Suddenly（2）Middle cassia twig, dried orange peel decocting liquid merge, and are concentrated into relative density as 1.27 (60 DEG C of heat are surveyed) clear creams, second is added in stirring
Alcohol, to determining alcohol 70%, less than 4 DEG C stand 24 hours, filtration, decompression filtrate recycling ethanol, are concentrated into relative density as 1.29
(60 DEG C of heat are surveyed) clear cream, with step（1）Alcohol extracting clear cream mixing, 67 DEG C drying；
（4）Powder is made according to a conventional method.
The discrimination method of rhizome of Chinese monkshood alkali cpd, this method is separated using high performance liquid chromatography to composition, with mass spectrum pair
Compound carries out discriminating and comprised the following steps：
A, the preparation of sample solution：Chinese medicinal composition preparation 1.2g is weighed, with 50% methanol ultrasonic extraction 30min, extract solution
It is evaporated, obtains drying sample, then weigh drying sample 10mg, add 5ml methanol ultrasonic dissolutions, centrifuges, take supernatant, add water,
Waters Oasis HLB SPE pillars, then eluted with 90% methanol, collect eluent, 40 DEG C of water-bath dryings, using ratio as 1:1
Methanol：Water redissolves, and obtains sample solution；
B, liquid phase chromatogram condition：Mobile phase is methanol and the H containing 0.1% formic acid2O, flow velocity：0.5ml/min；Applied sample amount is
10μL；Gradient program：0-2min, 90% contains the H of 0.1% formic acid2O, 10% methanol；2-13min, 90-60% containing 0.1% formic acid
H2O, 10%-40% methanol, 13-19min, the 60-30% H containing 0.1% formic acid2O, 40%-70% methanol, 19-20min, 30-0% contain
The H of 0.1% formic acid2O, 70%-100% methanol；40 DEG C of column temperature；
Mass Spectrometry Conditions：Ionization mode is ESI, and positive and negative ion scanning, capillary voltage is 3.5 kV, and CE voltages are
45 V, except solvent gas are nitrogen, flow velocity is 900 Lh1,Except solvent temperature is 550 DEG C, ion source temperature 120
DEG C, MS/MS mode-10 MS/MS at 80 ms each 50-1300 m/z, IDA, dynamic background deduct（DBS）Open；Follow
The ring time：1 second；Data processing：PeakViewSoftwareTMData processing software；
C, Components identification：The XIC that the molecular formula list of aconitine compounds containing is imported in data processing software
In Manager, the accurate mass number of molecular formula list is filtered out into the composition met with the Theoretical Mass number contrast calculated, according to
The cracking mode of patch information analysis of compounds in second order mses, identify 20 aconitine compounds containings.
Specific aconitine compounds containing is shown in Table 1
The mass spectrometric data for 20 compounds that the use of table 1. UPLC/Q-TOF-MS/MS identifies from QiLi capsule
1.1 diester-type alkaloids
Diester-type alkaloids are the main components in the monkshood without processing, and toxic component, but after processing
Major part can hydrolyze generation monoester alkaloid, toxicity be reduced to before 1/2000.
The mass spectrogram of composition 13 provides 646.3186 quasi-molecular ion peak, using Fomular Finder, according to diterpene
The general structure of alkaloid limits C number≤50, H≤200, N, O≤20, thus it is speculated that its possible chemical composition is C34H47NO11。
MS2There are [the M+H-AcOH-CH of m/z 554.2709 in spectrum3OH] +, 105.0332 [C7H5O] +, through with aconitine standard controlt R, accurate molecular quality it is consistent, be accredited as aconitine, see Fig. 2.
The mass spectrogram of composition 17 provides m/z 632.3026 quasi-molecular ion peak, speculates it using Fomular Finder
Possible chemical composition is C33H45NO11, MS2There is m/z 572.2832 [M+H- AcOH] in spectrum+, m/z 540.2532 [M+H-
AcOH-CH3OH]+, [the M+H-AcOH-CH of m/z 512.26363OH-CO]+, 105.0336[C7H5O] +Fragment ion, through with
Document is shown in Fig. 3 to being accredited as mesaconitine
The mass spectrogram of composition 19 provides m/z 616.3090 quasi-molecular ion peak, speculates it using Fomular Finder
Possible chemical composition is C33H45NO10, MS2There are [the M+H-CH of m/z 584.2851 in spectrum3OH]Ten, [the M+H- of m/z 556.2891
AcOH]+, [the M+H-AcOH-CH of m/z 524.26363OH]+, [the M+H-AcOH-CH of m/z 496.26693OH-CO]+, 105.0337
[C7H5O] +Fragment ion, be accredited as Hypaconitine through being compareed with document, see Fig. 4.
