CN103893234B - A kind of Sofflower injection, preparation method and content assaying method - Google Patents
A kind of Sofflower injection, preparation method and content assaying method Download PDFInfo
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Abstract
100 μ g/ml are less than the invention discloses a kind of Sofflower injection wherein P-hydroxybenzoic acid content, also disclose preparation method and content assaying method, the invention controls the content of P-hydroxybenzoic acid by preparation method and content assaying method, reduces the adverse reaction and side effect of finished product.
Description
Technical field
The present invention relates to field of medicaments, and in particular to a kind of Sofflower injection and preparation method thereof and assay method.
Background technology
Safflower be feverfew safflower (Carthamus tinctorius L. dry tubular flower), is that safflower category is unique
Cultigen.Sofflower injection is by the sterile water solution of the extracted yellow of Chinese medicine safflower to brownish red, with promoting blood circulation
The effect of stagnation resolvation, swelling and pain relieving, clinically it is mainly used in treating obliterated cerebral vascular disease, coronary heart disease, arteries and veins that various blood stasis cause
The diseases such as pipe inflammation.With extensive use clinically, its Reporting of harms also increases increasingly, and some adverse reactions are also more tight
Weight, so the quality standard improved and improve Sofflower injection is particularly significant.
P-hydroxybenzoic acid is a native chemical composition in flos carthami, and the component molecules amount is small, and simple structure is
The synthesis substrate of secondary metabolite.Containing the chemical composition of some P-hydroxybenzoic acid residue groups in flos carthami, for example：
Para hydroxybenzene formyl coumarinic anhydride, hydroxycinnamic acid, other polyphenols etc..In Sofflower injection production process, successively
Heating for multiple times can be experienced, these heating processes can cause part to the ingredient breakdown of thermally labile, at the same P-hydroxybenzoic acid from
These chemical compositions（Aldehydes matter）Middle degraded is out.
P-hydroxybenzoic acid, also known as nipalgin acid, for organic synthesis and manufacture dyestuff, makees preservative, bactericide, has
Poison, it is irritant.Molecular formula is C7H6O3, molecular weight 138.13, density 1.443g/cm3, 213-214 DEG C of fusing point, boiling point
199 oC.Structural formula is as follows：
Therefore, P-hydroxybenzoic acid is all contained in safflower injection of the prior art, content is not under control, its medicine
Product security cannot ensure.There is the content method for not yet reporting P-hydroxybenzoic acid in Sofflower injection in the prior art again.
The content of the invention
Present invention aim to overcome that the deficiencies in the prior art, there is provided a kind of Sofflower injection, contain in the Sofflower injection
There are extremely a small amount of P-hydroxybenzoic acid, adverse reaction reduction.
Another object of the present invention there are provided the preparation method of the Sofflower injection.
Another object of the present invention there are provided the assay method of P-hydroxybenzoic acid in the Sofflower injection.
The present invention is achieved by the following technical solutions：
A kind of Sofflower injection of the present invention, contained P-hydroxybenzoic acid content is less than 100 μ g/ml.
It is preferred that contained P-hydroxybenzoic acid content is less than 40 μ g/m1 in Sofflower injection.
It is preferred that contained P-hydroxybenzoic acid content is less than 10 μ g/m1 in Sofflower injection.
Sofflower injection described above, is obtained by following preparation methods：
（1）Flos carthami is taken, decocting is boiled, decoction liquor filtration, ethanol alcohol precipitation after filtrate concentration, filtration, reclaiming ethanol must soak
Cream；
（2）The medicinal extract water is sunk, and filtration, filtrate is crossed resin column and obtains eluent；
(3) step（2）Described in eluent with charcoal absorption adjust pH value after, add water for injection, filtration, nitrogen charging
Embedding, sterilizing, obtains final product.
Preferred steps（1）Described in flos carthami, with hot water decoct 2-5 time, merging decoction liquor, refrigerated overnight, with filter
Membrane filtration mistake, filtrate concentration plus 70%-95% ethanol alcohol precipitations, make alcohol content be 60-80%.Most preferably soaked three times with 80-90 DEG C of hydro-thermal,
Each 30-60min, merges decoction liquor, and refrigerated overnight is filtered with miillpore filter,（It is preferred that 10 μm of films of single filter, cascade filtration
0.05 μm of membrane filtration）Filtrate is concentrated into 50-60 DEG C of relative density for 1.1-1.3, plus 90%-95% ethanol, makes alcohol content up to 70%,
Refrigeration, static 24-72 hours, filtration, filtrate recycling ethanol is simultaneously concentrated into 50-60 DEG C of relative density 1.1-1.3.
Above-mentioned steps（1）Medicinal material extract uses 80-90 DEG C of hot dipping, while ensureing to extract active ingredient, most probable
Few extracts P-hydroxybenzoic acid.Decoction liquor refrigerated overnight, filtering uses micro-filtration.Impurity content in decoction liquor is reduced, is made dense
Contracting liquid product is reduced, and is conducive to removing water-soluble bad polyphenols（Polyphenols is heated in the later stage, it is likely that have portion
Divide and be degraded to P-hydroxybenzoic acid）；
Above-mentioned steps（2）Described in resin column be ion exchange resin, including cationic ion-exchange resin and anion exchange
Resin, preferably cationic ion-exchange resin.
Further preferred step（2）It is that medicinal extract plus 8-12 times measure water（It is preferred that 10 times of amounts）, refrigeration, standing 16-24 hours, filter
Cross, cationic ion-exchange resin removes potassium, obtains eluent.
Most preferably above-mentioned eluent is further concentrated into 600-800ml（50-60℃）Obtain concentrate.
Step of the present invention（3）It is preferred that eluent or concentrate charcoal absorption 2 times, for the first time with 0.1-0.5% activity
Charcoal, stirring at normal temperature, by carbon filtration mistake, pH value 7.5-8.0, heat treatment, refrigeration is adjusted with 10-30% aqueous alkalis；Add activity second
Charcoal 0.1-0.5%, is stirred evenly, filtering, uses milipore filter ultrafiltration, and pH value 7.5-8.0 is adjusted with 10-30% aqueous alkalis.
