CN103105371B

CN103105371B - Quality detection method of pharmaceutical composition injection

Info

Publication number: CN103105371B
Application number: CN201110354967.6A
Authority: CN
Inventors: 段宏泉; 翁红; 张子安; 李颖; 杨玲
Original assignee: CHANGBAISHAN PHARMACEUTICAL Co Ltd
Current assignee: Changbaishan Pharmaceutical Co ltd
Priority date: 2011-11-10
Filing date: 2011-11-10
Publication date: 2015-03-18
Anticipated expiration: 2031-11-10
Also published as: CN103105371A

Abstract

The invention discloses a quality detection method of a pharmaceutical composition injection. An analysis chromatographic column for the chromatographic fingerprint quality detection method is as below: Agilent XDB-C18 150*4.6mm 5 mum, detection wavelength of 205nm, column temperature of 30 DEG C, and flow rate of 0.8ml / min. Preparation of a sample solution comprises steps of: taking a pharmaceutical composition injection; evaporating to dryness by water bath; dissolving residue in an acetic acid aqueous solution; separating on a solid phase extraction column; conducting gradient elution; adding methanol to dissolve the residue; transferring the solution to a 2 ml measuring flask; adding methanol to a scale; shaking uniformly; filtering with a 0.45 mum microporous membrane; and introducing 10 muL of the sample for analysis to obtain a sample fingerprint (see figure 40). The invention also provides a method for determining content of total saponins and 9, 10-dimethoxy pterocarpan-3-o-beta-D-glucoside in the pharmaceutical composition injection.

Description

A kind of quality determining method of medicine composition injection

Technical field

The present invention relates to a kind of quality determining method of parenteral solution, in particular to the content assaying method of total saposins and 9,10-dimethoxy red sandalwood alkane-3-O-β-D-Glucose glycosides in the finger print quality detecting method of medicine composition injection of the present invention and parenteral solution thereof.

Background technology

The traditional Chinese medicine that medicine composition injection of the present invention is formed by the Radix Astragali, ginseng, kushenin 3 herbal medicine, adopted modern extraction and separation technology to refine, there is beneficial gas righting, effect of enhanced machine body immunity function, the leucocyte caused for primary carcinoma of liver, the carcinoma of the rectum, malignant lymphoma, gynecological tumor and a variety of causes is clinically low and reduce the treatment of disease and chronic hepatitis B.

High performance liquid chromatography (HPLC) technology builds the main method of traditional Chinese medicine fingerprint, plays an important role in the quality control of Chinese medicine; But traditional Chinese medicine fingerprint research at present is still in conceptual phase, also there is some problems to need constantly to explore, regulation and standardization as finger-print research has to be strengthened, owing to affecting by instrument, chromatographic column etc., the reappearance problem of finger-print is still needed raising, and fingerprint map analyzing evaluation method need perfect etc.

Ultra Performance Liquid Chromatography (Ultra Performance Liquid Chromatography, UPLC) chromatographic theory is thought and is improved the resolution (resolution) that the usefulness (efficiency) of chromatographic column just can increase instrument, and uses particle diameter lower than the good method of the granule increase usefulness beyond doubt of 2 μm; But reduce the granularity of Stationary liquid to increase the bottleneck of chromatographic column usefulness chromatographic apparatus science always, because granule not only requires that system can be born higher than current extreme pressure (such as 6000psi/400bar), need less system bulk (dead volume), and need to adapt to the fast detector that may only have several seconds peak width; Compared with traditional high performance liquid chromatography (HPLC), UPLC has high separation (ultra resolution), advantage such as high speed (ultra speed), sensitivity (sensitivity) etc.; At present, UPLC is applied to metabonomic analysis and some other biochemistry more, uses and also rise gradually in the analysis of natural products, because further investigation needs higher analysis precision in these fields.

Summary of the invention

The object of the invention is the quality determining method providing a kind of parenteral solution.

The present invention seeks to be achieved through the following technical solutions:

The quality determining method of medicine composition injection of the present invention, comprises following assay and/or fingerprint atlas detection method:

In A medicine composition injection, the content assaying method of total saposins composition is: prepared by reference substance solution, precision takes ginsenoside Rg1's standard items, is placed in volumetric flask, and methyl alcohol dissolves and constant volume, is mixed with the ginsenoside stock solution of 0.98 ~ 1.10mg/ml; Precision takes Astragaloside IV standard items, be placed in volumetric flask, methyl alcohol dissolves and constant volume, is mixed with the Astragaloside IV stock solution of 1.0 ~ 1.2mg/ml, the ratio of above two kinds of stock solutions according to 1: 1 (V/V) mixed, making concentration is 1.0 ~ 1.1mg/ml reference substance solution; Prepared by need testing solution, precision measures medicine composition injection 2 ~ 3ml of the present invention, injects the C18 SPE post after activation, adopts water 10ml successively, 1% acetic acid-water 15ml, water 15ml, 10% methanol-water 10ml, 80% methanol-water 15ml, wash-out, by 80% methanol-water elution fraction evaporate to dryness, get the appropriate constant volume of methyl alcohol in 2ml volumetric flask, make need testing solution; Accurate absorption need testing solution 200 μ L, be placed in test tube, nitrogen dries up, add vanillic aldehyde 0.2ml, perchloric acid 0.8ml mixes, and carries out chromogenic reaction 15min in 60 DEG C, adds glacial acetic acid 5ml and mixes, ice bath cooling 1min, make reference with blank solution, under 552nm wavelength, measure absorbance, substitute into the amount that typical curve calculates total saposins, to obtain final product;

In B medicine composition injection 9, the content assaying method of 10-dimethoxy red sandalwood alkane-3-O-β-D-Glucose glycosides is: prepared by reference substance solution, take 9,10-dimethoxy red sandalwood alkane-3-O-β-D-Glucose glycosides reference substance, be formulated as the reference substance solution of 15.0 ~ 16.5 μ g/ml, prepared by need testing solution, precision measures medicine composition injection 10ml of the present invention, water bath method, residue adds 1% glacial acetic acid aqueous solution 1 ~ 3ml makes dissolving, upper C-18, 500mg solid-phase extraction column, absolute methanol 5ml is first used before using, rinse with 5ml distilled water again and be separated, with 15ml 1% glacial acetic acid aqueous solution wash-out, again with 10ml water elution, then with 10% methanol aqueous solution 15ml wash-out, discard above-mentioned eluent, again with 80% methyl alcohol 18ml eluant solution, collect 80% meoh eluate, water bath method, residue adds methyl alcohol and dissolves and be transferred in 2ml measuring bottle, add methyl alcohol to scale, shake up, with 0.45 μm of filtering with microporous membrane, sample introduction 10 μ L analyzes, liquid-phase condition: Agilent Eclipse XDB-C18 (150 × 4.6mm, 5 μm), mobile phase: take acetonitrile as mobile phase A, take water as Mobile phase B, carry out gradient elution: 15%A (0min), 25%A (30min), 40%A (40min), determined wavelength 207nm, column temperature 30 DEG C, flow velocity 0.8ml/min, accurate absorption need testing solution 200 μ L, be placed in test tube, nitrogen dries up, add vanillic aldehyde 0.2ml, perchloric acid 0.8ml mixes, and carries out chromogenic reaction 15min in 60 DEG C, adds glacial acetic acid 5ml and mixes, ice bath cooling 1min, make reference with blank solution, under 552nm wavelength, measure absorbance, substitute into the amount that typical curve calculates total saposins, to obtain final product,

C medicine composition injection fingerprint atlas detection method is: chromatographic condition, analyze chromatographic column Agilent XDB-C18150 × 4.6mm 5 μm, determined wavelength 205nm, column temperature 30 DEG C, flow velocity 0.8ml/min, number of theoretical plate calculates should be not less than 200000 by ginsenoside Rf peak, mobile phase: take acetonitrile as mobile phase A, take water as Mobile phase B, carry out gradient elution: 15%A (0min), 19%A (20min), 25.5%A (36min), 50%A (50min), 70%A (65min), 70%A (85min), Mass Spectrometry Conditions: electron spray (ESI) ion gun, positive ion mode, gas curtain temperature degree (Gas temp) 350 DEG C, gas curtain gas velocity (Gas flow) 8 L/min, assisted gas pressure (Nebulizer) is 30psi, capillary voltage (Vcap): 4000V, level Four bar-flight time tandem mass spectrometer condition (MS QTOF conditions): cracked voltage (Fragmentor): 175V, intercept cone voltage (Skimmer): 65V, radio-frequency voltage (OCT1 RFVpp) 750V of first heavy eight grades of bars, prepared by need testing solution: precision measures medicine composition injection 10ml of the present invention, water bath method, residue adds 1% glacial acetic acid aqueous solution 2ml makes dissolving, upper solid-phase extraction column (C-18, 500mg, activation step: first use absolute methanol 5ml before use, rinse with 5ml distilled water again) be separated, first with 15ml 1% glacial acetic acid aqueous solution wash-out, again with 10ml water elution, then with 8% ~ 12% methanol aqueous solution 15ml wash-out, discard above-mentioned eluent, again with 75% ~ 85% methyl alcohol 18ml eluant solution, collect 75% ~ 85% meoh eluate, water bath method, residue adds methyl alcohol and dissolves and be transferred in 2ml measuring bottle, add methyl alcohol to scale, shake up, with 0.45 μm of filtering with microporous membrane, sample introduction 10 μ l analyzes, HPLC analysis is carried out to 10% and 100% meoh eluate, there is no saponins and flavonoids chromatographic peak in the retention time of correspondence, medicine composition injection finger-print of the present invention has 15 fingerprint peakses, and the relative retention time of total fingerprint peaks is respectively: 0.712 ± 0.071,0.740 ± 0.074,0.749 ± 0.075,0.769 ± 0.077,0.834 ± 0.083,1.000 ± 0.000,1.019 ± 0.102,1.031 ± 0.103,1.038 ± 0.104,1.045 ± 0.105,1.059 ± 0.106,1.072 ± 0.107,1.173 ± 0.117,1.191 ± 0.119,1.204 ± 0.120, relative reservation peak area is respectively: 1.616 ± 0.161,2.631 ± 0.263,2.036 ± 0.204,12.671 ± 1.267,3.346 ± 0.335,1.000 ± 0.000,0.868 ± 0.087,0.428 ± 0.043,0.566 ± 0.057,0.854 ± 0.085,0.633 ± 0.063,0.402 ± 0.040,0.417 ± 0.042,0.295 ± 0.030,0.461 ± 0.046.

The quality determining method of medicine composition injection of the present invention, is preferably as follows assay and/or fingerprint atlas detection method:

In A medicine composition injection, the content assaying method of total saposins composition is: prepared by reference substance solution, precision takes ginsenoside Rg1's standard items, is placed in volumetric flask, and methyl alcohol dissolves and constant volume, is mixed with the ginsenoside stock solution of 0.999mg/ml; Precision takes Astragaloside IV standard items, be placed in volumetric flask, methyl alcohol dissolves and constant volume, is mixed with the Astragaloside IV stock solution of 1.100mg/ml, the ratio of above two kinds of stock solutions according to 1: 1 (V/V) mixed, making concentration is 1.050mg/ml reference substance solution; Prepared by need testing solution, precision measures medicine composition injection 2.5ml of the present invention, injects the C18 SPE post after activation, adopts water 10ml successively, 1% acetic acid-water 15ml, water 15ml, 10% methanol-water 10ml, 80% methanol-water 15ml, wash-out, by 80% methanol-water elution fraction evaporate to dryness, get the appropriate constant volume of methyl alcohol in 2ml volumetric flask, make need testing solution; Accurate absorption need testing solution 200 μ L, be placed in test tube, nitrogen dries up, add vanillic aldehyde 0.2ml, perchloric acid 0.8ml mixes, and carries out chromogenic reaction 15min in 60 DEG C, adds glacial acetic acid 5ml and mixes, ice bath cooling 1min, make reference with blank solution, under 552nm wavelength, measure absorbance, substitute into the amount that typical curve calculates total saposins, to obtain final product;