The mass spectrogram of composition 20 provides m/z 630.3238 quasi-molecular ion peak, speculates it using Fomular Finder
Possible chemical composition is C34H47NO10, MS2There is m/z 570.3037 [M+H-AcOH] in spectrum+, [the M+H- of m/z 538.2769
AcOH-CH3OH] +, [the M+H-AcOH-CH of m/z 510.28093OH-CO] +With m/z 506.2556 [M+ H-AcOH-
2CH3OH] +Fragment ion, be inferred as deoxyaconitine through being compareed with document, see Fig. 5.
Same method simultaneously identifies composition 3 with reference to document（m/z 638.2712）, 18 (m/z 662.3136) be respectively outstanding person
This aconitine and 10- hydroxyl aconitines, are shown in Fig. 6, Fig. 7.
1.2 monoester alkaloid
Diester-type alkaloids can produce monoester alkaloid, C after hydrolysis or degraded8The R bases of position are changed into from acetyl group
8,9 double bonds of hydroxyl or formation.
Composition 1 provides m/z 486.2689 quasi-molecular ion peak, utilizes Fomular Finder combinations Aconitum biology
The general structure of alkali, it is C to infer its possible chemical composition24H40NO9。MS2There are [the M+H-H of m/z 468.2605 in spectrum2O]Ten, m/
z 454.2447 [M+H-CH3OH]Ten, [the M+H- H of m/z 436.23312O-CH3OH]Ten, [the M+H-2CH of m/z 422.21903OH
]Ten, and [the M+H-H of m/z 404.20732O-2CH3OH]TenFragment ion, with reference to the fracture of document and aconitine compounds containing
Rule is inferred as middle aconine, sees Fig. 8.
Composition 4 provides m/z 500.2844 quasi-molecular ion peak, infers its possibleization using Fomular Finder
It is C to learn composition25H42NO9。MS2There are [the M+H- H of m/z 468.2605 in spectrum2O]Ten, [the M+H-CH of m/z 454.24473OH]Ten, m/z
436.2331[M+H- H2O-CH3OH]Ten, [the M+H-2CH of m/z 422.21903OH]Ten, and [the M+H-H of m/z 404.20732O-
2CH3OH]TenFragment ion, be inferred as aconine with reference to the Fracture of document and aconitine compounds containing, see Fig. 9.
Same method identifies composition 5（m/z 470.2740）For secondary aconine, Figure 10 is seen.
Composition 12 provides m/z 590.2941 quasi-molecular ion peak, Fomular Finder combination aconite alkaloids
General structure, it is C to infer its possible chemical composition31H43NO10。MS2There are [the M+H- H of m/z 572.2851 in spectrum2O]Ten, m/z
558.2685 [M+H-CH3OH]Ten, [the M+H- H of m/z 540.25732O-CH3OH]Ten, [the M+H-2CH of m/z 526.24293OH
]Ten, and [the M+H-H of m/z 508.23212O-2CH3OH]Ten, 105.0331 [C7H5O] +, 77.0392 [C6H5] +Fragment ion,
14- Benzoylmesaconitines are inferred as with reference to document.There is composition 11 with similar fragment（m/z 606.2864）、14
（m/z 604.3097）、15（m/z 574.2978）、16（m/z 588.3130）, it is identified respectively as in 10- hydroxyl 14- benzoyls
Aconine, 14- benzoyl aconines, 14- benzoyl time aconines and 14- benzoyls deoxidation aconine.See respectively
Figure 11-15.