Above-mentioned steps（3）Described in aqueous alkali include but is not limited to sodium carbonate, sodium acid carbonate, NaOH, hydrogen-oxygen
Change potassium, potassium carbonate, saleratus etc., preferably sodium hydroxide solution.
Optimization procedure（3）For the eluent or concentrate add 0.1-0.5% activated carbons, stirring at normal temperature 30min, by charcoal
Filtration, pH value 7.5-8.0,110 DEG C of heat treatment 40-100min are adjusted with 20%NaOH solution, refrigerate 24-72h；Add activated carbon
0.1-0.5%, 50 DEG C stir evenly 20-40min, filtering, with the milipore filter ultrafiltration of molecular cut off 5KD-10KD, use 20% NaOH
Solution adjusts pH value 7.5-8.0, adds water for injection into 1000ml, and filtration, nitrogen charging embedding, 115 DEG C of sterilizings are obtained final product.
Above-mentioned steps（3）In, charcoal absorption twice is used in technique：The purpose of absorption --- absorption early stage is dense for the first time
The P-hydroxybenzoic acid produced in compression process；Second purpose of absorption --- the P-hydroxybenzoic acid that absorption heat treatment is produced.
Optimal Sofflower injection of the invention, is prepared by the following method：
Take flos carthami 500g, plus 80-90 DEG C of hot dipping of purified water three times, first time 30-60min, second 30-60min,
Third time 30-60min, merges decoction liquor, and refrigerated overnight is filtered, 10 μm of films of single filter, 0.05 μm of membrane filtration of cascade filtration,
Filtrate is concentrated into 50-60 DEG C of relative density for 1.1-1.3, plus 90%-95% ethanol, makes alcohol content up to 70%, refrigerates, static 24-72
Hour, filtration, filtrate recycling ethanol is simultaneously concentrated into 50-60 DEG C of relative density 1.1-1.3, plus 10 times of amount water, and refrigeration stands 16-
24 hours, filtration, cationic ion-exchange resin removed potassium, and filtrate is concentrated into 50-60 DEG C of 600-800ml, adds 0.1-0.5% activated carbons,
Stirring at normal temperature 30min, by carbon filtration mistake, pH value 7.5-8.0,110 DEG C of heat treatment 40-100min, refrigeration is adjusted with 20%NaOH solution
24-72h；Activated carbon 0.1-0.5% is added, 50 DEG C stir evenly 20-40min, filtering, with the milipore filter of molecular cut off 5KD-10KD
Ultrafiltration, pH value 7.5-8.0 is adjusted with 20% sodium hydroxide solution, adds water for injection into 1000ml, filtration, nitrogen charging embedding, 115 DEG C
Sterilizing, obtains final product.
The present invention also provides the preparation method of above-mentioned Sofflower injection, and step is as follows：
（1）Flos carthami is taken, decocting is boiled, decoction liquor filtration, ethanol alcohol precipitation after filtrate concentration, filtration, reclaiming ethanol must soak
Cream；
（2）The medicinal extract water is sunk, and filtration, filtrate is crossed resin column and obtains filtrate;
(3) step（2）Described in filtrate charcoal absorption regulation pH value after, add water for injection, filtration, nitrogen charging fill
Envelope, sterilizing, obtains final product.
Preferred steps（1）Described in flos carthami, with hot water decoct 2-5 time, merging decoction liquor, refrigerated overnight, with filter
Membrane filtration mistake, filtrate concentration plus 70%-95 ethanol alcohol precipitations, make alcohol content be 60-80%.Most preferably 80-90 DEG C of use hydro-thermal leaching three times, often
Secondary 30-60min, merges decoction liquor, and refrigerated overnight is filtered with miillpore filter,（It is preferred that 10 μm of films of single filter, cascade filtration
0.05 μm of membrane filtration）Filtrate is concentrated into 50-60 DEG C of relative density for 1.1-1.3, plus 90%-95% ethanol, makes alcohol content up to 70%,
Refrigeration, static 24-72 hours, filtration, filtrate recycling ethanol is simultaneously concentrated into 50-60 DEG C of relative density 1.1-1.3.
Above-mentioned steps（2）Described in resin column be ion exchange resin, including cationic ion-exchange resin and anion exchange
Resin, preferably cationic ion-exchange resin.
Further preferred step（2）It is that medicinal extract plus 8-12 times measure water（It is preferred that 10 times of amounts）, refrigeration, standing 16-24 hours, filter
Cross, cationic ion-exchange resin removes potassium, obtains eluent.
Most preferably above-mentioned eluent is further concentrated into 600-800ml（50-60℃）Obtain concentrate.
Above-mentioned steps（3）Described in charcoal absorption be 2 absorption, preferably filtrate or concentrate uses 0.1- for the first time
0.5% activated carbon, stirring at normal temperature, by carbon filtration mistake, pH value 7.5-8.0, heat treatment, refrigeration is adjusted with 10-30% aqueous alkalis；Second
Activated carbon 0.1-0.5% is added, is stirred evenly, filtered, use milipore filter ultrafiltration, pH value 7.5-8.0 is adjusted with 10-30% aqueous alkalis.
Above-mentioned steps（3）Described in aqueous alkali include but is not limited to sodium carbonate, sodium acid carbonate, NaOH, hydrogen-oxygen
Change potassium, potassium carbonate, saleratus etc., preferably sodium hydroxide solution.
Further preferred step（3）For described eluent or concentrate add 0.1-0.5% activated carbons, stirring at normal temperature
30min, by carbon filtration mistake, pH value 7.5-8.0,110 DEG C of heat treatment 40-100min is adjusted with 20%NaOH solution, refrigerates 24-72h；Plus
Enter activated carbon 0.1-0.5%, 50 DEG C stir evenly 20-40min, filter, with the milipore filter ultrafiltration of molecular cut off 5KD-10KD, with 20%
Sodium hydroxide solution adjusts pH value 7.5-8.0, adds water for injection into 1000ml, and filtration, nitrogen charging embedding, 115 DEG C of sterilizings are obtained final product.
