CN104345111A - Determination method for content of multiple active compositions in traditional Chinese medicine composition preparation - Google Patents
Determination method for content of multiple active compositions in traditional Chinese medicine composition preparation Download PDFInfo
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- CN104345111A CN104345111A CN201310343004.5A CN201310343004A CN104345111A CN 104345111 A CN104345111 A CN 104345111A CN 201310343004 A CN201310343004 A CN 201310343004A CN 104345111 A CN104345111 A CN 104345111A
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Abstract
The invention discloses a content determination method for multiple active compositions in a traditional Chinese medicine composition preparation, and belongs to the field of traditional Chinese medicines. The concrete preparation steps comprise preparation of a test sample solution, preparation of a reference sample solution, UPLC detection, making of a standard curve and result calculation. The determination method is short in period and high in sensitivity.
Description
Technical field
The invention belongs to Analysis of Chinese Traditional Medicine field, be specifically related to the content assaying method of traditional Chinese medicine ingredients.
Background technology
This Chinese medicine composition has antipyretic and detoxicated, effect of expelling the heat-evil of a surname's lung, and be applicable to influenza therapeutic, disease is seen: heating or high heat, aversion to cold, DOMS, nasal obstruction runny nose, cough, headache, dry throat pharyngalgia, and tongue is partially red, and tongue is yellow or yellow greasy etc.Principal ingredient comprises the capsule of weeping forsythia, honeysuckle, processs Chinese ephedra, fries semen armeniacae amarae, gypsum, Radix Isatidis, thick wood-fern rhizome, cordate houttuynia, Pogostemon cablin, rheum officinale, rhodiola root, menthol, Radix Glycyrrhizae.
In order to effectively product quality can be controlled, ensure the drug safety of the public, quality control must be carried out to the intermediate link of production technology, but in this Chinese medicine composition, comparison of ingredients is many, and adopting HPLC round of visits long, repeatability, sensitivity etc. cannot meet quantity-produced needs.
Summary of the invention
The object of the invention is to provide the method for Multiple components content in short, highly sensitive a kind of Chinese medicine composition of detection of a kind of cycle.
The technical solution adopted in the present invention is:
The assay method of various active component content in a kind of Chinese medicinal composition preparation, this Chinese medicinal composition preparation is made up of the bulk drug of following weight portion: capsule of weeping forsythia 200-300 honeysuckle 200-300 Radix Isatidis 200-300 rheum officinale 40-60 Pogostemon cablin 60-100 thick wood-fern rhizome 200-300 rhodiola root 60-100 menthol 5-9 Chinese ephedra 60-100 semen armeniacae amarae 60-100 cordate houttuynia 200-300 Radix Glycyrrhizae 60-100 gypsum 200-300, and this content assaying method is made up of following steps:
The preparation of A, need testing solution: take this Chinese medicinal composition preparation 1-2g, adds methyl alcohol 50mL, weighed weight, and ultrasonic extraction 10-30min, lets cool, weighs, supply weight with methyl alcohol, filters, shakes up, to obtain final product;
The preparation of B, control sample solution: take chlorogenic acid, forsythiaside A, Cryptochlorogenic acid, Isochlorogenic acid B, Isochlorogenic acid C reference substance respectively, add 70% methyl alcohol, shake up, obtains each reference substance solution of variable concentrations;
C, UPLC detect: chromatographic column is C
18, column temperature 40 DEG C, mobile phase is methanol-acetonitrile-0.1% phosphoric acid solution, gradient elution: 0-2min, 3% methyl alcohol, 3% acetonitrile, 0.1% phosphoric acid of 94%; 2-3min, 3%-5% methyl alcohol, 3%-5% acetonitrile, 0.1% phosphoric acid of 94%-90%; 3-10min, 5%-20% methyl alcohol, 5% acetonitrile, 0.1% phosphoric acid of 90%-75%; 10-13.5min, 20% methyl alcohol, 5% acetonitrile, 0.1% phosphoric acid of 75%; 13.5-22.5min, 85% methyl alcohol, 5% acetonitrile, 0.1% phosphoric acid of 10%; Determined wavelength 237nm, flow velocity is 0.2-0.4mL/min;
The formulation of D, typical curve and result of calculation: respectively by chlorogenic acid, forsythiaside A, Cryptochlorogenic acid, 3,4-dicaffeoylquinic acid, 4, analyze in ultra high efficiency chromatograph in 5-dicaffeoylquinic acid reference substance solution 0.5-10mL implantation step c, take chromatographic peak area as ordinate, sample size is horizontal ordinate, obtains the typical curve of each reference substance solution; Then test sample solution is injected ultra high efficiency chromatograph to analyze, each Component peak area is brought in typical curve, obtains the content of chlorogenic acid, forsythiaside A, Cryptochlorogenic acid, Isochlorogenic acid B, Isochlorogenic acid C.