2.3 other alkaloids
Composition 2 provides m/z 408.2727 quasi-molecular ion peak, infers its possibleization using Fomular Finder
It is C to learn composition23H37NO5。MS2There are [the M+H- H of m/z 390.2626 in spectrum2O]Ten, [the M+H- 2H of m/z 372.25412O]Ten, m/z
358.2373[M+H- H2O-CH3OH]Ten, Karma aconine is accredited as with reference to document.Also in conjunction with corresponding fragment information and text
Offer identification 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）Respectively Fuziline, neoline, tower draw ground Sa Min, Cha Simanding and 14- acetyl tower to draw ground Sa Min.See figure
16-21。
Claims (9)
1. the discrimination method of 20 kinds of aconitine compounds containings in a kind of Chinese medicinal composition preparation, the Chinese medicine composition are by following
The bulk drug of parts by weight is made：Radix Astragali 150-450 parts, monkshood 40-120 parts, ginseng or Radix Codonopsis 75-225 parts, red sage root 75-225
Part, lepidium seed 50-150 parts, cortex periplocae or CORTEX ACANTHOPANACIS 60-180 parts, rhizoma alismatis 75-225 parts, radix polygonati officinalis 25-75 parts, cassia twig 30-90
Part, safflower 30-90 parts, dried orange peel 25-75 parts, this method is separated using high performance liquid chromatography to composition, with mass spectrum to chemical combination
Thing is differentiated, it is characterised in that the discrimination method comprises the following steps：
A, the preparation of sample solution：Chinese medicinal composition preparation is weighed, with methanol aqueous solution ultrasonic extraction, centrifugation, supernatant is taken, adds
Water, SPE posts are crossed, the elution of the again with methanol aqueous solution, collect eluent, water-bath drying, with methanol：Water redissolves, and obtains sample solution；
B, liquid phase chromatogram condition：Mobile phase is methanol and the H containing 0.1% formic acid2O, flow velocity：0.5ml/min；Sample size is 10 μ L；
Gradient program：0-2min, 90% contains the H of 0.1% formic acid2O, 10% methanol；2-13min, 90-60% contain the H of 0.1% formic acid2O, 10%-
40% methanol, 13-19min, the 60-30% H containing 0.1% formic acid2O, 40%-70% methanol, 19-20min, 30-0% contain 0.1% formic acid
H2O, 70%-100% methanol；40 DEG C of column temperature；
Mass Spectrometry Conditions：Ionization mode is ESI, and positive and negative ion scanning, capillary voltage is 3.5 kV, and CE voltages are 45 V,
Except solvent gas are nitrogen, except solvent 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, which deducts, to be opened；Circulation time：1 second；Data processing：
PeakView SoftwareTMData processing software；
C, Components identification：The molecular formula list of aconitine compounds containing is imported in the XIC Manager in data processing software,
The accurate mass number of molecular formula list is filtered out into the composition met with the Theoretical Mass number contrast calculated, according in second order mses
Patch information analysis of compounds cracking mode, identify 20 kinds of aconitine compounds containings, described 20 kinds of aconitines
Compound is respectively：Middle aconine, Karma aconine, jesaconitine, aconine, secondary aconine, Fuziline, Ni Ao
Spirit, tower draw ground Sa Min, Cha Simanding, 14- acetyl tower to draw ground Sa Min, 10- hydroxyl 14- Benzoylmesaconitines, 14- benzoyls
Middle aconine, aconitine, 14- benzoyl aconines, 14- benzoyls time aconine, the 14- benzoyls deoxidation rhizome of Chinese monkshood are former
Alkali, mesaconitine, 10- hydroxyls aconitine, Hypaconitine, deoxyaconitine.
2. discrimination method according to claim 1, it is characterised in that methanol aqueous solution is 50-90% methanol in the step A
The aqueous solution, bath temperature are 40 DEG C, methanol：Water volume ratio is 1:1, SPE post is Waters Oasis HLB SPE pillars.