The preparation method of optimal Sofflower injection of the invention comprises the following steps：
Take flos carthami, plus 80-90 DEG C of hot dipping of purified water three times, first time 30-60min, second 30-60min, the 3rd
Secondary 30-60min, merges decoction liquor, refrigerated overnight, filtration, 10 μm of films of single filter, 0.05 μm of membrane filtration of cascade filtration, filtrate
50-60 DEG C of relative density is concentrated into for 1.1-1.3, plus 90%-95% ethanol, make alcohol content up to 70%, refrigerate, static 24-72 is small
When, filtration, filtrate recycling ethanol is simultaneously concentrated into 50-60 DEG C of relative density 1.1-1.3, plus 10 times of amount water, and refrigeration stands 16-24
Hour, filtration, cationic ion-exchange resin removes potassium, and filtrate is concentrated into 50-60 DEG C of 600-800ml, adds 0.1-0.5% activated carbons, often
Temperature stirring 30min, by carbon filtration mistake, pH value 7.5-8.0,110 DEG C of heat treatment 40-100min is adjusted with 20%NaOH solution, refrigerates 24-
72h；Activated carbon 0.1-0.5% is added, 50 DEG C stir evenly 20-40min, filtering is surpassed with the milipore filter of molecular cut off 5KD-10KD
Filter, pH value 7.5-8.0 is adjusted with 20% sodium hydroxide solution, adds water for injection into 1000ml, is filtered, nitrogen charging embedding, and 115 DEG C go out
Bacterium, obtains final product.
Present invention also offers P-hydroxybenzoic acid content assaying method in above-mentioned Sofflower injection, comprise the following steps：
It is prepared by step 1, reference substance solution：Take P-hydroxybenzoic acid reference substance appropriate, it is accurately weighed, it is made of 50% methyl alcohol
The solution containing 30 μ g, obtains final product in per 1ml；
Step 2, determination method：It is accurate respectively to draw reference substance solution and each 10 μ l of this product, liquid chromatograph is injected, determine,
Obtain final product；The high-efficient liquid phase chromatogram condition is as follows：
Chromatographic column： C18Column's length is 250mm, and internal diameter is 4.6mm, mobile phase：Methyl alcohol -0.02 ~ 0.05% sour water is molten
Liquid 5:90~100；Detection wavelength is 254nm；Column temperature is 20 ~ 30 DEG C；Flow velocity is 1.0 ~ 2.0ml/min.
The preferred high-efficient liquid phase chromatogram condition is in above-mentioned steps 2：Mobile phase：The trifluoroacetic acid aqueous solution of methyl alcohol -0.02%
5:95；Column temperature is 25 DEG C；Flow velocity is 1.5ml/min.
Mobile phase described in above-mentioned steps 2, aqueous acid includes but is not limited to phosphate aqueous solution, carbonate aqueous solution, trifluoro
Acetic acid, glacial acetic acid solution, acetic acid solution, formic acid solution etc., preferably trifluoroacetic acid solution.
Beneficial effect
Beneficial effects of the present invention are illustrated by following test examples
The measure of the median lethal dose of P-hydroxybenzoic acid content in the Sofflower injection of the invention of test example 1.
Purpose detects Sofflower injection by mouse tail vein injection various concentrations P-hydroxybenzoic acid Sofflower injection
The median lethal dose of middle P-hydroxybenzoic acid.
By P-hydroxybenzoic acid standard items, the Sofflower injection prepared with embodiment 1 dilutes and is configured to wait specific concentration method
The Sofflower injection P-hydroxybenzoic acid solution of gradient, with the solution mouse tail vein injection 0.5ml for being prepared, observation mouse
The death rate, and observe 7 days after mice organs change.
As a result mouse tail vein injection P-hydroxybenzoic acid Sofflower injection solution, median lethal dose is 34.6mg/kg,
95% fiducial limit scope is 31.2-38.6mg/kg.Dead and survival mice after 7 days is dissected, the subcutaneous, heart, liver, spleen, lung, kidney is observed
With the organ such as intestines and stomach, negative and positive mice is without substantially observing ANOMALOUS VARIATIONS.
Conclusion P-hydroxybenzoic acid has moderate toxicity.Need the content of P-hydroxybenzoic acid in control Sofflower injection.
1 materials and methods
1.1 medicines and reagent
Sofflower injection：Prepared by the method according to the embodiment of the present invention 1, yellowish red color transparency liquid, 20ml/ branch, lot number：Z-
130512, the pharmaceutcal corporation, Ltd's production of Ya'an Sichuan province three nine-day periods after the winter solstice；
P-hydroxybenzoic acid reference substance, purity more than 98%, lot number：Lot number：20110508, Shanghai source leaf biotechnology.
1.2 animals and test apparatus
Experimental animal：Kunming mouse, weight 17-20g, ♂ ♀ half and half, by Sanjiu Pharmaceutical Industry Co., Ltd., Ya'an City's quality pipe
Li Bu animals inspection center provides.Free choice feeding and drinking-water.The d of Mouse feeder 7, observes its health status before experiment, selects
Health, the mouse being in a good state of nutrition are tested.
Test apparatus：Assay balance（Plum Teller-support benefit Instrument Ltd.）, mouse cage (band feeder, water fountain and
Bedding and padding), mouse feed, disposable syringe, cotton ball soaked in alcohol, tweezers, scissors etc..
2 methods
2.1 preruns are tested
The nursing observation of 7 d is carried out before trial test to small white mouse, small white mouse free choice feeding, drinking-water in the observation period are daily empty
Abdomen is weighed, and eliminates natural death small white mouse.Small white mouse is randomly divided into 4 groups in the trial test phase, every group 8, male and female half and half,
Each group tail vein is administered, and determines the dosage range of formal test.