In B medicine composition injection 9, the content assaying method of 10-dimethoxy red sandalwood alkane-3-O-β-D-Glucose glycosides is: prepared by reference substance solution, take 9,10-dimethoxy red sandalwood alkane-3-O-β-D-Glucose glycosides reference substance, be formulated as the reference substance solution of 15.9 μ g/ml, prepared by need testing solution, precision measures medicine composition injection 10ml of the present invention, water bath method, residue adds 1% glacial acetic acid aqueous solution 2ml makes dissolving, upper C-18, 500mg solid-phase extraction column, absolute methanol 5ml is first used before using, rinse with 5ml distilled water again and be separated, with 15ml 1% glacial acetic acid aqueous solution wash-out, again with 10ml water elution, then with 10% methanol aqueous solution 15ml wash-out, discard above-mentioned eluent, again with 80% methyl alcohol 18ml eluant solution, collect 80% meoh eluate, water bath method, residue adds methyl alcohol and dissolves and be transferred in 2ml measuring bottle, add methyl alcohol to scale, shake up, with 0.45 μm of filtering with microporous membrane, sample introduction 10 μ L analyzes, liquid-phase condition: Agilent Eclipse XDB-C18 (150 × 4.6mm, 5 μm), mobile phase: take acetonitrile as mobile phase A, take water as Mobile phase B, carry out gradient elution: 15%A (0min), 25%A (30min), 40%A (40min), determined wavelength 207nm, column temperature 30 DEG C, flow velocity 0.8ml/min, accurate absorption need testing solution 200 μ L, be placed in test tube, nitrogen dries up, add vanillic aldehyde 0.2ml, perchloric acid 0.8ml mixes, and carries out chromogenic reaction 15min in 60 DEG C, adds glacial acetic acid 5ml and mixes, ice bath cooling 1min, make reference with blank solution, under 552nm wavelength, measure absorbance, substitute into the amount that typical curve calculates total saposins, to obtain final product,

The quality determining method of C medicine composition injection finger-print is, chromatographic condition: analyze chromatographic column: AgilentXDB-C18 150 × 4.6mm 5 μm, determined wavelength: 205nm, column temperature: 30 DEG C, flow velocity: 0.8ml/min, number of theoretical plate calculates should be not less than 200000 by ginsenoside Rf peak, mobile phase: take acetonitrile as mobile phase A, take water as Mobile phase B, carry out gradient elution: 0 minute 15%A, 20 minutes 19%A, 36 minutes 25.5%A, 50 minutes 50%A, 65 minutes 70%A, 85 minutes 70%A, Mass Spectrometry Conditions: electron spray (ESI) ion gun, positive ion mode, gas curtain temperature degree (Gas temp) 350 DEG C, gas curtain gas velocity (Gas flow) 8L/min, assisted gas pressure (Nebulizer) is 30psi, capillary voltage (Vcap) 4000V, level Four bar-flight time tandem mass spectrometer condition (MS QTOF conditions): cracked voltage (Fragmentor) 175V, intercept cone voltage (Skimmer) 65V, radio-frequency voltage (OCT1 RFVpp) 750V of first heavy eight grades of bars, prepared by need testing solution: precision measures medicine composition injection 10ml of the present invention, water bath method, residue adds 1% glacial acetic acid aqueous solution 2ml makes dissolving, upper solid-phase extraction column (C-18, 500mg, activation step: first use absolute methanol 5ml before use, rinse with 5ml distilled water again) be separated, first with 15ml 1% glacial acetic acid aqueous solution wash-out, again with 10ml water elution, then with 10% methanol aqueous solution 15ml wash-out, discard above-mentioned eluent, again with 80% methyl alcohol 18ml eluant solution, collect 80% meoh eluate, water bath method, residue adds methyl alcohol and dissolves and be transferred in 2ml measuring bottle, add methyl alcohol to scale, shake up, with 0.45 μm of filtering with microporous membrane, sample introduction 10 μ l analyzes, HPLC analysis is carried out to 10% and 100% meoh eluate, there is no saponins and flavonoids chromatographic peak in the retention time of correspondence, medicine composition injection finger-print of the present invention has 15 fingerprint peakses, and the relative retention time of total fingerprint peaks is respectively: 0.712,0.740,0.749,0.769,0.834,1.000,1.019,1.031,1.038,1.045,1.059,1.072,1.173,1.191,1.204, relative reservation peak area is respectively: 1.616,2.631,2.036,12.671,3.346,1.000,0.868,0.428,0.566,0.854,0.633,0.402,0.417,0.295,0.461.

In A medicine composition injection, the content assaying method of total saposins composition is: prepared by reference substance solution, precision takes ginsenoside Rg1's standard items, is placed in volumetric flask, and methyl alcohol dissolves and constant volume, is mixed with the ginsenoside stock solution of 0.98 ~ 1.10mg/ml; Precision takes Astragaloside IV standard items, be placed in volumetric flask, methyl alcohol dissolves and constant volume, is mixed with the Astragaloside IV stock solution of 1.0mg/ml, the ratio of above two kinds of stock solutions according to 1: 1 (V/V) mixed, making concentration is 1.0mg/ml reference substance solution; Prepared by need testing solution, precision measures medicine composition injection 2.1ml of the present invention, injects the C18 SPE post after activation, adopts water 10ml successively, 1% acetic acid-water 15ml, water 15ml, 10% methanol-water 10ml, 80% methanol-water 15ml, wash-out, by 80% methanol-water elution fraction evaporate to dryness, get the appropriate constant volume of methyl alcohol in 2ml volumetric flask, make need testing solution; Accurate absorption need testing solution 200 μ L, be placed in test tube, nitrogen dries up, add vanillic aldehyde 0.2ml, perchloric acid 0.8ml mixes, and carries out chromogenic reaction 15min in 60 DEG C, adds glacial acetic acid 5ml and mixes, ice bath cooling 1min, make reference with blank solution, under 552nm wavelength, measure absorbance, substitute into the amount that typical curve calculates total saposins, to obtain final product;

In B medicine composition injection 9, the content assaying method of 10-dimethoxy red sandalwood alkane-3-O-β-D-Glucose glycosides is: prepared by reference substance solution, take 9,10-dimethoxy red sandalwood alkane-3-O-β-D-Glucose glycosides reference substance, be formulated as the reference substance solution of 15.2 μ g/ml, prepared by need testing solution, precision measures medicine composition injection 10ml of the present invention, water bath method, residue adds 1% glacial acetic acid aqueous solution 1 ~ 3ml makes dissolving, upper C-18, 500mg solid-phase extraction column, absolute methanol 5ml is first used before using, rinse with 5ml distilled water again and be separated, with 15ml 1% glacial acetic acid aqueous solution wash-out, again with 10ml water elution, then with 10% methanol aqueous solution 15ml wash-out, discard above-mentioned eluent, again with 80% methyl alcohol 18ml eluant solution, collect 80% meoh eluate, water bath method, residue adds methyl alcohol and dissolves and be transferred in 2ml measuring bottle, add methyl alcohol to scale, shake up, with 0.45 μm of filtering with microporous membrane, sample introduction 10 μ L analyzes, liquid-phase condition: Agilent Eclipse XDB-C18 (150 × 4.6mm, 5 μm), mobile phase: take acetonitrile as mobile phase A, take water as Mobile phase B, carry out gradient elution: 15%A (0min), 25%A (30min), 40%A (40min), determined wavelength 207nm, column temperature 30 DEG C, flow velocity 0.8ml/min, accurate absorption need testing solution 200 μ L, be placed in test tube, nitrogen dries up, add vanillic aldehyde 0.2ml, perchloric acid 0.8ml mixes, and carries out chromogenic reaction 15min in 60 DEG C, adds glacial acetic acid 5ml and mixes, ice bath cooling 1min, make reference with blank solution, under 552nm wavelength, measure absorbance, substitute into the amount that typical curve calculates total saposins, to obtain final product,

C medicine composition injection fingerprint atlas detection method is: chromatographic condition, analyze chromatographic column Agilent XDB-C18150 × 4.6mm 5 μm, determined wavelength 205nm, column temperature 30 DEG C, flow velocity 0.8ml/min, number of theoretical plate calculates should be not less than 200000 by ginsenoside Rf peak, mobile phase: take acetonitrile as mobile phase A, take water as Mobile phase B, carry out gradient elution: 15%A (0min), 19%A (20min), 25.5%A (36min), 50%A (50min), 70%A (65min), 70%A (85min), Mass Spectrometry Conditions: electron spray (ESI) ion gun, positive ion mode, gas curtain temperature degree (Gas temp) 350 DEG C, gas curtain gas velocity (Gas flow) 8L/min, assisted gas pressure (Nebulizer) is 30psi, capillary voltage (Vcap): 4000V, level Four bar-flight time tandem mass spectrometer condition (MS QTOF conditions): cracked voltage (Fragmentor): 175V, intercept cone voltage (Skimmer): 65V, radio-frequency voltage (OCT1 RFVpp) 750V of first heavy eight grades of bars, prepared by need testing solution: precision measures medicine composition injection 10ml of the present invention, water bath method, residue adds 1% glacial acetic acid aqueous solution 2ml makes dissolving, upper solid-phase extraction column (C-18, 500mg, activation step: first use absolute methanol 5ml before use, rinse with 5ml distilled water again) be separated, first with 15ml 1% glacial acetic acid aqueous solution wash-out, again with 10ml water elution, then with 9% methanol aqueous solution 15ml wash-out, discard above-mentioned eluent, again with 78% methyl alcohol 18ml eluant solution, collect 78% meoh eluate, water bath method, residue adds methyl alcohol and dissolves and be transferred in 2ml measuring bottle, add methyl alcohol to scale, shake up, with 0.45 μm of filtering with microporous membrane, sample introduction 10 μ l analyzes, HPLC analysis is carried out to 10% and 100% meoh eluate, there is no saponins and flavonoids chromatographic peak in the retention time of correspondence, medicine composition injection finger-print of the present invention has 15 fingerprint peakses, and the relative retention time of total fingerprint peaks is respectively: 0.781,0.791,0.810,0.830,0.91,1.000,1.119,1.131,1.138,1.145,1159,1.172,1.273,1.291,1.304, relative reservation peak area is respectively: 1.776,2.891,2.236,13.671,3.676,1.000,0.948,0.471,0.616,0.939,0.696,0.442,0.459,0.325,0.507.

In A medicine composition injection, the content assaying method of total saposins composition is: prepared by reference substance solution, precision takes ginsenoside Rg1's standard items, is placed in volumetric flask, and methyl alcohol dissolves and constant volume, is mixed with the ginsenoside stock solution of 0.98 ~ 1.10mg/ml; Precision takes Astragaloside IV standard items, be placed in volumetric flask, methyl alcohol dissolves and constant volume, is mixed with the Astragaloside IV stock solution of 1.18mg/ml, the ratio of above two kinds of stock solutions according to 1: 1 (V/V) mixed, making concentration is 1.09mg/ml reference substance solution; Prepared by need testing solution, precision measures medicine composition injection 3ml of the present invention, injects the C18 SPE post after activation, adopts water 10ml successively, 1% acetic acid-water 15ml, water 15ml, 10% methanol-water 10ml, 80% methanol-water 15ml, wash-out, by 80% methanol-water elution fraction evaporate to dryness, get the appropriate constant volume of methyl alcohol in 2ml volumetric flask, make need testing solution; Accurate absorption need testing solution 200 μ L, be placed in test tube, nitrogen dries up, add vanillic aldehyde 0.2ml, perchloric acid 0.8ml mixes, and carries out chromogenic reaction 15min in 60 DEG C, adds glacial acetic acid 5ml and mixes, ice bath cooling 1min, make reference with blank solution, under 552nm wavelength, measure absorbance, substitute into the amount that typical curve calculates total saposins, to obtain final product;

