CN106198838B - A kind of detection method of bupleurum root and asarum herb parenteral solution - Google Patents
A kind of detection method of bupleurum root and asarum herb parenteral solution Download PDFInfo
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- CN106198838B CN106198838B CN201610511572.5A CN201610511572A CN106198838B CN 106198838 B CN106198838 B CN 106198838B CN 201610511572 A CN201610511572 A CN 201610511572A CN 106198838 B CN106198838 B CN 106198838B
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention discloses a kind of detection method of bupleurum root and asarum herb parenteral solution, this method includes the thin-layer chromatography inspection and/or content limit detection to safrole, thin-layer chromatography inspection：By contained Asarum medicinal materials gauge, when test sample point sample amount is not higher than 100 times of asarum control medicinal material point sample amounts, such as aobvious safrole spot in test sample chromatogram, compared with the safrole spot shown in asarum control medicinal material chromatogram, must not be deeper；Content limit is detected：Per 1ml parenteral solutions 8 μ g are must not exceed containing safrole.The detection method that the present invention is set up is conducive to controlling the security of bupleurum root and asarum herb parenteral solution.
Description
Technical field
The present invention relates to the field of Chinese medicines, more particularly to a kind of detection method of bupleurum root and asarum herb parenteral solution.
Background technology
Bupleurum root and asarum herb parenteral solution is recorded to collect internal medicine lung system (one) fascicle in national standard for traditional Chinese medicines, and it is by radix bupleuri and thin
What pungent two tastes medicinal material was made, have effects that inducing diaphoresis is brought down a fever, for caused nasal obstruction runny nose of catching a cold, sneeze, cough, headache are disliked
It is cold to generate heat, the disease such as general malaise.
It is to Asarum medicinal materials to differentiate (1) in bupleurum root and asarum herb parenteral solution current standard (WS-11114 (ZD-1114) -2002)
Differentiate, regulation：In bupleurum root and asarum herb parenteral solution test sample chromatogram, on position corresponding with asarum control medicinal material chromatogram, it should show identical
The principal spot of color.Applicant has found in the checkout procedure to multiple batches of product, test sample chromatogram and control medicinal material chromatogram phase
Compare, a few spot or the very unconspicuous situation of spot often occur, the judgement to product quality causes puzzlement.
The content of the invention
It is an object of the invention to provide a kind of detection method of bupleurum root and asarum herb parenteral solution, this method is favorably improved Chai Xin
The safely controllable property of cold treating injection.
Heretofore described bupleurum root and asarum herb parenteral solution be by radix bupleuri and the taste medicinal material of asarum two it is extracted be processed into go out
The bacterium aqueous solution.
The detection method for the bupleurum root and asarum herb parenteral solution that the present invention is provided, including thin-layer chromatography inspection to safrole and/or
Content limit detection, thin-layer chromatography inspection：By contained Asarum medicinal materials gauge, when test sample point sample amount is not higher than 100 times of asarums pair
During according to medicinal material point sample amount, such as aobvious safrole spot in test sample chromatogram, with the safrole spot shown in asarum control medicinal material chromatogram
Point compares, must not be deeper；Content limit is detected：Per 1ml parenteral solutions 8 μ g, preferably more than 3 μ g are must not exceed containing safrole.
Safrole thin-layer chromatography inspection method is preferred：Bupleurum root and asarum herb parenteral solution 20ml is taken, extraction 2 times is shaken with ether, often
Secondary 15ml, divides and takes ether solution, waves near and does, residue adds petroleum ether 0.5ml to make dissolving immediately, is used as need testing solution；It is another to take carefully
Pungent control medicinal material 1g, puts in round-bottomed flask, connects volatile oil determination apparatus, is added water from analyzer upper end, enters to overflow in flask and be
Only, petroleum ether 1ml is added, reflux condenser is connected, boiling is heated to, and keeps micro-boiling 2 hours, is let cool, takes petroleum ether liquid to make
For control medicinal material solution；According to《Chinese Pharmacopoeia》The B thin-layered chromatography of one annex of version in 2015 VI is tested, and draws need testing solution 10
The μ l of μ l, the μ l of control medicinal material solution 1~10, put on same silica gel g thin-layer plate respectively, n-hexane-vinegar using volume ratio as 15: 1
Acetoacetic ester is solvent, is deployed, and takes out, dries, and is sprayed with 5% vanillin-sulfuric acid solution, it is clear that hot blast is blown to spot development；For examination
Such as aobvious safrole spot in product chromatogram, compared with the safrole spot shown in asarum control medicinal material chromatogram, must not be deeper.