Compound dosage geometric progression 1: 0.5.Respectively:1 group of prerun, 2 groups of prerun, 3 groups of prerun, 4 groups of prerun,
Each compound dosage is shown in Table 1.
2.2 formal tests
Designed by Bliss methods, in the dosage containing Dm-Dn（Containing Dm and Dn）In the range of set 6 dosage groups（N）, according to as follows
Formula, calculates dose ratio（K）It is 0.76.
70 healthy mices are taken, after adaptability raises 7d, 7 groups, every group is divided into by weight, sex stratified random method
10, male and female half and half, each group is respectively：1 group is administered, 2 groups is administered, is administered 3 groups, is administered 4 groups, 5 groups, administration 6 groups and the moon is administered
Property control group, chemical composition dosage geometric progression be 1: 0.76.
Negative control group gives isometric Sofflower injection.Fasting（Can't help water）After 12 h, each group presses 25ml/kg
Disposable tail vein injection, records immediate reaction and the Continuous Observation 7d of animal after administration, record animal toxicity time of occurrence
And recovery time, death condition (the timely postmortem of dead animal), and dissected all survival small white mouses at the 8th day, observe abdominal cavity
Interior liquid situation and the pathological change of parenchymatous organ.
2.3 histopathologic examinations
After sacrifice, main organs carry out comprehensive postmortem, visually observe each internal organs whether there is lesion abnormal conditions;Core,
Then above-mentioned main organs are fixed, paraffin section by the internal organs such as liver, spleen, lung, kidney, brain with 10 % formaldehyde, HE dyeing, do disease
Reason histological examination.
3 the results are shown in Table 1 and table 2：
The dosage and chmice acute case fatality rate of the preliminary experiment of table 1
The dosage of the acute toxicity formal test of table 2 and mouse case fatality rate
4 discuss
Above experimental result shows that pharmacology is presented immediately instead after most animals administration, and different with dosage, animal is different
Chang Tizheng reaction time difference.Wherein performance muscle twitches breathing is tired during dead mouse after the injection of 34.6mg/ kg dosage
Difficult and dead, the not dead mandatory gap of mouse performance whole-body muscle is trembled respiratory distress, is tended to afterwards normal.The 1st after administration
My god, all not dead mouse feedings, drinking-water, autonomic activities and excrement all recover normal.Continuous Observation 7d, all mouse are by hair
Glossy, feeding, activity are normal.During off-test, administration group and control group mice visually observed after dissecting the subcutaneous, heart,
The organs such as liver, spleen, lung, kidney and intestines and stomach are without substantially observing ANOMALOUS VARIATIONS.Brain, the heart, liver, spleen, the lung of anatomic observation death mouse
With kidney compared with control group, have no significant change.It is computed, P-hydroxybenzoic acid tail vein administration half in Sofflower injection
Lethal dose（LD50）It is 34.6mg/kg, 95% fiducial limit scope is 31.2-38.6mg/kg.
P-hydroxybenzoic acid content control method in the production process of test example 2
1st, the control of raw material：P-hydroxybenzoic acid content standard in flos carthami
【Assay】According to high performance liquid chromatography（Annex VID of Chinese Pharmacopoeia 2010 edition）Determine
Chromatographic condition uses 5 μm of Grace VisionHT C18 HL with system suitability fixing phase（4.6mm×
250mm, 5 μm）；With the trifluoroacetic acid of methyl alcohol -0.02% (5:95) it is mobile phase；The ml/min of flow velocity 1.5；Detection wavelength 254
nm；25 DEG C of column temperature；In terms of P-hydroxybenzoic acid peak, number of theoretical plate is not less than 10000.
The preparation of reference substance solution takes P-hydroxybenzoic acid in right amount, with the trifluoroacetic acid of methyl alcohol -0.02% (5:95) it is solvent,
The solution that content is 20 μ g/ml concentration is made into, is obtained final product.
The preparation precision of need testing solution weighs saffron powder 2g and is placed in 150ml conical flask with stopper, and precision adds dilute second
Alcohol 50ml, close plug is weighed, ultrasonically treated（540W, 60kHZ）60min, is taken out, and lets cool, and the weight of less loss is supplied with Diluted Alcohol,
Shake up, filter, take subsequent filtrate, obtain final product.
Determination method is accurate respectively to draw reference substance solution and the μ l of need testing solution 10, injects liquid chromatograph, determines, i.e.,
.
In terms of dry product, every gram of medicinal powder contains P-hydroxybenzoic acid to this product（C7H6O3）40 μ g should be not higher than.
The control of technique（Safflower injection according to embodiment method prepare, assay according to test example 3 method）.
Medicinal material extract use 80-90 DEG C of hot dipping, ensure extract active ingredient while, most probable it is few extract it is right
Hydroxybenzoic acid.It is shown in Table 3
The contrast of P-hydroxybenzoic acid content under the different extracting modes of table 3
（1）
（2）Decoction liquor refrigerated overnight, filtering uses micro-filtration.Impurity content in decoction liquor is reduced, the volume of concentrate is reduced,
Be conducive to removing water-soluble bad polyphenols（Polyphenols is heated in the later stage, it is likely that it is to hydroxyl to have Partial digestion
Yl benzoic acid）It is shown in Table 4, table 5：
The contrast of the different processing mode precipitation solution total phenol content of the decoction liquor of table 4
The contrast of P-hydroxybenzoic acid content in the precipitation solution heat treatment solution of the different total phenol contents of table 5
（3）
（4）Charcoal absorption twice is used in technique：The purpose adsorbed for the first time --- produced in absorption early stage concentration process
Raw P-hydroxybenzoic acid；Second purpose of absorption --- the P-hydroxybenzoic acid that absorption heat treatment is produced, is shown in Table 6：
The content balance of P-hydroxybenzoic acid before and after the charcoal absorption twice of table 6
To sum up：
（1）The content of control P-hydroxybenzoic acid controls the content of the chemical composition from source in the medicinal material that feeds intake；
（2）Decoct by the way of temperature leaching, relative the boiling of the recovery rate of P-hydroxybenzoic acid extracts few；
（3）Decoction liquor refrigeration filtering is concentrated again, reduces the total phenol content in precipitation solution, has significantly cut off para hydroxybenzene first
The reaction source of acid；
（4）Using absorbing process twice, the P-hydroxybenzoic acid produced by heating can be progressively removed, make para hydroxybenzene first
Acid content is minimized.