Preferably, in described step C, chromatographic column is ACQUITY BEH C
18, specification is 1.7 μm, 2.1 × 100mm, and flow velocity is 0.4 mL/min.
As optimal way, in Chinese medicinal composition preparation, the assay method of various active component content has following steps to form:
The preparation of A, need testing solution: take this Chinese medicinal composition preparation 1-2g, adds methyl alcohol 50mL, weighed weight, and ultrasonic extraction 10-30min, lets cool, weighs, supply weight with methyl alcohol, filters, shakes up, to obtain final product;
The preparation of B, control sample solution: take chlorogenic acid, forsythiaside A, Cryptochlorogenic acid, Isochlorogenic acid B, Isochlorogenic acid C reference substance respectively, add 70% methyl alcohol, shake up, obtain each reference substance solution of variable concentrations, chlorogenic acid concentration is 0.70mg.mL
-1, forsythiaside A is 0.21mg.mL
-1, Cryptochlorogenic acid is 0.61mg.mL
-1, Isochlorogenic acid B is 0.83mg.mL
-1, Isochlorogenic acid C is 0.18mg.mL
-1;
C, UPLC detect: chromatographic column is C
18, column temperature 40 DEG C, mobile phase is methanol-acetonitrile-0.1% phosphoric acid solution, gradient elution: 0-2min, 3% methyl alcohol, 3% acetonitrile, 0.1% phosphoric acid of 94%; 2-3min, 3%-5% methyl alcohol, 3%-5% acetonitrile, 0.1% phosphoric acid of 94%-90%; 3-10min, 5-20% methyl alcohol, 5% acetonitrile, 0.1% phosphoric acid of 90%-75%; 10-13.5min, 20% methyl alcohol, 5% acetonitrile, 0.1% phosphoric acid of 75%; 13.5-22.5min, 85% methyl alcohol, 5% acetonitrile, 0.1% phosphoric acid of 10%; Determined wavelength 237nm, flow velocity is 0.2-0.4mL/min;
The formulation of D, typical curve and result of calculation: respectively by chlorogenic acid, forsythiaside A, Cryptochlorogenic acid, 3,4-dicaffeoylquinic acid, 4, analyze in ultra high efficiency chromatograph in 5-dicaffeoylquinic acid reference substance solution 0.5-10mL implantation step c, take chromatographic peak area as ordinate, sample size is horizontal ordinate, obtains the typical curve of each reference substance solution; Then test sample solution is injected ultra high efficiency chromatograph to analyze, each Component peak area is brought in typical curve, obtains the content of chlorogenic acid, forsythiaside A, Cryptochlorogenic acid, Isochlorogenic acid B, Isochlorogenic acid C.
As preferably, this Chinese medicine composition is made up of the bulk drug of following weight portion:
The capsule of weeping forsythia 200 honeysuckle 300 Radix Isatidis 200 rheum officinale 60 Pogostemon cablin 60
Thick wood-fern rhizome 300 rhodiola root 60 menthol 9 Chinese ephedra 60 semen armeniacae amarae 100
Cordate houttuynia 200 Radix Glycyrrhizae 100 gypsum 200.
As preferably, this Chinese medicine composition is made up of the bulk drug of following weight portion:
The capsule of weeping forsythia 300 honeysuckle 200 Radix Isatidis 300 rheum officinale 60 Pogostemon cablin 100
Thick wood-fern rhizome 200 rhodiola root 60 menthol 5 Chinese ephedra 100 semen armeniacae amarae 60
Cordate houttuynia 300 Radix Glycyrrhizae 60 gypsum 300.
As preferably, this Chinese medicine composition is made up of the bulk drug of following weight portion:
The capsule of weeping forsythia 255 honeysuckle 255 Radix Isatidis 255 rheum officinale 51 Pogostemon cablin 85
Thick wood-fern rhizome 255 rhodiola root 85 menthol 7.5 Chinese ephedra 85 semen armeniacae amarae 85
Cordate houttuynia 255 Radix Glycyrrhizae 85 gypsum 255.
In order to verify feasibility and the degree of accuracy of method of the present invention, do following test:
Precision is investigated
Draw 1 μ L chlorogenic acid, forsythiaside A, Cryptochlorogenic acid, 3 respectively, 4-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid reference substance solution, continuous sample introduction 6 times respectively, measure peak area, result chlorogenic acid, forsythiaside A, Cryptochlorogenic acid, Isochlorogenic acid B, 4, the RSD of 5-dicaffeoylquinic acid reference substance peak area is respectively 1.48%, 1.30%, 1.20,1.09%% and 2.06%, illustrates that instrument precision is good.
Stability test
Need testing solution in extraction embodiment 1, in 0,2,4,8,12,24h sample introduction, measure peak area, result test sample Content of Chlorogenic Acid, forsythiaside A, Cryptochlorogenic acid, 3,4-dicaffeoylquinic acid, 4, the RSD of 5-dicaffeoylquinic acid reference substance peak area is respectively 1.10%, 1.15%, 1.56%, 1.47% and 1.71%, illustrates that in 24h, need testing solution is stablized.