In B medicine composition injection 9, the content assaying method of 10-dimethoxy red sandalwood alkane-3-O-β-D-Glucose glycosides is: prepared by reference substance solution, take 9,10-dimethoxy red sandalwood alkane-3-O-β-D-Glucose glycosides reference substance, be formulated as the reference substance solution of 16.3 μ g/ml, prepared by need testing solution, precision measures medicine composition injection 10ml of the present invention, water bath method, residue adds 1% glacial acetic acid aqueous solution 3ml makes dissolving, upper C-18, 500mg solid-phase extraction column, absolute methanol 5ml is first used before using, rinse with 5ml distilled water again and be separated, with 15ml 1% glacial acetic acid aqueous solution wash-out, again with 10ml water elution, then with 10% methanol aqueous solution 15ml wash-out, discard above-mentioned eluent, again with 80% methyl alcohol 18ml eluant solution, collect 80% meoh eluate, water bath method, residue adds methyl alcohol and dissolves and be transferred in 2ml measuring bottle, add methyl alcohol to scale, shake up, with 0.45 μm of filtering with microporous membrane, sample introduction 10 μ L analyzes, liquid-phase condition: Agilent Eclipse XDB-C18 (150 × 4.6mm, 5 μm), mobile phase: take acetonitrile as mobile phase A, take water as Mobile phase B, carry out gradient elution: 15%A (0min), 25%A (30min), 40%A (40min), determined wavelength 207nm, column temperature 30 DEG C, flow velocity 0.8ml/min, accurate absorption need testing solution 200 μ L, be placed in test tube, nitrogen dries up, add vanillic aldehyde 0.2ml, perchloric acid 0.8ml mixes, and carries out chromogenic reaction 15min in 60 DEG C, adds glacial acetic acid 5ml and mixes, ice bath cooling 1min, make reference with blank solution, under 552nm wavelength, measure absorbance, substitute into the amount that typical curve calculates total saposins, to obtain final product,

C medicine composition injection fingerprint atlas detection method is: chromatographic condition, analyze chromatographic column Agilent XDB-C18150 × 4.6mm 5 μm, determined wavelength 205nm, column temperature 30 DEG C, flow velocity 0.8ml/min, number of theoretical plate calculates should be not less than 200000 by ginsenoside Rf peak, mobile phase: take acetonitrile as mobile phase A, take water as Mobile phase B, carry out gradient elution: 15%A (0min), 19%A (20min), 25.5%A (36min), 50%A (50min), 70%A (65min), 70%A (85min), Mass Spectrometry Conditions: electron spray (ESI) ion gun, positive ion mode, gas curtain temperature degree (Gas temp) 350 DEG C, gas curtain gas velocity (Gas flow) 8L/min, assisted gas pressure (Nebulizer) is 30psi, capillary voltage (Vcap): 4000V, level Four bar-flight time tandem mass spectrometer condition (MS QTOF conditions): cracked voltage (Fragmentor): 175V, intercept cone voltage (Skimmer): 65V, radio-frequency voltage (OCT1 RFVpp) 750V of first heavy eight grades of bars, prepared by need testing solution: precision measures medicine composition injection 10ml of the present invention, water bath method, residue adds 1% glacial acetic acid aqueous solution 2ml makes dissolving, upper solid-phase extraction column (C-18, 500mg, activation step: first use absolute methanol 5ml before use, rinse with 5ml distilled water again) be separated, first with 15ml 1% glacial acetic acid aqueous solution wash-out, again with 10ml water elution, then with 11% methanol aqueous solution 15ml wash-out, discard above-mentioned eluent, again with 82% methyl alcohol 18ml eluant solution, collect 82% meoh eluate, water bath method, residue adds methyl alcohol and dissolves and be transferred in 2ml measuring bottle, add methyl alcohol to scale, shake up, with 0.45 μm of filtering with microporous membrane, sample introduction 10 μ l analyzes, HPLC analysis is carried out to 10% and 100% meoh eluate, there is no saponins and flavonoids chromatographic peak in the retention time of correspondence, medicine composition injection finger-print of the present invention has 15 fingerprint peakses, and the relative retention time of total fingerprint peaks is respectively: 0.642,0.670,0.679,0.700,0.75,1.000,1.009,1.008,1.008,1.005,1.009,1.007,1156,1.072,1.084, relative reservation peak area is respectively: 1.455,2.368,1.832,11.404,3.311,1.000,0.781,0.385,0.509,0.769,0.570,0.362,0.375,0.265,0.415.

The bulk drug of the invention described above medicine composition injection consists of: Radix Astragali 25-35 weight portion, ginseng 8-12 weight portion, kushenin 0.5-1.5 weight portion; The preparation method of medicine composition injection of the present invention is: above three taste medicines, the ethanol of ginseng 60-80%, refluxing extraction 1-3 time, each 1-2 hour, merges extract, is evaporated to the clear cream that relative density is 65 DEG C 1.10 ~ 1.20, for subsequent use; Radix Astragali boiling 1-3 time, each 1-3 hour, filters, merging filtrate, be evaporated to the clear cream that relative density is 65 DEG C 1.10 ~ 1.20, merge with the clear cream of ginseng, add ethanol and make alcohol content reach 75%, adjust pH value to 6 ~ 7 with NaOH, leave standstill 10-18 hour, get supernatant, reclaim ethanol, be evaporated to the clear cream that relative density is 65 DEG C 1.10 ~ 1.15; Adding ethanol again makes alcohol content reach 85%, and with NaOH tone pitch PH to 6 ~ 7, leave standstill, filter, filtrate recycling ethanol to without alcohol taste, injects with water to 400ml, and with NaOH tune pH value to 6 ~ 7,100 DEG C of sterilizings 30 minutes, refrigerate, suction filtration; Adjust pH value to 6 ~ 7 with NaOH, add activated charcoal appropriate, stir evenly, boil 15 minutes, filter, filtrate is for subsequent use; Separately get kushenin to dissolve, adjust pH value to 6 ~ 7 with watery hydrochloric acid, 100--120 DEG C of sterilizing 30 minutes, refrigeration, suction filtration, merges with the liquid after de-charcoal, and mixing, injects water to ormal weight, and filtration is filling, to obtain final product.

The bulk drug of the invention described above medicine composition injection consists of: the Radix Astragali 30 weight portion, ginseng 10 weight portion, kushenin 1 weight portion; The preparation method of medicine composition injection of the present invention is: above three taste medicines, ginseng with 75% ethanol, refluxing extraction three times, each 2 hours, merge extract, be evaporated to the clear cream that relative density is 1.10 ~ 1.20 (65 DEG C), for subsequent use; Radix Astragali boiling secondary, each 2 hours, filter, merging filtrate, be evaporated to the clear cream that relative density is 1.10 ~ 1.20 (65 DEG C), merge with the clear cream of ginseng, add ethanol and make alcohol content reach 75%, adjust pH value to 6 ~ 7 with NaOH, leave standstill 12 hours, get supernatant, reclaim ethanol, be evaporated to the clear cream that relative density is 1.10 ~ 1.15 (65 DEG C); Adding ethanol again makes alcohol content reach 85%, and with NaOH tone pitch PH to 6 ~ 7, leave standstill, filter, filtrate recycling ethanol to without alcohol taste, injects with water to 400ml, and with NaOH tune pH value to 6 ~ 7,100 DEG C of sterilizings 30 minutes, refrigerate, suction filtration; Adjust pH value to 6 ~ 7 with NaOH, add activated charcoal appropriate, stir evenly, boil 15 minutes, filter, filtrate is for subsequent use; Separately get kushenin to dissolve, adjust pH value to 6 ~ 7 with watery hydrochloric acid, 100 DEG C of sterilizings 30 minutes, refrigeration, suction filtration, merges with the liquid after de-charcoal, and mixing, injects water to ormal weight, and filtration is filling, to obtain final product.Compared with prior art, the present invention has following beneficial effect:

Medicine composition injection content assaying method of the present invention, definitely the effective constituent of product and content assaying method thereof, can better control product quality, ensure that product is safer, effective;

The present invention uses fingerprint pattern technology effectively to control the quality of medicine composition injection of the present invention; Prove that finger print measuring method precision of the present invention is good by experiment, reappearance is better, and need testing solution is good at 24h internal stability;

The present invention is by the qualification of high resolution mass spectrum to chemical composition, and experimental basis more fully leans on examining; The mass spectrogram of the chromatographic peak identified through this chromatographic peak (TIC) retention time region compares, and proves that this chromatographic peak shows single compound; Identify 16 chemical compositions altogether in medicine composition injection chromatographic fingerprinting (except kushenin) of the present invention, above chemical composition has carried out structural confirmation further across high resolution mass spectrum; Four chemical compositions derive from the medicinal material Radix Astragali, be respectively (6aR, 11aR)-3-hydroxy-9,10-dimethoxypterocarpan-3-O-β-D-sambubioside, 9,10-dimethoxy red sandalwood alkane-3-O-β-D-Glucose glycosides, 2 '-hydroxyl-3 ', 4 '-dimethoxy isoflavan-7-O-β-D-Glucose glycosides and Astragaloside IV; 12 chemical compositions derive from medicinal material ginseng, wherein 8 chemical composition (ginsenoside Res, Rg1, Rf, Rb1, Rc, Rg2, Rb2 and Rd) derive from prototype composition in ginseng crude drug, 4 chemical compositions derive from the high prototype composition of content in ginseng crude drug and are producing and (sour environment of medicinal material extract solution, higher temperature heating 80 degree in preparation process, preparation sterilizing 100 degree heating) decompose the secondary ginsenoside of generation under specific process conditions, but itself be also active chemical components.

Accompanying drawing:

1. do not carry out the medicine composition injection test sample of the present invention of Solid-Phase Extraction process

2. the mass spectrogram of ESI (+) MS of oxymatrine

3. oxymatrine is that UV 205nm detects 1

4. oxymatrine is that UV 205nm detects 2

5. in medicine composition injection of the present invention, chemical composition low resolution LC-MS identifies

6. in medicine composition injection of the present invention, chemical composition low resolution LC-MS identifies ginsenoside Rg1

7. in medicine composition injection of the present invention, chemical composition low resolution LC-MS identifies ginsenoside Re

8.9,10-dimethoxy red sandalwood alkane-3-O-β-D-Glucose glycosides (No.4)

9.2 '-hydroxyl-3 ', 4 '-dimethoxy isoflavan-7-O-β-D-Glucose glycosides (No.5)

10. ginsenoside Rf (No.6)

11. ginsenoside Rb1s (No.7)

12. Ginsenoside Rcs (No.8)

13. ginsenoside Rg2s (No.9)

14. ginsenoside Rb2s (No.10)

15. ginsenoside Rh 1s (No.11)

16. Astragaloside IVs (No.12)

17. ginsenoside Rds (No.13)

18. ginsenoside Rgs 5, Rg6, RK1, F4

19. Ginsenoside Rh4s or Rk3 (No.15)

20. 3beta,6alpha,12beta-Trihydroxydammar-20(21),24-diene-6-O-beta-D-glucopyranosides or Rh4 (No.16)

The total ion current figure (TIC) of 21-1.ESI (-) negative ion

21-2.DAD detecting device collection of illustrative plates (205nm)

22. medicine composition injection component high-resolution LC-MS of the present invention identify (No.1)

23. ginsenoside Rg1s (No.2)

24. ginsenoside Res (No.3)

25.9,10-dimethoxy red sandalwood alkane-3-O-β-D-Glucose glycosides (No.4)

26.2 '-hydroxyl-3 ', 4 '-dimethoxy isoflavan-7-O-β-D-Glucose glycosides (No.5)

27. ginsenoside Rfs (No.6)

28. ginsenoside Rb1s (No.7)

29. Ginsenoside Rcs (No.8)

30. ginsenoside Rg2s (No.9)

31. ginsenoside Rb2s (No.10)

32. ginsenoside Rh 1s (No.11)

33. Astragaloside IVs (No.12)

34. ginsenoside Rds (No.13)

35. ginsenoside Rgs 5, F4, Rk1 or Rg6 (No.14)

36. Ginsenoside Rh4s or Rk3 (No.15)

37. 3beta,6alpha,12beta-Trihydroxydammar-20(21),24-diene-6-O-beta-D-glucopyranosides or Rh4 (No.16)

38. medicine composition injection 150min collection of illustrative plates of the present invention

39. blank gradient collection of illustrative plates

40. medicine composition injection finger-prints of the present invention

Following experimental example and embodiment are used for further illustrating but are not limited to the present invention; Experimental example Chinese traditional medicine composite injection of the present invention is prepared by embodiment 1 method.