The screening of the P-hydroxybenzoic acid content assaying method of test example 3
Below by way of methodological study and the screening of condition determination, research process of the invention and its advantage are illustrated.
1 instrument and reagent
The high performance liquid chromatographs of Agilent 1100；P-hydroxybenzoic acid reference substance（Shanghai source leaf biotechnology, lot number：
20110508）；Sofflower injection（Prepared according to 1 method is implemented）；Methyl alcohol is chromatographically pure；Water is water for injection；Other reagents are equal
It is pure to analyze.
2 experimental techniques and result
The foundation of 2.1 optimum chromatogram conditions
The chromatographic column that this method is used：Grace VisionHT C18HL 5µm （4.6mm × 250mm, 5 μm）.
2.1.1 the selection of Detection wavelength
P-hydroxybenzoic acid has absorption maximum at 254nm, therefore 254 nm of selection are best detection wavelength.
2.1.2 the selection of mobile phase
Be respectively adopted under the conditions of flow velocity 1.0ml/min, 25 DEG C of temperature trifluoroacetic acid-methyl alcohol, glacial acetic acid-methyl alcohol, phosphoric acid-
Methyl alcohol, trifluoroacetic acid-acetonitrile system are tested, result of the test such as table 7：
Table 7, different flow visualizing experimental results
| Mobile phase | Symmetrical factor | The number of plates | Peak purity |
| 0.05% trifluoroacetic acid：Methyl alcohol（95：5） | 0.58 | 16751 | 681.897 |
| 0.05% glacialacetic acid：Methyl alcohol（95：5） | 0.46 | 9846 | 936.006 |
| 0.05% phosphoric acid：Methyl alcohol（95：5） | 0.44 | 4300 | 965.492 |
| 0.02% trifluoroacetic acid：Methyl alcohol（95：5） | 0.76 | 17574 | 995.152 |
| 0.02% trifluoroacetic acid：Methyl alcohol（92：8） | 0.53 | 16572 | 672.100 |
| 0.02% trifluoroacetic acid：Acetonitrile（95：5） | 0.69 | 11672 | 997.169 |
As seen from Table 1, trifluoroacetic acid-methyl alcohol and trifluoroacetic acid-acetonitrile system are better than other systems, consider symmetrical
The factor, the number of plates and peak purity, optimal flow phase system are 0.02% trifluoroacetic acid：Methyl alcohol（95：5）.
2.1.3 the selection of column temperature
Column temperature changes influence makes the separating degree at peak and peak shape, in 0.02% trifluoroacetic acid：Methyl alcohol（95：5）, flow velocity 1.0ml/
Investigated under the conditions of min column temperature be 25 DEG C, 30 DEG C, 35 DEG C when peak separating degree, it is right test result indicate that as column temperature is raised
Hydroxybenzoic acid peak symmetry is deteriorated and peak purity reduction, therefore with 25 DEG C for optimized analysis column temperature, result of the test is shown in Table 8：
The experimental result of the different temperatures of table 8
| Temperature | Symmetrical factor | Peak purity |
| 25 | 0.7 | 994.848 |
| 30 | 0.69 | 981.796 |
| 35 | 0.64 | 990.174 |
2.1.4 flow velocity
It is 0.02% trifluoroacetic acid in mobile phase：Methyl alcohol（95：5）, under the conditions of 25 DEG C of temperature, try different in flow rate
Test, experimental result such as table 3.As can be seen that when flow velocity is 1.5 ml/min, peak symmetry has slight decrease, but peak purity is improved,
And flow velocity increases, sample retention time reduces, and shortens analysis time, therefore 1.5 ml/min are optimum flow rate, and result of the test is shown in
Table 9：
The experimental result different in flow rate of table 9.
| Flow velocity（ml/min） | Symmetrical factor | The number of plates | Peak purity |
| 1.0 | 0.76 | 17574 | 995.152 |
| 1.5 | 0.74 | 15210 | 998.794 |
2.1.5 optimum chromatogram condition
Experiment determines that optimal chromatographic condition is more than：Chromatographic column, Grace VisionHT C18HL 5µm （4.6mm
× 250mm, 5 μm）；Mobile phase, the trifluoroacetic acid of methyl alcohol -0.02% (5:95)；The ml/min of flow velocity 1.5；The nm of Detection wavelength 254；