Replica test
Get same batch sample, 6 parts are prepared according to the preparation method of need testing solution in embodiment 1, measure, the RSD of results sample Content of Chlorogenic Acid, forsythiaside A, Cryptochlorogenic acid, Isochlorogenic acid B, Isochlorogenic acid C is respectively 1.77%, 1.94%, 1.48%, 0.97% and 1.07%, illustration method repeatability is good.
Average recovery is tested
Adopt application of sample recovery test, precision takes the Chinese traditional medicine composition matter sample 6 parts in embodiment 1, every part all adds chlorogenic acid, forsythiaside A, Cryptochlorogenic acid, 3,4-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid reference substance solution, measure, calculate the recovery, the recovery of result chlorogenic acid, Cryptochlorogenic acid, forsythiaside A, Isochlorogenic acid B, Isochlorogenic acid C is respectively: 97.2%, 98.2%, 98.1%, 97.5% and 96.5%, RSD is respectively 1.77%, 1.94%, 1.48%, 0.97% and 1.07%.
Owing to have employed technique scheme, the technical progress that the present invention obtains is:
The present invention adopts the content of Multiple components in this Chinese medicine composition of UPLC Simultaneously test, reproducible, precision is high, analysis speed is fast, can detect the content of each composition, can control the quality of medicine more easily within the shorter cycle.
Embodiment
Below in conjunction with embodiment, the present invention is described in further details:
instrument and reagent
Instrument
Ultra Performance Liquid Chromatography instrument (model ACQUITY UPLC H CLASS, Waters, US, comprises quaternary geopressure gradient pump, vacuum degassing machine, automatic sampler, column oven, diode array detector, Empower3 chromatographic work station); Ultrasonic cleaner (KQ5200B, Kunshan Ultrasonic Instruments Co., Ltd.); Analytical balance (AG135, METTLER TO-LEDO).
Reagent
Reference substance: chlorogenic acid, forsythiaside A, (lot number is respectively: 110753-200413,111810-201001), be all purchased from Nat'l Pharmaceutical & Biological Products Control Institute.Cryptochlorogenic acid, 3.4 dicaffeoylquinic acids, 4.5-dicaffeoylquinic acid is purchased from Man Site bio tech ltd, Chengdu (purity >98%).This Chinese medicine composition (Shijiazhuang Yiling Pharmaceutical Co., Ltd provides, lot number 110121,110601,110807,110610,110710,110220,110725,110147,110817,110910,110241,120515).Acetonitrile, methyl alcohol (chromatographically pure, Fisher company of the U.S.), phosphoric acid (analyzing pure, Tianjin Kermel Chemical Reagent Co., Ltd.), water is double distilled water, and it is pure that other reagent are analysis.
Embodiment 1:
Bulk drug formula is:
The capsule of weeping forsythia 255 g honeysuckle 255 g Radix Isatidis 255 g rheum officinale 51 g Pogostemon cablin 85 g
Thick wood-fern rhizome 255 g rhodiola root 85 g menthol 7.5 g Herba ephedrae 85 g
Semen armeniacae amarae 85 g cordate houttuynia 255 g Radix Glycyrrhizae 85 g gypsum 255 g
The preparation method of Chinese medicinal composition capsules agent is:
(1) take Chinese crude drug according to above-mentioned prescription, clean;
(2) Pogostemon cablin is cataclasm, adds 6 times amount water extractions and gets volatile oil, carries the 4 hours time of oil, collects volatile oil, for subsequent use; After extracting liquid filtering, residue discards, and filtrate is for subsequent use;
(3) capsule of weeping forsythia, Chinese ephedra, cordate houttuynia, rheum officinale, by the alcohol extract 2 times of 8 times amount 70%, 2 hours first times, second time 1.5 hours, extract merges and filters, and reclaim ethanol, filtrate is for subsequent use;
(4) honeysuckle, gypsum, Radix Isatidis, thick wood-fern rhizome, Radix Glycyrrhizae, rhodiola root, add 9 times amount soak by water to boiling, add semen armeniacae amarae and decoct 2 times, 1.5 hours first times, second time 1 hour, extract merges filtration, filtrate after oil carried by gained filtrate and step (2) Pogostemon cablin merges, be condensed into and measure the clear cream that relative density is 1.10 60 DEG C time, add ethanol, being adjusted to determining alcohol is 70%, refrigeration placement 24 hours, filter, reclaim ethanol extremely without alcohol taste, gained filtrate and step (3) gained alcohol extract merge, be concentrated into and measure the clear cream that relative density is 1.15 60 DEG C time, spraying dry, get dry extract powder, for subsequent use,
(5) step (4) gained dried cream powder is added starch 138 grams, use 85% alcohol granulation;
(6) menthol, step (2) gained volatile oil are added ethanol and dissolves, spray into step (5) gained particle, airtight, mixing, loads 1000 capsules, to obtain final product.