Chemical composition in experimental example 1 HPLC-ESI-Iontrap Analysis and Identification medicine composition injection of the present invention

1 instrument and chromatographic mass spectrometry condition

1.1 instruments and material

HPLC-MS instrument:

Efficient liquid phase: Agilent 1200 series; Mass spectrum: Bruker HCT Iontrap (three-dimensional ion trap mass spectrometer)

Trifluoroacetic acid aqueous solution (Acetonitrile): HPLC grade, Fisher Scientific

Chromatographic Pure Methanol (Methanol): HPLC grade, Tianjin Jizhun Chemicals

Ultrapure water (Water): ultra-pure water

Solid-phase extraction column (SPE): Tianjin Shuangji chromatography scientific

1.2 chromatographic condition

Analyze chromatographic column: Agilent XDB-C18 150 × 4.6mm 5 μm

Determined wavelength: 205nm, column temperature: 30 DEG C, flow velocity: 0.8ml/min

Number of theoretical plate calculates should be not less than 200000 by ginsenoside Rf peak

Timetable：

1.3 Mass Spectrometry Conditions

ESI conditions：

Gas Temp：350℃

Drying Gas：8L/min

Nebulizer：30psi

Vcap：4000V

MS Iontrap conditions：

Capillary Exit：145V

Skimmer：40V

1.4 workstation

Bruker ESI Compass 1.3 for HCT/Esquire

Data Analysis version 4.0

2 need testing solution preparations

2.1 sample pre-treatments

In medicine composition injection of the present invention, (prepared by embodiment 1) is containing a large amount of kushenins (10mg/mL), and kushenin is the mixed base of oxymatrine (Oxymatrine) and minute quantity N-Oxysophocarpine (Oxysophocarpine), this experiment identifies the existence of a large amount of oxymatrine with LC-MS (LC-MS) method;

HPLC-ESI-MS analyzes:

Start 2-5min at chromatographic fingerprinting and occur a very large chromatographic peak (Fig. 1), be accredited as kushenin through HPLC-ESI-MS; The molecular weight of oxymatrine is 264.4, and 265.1 in mass spectrogram is [M+H] of oxymatrine ⁺peak, 529.2 is [2M+H] of oxymatrine ⁺peak (Fig. 2);

In order to avoid the existence of a large amount of kushenin is to the interference of ginseng and Radix Astragali chemical composition analysis, we adopt solid phase extraction method to remove a large amount of kushenins.

2.2 sample pre-treatments experimental procedures:

Precision measures medicine composition injection 10ml of the present invention, water bath method, and residue adds 1% glacial acetic acid aqueous solution 2ml makes dissolving, upper solid-phase extraction column (C-18,500mg; Activation step: first use absolute methanol 5ml before use, rinse with 5ml distilled water again) be separated, first with 15ml 1% glacial acetic acid aqueous solution wash-out, then with 10ml water elution, then with 10% methanol aqueous solution 15ml wash-out, discard above-mentioned eluent, then with 80% methyl alcohol 18ml eluant solution, collect 80% meoh eluate, water bath method, residue adds methyl alcohol and dissolves and be transferred in 2ml measuring bottle, adds methyl alcohol to scale, shakes up, with 0.45 μm of filtering with microporous membrane, sample introduction 10 μ l analyzes;

Remarks: utilize that thin-layered chromatography (TLC) identifies sour water eluent, water elution liquid contains a large amount of kushenin, kushenin containing minute quantity in 10% meoh eluate, 80% meoh eluate has no kushenin spot, and shows the spot of aubergine saponin(e; 100% meoh eluate has no obvious spot;

Further HPLC analysis is carried out to 10% and 100% meoh eluate, there is no saponins and flavonoids chromatographic peak in the retention time of correspondence.

The structural formula of oxymatrine

3 experimental results

3.1 medicine composition injection HPLC-ESI-Iontrap of the present invention analyze collection of illustrative plates

Fig. 2 is the total ion current figure (TIC) of ESI (+) positive ion, and Fig. 3, Fig. 4 are that UV 205nm detects;

The compound structure qualification of 3.2 each chromatographic peaks (UV figure)

In 4 medicine composition injections of the present invention, chemical composition low resolution LC-MS identifies

4.1 (6aR, 11aR)-3-hydroxy-9,10-dimethoxypterocarpan-3-O-β-D-sambubioside (No.1) (see Fig. 5)

(6aR，11aR)-3-hydroxy-9，10-dimethoxypterocarpan-3-O-B-D-sambubioside

4.2 ginsenoside Rg1s (No.2) (see Fig. 6)

Molecular formula	Ionized form	Measured value	Qualification
				C ₄₂H ₇₂O ₁₄	[M+Na] ⁺	823.6	Ginsenoside Rg1

Chemical constitution:

4.3 ginsenoside Res (No.3) (see Fig. 7)

Molecular formula	Ionized form	Measured value	Qualification
				C ₄₈H ₈₂O ₁₈	[M+Na] ⁺	969.6	Ginsenoside Re

Chemical constitution:

4.4 9,10-dimethoxy red sandalwood alkane-3-O-β-D-Glucose glycosides (No.4) (see Fig. 8)

Chemical constitution:

9，10-Dimethoxypterocarpan-3-O-B-D-glucoside

4.5 2 '-hydroxyl-3 ', 4 '-dimethoxy isoflavan-7-O-β-D-Glucose glycosides (No.5) (see Fig. 9)

Chemical constitution:

No. 6 chromatographic peaks

Chromatographic fingerprinting through medicine composition injection of the present invention, ginseng crude drug, Milkvetch Root compares, this chromatographic peak is not the chemical composition contained in original medicinal material, should be at the decomposition product produced and produce in preparation process, impure through mass spectrophotometry, containing a small amount of saponin compound and other impurity; And peak area changes greatly, so do not have peak as finger-print in multiple batches of medicine composition injection of the present invention is analyzed.

4.6 ginsenoside Rfs (No.6) (see Figure 10)

Molecular formula	Ionized form	Measured value	Qualification
				C ₄₂H ₇₂O ₁₄	[M+Na] ⁺	823.6	Ginsenoside Rf

Chemical constitution:

4.7 ginsenoside Rb1s (No.7) (see Figure 11)

Molecular formula	Ionized form	Measured value	Qualification
				C ₅₄H ₉₂O ₂₃	[M+Na] ⁺	1131.6	Ginsenoside Rb1

Chemical constitution:

4.8 Ginsenoside Rcs (No.8) (see Figure 12)

Molecular formula	Ionized form	Measured value	Qualification
				C ₅₃H ₉₀O ₂₂	[M+Na] ⁺	1101.6	Ginsenoside Rc

Chemical constitution:

4.9 ginsenoside Rg2s (No.9) (see Figure 13)

Molecular formula	Ionized form	Measured value	Qualification
				C ₄₂H ₇₂O ₁₃	[M+Na] ⁺	807.6	Ginsenoside-Rg2

Chemical constitution:

4.10 ginsenoside Rb2s (No.10) (see Figure 14)

Molecular formula	Ionized form	Measured value	Qualification
				C ₅₃H ₉₀O ₂₂	[M+Na] ⁺	1101.6	Ginsenoside Rb2

Chemical constitution:

4.11 ginsenoside Rh 1s (No.11) (see Figure 15)

Molecular formula	Ionized form	Measured value	Qualification
				C ₃₆H ₆₂O ₉	[M+Na] ⁺	661.5	Ginsenoside Rh 1

Chemical constitution:

4.12 Astragaloside IVs (No.12) (see Figure 16)

Molecular formula	Ionized form	Measured value	Qualification
				C ₄₁H ₆₈O ₁₄	[M+Na] ⁺	807.6	Astragaloside IV

Chemical constitution:

4.13 ginsenoside Rds (No.13) (see Figure 17)

Molecular formula	Ionized form	Measured value	Qualification
				C ₄₈H ₈₂O ₁₈	[M+Na] ⁺	969.6	Ginsenoside Rd

Chemical constitution:

4.14 No. 14 chromatographic peaks:

Be accredited as one of molecular weight following four compounds identical with molecular formula: ginsenoside Rg 5, Rg6, RK1, F4; (see Figure 18)

Owing to being the decomposition product-secondary ginsenoside produced and produce in production process, do not confirm particular compound further;

Molecular formula	Ionized form	Measured value	Qualification
				C ₄₂H ₇₀O ₁₂	[M+Na] ⁺	789.6	Ginsenoside Rg 5 Rg6, RK1, or

F4；

Chemical constitution:

4.15 Ginsenoside Rh4s or Rk3 (No.15) (see Figure 19)

Note: under structural formula is shown in 4.16;

4.16 3beta,6alpha,12beta-Trihydroxydammar-20(21),24-diene-6-O-beta-D-glucopyranosides or Rh4 (No.16) (see Figure 20)

Chemical constitution:

Chemical composition in experimental example 2 UHPLC-ESI-QTOF Analysis and Identification medicine composition injection of the present invention

1 instrument and chromatographic mass spectrometry condition

1.1 instrument

UHV (ultra-high voltage) high performance liquid chromatograph (UHPLC): Agilent 1290 series-DAD UV-Detection

Mass spectrum: Agilent 6520 QTOF (quadrupole rod-flight time tandem mass spectrometer)

Acetonitrile (Acetonitrile): HPLC grade, Fisher Scientific

Methyl alcohol (Methanol): HPLC grade, Tianjin Jizhun Chemicals

Ultrapure water (ultra-pure water)

Solid-phase extraction column (SPE): Tianjin Shuangji chromatography scientific

1.2 chromatographic condition

Analytical column:Agilent XDB-C18 150 × 4.6mm 5 μm is (in order to corresponding with the chromatogram of ion trap mass spectrometry above, select same root chromatogram column, mass detector analysis is entered) with shunting mode, determined wavelength: 205nm, column temperature: 30 DEG C, flow velocity: 0.8ml/min, theoretical version number calculates should be not less than 200000 by Rf peak;

Timetable：

1.3 Mass Spectrometry Conditions

ESI conditions：

Gas Temp：350℃；Drying Gas：8L/min；Nebulizer：30psi；Vcap：4000V

MS QTOF conditions：

Fragmentor：175V；Skimmer：65V；OCT1 RF Vpp：750V

1.4 workstation

Agilent MassHunter Workstation Qualitative Analysis

Version B.03.01 Build 3.1.346.0，Agilent Technologies Inc.2003-2009

2 need testing solution preparations

With under " experimental example 1,2 "

3 experimental results

3.1 medicine composition injection UHPLC-ESI-QTOF of the present invention analyze collection of illustrative plates (see Figure 21)

3.2 qualification structures

In order to chromatographic peak is number corresponding with liquid phase-Low Resolution Mass Spectra combination analysis result, have employed the chromatographic peak identical with experimental example 1 lower collection of illustrative plates and number;

^*the compound identification that molecular formula is the same with molecular weight is the chromatographic retention determination compounds belong in conjunction with chemical reference substance;

4 medicine composition injection component high-resolution LC-MS of the present invention identify

4.1 (6aR, 11aR)-3-hydroxy-9,10-dimethoxypterocarpan-3-O-β-D-sambubioside (No.1) (see Figure 22)

4.2 ginsenoside Rg1s (No.2) (see Figure 23)

Chemical constitution:

4.3 ginsenoside Res (No.3) (see Figure 24)

Chemical constitution:

4.4 9,10-dimethoxy red sandalwood alkane-3-O-β-D-Glucose glycosides (No.4) (see Figure 25)

Chemical constitution:

4.5 2 '-hydroxyl-3 ', 4 '-dimethoxy isoflavan-7-O-β-D-Glucose glycosides (No.5) (see Figure 26)

Chemical constitution:

4.6 ginsenoside Rfs (No.6) (see Figure 27)

Chemical constitution:

4.7 ginsenoside Rb1s (No.7) (see Figure 28)

Molecular formula	Molion	Calculated value	Measured value	Error (ppm)	Qualification
						C ₅₄H ₉₂O ₂₃	[M-H] ^-	1107.5957	1107.5960	-0.39	Ginsenoside Rb1

Chemical constitution:

4.8 Ginsenoside Rcs (No.8) (see Figure 29)

Molecular formula	Molion	Calculated value	Measured value	Error (ppm)	Qualification
						C ₅₃H ₉₀O ₂₂	[M-H] ^-	1077.5851	1077.5853	-0.27	Ginsenoside Rc

Chemical constitution:

4.9 ginsenoside Rg2s (No.9) (see Figure 30)

Chemical constitution:

4.10 ginsenoside Rb2s (No.10) (see Figure 31)

Molecular formula	Molion	Calculated value	Measured value	Error (ppm)	Qualification
						C ₅₃H ₉₀O ₂₂	[M-H] ^-	1077.5851	1077.5855	-0.24	Ginsenoside Rb2

Chemical constitution:

4.11 ginsenoside Rh 1s (No.11) (see Figure 32)

Molecular formula	Molion	Calculated value	Measured value	Error (ppm)	Qualification
						C ₃₆H ₆₂O ₉	[M+HCOO] ^-	683.4376	683.4383	-1.12	Ginsenoside Rh 1