25 DEG C of column temperature；The μ l of sample size 10.P-hydroxybenzoic acid theoretical cam curve is up to 14000.
It is prepared by 2.2 solution
2.2.1 the preparation of reference substance solution
Precision weighs the mg of reference substance 16.09, and in putting 100 ml volumetric flasks, plus 50% methanol dilution shakes up to scale, is made
Concentration is the P-hydroxybenzoic acid reference substance stock solution of 0.1609 mg/ml.According to sample size, take stock solution plus 50% methyl alcohol is dilute
Release to required concentration, as reference substance solution；
2.2.2 the preparation of need testing solution
Sofflower injection is taken, as need testing solution；
2.3 linear relationships are investigated
Precision measures the ml of reference substance stock solution 0.5,1,2,4,6,8,10 under " 2.2.1 " item, in putting 10 ml measuring bottles respectively,
Plus 50% methanol solution be diluted to scale, shake up, by chromatographic condition sample introduction under " 2.1.5 " item, chromatogram is recorded, with reference substance peak
Area is ordinate Y, the concentration X of reference substance solution（µg/ml）It is abscissa, carries out linear regression, obtains regression equation：y=
39.905x + 11.057.Result shows that in the range of 8.045-160.9 μ g/ml reference substance peak area is with detectable concentration in good
Good linear relationship（See Fig. 1）.Result of the test is shown in Table 10：
The linear relationship of table 10. investigates experimental result
| Control numbering | Concentration（µg/ml） | Peak area |
| 1 | 8.045 | 323.82532 |
| 2 | 16.09 | 649.59802 |
| 3 | 32.18 | 1295.66736 |
| 4 | 64.36 | 2593.79321 |
| 5 | 96.54 | 3856.67212 |
| 6 | 128.72 | 5171.43115 |
| 7 | 160.9 | 6411.82422 |
2.4 Precision Experiments
The reference substance solution of concentration known is taken, by continuous sample introduction under " 2.1.5 " item chromatographic condition 6 times, chromatogram is recorded.
It is 0.12% to measure the RSD of P-hydroxybenzoic acid peak area, shows that instrument precision is good, and result of the test is shown in Table 11：
The Precision Experiment result of table 11.
2.5 repeated experiments
6 parts of same batch need testing solution is taken, by sample introduction under " 2.1.5 " item chromatographic condition, chromatogram is recorded.Measure to hydroxyl
The RSD of yl benzoic acid peak area is 3.35%, shows that the method repeatability is good, and result of the test is shown in Table 12：
The repeated experiment result of table 12.
2.6 stability tests
Take with portion need testing solution, place at room temperature, respectively at the analysis of 0,2,4,12,27 h sample introductions, note
Record chromatogram, the RSD for calculating P-hydroxybenzoic acid peak area is 0.85%, shows need testing solution stabilization in 27 hours, examination
Test and the results are shown in Table 13：
The stability experiment result of table 13.
2.7 average recoveries are tested
6 parts of the need testing solution of same batch concentration known is taken, the reference substance solution for being separately added into concentration known is appropriate, presses
Sample introduction analysis under " 2.1.5 " item chromatographic condition, calculates the rate of recovery, and result of the test is shown in Table 14：
Table 14. is loaded recovery experiment result
3 quality standards are set up
The Sofflower injection of different lot numbers is taken respectively, according to sample introduction under " 2.1.5 " item chromatographic condition, measures peak area,
The content of P-hydroxybenzoic acid in sample is calculated, 15 are the results are shown in Table；P-hydroxybenzoic acid in Sofflower injection can be made from table
Content must not be higher than 40 μ g/ml：
The sample determination experimental result of table 15.
4 conclusions
Through methodological study, P-hydroxybenzoic acid content assaying method is accurate, reproducible, and precision is high, can be used for red
The measure of P-hydroxybenzoic acid content in flower parenteral solution；Simultaneously through the assay to testing sample preparation, find per the injection of 1ml safflowers
P-hydroxybenzoic acid content is in the scope less than 30 μ g in liquid, it is considered to certain secure border, therefore thinks for standard to be set to every 1ml red
P-hydroxybenzoic acid must not be higher than 35 μ g in flower parenteral solution.
The assay method of P-hydroxybenzoic acid is external standard method in a kind of Sofflower injection of the present invention.Determine red using this method
Toxic component P-hydroxybenzoic acid content in flower parenteral solution, with simplicity easy to operate, P-hydroxybenzoic acid good separating effect, peak
The features such as purity, precision and repeatability higher, meet detection and require.Sofflower injection is determined to hydroxyl by the method
Benzoic acid content, can be used for the quality control standard of Sofflower injection.
Brief description of the drawings
Fig. 1, P-hydroxybenzoic acid concentration and peak area linear relationship chart
The high-efficient liquid phase chromatogram of Fig. 2, P-hydroxybenzoic acid contrast solution
Fig. 3, Sofflower injection（Prepared according to the method for embodiment 1）High-efficient liquid phase chromatogram
Fig. 4, Sofflower injection（Prepared according to the method for embodiment 1）High-efficient liquid phase chromatogram after sample-adding
Fig. 5, P-hydroxybenzoic acid control spectrum figure
Fig. 6, Sofflower injection（Prepared according to the method for embodiment 1）The spectrogram at 23.3min peaks
Fig. 7, Sofflower injection（Prepared according to the method for embodiment 1）The spectrogram at 23.3min peaks after sample-adding
Following specific embodiments are only to be better understood from the present invention, do not have restriction effect to protection scope of the present invention.
Specific embodiment
Embodiment 1（The preparation of Sofflower injection）
Take flos carthami 500g, plus 80-90 DEG C of hot dipping of purified water three times, first time 30-60min, second 30-60min,
Third time 30-60min, merges decoction liquor, and refrigerated overnight is filtered, 10 μm of films of single filter, 0.05 μm of membrane filtration of cascade filtration,
Filtrate is concentrated into 50-60 DEG C of relative density for 1.1-1.3, plus 90%-95% ethanol, makes alcohol content up to 70%, refrigerates, static 24-72
Hour, filtration, filtrate recycling ethanol is simultaneously concentrated into 50-60 DEG C of relative density 1.1-1.3, plus 10 times of amount water, and refrigeration stands 16-
24 hours, filtration, cationic ion-exchange resin removed potassium, and filtrate is concentrated into 50-60 DEG C of 600-800ml, adds 0.1-0.5% activated carbons,
Stirring at normal temperature 30min, by carbon filtration mistake, pH value 7.5-8.0,110 DEG C of heat treatment 40-100min, refrigeration is adjusted with 20%NaOH solution
24-72h；Activated carbon 0.1-0.5% is added, 50 DEG C stir evenly 20-40min, filtering, with the milipore filter of molecular cut off 5KD-10KD
Ultrafiltration, pH value 7.5-8.0 is adjusted with 20% sodium hydroxide solution, adds water for injection into 1000ml, filtration, nitrogen charging embedding, 115 DEG C
Sterilizing, obtains final product.