3. discrimination method according to claim 1, it is characterised in that liquid phase post is Brownlee SPP liquid in the step B
Xiang Zhu, specification are 2.7 μm, 2.1mm × 100mm, and nitrogen flow rate is 900 Lh−1。
4. discrimination method according to claim 1, it is characterised in that the discrimination method is made up of following steps：
A, the preparation of sample solution：Chinese medicinal composition preparation is weighed, with 50% methanol aqueous solution ultrasonic extraction 30min, extract solution steams
It is dry, drying sample is obtained, then drying sample is weighed, add methanol ultrasonic dissolution, centrifuge, take supernatant, add water, Waters Oasis
HLB SPE pillars, then eluted with 90% methanol aqueous solution, collect eluent, 40 DEG C of water-bath dryings, using volume ratio as 1:1 first
Alcohol：Water redissolves, and obtains sample solution；
B, liquid phase chromatogram condition：Mobile phase is methanol and the H containing 0.1% formic acid2O, flow velocity：0.5ml/min；Sample size is 10 μ L；
Gradient program：0-2min, 90% contains the H of 0.1% formic acid2O, 10% methanol；2-13min, 90-60% contain the H of 0.1% formic acid2O, 10%-
40% methanol, 13-19min, the 60-30% H containing 0.1% formic acid2O, 40%-70% methanol, 19-20min, 30-0% contain 0.1% formic acid
H2O, 70%-100% methanol；40 DEG C of column temperature；
Mass Spectrometry Conditions：Ionization mode is ESI, and positive and negative ion scanning, capillary voltage is 3.5 kV, and CE voltages are 45 V,
Except solvent gas are nitrogen, except solvent 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, which deducts, to be opened；Circulation time：1 second；Data processing：
PeakView SoftwareTMData processing software；
C, Components identification：The molecular formula list of aconitine compounds containing is imported in the XIC Manager in data processing software,
The accurate mass number of molecular formula list is filtered out into the composition met with the Theoretical Mass number contrast calculated, according in second order mses
Patch information analysis of compounds cracking mode, identify 20 kinds of aconitine compounds containings.
5. discrimination method according to claim 1, it is characterised in that the Chinese medicine composition by following parts by weight raw material
Medicine is made：
225 parts of 450 parts of the Radix Astragali, 112.5 parts of monkshood, ginseng or Radix Codonopsis, 225 parts of the red sage root, 150 parts of lepidium seed, cortex periplocae or south five
Add 180 parts of skin, 225 parts of rhizoma alismatis, 75 parts of radix polygonati officinalis, 90 parts of cassia twig, 90 parts of safflower, 75 parts of dried orange peel.
6. discrimination method according to claim 1, it is characterised in that the Chinese medicine composition by following parts by weight raw material
Medicine is made：
225 parts of 150 parts of the Radix Astragali, 40 parts of monkshood, ginseng or Radix Codonopsis, 225 parts of the red sage root, 50 parts of lepidium seed, cortex periplocae or CORTEX ACANTHOPANACIS
180 parts, 75 parts of rhizoma alismatis, 75 parts of radix polygonati officinalis, 30 parts of cassia twig, 90 parts of safflower, 25 parts of dried orange peel.
7. discrimination method according to claim 1, it is characterised in that the Chinese medicine composition by following parts by weight raw material
Medicine is made：
200 parts of 250 parts of the Radix Astragali, 112.5 parts of monkshood, ginseng or Radix Codonopsis, 120 parts of the red sage root, 135 parts of lepidium seed, cortex periplocae or south five
Add 150 parts of skin, 200 parts of rhizoma alismatis, 60 parts of radix polygonati officinalis, 75 parts of cassia twig, 75 parts of safflower, 60 parts of dried orange peel.
8. according to the discrimination method described in claim any one of 1-7, it is characterised in that the active component of the Chinese medicine composition
It is made up of the following steps：
（1）The Radix Astragali, lepidium seed, rhizoma alismatis, ginseng or Radix Codonopsis, cortex periplocae or CORTEX ACANTHOPANACIS are measured into 70% ethanol with 7-9 times to extract, filter
Cross, concentrated extracting solution to the clear cream that relative density is 1.25-1.30 when 60 DEG C of heat are surveyed；
（2）Extract cassia twig, the volatile oil of dried orange peel；
（3）Monkshood, the red sage root, radix polygonati officinalis, safflower add 6-10 times to measure decocting to boil 2 times, 2 hours every time, merge extract solution, filtration；Cassia twig,
Dried orange peel proposes the aqueous solution filtration after oil, collects aqueous solution filtrate, then adds 6-10 times to measure decocting and boil residue 1 hour, filters, merges water
Solution；The various aqueous solution of above-mentioned gained are merged, it is 1.25-1.30 clear creams to be concentrated into relative density, and ethanol is added in stirring,
To determining alcohol 70%, stand, filtration, it is 1.25-1.30 clear creams that filtrate, which is concentrated into the relative density when 60 DEG C of heat are surveyed,.
9. according to the discrimination method any one of claim 1-7, it is characterised in that the Chinese medicine composition is as activity
The pharmaceutical preparation of composition is capsule, tablet, granule, powder or pill.
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