The assay method of Multiple components content in Chinese medicinal composition preparation, is made up of following steps:
The preparation of A, need testing solution: get this Chinese medicinal composition capsules, pour out content, porphyrize, take 1g, adds methyl alcohol 50mL, weighed weight, and ultrasonic extraction 30min, lets cool, weighs, supply weight with methyl alcohol, filters, shakes up, to obtain final product;
The preparation of B, control sample solution: take chlorogenic acid, forsythiaside A, Cryptochlorogenic acid, Isochlorogenic acid B, Isochlorogenic acid C reference substance respectively, add 70% methyl alcohol, shake up, obtain each reference substance solution of variable concentrations, chlorogenic acid concentration is 0.70mg.mL
-1, forsythiaside A is 0.21mg.mL
-1, Cryptochlorogenic acid is 0.61mg.mL
-1, Isochlorogenic acid B is 0.83mg.mL
-1, Isochlorogenic acid C is 0.18mg.mL
-1;
C, UPLC detect: chromatographic column is C18, and chromatographic column is ACQUITY BEH C
18, specification is 1.7 μm, 2.1 × 100mm, column temperature 40 DEG C, and mobile phase is methanol-acetonitrile-0.1% phosphoric acid solution, gradient elution: 0-2min, 3% methyl alcohol, 3% acetonitrile, 0.1% phosphoric acid of 94%; 2-3min, 3%-5% methyl alcohol, 3%-5% acetonitrile, 0.1% phosphoric acid of 94%-90%; 3-10min, 5%-20% methyl alcohol, 5% acetonitrile, 0.1% phosphoric acid of 90%-75%; 10-13.5min, 20% methyl alcohol, 5% acetonitrile, 0.1% phosphoric acid of 75%; 13.5-22.5min, 85% methyl alcohol, 5% acetonitrile, 0.1% phosphoric acid of 10%; Determined wavelength 237nm, flow velocity is 0.4mL/min;
D, the formulation of typical curve and result of calculation: the accurate chlorogenic acid drawing 0.5mL respectively, forsythiaside A, Cryptochlorogenic acid, 3, 4-dicaffeoylquinic acid, 4, analyze in ultra high efficiency chromatograph in 5-dicaffeoylquinic acid reference substance solution implantation step c, then each reference substance solution of 1.0mL is drawn respectively, each reference substance solution of 2.0 mL, each reference substance solution of 5.0 mL, each reference substance solution of 8.0 mL, each reference substance solution of 10 mL, inject ultra high efficiency chromatograph and carry out stratographic analysis, take chromatographic peak area as ordinate, sample size is horizontal ordinate, obtain the typical curve of each control sample solution, then test sample solution is injected ultra high efficiency chromatograph to analyze, each Component peak area is brought in typical curve, obtain chlorogenic acid, forsythiaside A, Cryptochlorogenic acid, 3,4-dicaffeoylquinic acid, 4, the content of 5-dicaffeoylquinic acid, is respectively 3.29mg/g, 4.21mg/g, 0.95mg/g, 2.19 mg/g, 1.15 mg/g.
Table 1
Get 12 batches of these Chinese medicinal composition capsules samples, measure according to the method described above, result is as table 2
Embodiment 2:
Bulk drug formula is:
The capsule of weeping forsythia 300 g honeysuckle 200 g Radix Isatidis 300 g rheum officinale 60 g Pogostemon cablin 100 g
Thick wood-fern rhizome 200 g rhodiola root 60 g menthol 5 g Chinese ephedra 100 g
Semen armeniacae amarae 60 g cordate houttuynia 300 g Radix Glycyrrhizae 60 g gypsum 300 g
The preparation method of Chinese medicine composition tablet is:
(1) take Chinese crude drug according to above-mentioned prescription, clean;
(2) Pogostemon cablin is cataclasm, adds 5 times amount water extractions and gets volatile oil, carries the 4 hours time of oil, collects volatile oil, for subsequent use; After extracting liquid filtering, residue discards, and filtrate is for subsequent use;
(3) capsule of weeping forsythia, Chinese ephedra, cordate houttuynia, rheum officinale, by the alcohol extract 2 times of 6 times amount 50%, 3 hours first times, second time 1 hour, extract merges and filters, and reclaim ethanol, filtrate is for subsequent use;
(4) honeysuckle, gypsum, Radix Isatidis, thick wood-fern rhizome, Radix Glycyrrhizae, rhodiola root, add 7 times amount soak by water to boiling, add semen armeniacae amarae and decoct 2 times, 0.5 hour first time, second time 2.5 hours, extract merges filtration, filtrate after oil carried by gained filtrate and step (2) Pogostemon cablin merges, be condensed into and measure the clear cream that relative density is 1.10 60 DEG C time, add ethanol, being adjusted to determining alcohol is 70%, refrigeration placement 24 hours, filter, reclaim ethanol extremely without alcohol taste, gained filtrate and step (3) gained alcohol extract merge, be concentrated into and measure the clear cream that relative density is 1.15 60 DEG C time, spraying dry, get dry extract powder, for subsequent use,
(5) step (4) gained dried cream powder is added starch 134 grams, use 85% alcohol granulation;
(6) menthol, step (2) gained volatile oil are added ethanol and dissolves, spray into step (5) gained particle, formulation method is made tablet and be get final product routinely.