Chemical constitution:

4.12 Astragaloside IVs (No.12) (see Figure 33)

Molecular formula	Molion	Calculated value	Measured value	Error (ppm)	Qualification
						C ₄₁H ₆₈O ₁₄	[M+HCOO] ^-	829.4591	829.4587	0.61	Astragaloside IV

Chemical constitution:

4.13 ginsenoside Rds (No.13) (see Figure 34)

Molecular formula	Molion	Calculated value	Measured value	Error (ppm)	Qualification
						C ₄₈H ₈₂O ₁₈	[M-H] ^-	945.5428	945.5422	0.85	Ginsenoside Rd
	[M+HCOO] ^-	991.5483	991.5466	1.89

Chemical constitution:

4.14 ginsenoside Rgs 5, F4, Rk1 or Rg6 (No.14) (see Figure 35)

Chemical constitution:

4.15 Ginsenoside Rh4s or Rk3 (No.15) (see Figure 36)

Chemical constitution is shown in 4.16

4.16 3beta,6alpha,12beta-Trihydroxydammar-20(21),24-diene-6-O-beta-D-glucopyranosides or Rh4 (No.16) (see Figure 37)

Chemical constitution:

Experimental example 3 Precision Experiment

Instrument and material: efficient liquid phase: Agilent 1200 series, trifluoroacetic acid aqueous solution (Acetonitrile): HPLC grade, Fisher Scientific, Chromatographic Pure Methanol (Methanol): HPLC grade, Tianjin Jizhun Chemicals, ultrapure water (Water): ultra-pure water, solid-phase extraction column (SPE): Tianjin Shuangji chromatography scientific

Chromatographic condition: analyze chromatographic column: Agilent XDB-C18 150 × 4.6mm 5 μm, determined wavelength: 205nm, column temperature: 30 DEG C, flow velocity: 0.8ml/min, number of theoretical plate calculates should be not less than 200000 by ginsenoside Rf peak

Timetable：

Prepared by test liquid: precision measures medicine composition injection 10ml of the present invention, water bath method, and residue adds 1% glacial acetic acid aqueous solution 2ml makes dissolving, upper solid-phase extraction column (C-18,500mg; Activation step: first use absolute methanol 5ml before use, rinse with 5ml distilled water again) be separated, first with 15ml 1% glacial acetic acid aqueous solution wash-out, then with 10ml water elution, then with 10% methanol aqueous solution 15ml wash-out, discard above-mentioned eluent, then with 80% methyl alcohol 18ml eluant solution, collect 80% meoh eluate, water bath method, residue adds methyl alcohol and dissolves and be transferred in 2ml measuring bottle, adds methyl alcohol to scale, shakes up, with 0.45 μm of filtering with microporous membrane, sample introduction 10 μ l analyzes;

Get above-mentioned same test liquid, continuous sample introduction 6 times, measures by above-mentioned chromatographic condition, 15 total peaks are selected to compare, result (see table 1,2) shows each total peak relative retention time RSD≤0.099%, and each total peak relative peak area RSD≤2.962%, shows that precision is good.

Table 1 Precision Experiment result (relative retention time)

Table 2 Precision Experiment result (relative Retention area)

Experimental example 4 reappearance is tested

Operate preparation 6 parts of test liquids according to the preparation method of experimental example 4, measure by above-mentioned chromatographic condition, result (see table 3,4) shows each total peak relative retention time RSD≤0.145%, each total peak relative peak area RSD≤2.806%; Show that its reappearance is better.

Table 3 reappearance experimental result (relative retention time)

Table 4 reappearance experimental result (relative Retention area)

Experimental example 5 stability experiment

Test liquid is prepared according to preparation method's operation of experimental example 4, get same need testing solution, respectively at 0,2,4,8,12,24h measures by above-mentioned chromatographic condition, result (see table 5,6) each total peak relative retention time RSD≤0.029%, each total peak relative peak area RSD≤2.352%; Show that need testing solution is good at 24h internal stability.

Table 5 stability experiment result (relative retention time)

Table 6 stability experiment result (relative Retention area)

The foundation of experimental example 6 medicine composition injection finger-print of the present invention

The foundation of 1 HPLC finger-print and the demarcation of total fingerprint peaks

Medicine composition injection HPLC/UV finger-print of the present invention is set up with " technical requirement (provisional) of traditional Chinese medicine finger-print research "; By record 150min collection of illustrative plates (see Figure 38), occur without chromatographic peak again after can seeing 80min, thus determine that the acquisition time of finger-print is 80min, and wherein before two peaks in 2min be respectively solvent peak and kushenin peak, the solvent peak that about 68min causes for eluent gradient wash-out, blank collection of illustrative plates also this peak provable is solvent peak (see Figure 39); Operate according to need testing solution preparation method, prepare 10 batches of Kang ' ai injection need testing solutions, by comparing the analysis testing result of 10 batches of test sample HPLC chromatograms, in conjunction with the chemical composition ownership of each chromatographic peak of " in Kang ' ai injection constituent analysis qualification " research qualification, selected wherein 15 total chromatographic peaks are total fingerprint peaks, establish the HPLC chromatographic fingerprinting (see Figure 40) of Kang ' ai injection;

In finger-print, No. 6 chromatographic peaks are through interpretation of mass spectra, chemical reference substance analysis contrast, be accredited as ginsenoside Rf, its peak area accounts for 4% of total peak area, more stable, therefore select it to be with reference to peak, be designated as S, each chromatographic peak retention time is compared with the retention time with reference to peak in same collection of illustrative plates, its ratio is the relative retention time of each chromatographic peak, and by the peak area ratio at reference peak in each chromatogram peak-to-peak area and same collection of illustrative plates comparatively, its ratio is the relative peak area of each chromatographic peak;

The chemical composition ownership of 2 each chromatographic peaks

The qualification at each total peak is with the HPLC retention time analysis of chemical reference substance contrast in conjunction with high-resolution and low resolution LC-MS, then combination is compared seminar's antecedent chemical composition Study result and finally determines the chemical composition ownership of following 15 total chromatographic peaks;

* No. 6 peak Ginsenoside-Rf stablize, the behavior of degree of separation isochromatic spectrum is better, therefore it can be used as with reference to peak;

* can not reach baseline separation due to Ginsenoside-Rh1 and astragaloside IV two chromatographic peaks, therefore calculates with its total peak area, carries out evaluation of methodology.

In experimental example 7 medicine composition injection of the present invention, total saposins component content measures

In medicine composition injection of the present invention, saponin component mainly comprises ginsenoside and astragaloside, wherein ginsenoside is ginsenoside Re,, Rg1, Rf, Rb1, Rc,, Rg2, Rb2, Rd, Rh1, Rg5, Rh4 and Rk3, astragaloside take Astragaloside IV as the Radix Astragali of principal ingredient; Formulate medicine composition injection total saposins of the present invention method for measuring: the total content measuring ginsenoside and astragaloside after preparation mixing reference substance under the same conditions;

Prepared by reference substance solution: precision takes ginsenoside Rg1's standard items, is placed in volumetric flask, and methyl alcohol dissolves and constant volume, is mixed with the ginsenoside stock solution of 0.999mg/ml; Precision takes Astragaloside IV standard items, be placed in volumetric flask, methyl alcohol dissolves and constant volume, is mixed with the Astragaloside IV stock solution of 1.100mg/ml, the ratio of above two kinds of stock solutions according to 1: 1 (V/V) mixed, making concentration is 1.050mg/ml reference substance solution;

Need testing solution: precision measures medicine composition injection 2.5ml of the present invention, injects the C18 SPE post after activation, adopts water 10ml successively, 1% acetic acid-water 15ml, water 15ml, 10% methanol-water 10ml, 80% methanol-water 15ml, wash-out; By 80% methanol-water elution fraction evaporate to dryness, get the appropriate constant volume of methyl alcohol in 2ml volumetric flask, make need testing solution;

Assay method: accurate absorption need testing solution 200 μ L, be placed in test tube, nitrogen dries up, and adds vanillic aldehyde 0.2ml, and perchloric acid 0.8ml mixes, and carries out chromogenic reaction 15min in 60 DEG C, adds glacial acetic acid 5ml and mixes, ice bath cooling 1min; Make reference with blank solution, under 552nm wavelength, measure absorbance, substitute into the amount that typical curve calculates total saposins;

Ginseng and astragalus root total saponin measure: getting lot number is 110440,110441,110445 totally 3 batches of medicine composition injections of the present invention, and prepare need testing solution as stated above, under these conditions sample analysis, record absorbance, calculates content, the results are shown in Table 7.

The assay result (n=3) of table 7 total saposins

9,10-dimethoxy red sandalwood alkane-3-O-β-D-Glucose glycosides assays in experimental example 8 medicine composition injection of the present invention

Prepared by reference substance solution: take 9,10-dimethoxy red sandalwood alkane-3-O-β-D-Glucose glycosides reference substance, be formulated as the reference substance solution of 15.9 μ g/ml

Prepared by need testing solution: precision measures medicine composition injection 10ml of the present invention, water bath method, and residue adds 1% glacial acetic acid aqueous solution 2ml makes dissolving, upper solid-phase extraction column (C-18,500mg; Activation step: first use absolute methanol 5ml before use, rinse with 5ml distilled water again) be separated, first with 15ml 1% glacial acetic acid aqueous solution wash-out, then with 10ml water elution, then with 10% methanol aqueous solution 15ml wash-out, discard above-mentioned eluent, then with 80% methyl alcohol 18ml eluant solution, collect 80% meoh eluate, water bath method, residue adds methyl alcohol and dissolves and be transferred in 2ml measuring bottle, adds methyl alcohol to scale, shakes up, with 0.45 μm of filtering with microporous membrane, sample introduction 10 μ l analyzes;

Liquid-phase condition: Agilent Eclipse XDB-C18 (150 × 4.6mm, 5 μm), mobile phase is

Determined wavelength 207nm, column temperature 30 DEG C, flow velocity 0.8ml/min

Assay: accurate absorption need testing solution 200 μ L, be placed in test tube, nitrogen dries up, and adds vanillic aldehyde 0.2ml, and perchloric acid 0.8ml mixes, and carries out chromogenic reaction 15min in 60 DEG C, adds glacial acetic acid 5ml and mixes, ice bath cooling 1min; Make reference with blank solution, under 552nm wavelength, measure absorbance, substitute into the amount that typical curve calculates total saposins, the results are shown in table 8.

The assay result (n=3) of table 89,10-dimethoxy red sandalwood alkane-3-O-β-D-Glucose glycosides

Active constituent content analysis in medicine composition injection of the present invention: in parenteral solution, the content that adds of kushenin is 10mg/ml; Medicine composition injection effective constituent of the present invention comprises kushenin, ginseng and astragalus root total saponin and flavones ingredient; Lot number is that in 110440 Kang ' ai injections, every 10ml total solid quality is 104.9mg, and Determination of Oxymatrine is 97mg/10ml, and total saponin content is 4.83mg/10ml, and in flavones, 9,10-dimethoxy red sandalwood alkane-3-O-β-D-Glucose glycosides content are 0.13mg/10ml; So effective constituent accounts for 97.19% of total total solid in medicine composition injection of the present invention, meet traditional Chinese medicine injection relevant criterion.