The detection of safrole content limit can be using detection methods such as high performance liquid chromatography, gas chromatographies.Work as use
During high effective liquid chromatography for measuring safrole content, isocratic elution can also be used using gradient elution, be washed using gradient
De- chromatographic condition is preferred：Using octadecylsilane chemically bonded silica as filler；Using acetonitrile as mobile phase A, using water as mobile phase
B, gradient elution, elution process mobile phase A and B ratio, which become, to be turned to：0~20min, A phase 40%, B phases 60%；20~30min,
A phases 40%-45%, B phase 60%-55%；30~35min, A phase 45%-47%, B phase 55%-53%；Column temperature：25-35℃；Inspection
Survey wavelength is 230nm~240nm；It is preferred using the chromatographic condition of isocratic elution：Using octadecylsilane chemically bonded silica as filling
Agent；Using volume ratio as 40:60~44:56 acetonitrile-water is mobile phase；Column temperature：25-35℃；Detection wavelength be 230nm~
240nm.When determining safrole content using gas chromatography, preferably chromatographic condition is：Nonpolar or middle polarity capillary
Post, temperature programming：100 DEG C of initial temperature, is kept for 12 minutes, 130 DEG C is warming up to 5 DEG C per minute of speed, then with per minute
25 DEG C of speed is warming up to 290 DEG C, is kept for 3 minutes, 150 DEG C~300 DEG C of injector temperature, detector temperature 200 DEG C~300
℃。
The innovation of the present invention is：The present invention, which is directed in primary standard, to be differentiated in the test sample chromatogram that (1) is present often
A spot fewer than control medicinal material chromatogram or the unconspicuous problem of spot, by preparing thin-layer chromatography and silica gel column chromatogram method
Isolated and purified, and use Gc-mss, it is safrole to determine the corresponding chemical composition of the spot, and establishes yellow camphor tree
The thin layer inspection method and content limit detection method of ether, the content to safrole in bupleurum root and asarum herb parenteral solution are controlled,
Improve the safely controllable property of product.
Below will the invention will be further described by experimental example.
The ownership research of the chemical composition of experimental example 1
1st, target component is collected using thin-layer chromatography
By Herba Asari volatile oil point in preparing on silica gel g thin-layer plate, with n-hexane-ethyl acetate (15: 1) for solvent, on
Row method is deployed, and dries, scraper plate, elutes, obtains target component crude product.
2nd, target component crude product is purified
Target component crude product, through silica gel column chromatography, n-hexane-ethyl acetate (30: 1) is eluted, thin-layer chromatography inspection,
Merge identical flow point, obtain target component.
3rd, target component chemical composition belongs to
Target component is analyzed through GC-MS, and the molecular weight of target component is consistent with the safrole of document report, then through thin layer color
Spectrum, gas-chromatography and liquid chromatographic detection checking, target component are defined as safrole.
The liquid chromatography for measuring safrole content of experimental example 2
First, sample and reagent
Safrole reference substance；Bupleurum root and asarum herb parenteral solution (lot number：20150826-1,20150826-2,20150826-3, river
Western Paradise Shi Kang Chinese medicines limited company provides)；Methanol (analysis is pure)
2nd, the preparation of reference substance solution and sample
1. reference substance solution is prepared：Precision weighs safrole reference substance 11.15mg and is placed in 10ml volumetric flasks, fixed with methanol
Hold to scale, produce 1.115mg/ml storing solutions I；Precision measures the 1ml of storing solution I in 50ml volumetric flasks, plus methanol constant volume is extremely
Scale, shakes up, and produces 22.3 μ g/ml storing solutions II；Precision measures the 1ml of storing solution II in 10ml volumetric flasks, plus methanol constant volume
To scale, shake up, produce 2.23 μ g/ml reference substance solutions.
2. the preparation of sample：Take sample to cross 0.22 μm of miillpore filter, take subsequent filtrate, produce.
3rd, chromatographic condition
Instrument：Shimadzu HPLC (LC-20A)
Chromatographic column：Yi Lite Hypersil ODS C18 posts (4.6mm × 250mm, 5 μm)
Column temperature：30℃
Flow velocity：1ml/min
Detection wavelength：235nm
Sample size：20μl
Mobile phase：Acetonitrile (A)-water (B)
| Time/min | A% | B% |
| 0 | 40 | 60 |
| 20 | 40 | 60 |
| 30 | 45 | 55 |
| 35 | 47 | 53 |
4th, methodological study
1. system suitability
Determined by above-mentioned chromatographic condition, negative control is noiseless.