The assay method of Multiple components content in Chinese medicinal composition preparation, is made up of following steps:
The preparation of A, need testing solution: get this Chinese medicine composition tablet, porphyrize, take 1.5g, adds methyl alcohol 50mL, weighed weight, and ultrasonic extraction 10min, lets cool, weighs, supply weight with methyl alcohol, filters, shakes up, to obtain final product;
The preparation of B, control sample solution: take chlorogenic acid, forsythiaside A, Cryptochlorogenic acid, Isochlorogenic acid B, Isochlorogenic acid C reference substance respectively, add 70% methyl alcohol, shake up, obtain each reference substance solution of variable concentrations, chlorogenic acid concentration is 0.70mg.mL
-1, forsythiaside A is 0.21mg.mL
-1, Cryptochlorogenic acid is 0.61mg.mL
-1, Isochlorogenic acid B is 0.83mg.mL
-1, Isochlorogenic acid C is 0.18mg.mL
-1;
C, UPLC detect: chromatographic column is C
18, chromatographic column is ACQUITY BEH C
18, specification is 1.7 μm, 2.1 × 100mm, column temperature 40 DEG C, and mobile phase is methanol-acetonitrile-0.1% phosphoric acid solution, gradient elution: 0-2min, 3% methyl alcohol, 3% acetonitrile, 0.1% phosphoric acid of 94%; 2-3min, 3%-5% methyl alcohol, 3%-5% acetonitrile, 0.1% phosphoric acid of 94%-90%; 3-10min, 5%-20% methyl alcohol, 5% acetonitrile, 0.1% phosphoric acid of 90%-75%; 10-13.5min, 20% methyl alcohol, 5% acetonitrile, 0.1% phosphoric acid of 75%; 13.5-22.5min, 85% methyl alcohol, 5% acetonitrile, 0.1% phosphoric acid of 10%; Determined wavelength 237nm, flow velocity is 0.2mL/min;
D, the formulation of typical curve and result of calculation: the accurate chlorogenic acid drawing 0.5mL respectively, forsythiaside A, Cryptochlorogenic acid, 3, 4-dicaffeoylquinic acid, 4, analyze in ultra high efficiency chromatograph in 5-dicaffeoylquinic acid reference substance solution implantation step c, then each reference substance solution of 1.0mL is drawn respectively, each reference substance solution of 2.0 mL, each reference substance solution of 5.0 mL, each reference substance solution of 8.0 mL, each reference substance solution of 10 mL, inject ultra high efficiency chromatograph and carry out stratographic analysis, take chromatographic peak area as ordinate, sample size is horizontal ordinate, obtain the typical curve of each control sample solution, then test sample solution is injected ultra high efficiency chromatograph to analyze, each Component peak area is brought in typical curve, obtain chlorogenic acid, forsythiaside A, Cryptochlorogenic acid, 3,4-dicaffeoylquinic acid, 4, the content of 5-dicaffeoylquinic acid, is respectively 2.79mg/g, 4.01mg/g, 0.85mg/g, 2.09 mg/g, 1.12 mg/g.。
Precision is investigated
Draw 1 μ L chlorogenic acid, forsythiaside A, Cryptochlorogenic acid, 3 respectively, 4-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid reference substance solution, continuous sample introduction 6 times respectively, measure peak area, result chlorogenic acid, forsythiaside A, Cryptochlorogenic acid, Isochlorogenic acid B, 4, the RSD of 5-dicaffeoylquinic acid reference substance peak area is respectively 1.47%, 1.30%, 1.21,1.09%% and 2.05%, illustrates that instrument precision is good.
Stability test
Draw the need testing solution in the present embodiment, in 0,2,4,8,12,24h sample introduction, measure peak area, result test sample Content of Chlorogenic Acid, forsythiaside A, Cryptochlorogenic acid, 3,4-dicaffeoylquinic acid, 4, the RSD of 5-dicaffeoylquinic acid reference substance peak area is respectively 1.12%, 1.13%, 1.55%, 1.47% and 1.73%, illustrates that in 24h, need testing solution is stablized.
Replica test
Get same batch sample, 6 parts are prepared according to the preparation method of need testing solution in the present embodiment, measure, the RSD of results sample Content of Chlorogenic Acid, forsythiaside A, Cryptochlorogenic acid, Isochlorogenic acid B, Isochlorogenic acid C is respectively 1.76%, 1.95%, 1.47%, 0.97% and 1.06%, illustration method repeatability is good.