Embodiment 2（The preparation of Sofflower injection）
Take flos carthami 500g, plus 80-90 DEG C of hot dipping of purified water three times, first time 30-60min, second 30-60min,
Third time 30-60min, merges decoction liquor, and refrigerated overnight is filtered, 10 μm of films of single filter, 0.05 μm of membrane filtration of cascade filtration,
Filtrate is concentrated into 50-60 DEG C of relative density for 1.1-1.3, plus 80%-90% ethanol, makes alcohol content up to 70%, refrigerates, static 24-72
Hour, filtration, filtrate recycling ethanol is simultaneously concentrated into 50-60 DEG C of relative density 1.1-1.3, plus 8 times of amount water, and refrigeration stands 16-24
Hour, filtration, cationic ion-exchange resin removes potassium, and filtrate is concentrated into 50-60 DEG C of 600-800ml, adds 0.1-0.5% activated carbons, often
Temperature stirring 30min, by carbon filtration mistake, pH value 7.5-8.0,110 DEG C of heat treatment 40-100min is adjusted with 10%NaOH solution, refrigerates 24-
72h；Activated carbon 0.1-0.5% is added, 50 DEG C stir evenly 20-40min, filtering is surpassed with the milipore filter of molecular cut off 5KD-10KD
Filter, pH value 7.5-8.0 is adjusted with 10% sodium hydroxide solution, adds water for injection into 1000ml, is filtered, nitrogen charging embedding, and 115 DEG C go out
Bacterium, obtains final product.
Embodiment 3（P-hydroxybenzoic acid assay）
According to high performance liquid chromatography（One D of annex VI of China's coastal port）Determine：
It is prepared by step 1, reference substance solution：Take P-hydroxybenzoic acid reference substance appropriate, it is accurately weighed, it is made of 50% methyl alcohol
The solution containing 30 μ g, obtains final product in per 1ml；
Step 2, determination method：It is accurate respectively to draw reference substance solution and each 10 μ l of this product, liquid chromatograph is injected, determine,
Obtain final product；
High-efficient liquid phase chromatogram condition：
Chromatographic column：Grace VisionHT C18 5 μm of chromatographic columns of HL（Column length is 250mm, and internal diameter is 4.6mm）；With first
The trifluoroacetic acid aqueous solution of alcohol -0.02%（5:95）It is mobile phase；Detection wavelength is 254nm；Column temperature is 25 DEG C；Flow velocity is 1.5ml/
min.Number of theoretical plate is calculated by P-hydroxybenzoic acid peak and should be not less than 10000；
This product contains P-hydroxybenzoic acid per 1ml（C7H6O3）, 25 μ g.
Embodiment 4（P-hydroxybenzoic acid assay）
According to high performance liquid chromatography（One D of annex VI of China's coastal port）Determine：
It is prepared by step 1, reference substance solution：Take P-hydroxybenzoic acid reference substance appropriate, it is accurately weighed, it is made of 50% methyl alcohol
The solution containing 30 μ g, obtains final product in per 1ml；
Step 2, determination method：It is accurate respectively to draw reference substance solution and each 10 μ l of this product, liquid chromatograph is injected, determine,
Obtain final product；
High-efficient liquid phase chromatogram condition：
Chromatographic column：Grace VisionHT C18 5 μm of chromatographic columns of HL（Column length is 250mm, and internal diameter is 4.6mm）；With first
The trifluoroacetic acid aqueous solution of alcohol -0.02%（5:90）It is mobile phase；Detection wavelength is 254nm；Column temperature is 20 DEG C；Flow velocity is 1.0ml/
min.Number of theoretical plate is calculated by P-hydroxybenzoic acid peak and should be not less than 10000；
This product contains P-hydroxybenzoic acid per 1ml（C7H6O3）, 26 μ g.
Embodiment 5（P-hydroxybenzoic acid assay）
According to high performance liquid chromatography（One D of annex VI of China's coastal port）Determine：
It is prepared by step 1, reference substance solution：Take P-hydroxybenzoic acid reference substance appropriate, it is accurately weighed, it is made of 50% methyl alcohol
The solution containing 30 μ g, obtains final product in per 1ml；
Step 2, determination method：It is accurate respectively to draw reference substance solution and each 10 μ l of this product, liquid chromatograph is injected, determine,
Obtain final product；
High-efficient liquid phase chromatogram condition：
Chromatographic column：Grace VisionHT C18 5 μm of chromatographic columns of HL（Column length is 250mm, and internal diameter is 4.6mm）；With first
The trifluoroacetic acid aqueous solution of alcohol -0.02%（5:100）It is mobile phase；Detection wavelength is 254nm；Column temperature is 30 DEG C；Flow velocity is 2.0ml/
min.Number of theoretical plate is calculated by P-hydroxybenzoic acid peak and should be not less than 10000；
This product contains P-hydroxybenzoic acid per 1ml（C7H6O3）, 26 μ g.
Embodiment 6（P-hydroxybenzoic acid assay）
According to high performance liquid chromatography（One D of annex VI of China's coastal port）Determine：
It is prepared by step 1, reference substance solution：Take P-hydroxybenzoic acid reference substance appropriate, it is accurately weighed, it is made of 50% methyl alcohol
The solution containing 30 μ g, obtains final product in per 1ml；
Step 2, determination method：It is accurate respectively to draw reference substance solution and each 10 μ l of this product, liquid chromatograph is injected, determine,
Obtain final product；
High-efficient liquid phase chromatogram condition：
Chromatographic column：Grace VisionHT C18 5 μm of chromatographic columns of HL（Column length is 250mm, and internal diameter is 4.6mm）；With first
The trifluoroacetic acid aqueous solution of alcohol -0.02%（5:98）It is mobile phase；Detection wavelength is 254nm；Column temperature is 28 DEG C；Flow velocity is 1.8ml/
min.Number of theoretical plate is calculated by P-hydroxybenzoic acid peak and should be not less than 10000；
This product contains P-hydroxybenzoic acid per 1ml（C7H6O3）, 25 μ g.