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

Embodiment 1 UHPLC-ESI-QTOF medicine composition injection thin-layer identification method of the present invention

The preparation method of medicine composition injection of the present invention is: Radix Astragali 28kg, ginseng 12kg, kushenin 0.9kg, ginseng with 75% ethanol, refluxing extraction three times, each 2 hours, merge extract, be evaporated to the clear cream that relative density is 1.10 (65 DEG C), for subsequent use; Radix Astragali boiling secondary, each 2 hours, filter, merging filtrate, be evaporated to the clear cream that relative density is 1.20 (65 DEG C), merge with the clear cream of ginseng, add ethanol and make alcohol content reach 75%, adjust pH value to 6 with NaOH, leave standstill 12 hours, get supernatant, reclaim ethanol, be evaporated to the clear cream that relative density is 1.10 (65 DEG C); Adding ethanol again makes alcohol content reach 85%, and with NaOH tone pitch PH to 6, leave standstill, filter, filtrate recycling ethanol to without alcohol taste, injects with water to 400ml, and with NaOH tune pH value to 6,100 DEG C of sterilizings 30 minutes, refrigerate, suction filtration; Adjust pH value to 6 with NaOH, add activated charcoal appropriate, stir evenly, boil 15 minutes, filter, filtrate is for subsequent use; Separately get kushenin to dissolve, adjust pH value to 6 with watery hydrochloric acid, 100 DEG C of sterilizings 30 minutes, refrigeration, suction filtration, merges with the liquid after de-charcoal, and mixing, injects water to ormal weight, and filtration is filling, to obtain final product; Chromatographic condition: analyze chromatographic column: Agilent XDB-C18150 × 4.6mm 5 μm, determined wavelength: 205nm, column temperature: 30 DEG C, flow velocity: 0.8ml/min, number of theoretical plate calculates should be not less than 200000 by ginsenoside Rf peak, mobile phase: take acetonitrile as mobile phase A, take water as Mobile phase B, carry out gradient elution: 15%A (0min), 19%A (20min), 25.5%A (36min), 50%A (50min), 70%A (65min), 70%A (85min);

Mass Spectrometry Conditions: electron spray (ESI) ion gun, positive ion mode; Gas curtain temperature degree (Gas temp) 350 DEG C; Gas curtain gas velocity (Gas flow) 8L/min; Assisted gas pressure (Nebulizer) is 30psi; Vcap (capillary voltage): 4000V; MS QTOF conditions (level Four bar-flight time tandem mass spectrometer condition): Fragmentor (cracked voltage): 175V; Skimmer (intercepting cone voltage): 65V; OCT1 RF Vpp (radio-frequency voltages of first heavy eight grades of bars): 750V;

Prepared by need testing solution: precision measures medicine composition injection 10ml of the present invention, water bath method, residue adds 1% glacial acetic acid aqueous solution 2ml makes dissolving, upper solid-phase extraction column (C-18, 500mg, activation step: first use absolute methanol 5ml before use, rinse with 5ml distilled water again) be separated, first with 15ml 1% glacial acetic acid aqueous solution wash-out, again with 10ml water elution, then with 10% methanol aqueous solution 15ml wash-out, discard above-mentioned eluent, again with 80% methyl alcohol 18ml eluant solution, collect 80% meoh eluate, water bath method, residue adds methyl alcohol and dissolves and be transferred in 2ml measuring bottle, add methyl alcohol to scale, shake up, with 0.45 μm of filtering with microporous membrane, sample introduction 10 μ l analyzes, HPLC analysis is carried out to 10% and 100% meoh eluate, there is no saponins and flavonoids chromatographic peak in the retention time of correspondence,

Medicine composition injection finger-print of the present invention has 15 fingerprint peakses, and the relative retention time of total fingerprint peaks is respectively: 0.712,0.740,0.749,0.769,0.834,1.000,1.019,1.031,1.038,1.045,1.059,1.072,1.173,1.191,1.204; Relative reservation peak area is respectively: 1.616,2.631,2.036,12.671,3.346,1.000,0.868,0.428,0.566,0.854,0.633,0.402,0.417,0.295,0.461.

(the results are shown in accompanying drawing 40)

Embodiment 2 medicine composition injection quality determining method of the present invention

In B medicine composition injection 9, the content assaying method of 10-dimethoxy red sandalwood alkane-3-O-β-D-Glucose glycosides is: prepared by reference substance solution, take 9,10-dimethoxy red sandalwood alkane-3-O-β-D-Glucose glycosides reference substance, be formulated as the reference substance solution of 15.9 μ g/ml, prepared by need testing solution, precision measures medicine composition injection 10ml of the present invention, water bath method, residue adds 1% glacial acetic acid aqueous solution 2ml makes dissolving, upper C-18, 500mg solid-phase extraction column, absolute methanol 5ml is first used before using, use 5ml again) distilled water flushing separation, with 15ml 1% glacial acetic acid aqueous solution wash-out, again with 10ml water elution, then with 10% methanol aqueous solution 15ml wash-out, discard above-mentioned eluent, again with 80% methyl alcohol 18ml eluant solution, collect 80% meoh eluate, water bath method, residue adds methyl alcohol and dissolves and be transferred in 2ml measuring bottle, add methyl alcohol to scale, shake up, with 0.45 μm of filtering with microporous membrane, sample introduction 10 μ L analyzes, liquid-phase condition: Agilent Eclipse XDB-C18 (150 × 4.6mm, 5 μm), mobile phase: take acetonitrile as mobile phase A, take water as Mobile phase B, carry out gradient elution: 15%A (0min), 25%A (30min), 40%A (40min) determined wavelength 207nm, column temperature 30 DEG C, flow velocity 0.8ml/min, accurate absorption need testing solution 200 μ L, be placed in test tube, nitrogen dries up, add vanillic aldehyde 0.2ml, perchloric acid 0.8ml mixes, and carries out chromogenic reaction 15min in 60 DEG C, adds glacial acetic acid 5ml and mixes, ice bath cooling 1min, make reference with blank solution, under 552nm wavelength, measure absorbance, substitute into the amount that typical curve calculates total saposins, to obtain final product,

The quality determining method of C medicine composition injection finger-print is, chromatographic condition: analyze chromatographic column: AgilentXDB-C18 150 × 4.6mm 5 μm, determined wavelength: 205nm, column temperature: 30 DEG C, flow velocity: 0.8ml/min, number of theoretical plate calculates should be not less than 200000 by ginsenoside Rf peak, mobile phase: take acetonitrile as mobile phase A, take water as Mobile phase B, carry out gradient elution: 15%A (0min), 19%A (20min), 25.5%A (36min), 50%A (50min), 70%A (65min), 70%A (85min), Mass Spectrometry Conditions: electron spray (ESI) ion gun, positive ion mode, gas curtain temperature degree (Gas temp) 350 DEG C, gas curtain gas velocity (Gas flow) 8L/min, assisted gas pressure (Nebulizer) is 30psi, Vcap (capillary voltage): 4000V, MS QTOF conditions (level Four bar-flight time tandem mass spectrometer condition): Fragmentor (cracked voltage): 175V, Skimmer (intercepting cone voltage): 65V, OCT1 RF Vpp (radio-frequency voltages of first heavy eight grades of bars): 750V, prepared by need testing solution: precision measures medicine composition injection 10ml of the present invention, water bath method, residue adds 1% glacial acetic acid aqueous solution 2ml makes dissolving, upper solid-phase extraction column (C-18, 500mg, activation step: first use absolute methanol 5ml before use, rinse with 5ml distilled water again) be separated, first with 15ml 1% glacial acetic acid aqueous solution wash-out, again with 10ml water elution, then with 10% methanol aqueous solution 15ml wash-out, discard above-mentioned eluent, again with 80% methyl alcohol 18ml eluant solution, collect 80% meoh eluate, water bath method, residue adds methyl alcohol and dissolves and be transferred in 2ml measuring bottle, add methyl alcohol to scale, shake up, with 0.45 μm of filtering with microporous membrane, sample introduction 10 μ l analyzes, HPLC analysis is carried out to 10% and 100% meoh eluate, there is no saponins and flavonoids chromatographic peak in the retention time of correspondence,

Medicine composition injection finger-print of the present invention has 15 fingerprint peakses, and the relative retention time of total fingerprint peaks is respectively: 0.712,0.740,0.749,0.769,0.834,1.000,1.019,1.031,1.038,1.045,1.059,1.072,1.173,1.191,1.204; Relative reservation peak area is respectively: 1.616,2.631,2.036,12.671,3.346,1.000,0.868,0.428,0.566,0.854,0.633,0.402,0.417,0.295,0.461;

The preparation method of medicine composition injection of the present invention is: Radix Astragali 28kg, ginseng 12kg, kushenin 0.9kg, ginseng with 75% ethanol, refluxing extraction three times, each 2 hours, merge extract, be evaporated to the clear cream that relative density is 1.10 (65 DEG C), for subsequent use; Radix Astragali boiling secondary, each 2 hours, filter, merging filtrate, be evaporated to the clear cream that relative density is 1.20 (65 DEG C), merge with the clear cream of ginseng, add ethanol and make alcohol content reach 75%, adjust pH value to 6 with NaOH, leave standstill 12 hours, get supernatant, reclaim ethanol, be evaporated to the clear cream that relative density is 1.10 (65 DEG C); Adding ethanol again makes alcohol content reach 85%, and with NaOH tone pitch PH to 6, leave standstill, filter, filtrate recycling ethanol to without alcohol taste, injects with water to 400ml, and with NaOH tune pH value to 6,100 DEG C of sterilizings 30 minutes, refrigerate, suction filtration; Adjust pH value to 6 with NaOH, add activated charcoal appropriate, stir evenly, boil 15 minutes, filter, filtrate is for subsequent use; Separately get kushenin to dissolve, adjust pH value to 6 with watery hydrochloric acid, 100 DEG C of sterilizings 30 minutes, refrigeration, suction filtration, merges with the liquid after de-charcoal, and mixing, injects water to ormal weight, and filtration is filling, to obtain final product.

In embodiment 3 medicine composition injection of the present invention, total saposins component content measures

The preparation method of medicine composition injection of the present invention is: Radix Astragali 28kg, ginseng 12kg, kushenin 0.9kg, ginseng with 75% ethanol, refluxing extraction three times, each 2 hours, merge extract, be evaporated to the clear cream that relative density is 1.10 (65 DEG C), for subsequent use; Radix Astragali boiling secondary, each 2 hours, filter, merging filtrate, be evaporated to the clear cream that relative density is 1.20 (65 DEG C), merge with the clear cream of ginseng, add ethanol and make alcohol content reach 75%, adjust pH value to 6 with NaOH, leave standstill 12 hours, get supernatant, reclaim ethanol, be evaporated to the clear cream that relative density is 1.10 (65 DEG C); Adding ethanol again makes alcohol content reach 85%, and with NaOH tone pitch PH to 6, leave standstill, filter, filtrate recycling ethanol to without alcohol taste, injects with water to 400ml, and with NaOH tune pH value to 6,100 DEG C of sterilizings 30 minutes, refrigerate, suction filtration; Adjust pH value to 6 with NaOH, add activated charcoal appropriate, stir evenly, boil 15 minutes, filter, filtrate is for subsequent use; Separately get kushenin to dissolve, adjust pH value to 6 with watery hydrochloric acid, 100 DEG C of sterilizings 30 minutes, refrigeration, suction filtration, merges with the liquid after de-charcoal, and mixing, injects water to ormal weight, and filtration is filling, to obtain final product;

Assay method: accurate absorption need testing solution 200 μ L, be placed in test tube, nitrogen dries up, add vanillic aldehyde 0.2ml, perchloric acid 0.8ml mixes, and carries out chromogenic reaction 15min in 60 DEG C, adds glacial acetic acid 5ml and mixes, ice bath cooling 1min, make reference with blank solution, under 552nm wavelength, measure absorbance, substitute into the amount that typical curve calculates total saposins, to obtain final product.

9,10-dimethoxy red sandalwood alkane-3-O-β-D-Glucose glycosides assays in embodiment 4 medicine composition injection of the present invention

Liquid-phase condition: Agilent Eclipse XDB-C18 (150 × 4.6mm, 5 μm), mobile phase: take acetonitrile as mobile phase A, take water as Mobile phase B, carry out gradient elution: 15%A (0min), 25%A (30min), 40%A (40min) determined wavelength 207nm, column temperature 30 DEG C, flow velocity 0.8ml/min

Assay: accurate absorption need testing solution 200 μ L, be placed in test tube, nitrogen dries up, and adds vanillic aldehyde 0.2ml, and perchloric acid 0.8ml mixes, and carries out chromogenic reaction 15min in 60 DEG C, adds glacial acetic acid 5ml and mixes, ice bath cooling 1min; Make reference with blank solution, under 552nm wavelength, measure absorbance, substitute into the amount that typical curve calculates total saposins, to obtain final product;

The preparation method of medicine composition injection of the present invention is: Radix Astragali 30kg, ginseng 10kg, kushenin 1kg, ginseng with 75% ethanol, refluxing extraction three times, each 2 hours, merge extract, be evaporated to the clear cream that relative density is 1.15 (65 DEG C), for subsequent use; Radix Astragali boiling secondary, each 2 hours, filter, merging filtrate, be evaporated to the clear cream that relative density is 1.15 (65 DEG C), merge with the clear cream of ginseng, add ethanol and make alcohol content reach 75%, adjust pH value to 7 with NaOH, leave standstill 12 hours, get supernatant, reclaim ethanol, be evaporated to the clear cream that relative density is 1.12 (65 DEG C); Adding ethanol again makes alcohol content reach 85%, and with NaOH tone pitch PH to 7, leave standstill, filter, filtrate recycling ethanol to without alcohol taste, injects with water to 400ml, and with NaOH tune pH value to 7,100 DEG C of sterilizings 30 minutes, refrigerate, suction filtration; Adjust pH value to 7 with NaOH, add activated charcoal appropriate, stir evenly, boil 15 minutes, filter, filtrate is for subsequent use; Separately get kushenin to dissolve, adjust pH value to 7 with watery hydrochloric acid, 100 DEG C of sterilizings 30 minutes, refrigeration, suction filtration, merges with the liquid after de-charcoal, and mixing, injects water to ormal weight, and filtration is filling, to obtain final product.