Average recovery is tested
Adopt application of sample recovery test, precision takes the Chinese traditional medicine composition matter sample 6 parts in the present embodiment, every part all adds chlorogenic acid, forsythiaside A, Cryptochlorogenic acid, 3,4-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid reference substance solution, measure, calculate the recovery, the recovery of result chlorogenic acid, Cryptochlorogenic acid, forsythiaside A, Isochlorogenic acid B, Isochlorogenic acid C is respectively: 97.1%, 98.1%, 98.2%, 97.6% and 96.4%, RSD is respectively 1.72%, 1.91%, 1.47%, 0.97% and 1.08%
Embodiment 3:
Bulk drug formula is:
The capsule of weeping forsythia 200 g honeysuckle 300g Radix Isatidis 200g rheum officinale 60g Pogostemon cablin 60g
Thick wood-fern rhizome 300g rhodiola root 60g menthol 9g Chinese ephedra 60g semen armeniacae amarae 100g
Cordate houttuynia 200g Radix Glycyrrhizae 100g gypsum 200g.
The pill preparation method of Chinese medicine composition is:
(1) take Chinese crude drug according to above-mentioned prescription, clean;
(2) Pogostemon cablin is cataclasm, adds 10 times amount water extractions and gets volatile oil, carries the 4 hours time of oil, collects volatile oil, for subsequent use; After extracting liquid filtering, residue discards, and filtrate is for subsequent use;
(3) capsule of weeping forsythia, Chinese ephedra, cordate houttuynia, rheum officinale, by the alcohol extract 2 times of 10 times amount 90%, 1 hour first time, second time 3 hours, extract merges and filters, and reclaim ethanol, filtrate is for subsequent use;
(4) honeysuckle, gypsum, Radix Isatidis, thick wood-fern rhizome, Radix Glycyrrhizae, rhodiola root, add 11 times amount soak by water to boiling, add semen armeniacae amarae and decoct 2 times, 2.5 hours first times, second time 0.5 hour, extract merges filtration, filtrate after oil carried by gained filtrate and step (2) Pogostemon cablin merges, be condensed into and measure the clear cream that relative density is 1.12 60 DEG C time, add ethanol, being adjusted to determining alcohol is 70%, refrigeration placement 24 hours, filter, reclaim ethanol extremely without alcohol taste, gained filtrate and step (3) gained alcohol extract merge, be concentrated into and measure the clear cream that relative density is 1.17 60 DEG C time, spraying dry, get dry extract powder, for subsequent use,
(5) menthol, step (2) gained volatile oil are added ethanol and dissolves, spray into step (4) gained dried cream powder, formulation method is made pill and be get final product routinely.
The assay method of Multiple components content in Chinese medicinal composition preparation, is made up of following steps:
The preparation of A, need testing solution: get this Chinese medicine composition pill, porphyrize, takes 2g, adds methyl alcohol 50mL, weighed weight, ultrasonic extraction 20min, lets cool, weighs, supply weight with methyl alcohol, filters, shakes up, to obtain final product;
The preparation of B, control sample solution: take chlorogenic acid, forsythiaside A, Cryptochlorogenic acid, Isochlorogenic acid B, Isochlorogenic acid C reference substance respectively, add 70% methyl alcohol, shake up, obtain each reference substance solution of variable concentrations, chlorogenic acid concentration is 0.70mg.mL
-1, forsythiaside A is 0.21mg.mL
-1, Cryptochlorogenic acid is 0.61mg.mL
-1, Isochlorogenic acid B is 0.83mg.mL
-1, Isochlorogenic acid C is 0.18mg.mL
-1;
C, UPLC detect: chromatographic column is C
18, chromatographic column is ACQUITY BEH C
18, specification is 1.7 μm, 2.1 × 100mm, column temperature 40 DEG C, and mobile phase is methanol-acetonitrile-0.1% phosphoric acid solution, gradient elution: 0-2min, 3% methyl alcohol, 3% acetonitrile, 0.1% phosphoric acid of 94%; 2-3min, 3%-5% methyl alcohol, 3%-5% acetonitrile, 0.1% phosphoric acid of 94%-90%; 3-10min, 5%-20% methyl alcohol, 5% acetonitrile, 0.1% phosphoric acid of 90%-75%; 10-13.5min, 20% methyl alcohol, 5% acetonitrile, 0.1% phosphoric acid of 75%; 13.5-22.5min, 85% methyl alcohol, 5% acetonitrile, 0.1% phosphoric acid of 10%; Determined wavelength 237nm, flow velocity is 0.3mL/min;
D, the formulation of typical curve and result of calculation: the accurate chlorogenic acid drawing 0.5ml respectively, forsythiaside A, Cryptochlorogenic acid, 3, 4-dicaffeoylquinic acid, 4, analyze in ultra high efficiency chromatograph in 5-dicaffeoylquinic acid reference substance solution implantation step C, then each reference substance solution of 1.0mL is drawn respectively, each reference substance solution of 2.0 mL, each reference substance solution of 5.0 mL, each reference substance solution of 8.0 mL, each reference substance solution of 10 mL, inject ultra high efficiency chromatograph and carry out stratographic analysis, take chromatographic peak area as ordinate, sample size is horizontal ordinate, obtain the typical curve of each control sample solution, then test sample solution is injected ultra high efficiency chromatograph to analyze, each Component peak area is brought in typical curve, obtain chlorogenic acid, forsythiaside A, Cryptochlorogenic acid, 3,4-dicaffeoylquinic acid, 4, the content of 5-dicaffeoylquinic acid, is respectively 2.99mg/g, 3.21mg/g, 0.85mg/g, 2.49 mg/g, 0.95 mg/g.