Claims (9)
1. a kind of Sofflower injection, it is characterised in that contained P-hydroxybenzoic acid content is less than 40 in Sofflower injection
μ g/m1, are prepared by the following method：
Step（1）, flos carthami is taken, decocted 2-5 times with warm water, merge decoction liquor, refrigerated overnight uses filter membrane
Filtration, filtrate concentration plus 70%-95% ethanol alcohol precipitations, make alcohol content for 60-80%, and filtration is reclaimed ethanol and obtained
Medicinal extract；
Step（2）, described medicinal extract adds water, and refrigerates, and stands, filtration, with cationic ion-exchange resin except potassium obtains eluent；
Step（3）, described 2 charcoal absorptions of eluent adjust pH, 0.1-0.5% activated carbons are used for the first time, and stirring at normal temperature will
Carbon filtration mistake, pH value 7.5-8.0, heat treatment, refrigeration are adjusted with 10-30% aqueous alkalis；Add activated carbon 0.1-0.5% second, stir
Even, milipore filter ultrafiltration is used in filtering, and pH value 7.5-8.0 is adjusted with 10-30% aqueous alkalis, adds water for injection, and filtration, nitrogen charging is filled
Envelope, sterilizing, obtains final product.
2. a kind of Sofflower injection as claimed in claim 1, it is characterised in that contained to hydroxyl in Sofflower injection
Yl benzoic acid content is less than 10 μ g/m1.
3. Sofflower injection as claimed in claim 1, it is characterised in that the step（1）Middle 80-90 DEG C of hydro-thermal is soaked three times,
Each 30-60min, merges decoction liquor, and refrigerated overnight is filtered with miillpore filter, and filtrate is concentrated into 50-60 DEG C of relative density and is
1.1-1.3, plus 90%-95% ethanol, make alcohol content up to 70%, refrigeration, static 24-72 hours, and filtration, filtrate recycling ethanol is simultaneously dense
It is reduced to 50-60 DEG C of relative density 1.1-1.3.
4. Sofflower injection as claimed in claim 3, it is characterised in that 10 μm of described filtering with microporous membrane single filter
Film, 0.05 μm of membrane filtration of cascade filtration.
5. Sofflower injection as claimed in claim 1, it is characterised in that step（2）In add 8-12 times to measure water for medicinal extract, it is cold
Hide, stand 16-24 hours, filtration, cationic ion-exchange resin removes potassium, obtains eluent.
6. Sofflower injection as claimed in claim 1, it is characterised in that step（3）For described eluent adds 0.1-
0.5% activated carbon, stirring at normal temperature 30min, by carbon filtration mistake, pH value 7.5-8.0,110 DEG C of heat treatment 40- is adjusted with 20%NaOH solution
100min, refrigerates 24-72h；Activated carbon 0.1-0.5% is added, 50 DEG C stir evenly 20-40min, molecular cut off 5KD- is used in filtering
The milipore filter ultrafiltration of 10KD, pH value 7.5-8.0 is adjusted with 20% sodium hydroxide solution, adds water for injection into 1000ml, and filtration is filled
Nitrogen embedding, 115 DEG C of sterilizings, obtains final product.
7. a kind of preparation method of Sofflower injection, it is characterised in that comprise the following steps：
Take flos carthami, plus 80-90 DEG C of hot dipping of purified water three times, first time 30-60min, second 30-60min, third time
30-60min, merges decoction liquor, and refrigerated overnight, filtration, 10 μm of films of single filter, 0.05 μm of membrane filtration of cascade filtration, filtrate is dense
50-60 DEG C of relative density is reduced to for 1.1-1.3, plus 90%-95% ethanol, make alcohol content up to 70%, refrigerate, it is static 24-72 hours,
Filtration, filtrate recycling ethanol is simultaneously concentrated into 50-60 DEG C of relative density 1.1-1.3, plus 10 times of amount water, and refrigeration stands 16-24 small
When, filtration, cationic ion-exchange resin removes potassium, and filtrate is concentrated into 50-60 DEG C of 600-800ml, adds 0.1-0.5% activated carbons, normal temperature
Stirring 30min, by carbon filtration mistake, pH value 7.5-8.0,110 DEG C of heat treatment 40-100min is adjusted with 20%NaOH solution, refrigerates 24-
72h；Activated carbon 0.1-0.5% is added, 50 DEG C stir evenly 20-40min, filtering is surpassed with the milipore filter of molecular cut off 5KD-10KD
Filter, pH value 7.5-8.0 is adjusted with 20% sodium hydroxide solution, adds water for injection into 1000ml, is filtered, nitrogen charging embedding, and 115 DEG C go out
Bacterium, obtains final product.
8. P-hydroxybenzoic acid content assaying method in Sofflower injection as claimed in claim 1, comprises the following steps：
It is prepared by step 1, reference substance solution：Take P-hydroxybenzoic acid reference substance appropriate, it is accurately weighed, it is made every 1ml with 50% methyl alcohol
In containing 30 μ g solution, obtain final product；
Step 2, determination method：It is accurate respectively to draw reference substance solution and each 10 μ l of this product, high performance liquid chromatograph is injected, determine,
Obtain final product；
The high-efficient liquid phase chromatogram condition is as follows：
Chromatographic column： C18Column's length is 250mm, and internal diameter is 4.6mm, mobile phase：Methyl alcohol -0.02~0.05% aqueous acid 5:
90~100；Detection wavelength is 254nm；Column temperature is 20~30 DEG C；Flow velocity is 1.0~2.0ml/min.
9. content assaying method as claimed in claim 8, it is characterised in that：High-efficient liquid phase chromatogram condition is described in step 2,
Mobile phase：The trifluoroacetic acid aqueous solution of methyl alcohol -0.02% 5:95；Column temperature is 25 DEG C；Flow velocity is 1.5ml/min.
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