Embodiment 5 medicine composition injection thin-layer identification method of the present invention

The preparation method of medicine composition injection of the present invention is: Radix Astragali 28kg, ginseng 12kg, kushenin 0.9kg, ginseng with 75% ethanol, refluxing extraction three times, each 2 hours, merge extract, be evaporated to the clear cream that relative density is 1.10 (65 DEG C), for subsequent use; Radix Astragali boiling secondary, each 2 hours, filter, merging filtrate, be evaporated to the clear cream that relative density is 1.20 (65 DEG C), merge with the clear cream of ginseng, add ethanol and make alcohol content reach 75%, adjust pH value to 6 with NaOH, leave standstill 12 hours, get supernatant, reclaim ethanol, be evaporated to the clear cream that relative density is 1.10 (65 DEG C); Adding ethanol again makes alcohol content reach 85%, and with NaOH tone pitch PH to 6, leave standstill, filter, filtrate recycling ethanol to without alcohol taste, injects with water to 400ml, and with NaOH tune pH value to 6,100 DEG C of sterilizings 30 minutes, refrigerate, suction filtration; Adjust pH value to 6 with NaOH, add activated charcoal appropriate, stir evenly, boil 15 minutes, filter, filtrate is for subsequent use; Separately get kushenin to dissolve, adjust pH value to 6 with watery hydrochloric acid, 100 DEG C of sterilizings 30 minutes, refrigeration, suction filtration, merges with the liquid after de-charcoal, and mixing, injects water to ormal weight, and filtration is filling, to obtain final product; In A medicine composition injection, the content assaying method of total saposins composition is: prepared by reference substance solution, precision takes ginsenoside Rg1's standard items, is placed in volumetric flask, and methyl alcohol dissolves and constant volume, is mixed with the ginsenoside stock solution of 0.98 ~ 1.10mg/ml; Precision takes Astragaloside IV standard items, be placed in volumetric flask, methyl alcohol dissolves and constant volume, is mixed with the Astragaloside IV stock solution of 1.0mg/ml, the ratio of above two kinds of stock solutions according to 1: 1 (V/V) mixed, making concentration is 1.0mg/ml reference substance solution; Prepared by need testing solution, precision measures medicine composition injection 2.1ml of the present invention, injects the C18 SPE post after activation, adopts water 10ml successively, 1% acetic acid-water 15ml, water 15ml, 10% methanol-water 10ml, 80% methanol-water 15ml, wash-out, by 80% methanol-water elution fraction evaporate to dryness, get the appropriate constant volume of methyl alcohol in 2ml volumetric flask, make need testing solution; Accurate absorption need testing solution 200 μ L, be placed in test tube, nitrogen dries up, add vanillic aldehyde 0.2ml, perchloric acid 0.8ml mixes, and carries out chromogenic reaction 15min in 60 DEG C, adds glacial acetic acid 5ml and mixes, ice bath cooling 1min, make reference with blank solution, under 552nm wavelength, measure absorbance, substitute into the amount that typical curve calculates total saposins, to obtain final product;

In B medicine composition injection 9, the content assaying method of 10-dimethoxy red sandalwood alkane-3-O-β-D-Glucose glycosides is: prepared by reference substance solution, take 9,10-dimethoxy red sandalwood alkane-3-O-β-D-Glucose glycosides reference substance, be formulated as the reference substance solution of 15.2 μ g/ml, prepared by need testing solution, precision measures medicine composition injection 10ml of the present invention, water bath method, residue adds 1% glacial acetic acid aqueous solution 1 ~ 3ml makes dissolving, upper C-18, 500mg solid-phase extraction column, absolute methanol 5ml is first used before using, rinse with 5ml distilled water again and be separated, with 15ml 1% glacial acetic acid aqueous solution wash-out, again with 10ml water elution, then with 10% methanol aqueous solution 15ml wash-out, discard above-mentioned eluent, again with 80% methyl alcohol 18ml eluant solution, collect 80% meoh eluate, water bath method, residue adds methyl alcohol and dissolves and be transferred in 2ml measuring bottle, add methyl alcohol to scale, shake up, with 0.45 μm of filtering with microporous membrane, sample introduction 10 μ L analyzes, liquid-phase condition: Agilent Eclipse XDB-C18 (150 × 4.6mm, 5 μm), mobile phase: take acetonitrile as mobile phase A, take water as Mobile phase B, carry out gradient elution: 15%A (0min), 25%A (30min), 40%A (40min), determined wavelength 207nm, column temperature 30 DEG C, flow velocity 0.8ml/min, accurate absorption need testing solution 200 μ L, be placed in test tube, nitrogen dries up, add vanillic aldehyde 0.2ml, perchloric acid 0.8ml mixes, and carries out chromogenic reaction 15min in 60 DEG C, adds glacial acetic acid 5ml and mixes, ice bath cooling lmin, make reference with blank solution, under 552nm wavelength, measure absorbance, substitute into the amount that typical curve calculates total saposins, to obtain final product,

Embodiment 6 medicine composition injection thin-layer identification method of the present invention

C medicine composition injection fingerprint atlas detection method is: chromatographic condition, analyze chromatographic column Agilent XDB-C18150 × 4.6mm 5 μm, determined wavelength 205nm, column temperature 30 DEG C, flow velocity 0.8ml/min, number of theoretical plate calculates should be not less than 200000 by ginsenoside Rf peak, mobile phase: take acetonitrile as mobile phase A, take water as Mobile phase B, carry out gradient elution: 15%A (0min), 19%A (20min), 25.5%A (36min), 50%A (50min), 70%A (65min), 70%A (85min), Mass Spectrometry Conditions: electron spray (ESI) ion gun, positive ion mode, gas curtain temperature degree (Gas temp) 350 DEG C, gas curtain gas velocity (Gas flow) 8L/min, assisted gas pressure (Nebulizer) is 30psi, capillary voltage (Vcap): 4000V, level Four bar-flight time tandem mass spectrometer condition (MS QTOF conditions): cracked voltage (Fragmentor): 175V, intercept cone voltage (Skimmer): 65V, radio-frequency voltage (OCT1 RFVpp) 750V of first heavy eight grades of bars, prepared by need testing solution: precision measures medicine composition injection 10ml of the present invention, water bath method, residue adds 1% glacial acetic acid aqueous solution 2ml makes dissolving, upper solid-phase extraction column (C-18, 500mg, activation step: first use absolute methanol 5ml before use, rinse with 5ml distilled water again) be separated, first with 15ml 1% glacial acetic acid aqueous solution wash-out, again with 10ml water elution, then with 11% methanol aqueous solution 15ml wash-out, discard above-mentioned eluent, again with 82% methyl alcohol 18ml eluant solution, collect 82% meoh eluate, water bath method, residue adds methyl alcohol and dissolves and be transferred in 2ml measuring bottle, add methyl alcohol to scale, shake up, with 0.45 μm of filtering with microporous membrane, sample introduction 10 μ l analyzes, HPLC analysis is carried out to 10% and 100% meoh eluate, there is no saponins and flavonoids chromatographic peak in the retention time of correspondence, medicine composition injection finger-print of the present invention has 15 fingerprint peakses, and the relative retention time of total fingerprint peaks is respectively: 0.642,0.670,0.679,0.700,0.75,1.000,1.009,1.008,1.008,1.005,1.009,1.007,1156,1.072,1.084, relative reservation peak area is respectively: 1.455,2.368,1.832,11.404,3.311,1.000,0.781,0.385,0.509,0.769,0.570,0.362,0.375,0.265,0.415,

The preparation method of invention medicine composition injection is: Radix Astragali 28kg, ginseng 12kg, kushenin 0.9kg, ginseng with 75% ethanol, refluxing extraction three times, each 2 hours, merge extract, be evaporated to the clear cream that relative density is 1.10 (65 DEG C), for subsequent use; Radix Astragali boiling secondary, each 2 hours, filter, merging filtrate, be evaporated to the clear cream that relative density is 1.20 (65 DEG C), merge with the clear cream of ginseng, add ethanol and make alcohol content reach 75%, adjust pH value to 6 with NaOH, leave standstill 12 hours, get supernatant, reclaim ethanol, be evaporated to the clear cream that relative density is 1.10 (65 DEG C); Adding ethanol again makes alcohol content reach 85%, and with NaOH tone pitch PH to 6, leave standstill, filter, filtrate recycling ethanol to without alcohol taste, injects with water to 400ml, and with NaOH tune pH value to 6,100 DEG C of sterilizings 30 minutes, refrigerate, suction filtration; Adjust pH value to 6 with NaOH, add activated charcoal appropriate, stir evenly, boil 15 minutes, filter, filtrate is for subsequent use; Separately get kushenin to dissolve, adjust pH value to 6 with watery hydrochloric acid, 100 DEG C of sterilizings 30 minutes, refrigeration, suction filtration, merges with the liquid after de-charcoal, and mixing, injects water to ormal weight, and filtration is filling, to obtain final product.

Claims

1. the quality determining method of a medicine composition injection, it is characterized in that the method only comprises following 9, the content assaying method of 10-dimethoxy red sandalwood alkane-3-O-β-D-Glucose glycosides, or the method comprises the following content assaying method of 9,10-dimethoxy red sandalwood alkane-3-O-β-D-Glucose glycosides and the content assaying method of total saposins composition and/or fingerprint atlas detection method:

In A, medicine composition injection, the content assaying method of total saposins composition is: prepared by reference substance solution, precision takes ginsenoside Rg1's standard items, is placed in volumetric flask, and methyl alcohol dissolves and constant volume, is mixed with the ginsenoside stock solution of 0.98 ~ 1.10mg/ml; Precision takes Astragaloside IV standard items, be placed in volumetric flask, methyl alcohol dissolves and constant volume, is mixed with the Astragaloside IV stock solution of 1.0 ~ 1.2mg/ml, mixed according to the ratio of 1:1V/V by above two kinds of stock solutions, making concentration is 1.0 ~ 1.1mg/ml reference substance solution; Prepared by need testing solution, precision measures medicine composition injection 2 ~ 3ml of the present invention, injects the C18SPE post after activation, adopts water 10ml successively, 1% acetic acid-water 15ml, water 15ml, 10% methanol-water 10ml, 80% methanol-water 15ml, wash-out, by 80% methanol-water elution fraction evaporate to dryness, get the appropriate constant volume of methyl alcohol in 2ml volumetric flask, make need testing solution; Accurate absorption need testing solution 200 μ L, be placed in test tube, nitrogen dries up, add vanillic aldehyde 0.2ml, perchloric acid 0.8ml mixes, and carries out chromogenic reaction 15min in 60 DEG C, adds glacial acetic acid 5ml and mixes, ice bath cooling 1min, make reference with blank solution, under 552nm wavelength, measure absorbance, substitute into the amount that typical curve calculates total saposins, to obtain final product;