Precision is investigated
Draw 1 μ L chlorogenic acid, forsythiaside A, Cryptochlorogenic acid, 3 respectively, 4-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid reference substance solution, continuous sample introduction 6 times respectively, measure peak area, result chlorogenic acid, forsythiaside A, Cryptochlorogenic acid, Isochlorogenic acid B, 4, the RSD of 5-dicaffeoylquinic acid reference substance peak area is respectively 1.49%, 1.30%, 1.20,1.08% and 2.05%, illustrates that instrument precision is good.
Stability test
Draw the need testing solution in the present embodiment, in 0,2,4,8,12,24h sample introduction, measure peak area, result test sample Content of Chlorogenic Acid, forsythiaside A, Cryptochlorogenic acid, 3,4-dicaffeoylquinic acid, 4, the RSD of 5-dicaffeoylquinic acid reference substance peak area is respectively 1.11%, 1.14%, 1.55%, 1.48% and 1.71%, illustrates that in 24h, need testing solution is stablized.
Replica test
Get same batch sample, 6 parts are prepared according to the preparation method of need testing solution in the present embodiment, measure, the RSD of results sample Content of Chlorogenic Acid, forsythiaside A, Cryptochlorogenic acid, Isochlorogenic acid B, Isochlorogenic acid C is respectively 1.76%, 1.93%, 1.45%, 0.96% and 1.08%, illustration method repeatability is good.
Average recovery is tested
Adopt application of sample recovery test, precision takes the Chinese traditional medicine composition matter sample 6 parts in the present embodiment, every part all adds chlorogenic acid, forsythiaside A, Cryptochlorogenic acid, 3,4-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid reference substance solution, measure, calculate the recovery, the recovery of result chlorogenic acid, Cryptochlorogenic acid, forsythiaside A, Isochlorogenic acid B, Isochlorogenic acid C is respectively: 97.3%, 98.3%, 98.2%, 97.4% and 96.4%, RSD is respectively 1.76%, 1.93%, 1.47%, 0.96% and 1.06%.
Claims (7)
1. the assay method of various active component content in a Chinese medicinal composition preparation, this Chinese medicinal composition preparation is made up of the bulk drug of following weight portion: capsule of weeping forsythia 200-300 honeysuckle 200-300 Radix Isatidis 200-300 rheum officinale 40-60 Pogostemon cablin 60-100 thick wood-fern rhizome 200-300 rhodiola root 60-100 menthol 5-9 Chinese ephedra 60-100 semen armeniacae amarae 60-100 cordate houttuynia 200-300 Radix Glycyrrhizae 60-100 gypsum 200-300, is characterized in that this assay method is made up of following steps:
The preparation of A, need testing solution: take this Chinese medicinal composition preparation 1-2g, adds methyl alcohol 50mL, weighed weight, and ultrasonic extraction 10-30min, lets cool, weighs, supply weight with methyl alcohol, filters, shakes up, to obtain final product;
The preparation of B, control sample solution: take chlorogenic acid, forsythiaside A, Cryptochlorogenic acid, Isochlorogenic acid B, Isochlorogenic acid C reference substance respectively, add 70% methyl alcohol, shake up, obtains each reference substance solution of variable concentrations;
C, UPLC detect: chromatographic column is C18, column temperature 40 DEG C, and mobile phase is methanol-acetonitrile-0.1% phosphoric acid solution, gradient elution: 0-2min, 3% methyl alcohol, 3% acetonitrile, 0.1% phosphoric acid of 94%; 2-3min, 3%-5% methyl alcohol, 3%-5% acetonitrile, 0.1% phosphoric acid of 94%-90%; 3-10min, 5%-20% methyl alcohol, 5% acetonitrile, 0.1% phosphoric acid of 90%-75%; 10-13.5min, 20% methyl alcohol, 5% acetonitrile, 0.1% phosphoric acid of 75%; 13.5-22.5min, 85% methyl alcohol, 5% acetonitrile, 0.1% phosphoric acid of 10%; Determined wavelength 237nm, flow velocity is 0.2-0.4mL/min;
The formulation of D, typical curve and result of calculation: respectively by chlorogenic acid, forsythiaside A, Cryptochlorogenic acid, 3,4-dicaffeoylquinic acid, 4, analyze in ultra high efficiency chromatograph in 5-dicaffeoylquinic acid reference substance solution 0.5-10mL implantation step C, take chromatographic peak area as ordinate, sample size horizontal ordinate, obtains the typical curve of each reference substance solution; Then test sample solution is injected ultra high efficiency chromatograph to analyze, each Component peak area is brought in typical curve, obtains the content of chlorogenic acid, forsythiaside A, Cryptochlorogenic acid, Isochlorogenic acid B, Isochlorogenic acid C.