2. the investigation of chromatographic condition
The investigation of 2.1 mobile phases
Instrument：Shimadzu HPLC (LC-20A)
Chromatographic column：Yi Lite Hypersil ODS C18 posts (4.6mm × 250mm, 5 μm)
Column temperature：30℃
Flow velocity：1ml/min
Detection wavelength：235nm
Sample size：20μl
Mobile phase is as follows：
During from mobile phase 6, separating degree is best, so selective flow phase 6 is used as the mobile phase for determining safrole content.
The selection of 2.2 Detection wavelengths
Because safrole has absorption maximum at 287nm and 235nm, there is interference at sample peak of safrole at 287nm, and
Do not interfered with 235nm, so selection Detection wavelength is 235nm.
3. the range of linearity
Reference substance storing solution II 2ml, 1ml, 1ml, 2ml, 5ml, 1ml are taken respectively to 5ml, 5ml, 10ml, 25ml, 100ml,
In 25ml volumetric flasks, with methanol dilution to scale, constant volume shakes up, i.e., reference substance storing solution II is diluted into 2.5,5 respectively,
10.12.5,20,25 times, detected according to chromatographic condition, record chromatogram.With peak area (A) for ordinate (Y), safrole
Concentration (X, μ g/mL) is that abscissa draws standard curve, obtains regression equation Y=28175X-4643, r=0.99985.Safrole
With peak area it is in good linear relationship in the μ g/ml concentration ranges of 0.89 μ g/ml~8.92.
4. precision test
Same reference substance solution is taken, according to the pin of chromatographic condition continuous sample introduction 6, retention time and the peak face of safrole is determined
Product, the results are shown in Table 1.
The precision test measurement result of table 1
Brief summary：Precision test result RSD<2% (RSD=1.30%, n=6), shows that instrument precision is preferable.
5.. need testing solution stability test
Need testing solution is determined by chromatographic condition a, the sample introduction respectively at 0, after 2,4,8,12,24 hours the results are shown in Table 2.
The solution stability testing result of table 2
Brief summary：Need testing solution is not fine at 24 hours internal stabilities, but, RSD. in 12 hours internal stabilities preferably
=1.76%, n=5.
6. average recovery is tested
Respectively precision measure 0.6,0.8,0.9ml concentration be 2.23 μ g/ml reference substance solutions put in 2ml measuring bottles (totally 6 parts,
Each 2 parts of concentration), then accurate addition bupleurum root and asarum herb parenteral solution 1ml, plus methanol constant volume is to scale, shakes up, according to above-mentioned chromatostrip
Part is detected, the results are shown in Table 3.
The average recovery result of the test of table 3
Brief summary：Average recovery result of the test, mean sample recovery rate is 110.70%, RSD=3.42%.
7. durability is investigated
The durability of three different brands chromatographic columns and three kinds of different brands liquid chromatographs has been investigated in this experiment, is as a result seen
Table 4.
The chromatographic column of table 4 investigates result with instrument durability
Same instrument, the result RSD values that three different chromatographic columns are measured are 3.7%, and three same chromatographic columns of instrument are measured
Result RSD values be 4.5%.
5th, sample is determined
Take each 2 parts of 3 different batches parenteral solutions to be measured as stated above, the results are shown in Table 5.
The assay result of table 5
The gas chromatography of experimental example 3 determines safrole content
First, sample and reagent
Safrole reference substance；Bupleurum root and asarum herb parenteral solution (Tianshikang Chinese Medicines Co., Ltd., Jiangxi's offer)；N-hexane (point
Analysis is pure)；N-tetradecane (analysis is pure)；Petroleum ether (analysis is pure)；Ether (analysis is pure)
2nd, the preparation of sample and reference substance solution
1. the preparation of sample solution：Precision measures 10ml bupleurum root and asarum herb parenteral solutions, and with petroleum ether, (30-60 °) extracts 3 times,
Each 10ml, takes upper liquid, is volatilized in 20 DEG C of water-baths, and n-hexane dissolution simultaneously adds 1ml inner mark solution and is settled to 5ml and obtains sample
Solution.
2. the preparation of inner mark solution：Precision weighs n-tetradecane 0.05876g, and 10ml, then accurate amount are settled to n-hexane
Take 1ml to be settled to 100ml with n-hexane, obtain inner mark solution.
3. the preparation of reference substance solution：Safrole reference substance sampling amount 0.01038g, with n-hexane dissolution and is settled to
10ml, then precision measure 1ml 10ml are settled to n-hexane and obtain safrole reference substance solution；By safrole reference substance solution with it is interior
Both mark solution takes 1ml in 5ml volumetric flasks respectively, mixes, is settled to scale with n-hexane, obtains reference substance solution.