In B, medicine composition injection 9, the content assaying method of 10-dimethoxy red sandalwood alkane-3-O-β-D-Glucose glycosides is: prepared by reference substance solution, take 9,10-dimethoxy red sandalwood alkane-3-O-β-D-Glucose glycosides reference substance, be formulated as the reference substance solution of 15.0 ~ 16.5 μ g/ml, prepared by need testing solution, precision measures medicine composition injection 10ml of the present invention, water bath method, residue adds 1% glacial acetic acid aqueous solution 1 ~ 3ml makes dissolving, upper C-18, 500mg solid-phase extraction column, absolute methanol 5ml is first used before using, rinse with 5ml distilled water again and be separated, with 15ml 1% glacial acetic acid aqueous solution wash-out, again with 10ml water elution, then with 10% methanol aqueous solution 15ml wash-out, discard above-mentioned eluent, again with 80% methyl alcohol 18ml eluant solution, collect 80% meoh eluate, water bath method, residue adds methyl alcohol and dissolves and be transferred in 2ml measuring bottle, add methyl alcohol to scale, shake up, with 0.45 μm of filtering with microporous membrane, sample introduction 10 μ L analyzes, liquid-phase condition: Agilent EclipseXDB-C18:150 × 4.6mm, 5 μm, mobile phase: take acetonitrile as mobile phase A, take water as Mobile phase B, carry out gradient elution: 0 minute: 15%A, 30 minutes: 25%A, 40 minutes: 40%A, determined wavelength 207nm, column temperature 30 DEG C, flow velocity 0.8ml/min, accurate absorption need testing solution 200 μ L, be placed in test tube, nitrogen dries up, add vanillic aldehyde 0.2ml, perchloric acid 0.8ml mixes, and carries out chromogenic reaction 15min in 60 DEG C, add glacial acetic acid 5ml to mix, ice bath cooling 1min, makes reference with blank solution, under 552nm wavelength, measure absorbance, substitute into the amount that typical curve calculates 9,10-dimethoxy red sandalwood alkane-3-O-β-D-Glucose glycosides, to obtain final product,

C, medicine composition injection fingerprint atlas detection method are: chromatographic condition, analyze chromatographic column Agilent XDB-C18:150 × 4.6mm, 5 μm, determined wavelength 205nm, column temperature 30 DEG C, flow velocity 0.8ml/min, number of theoretical plate calculates by ginsenoside Rf peak should be not less than 200000, mobile phase: being mobile phase A with acetonitrile, take water as Mobile phase B, carry out gradient elution: 0 minute: 15%A, 20 minutes: 19%A, 36 minutes: 25.5%A, 50 minutes: 50%A, 65 minutes: 70%A, 85 minutes: 70%A, Mass Spectrometry Conditions: electron spray (ESI) ion gun, positive ion mode, gas curtain temperature degree (Gas temp) 350 DEG C, gas curtain gas velocity (Gas flow) 8L/min, assisted gas pressure (Nebulizer) is 30psi, capillary voltage (Vcap): 4000V, level Four bar-flight time tandem mass spectrometer condition (MS QTOF conditions): cracked voltage (Fragmentor): 175V, intercept cone voltage (Skimmer): 65V, radio-frequency voltage (OCT1RF Vpp) 750V of first heavy eight grades of bars, prepared by need testing solution: precision measures medicine composition injection 10ml of the present invention, water bath method, residue adds 1% glacial acetic acid aqueous solution 2ml makes dissolving, upper C-18, 500mg solid-phase extraction column, absolute methanol 5ml is first used before using, rinse with 5ml distilled water again and be separated, with 15ml 1% glacial acetic acid aqueous solution wash-out, again with 10ml water elution, then with 8% ~ 12% methanol aqueous solution 15ml wash-out, discard above-mentioned eluent, again with 75% ~ 85% methyl alcohol 18ml eluant solution, collect 75% ~ 85% meoh eluate, water bath method, residue adds methyl alcohol and dissolves and be transferred in 2ml measuring bottle, add methyl alcohol to scale, shake up, with 0.45 μm of filtering with microporous membrane, sample introduction 10 μ l analyzes, HPLC analysis is carried out to 10% and 100% meoh eluate, there is no saponins and flavonoids chromatographic peak in the retention time of correspondence, medicine composition injection finger-print has 15 fingerprint peakses, and the relative retention time of total fingerprint peaks is respectively: 0.712 ± 0.071,0.740 ± 0.074,0.749 ± 0.075,0.769 ± 0.077,0.834 ± 0.083,1.000 ± 0.000,1.019 ± 0.102,1.031 ± 0.103,1.038 ± 0.104,1.045 ± 0.105,1.059 ± 0.106,1.072 ± 0.107,1.173 ± 0.117,1.191 ± 0.119,1.204 ± 0.120, relative reservation peak area is respectively: 1.616 ± 0.161,2.631 ± 0.263,2.036 ± 0.204,12.671 ± 1.267,3.346 ± 0.335,1.000 ± 0.000,0.868 ± 0.087,0.428 ± 0.043,0.566 ± 0.057,0.854 ± 0.085,0.633 ± 0.063,0.402 ± 0.040,0.417 ± 0.042,0.295 ± 0.030,0.461 ± 0.046,

Medicine composition injection is made by the following method: more than Radix Astragali 25-35 weight portion, ginseng 8-12 weight portion, kushenin 0.5-1.5 weight portion three taste medicines, the ethanol of ginseng 60-80%, refluxing extraction 1-3 time, each 1-2 hour, merge extract, be evaporated to the clear cream that relative density is 65 DEG C 1.10 ~ 1.20, for subsequent use; Radix Astragali boiling 1-3 time, each 1-3 hour, filters, merging filtrate, be evaporated to the clear cream that relative density is 65 DEG C 1.10 ~ 1.20, merge with the clear cream of ginseng, add ethanol and make alcohol content reach 75%, adjust pH value to 6 ~ 7 with NaOH, leave standstill 10-18 hour, get supernatant, reclaim ethanol, be evaporated to the clear cream that relative density is 65 DEG C 1.10 ~ 1.15; Adding ethanol again makes alcohol content reach 85%, and with NaOH tone pitch PH to 6 ~ 7, leave standstill, filter, filtrate recycling ethanol to without alcohol taste, injects with water to 400ml, and with NaOH tune pH value to 6 ~ 7,100 DEG C of sterilizings 30 minutes, refrigerate, suction filtration; Adjust pH value to 6 ~ 7 with NaOH, add activated charcoal appropriate, stir evenly, boil 15 minutes, filter, filtrate is for subsequent use; Separately get kushenin to dissolve, adjust pH value to 6 ~ 7 with watery hydrochloric acid, 100--120 DEG C of sterilizing 30 minutes, refrigeration, suction filtration, merges with the liquid after de-charcoal, and mixing, injects water to ormal weight, and filtration is filling, to obtain final product.

2. the quality determining method of medicine composition injection as claimed in claim 1, it is characterized in that the method only comprises following 9, the content assaying method of 10-dimethoxy red sandalwood alkane-3-O-β-D-Glucose glycosides, or the method comprises the following content assaying method of 9,10-dimethoxy red sandalwood alkane-3-O-β-D-Glucose glycosides and the content assaying method of total saposins composition and/or fingerprint atlas detection method:

In A, medicine composition injection, the content assaying method of total saposins composition is: prepared by reference substance solution, precision takes ginsenoside Rg1's standard items, is placed in volumetric flask, and methyl alcohol dissolves and constant volume, is mixed with the ginsenoside stock solution of 0.999mg/ml; Precision takes Astragaloside IV standard items, is placed in volumetric flask, and methyl alcohol dissolves and constant volume, and be mixed with the Astragaloside IV stock solution of 1.100mg/ml, mixed by above two kinds of stock solutions according to the ratio of 1:1V/V, making concentration is 1.050mg/ml reference substance solution; Prepared by need testing solution, precision measures medicine composition injection 2.5ml of the present invention, injects the C18SPE post after activation, adopts water 10ml successively, 1% acetic acid-water 15ml, water 15ml, 10% methanol-water 10ml, 80% methanol-water 15ml, wash-out, by 80% methanol-water elution fraction evaporate to dryness, get the appropriate constant volume of methyl alcohol in 2ml volumetric flask, make need testing solution; Accurate absorption need testing solution 200 μ L, be placed in test tube, nitrogen dries up, add vanillic aldehyde 0.2ml, perchloric acid 0.8ml mixes, and carries out chromogenic reaction 15min in 60 DEG C, adds glacial acetic acid 5ml and mixes, ice bath cooling 1min, make reference with blank solution, under 552nm wavelength, measure absorbance, substitute into the amount that typical curve calculates total saposins, to obtain final product;

In B, medicine composition injection 9, the content assaying method of 10-dimethoxy red sandalwood alkane-3-O-β-D-Glucose glycosides is: prepared by reference substance solution, take 9,10-dimethoxy red sandalwood alkane-3-O-β-D-Glucose glycosides reference substance, be formulated as the reference substance solution of 15.9 μ g/ml, prepared by need testing solution, precision measures medicine composition injection 10ml of the present invention, water bath method, residue adds 1% glacial acetic acid aqueous solution 2ml makes dissolving, upper C-18, 500mg solid-phase extraction column, absolute methanol 5ml is first used before using, rinse with 5ml distilled water again and be separated, with 15ml 1% glacial acetic acid aqueous solution wash-out, again with 10ml water elution, then with 10% methanol aqueous solution 15ml wash-out, discard above-mentioned eluent, again with 80% methyl alcohol 18ml eluant solution, collect 80% meoh eluate, water bath method, residue adds methyl alcohol and dissolves and be transferred in 2ml measuring bottle, add methyl alcohol to scale, shake up, with 0.45 μm of filtering with microporous membrane, sample introduction 10 μ L analyzes, liquid-phase condition: Agilent Eclipse XDB-C18:150 × 4.6mm, 5 μm, mobile phase: take acetonitrile as mobile phase A, take water as Mobile phase B, carry out gradient elution: 0 minute: 15%A, 30 minutes: 25%A, 40 minutes: 40%A, determined wavelength 207nm, column temperature 30 DEG C, flow velocity 0.8ml/min, accurate absorption need testing solution 200 μ L, be placed in test tube, nitrogen dries up, add vanillic aldehyde 0.2ml, perchloric acid 0.8ml mixes, and carries out chromogenic reaction 15min in 60 DEG C, add glacial acetic acid 5ml to mix, ice bath cooling 1min, makes reference with blank solution, under 552nm wavelength, measure absorbance, substitute into the amount that typical curve calculates 9,10-dimethoxy red sandalwood alkane-3-O-β-D-Glucose glycosides, to obtain final product,

The quality determining method of C, medicine composition injection finger-print is, chromatographic condition: analyze chromatographic column: AgilentXDB-C18:150 × 4.6mm, 5 μm, determined wavelength: 205nm, column temperature: 30 DEG C, flow velocity: 0.8ml/min, number of theoretical plate calculates by ginsenoside Rf peak should be not less than 200000, mobile phase: being mobile phase A with acetonitrile, take water as Mobile phase B, carry out gradient elution: 0 minute 15%A, 20 minutes 19%A, 36 minutes 25.5%A, 50 minutes 50%A, 65 minutes 70%A, 85 minutes 70%A, Mass Spectrometry Conditions: electron spray (ESI) ion gun, positive ion mode, gas curtain temperature degree (Gas temp) 350 DEG C, gas curtain gas velocity (Gas flow) 8L/min, assisted gas pressure (Nebulizer) is 30psi, capillary voltage (Vcap) 4000V, level Four bar-flight time tandem mass spectrometer condition (MS QTOF conditions): cracked voltage (Fragmentor) 175V, intercept cone voltage (Skimmer) 65V, radio-frequency voltage (OCT1RFVpp) 750V of first heavy eight grades of bars, prepared by need testing solution: precision measures medicine composition injection 10ml of the present invention, water bath method, residue adds 1% glacial acetic acid aqueous solution 2ml makes dissolving, upper C-18, 500mg solid-phase extraction column, absolute methanol 5ml is first used before using, rinse with 5ml distilled water again and be separated, with 15ml 1% glacial acetic acid aqueous solution wash-out, again with 10ml water elution, then with 10% methanol aqueous solution 15ml wash-out, discard above-mentioned eluent, again with 80% methyl alcohol 18ml eluant solution, collect 80% meoh eluate, water bath method, residue adds methyl alcohol and dissolves and be transferred in 2ml measuring bottle, add methyl alcohol to scale, shake up, with 0.45 μm of filtering with microporous membrane, sample introduction 10 μ l analyzes, HPLC analysis is carried out to 10% and 100% meoh eluate, there is no saponins and flavonoids chromatographic peak in the retention time of correspondence, medicine composition injection finger-print has 15 fingerprint peakses, and the relative retention time of total fingerprint peaks is respectively: 0.712,0.740,0.749,0.769,0.834,1.000,1.019,1.031,1.038,1.045,1.059,1.072,1.173,1.191,1.204, relative reservation peak area is respectively: 1.616,2.631,2.036,12.671,3.346,1.000,0.868,0.428,0.566,0.854,0.633,0.402,0.417,0.295,0.461.