2. assay method according to claim 1, is characterized in that: in described step C, chromatographic column is ACQUITY BEH C
18, specification is 1.7 μm, 2.1 × 100mm, and flow velocity is 0.4 mL/min.
3. assay method according to claim 1, is characterized in that:
The preparation of A, need testing solution: take this Chinese medicinal composition preparation 1-2g, adds methyl alcohol 50mL, weighed weight, and ultrasonic extraction 10-30min, lets cool, weighs, supply weight with methyl alcohol, filters, shakes up, to obtain final product;
The preparation of B, control sample solution: take chlorogenic acid, forsythiaside A, Cryptochlorogenic acid, Isochlorogenic acid B, Isochlorogenic acid C reference substance respectively, add 70% methyl alcohol, shake up, obtain each reference substance solution of variable concentrations, chlorogenic acid concentration is 0.70mg.mL
-1, forsythiaside A is 0.21mg.mL
-1, Cryptochlorogenic acid is 0.61mg.mL
-1, Isochlorogenic acid B is 0.83mg.mL
-1, Isochlorogenic acid C is 0.18mg.mL
-1;
C, UPLC detect: chromatographic column is C
18, column temperature 40 DEG C, mobile phase is methanol-acetonitrile-0.1% phosphoric acid solution, gradient elution: 0-2min, 3% methyl alcohol, 3% acetonitrile, 0.1% phosphoric acid of 94%; 2-3min, 3%-5% methyl alcohol, 3%-5% acetonitrile, 0.1% phosphoric acid of 94%-90%; 3-10min, 5%-20% methyl alcohol, 5% acetonitrile, 0.1% phosphoric acid of 90%-75%; 10-13.5min, 20% methyl alcohol, 5% acetonitrile, 0.1% phosphoric acid of 75%; 13.5-22.5min, 85% methyl alcohol, 5% acetonitrile, 0.1% phosphoric acid of 10%; Determined wavelength 237nm, flow velocity is 0.2-0.4ml/min;
The formulation of D, typical curve and result of calculation: respectively by chlorogenic acid, forsythiaside A, Cryptochlorogenic acid, 3,4-dicaffeoylquinic acid, 4, analyze in ultra high efficiency chromatograph in 5-dicaffeoylquinic acid reference substance solution 0.5-10mL implantation step C, take chromatographic peak area as ordinate, sample size horizontal ordinate, obtains the typical curve of each reference substance solution; Then test sample solution is injected ultra high efficiency chromatograph to analyze, each Component peak area is brought in typical curve, obtains the content of chlorogenic acid, forsythiaside A, Cryptochlorogenic acid, Isochlorogenic acid B, Isochlorogenic acid C.
4. the assay method according to any one of claim 1-3, is characterized in that this Chinese medicine composition is made up of the bulk drug of following weight portion:
The capsule of weeping forsythia 200 honeysuckle 300 Radix Isatidis 200 rheum officinale 60 Pogostemon cablin 60
Thick wood-fern rhizome 300 rhodiola root 60 menthol 9 Chinese ephedra 60 semen armeniacae amarae 100
Cordate houttuynia 200 Radix Glycyrrhizae 100 gypsum 200.
5. the assay method according to any one of claim 1-3, is characterized in that this Chinese medicine composition is made up of the bulk drug of following weight portion:
The capsule of weeping forsythia 300 honeysuckle 200 Radix Isatidis 300 rheum officinale 60 Pogostemon cablin 100
Thick wood-fern rhizome 200 rhodiola root 60 menthol 5 Chinese ephedra 100 semen armeniacae amarae 60
Cordate houttuynia 300 Radix Glycyrrhizae 60 gypsum 300.
6. the assay method according to any one of claim 1-3, is characterized in that this Chinese medicine composition is made up of the bulk drug of following weight portion:
The capsule of weeping forsythia 255 honeysuckle 255 Radix Isatidis 255 rheum officinale 51 Pogostemon cablin 85
Thick wood-fern rhizome 255 rhodiola root 85 menthol 7.5 Chinese ephedra 85 semen armeniacae amarae 85
Cordate houttuynia 255 Radix Glycyrrhizae 85 gypsum 255.
7. the assay method according to any one of claim 1-3, is characterized in that described Chinese medicine composition formulation is capsule, tablet or pill.
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