3rd, chromatographic condition
Instrument：Agilent 6890N gas chromatographs
Chromatographic column：HP-1 capillary column 0.25um 320um*30m
Injector temperature：270℃
Detector temperature：300℃
Flow rate of carrier gas：1.0ml/min
Split ratio：Reference substance, sample (2：1)
Sample size：1ul
Temperature programming：100 DEG C of initial temperature, is kept for 12 minutes, 130 DEG C is warming up to 5 DEG C per minute of speed, then with every
The speed of 25 DEG C of minute is warming up to 290 DEG C, is kept for 3 minutes.
4th, methodological study
The investigation of 4.1 chromatographic columns
This experiment has investigated two kinds of capillary columns of HP-1 and DB-WAX test result indicates that HP-1 and DB-WAX can be used for
Determine safrole content.
4.2 system suitability
Determined by above-mentioned chromatographic condition, negative control is noiseless.
4.3 investigate extractant
Precision measures 10ml bupleurum root and asarum herb parenteral solutions, respectively with petroleum ether (30-60 °), n-hexane, ether extract 3 times, often
Secondary 10ml, takes upper liquid, is volatilized in 20 DEG C of water-baths, and n-hexane, which redissolves and adds 1ml inner mark solution, to be settled to 5ml and obtain petroleum ether
(30-60 °) extraction sample solution, n-hexane extraction sample solution, ether extraction sample solution；Determined by above-mentioned chromatographic condition,
It the results are shown in Table 6.
The extractant of table 6 investigates result
Brief summary：By comparing petroleum ether (30-60 °), n-hexane, ether three's effect of extracting, selection small toxicity, low boiling point
Petroleum ether (30-60) be used as extractant.
4.4 investigate extraction times
Precision measures 10ml bupleurum root and asarum herb parenteral solutions, respectively with the extraction 2 times, 3 times, 4 times of (30-60 °) of petroleum ether, every time
10ml；Upper liquid is taken, waves near dry in 20 DEG C of water-baths, n-hexane, which redissolves and adds 1ml inner mark solution, to be settled to 5ml and obtain petroleum ether
2 sample solutions of (30-60 °) extraction, 3 sample solutions of extraction, 4 sample solutions of extraction, are determined, knot by above-mentioned chromatographic condition
Fruit is shown in Table 7.
The extraction times of table 7 investigate result
Brief summary：By comparing 2 extractions, 3 extractions, 4 extraction threes, slective extraction prepares sample solution 3 times.
4.5 investigations volatilize temperature
Precision measures 10ml bupleurum root and asarum herb parenteral solutions, and with petroleum ether, (30-60 °) extracts 3 times, and each 10ml takes upper liquid,
Volatilized respectively in 20 DEG C, 30 DEG C, 40 DEG C of water-baths, n-hexane, which redissolves and adds 1ml inner mark solution, to be settled to 5ml and obtain 20 DEG C and volatilize
Sample solution, 30 DEG C volatilize sample solution, 40 DEG C volatilize sample solution, by above-mentioned chromatographic condition determine, the results are shown in Table 8.
Table 8 volatilizes temperature and investigates result
Brief summary：By compare 20 DEG C, 30 DEG C, 40 DEG C of threes volatilize temperature, 20 DEG C of selection, which is volatilized, prepares sample solution
4.6 the range of linearity
The preparation of reference substance solution：Precision weighs safrole reference substance 0.01038g, and n-hexane constant volume is in 10ml, then precision
Measure 1ml and 10ml is settled to n-hexane as stock solution I (concentration is 103.8 μ g/ml)；Precision measures reference substance stock solution I
1ml is put in 10ml measuring bottles, plus n-hexane dissolution and is diluted to scale, is shaken up, and is used as stock solution II (concentration is 10.38 μ g/ml)；
The preparation of inner mark solution：Precision weighs n-tetradecane 0.05876g, and 10ml is settled to n-hexane, then precision is measured
1ml is settled to 100ml with n-hexane, obtains inner mark solution；
Precision measures the 1ml of reference substance storing solution II and put in 5ml measuring bottles, plus 1ml inner mark solutions, and n-hexane is diluted to scale,
Shake up, be used as solution 1 (concentration is 2.076 μ g/ml)；
Precision measures the 2ml of reference substance storing solution II and put in 5ml measuring bottles, plus 1ml inner mark solutions, and n-hexane is diluted to scale,
Shake up, be used as solution 2 (concentration is 4.152 μ g/ml)
Precision measures 1ml inner mark solutions and put in 5ml measuring bottles, plus reference substance storing solution II shakes up to scale, is used as solution 3
(concentration is 8.304 μ g/ml)；
Precision measures the 1ml of reference substance storing solution I and put in 5ml measuring bottles, plus 1ml inner mark solutions, and n-hexane is diluted to scale, shaken
It is even, it is used as solution 4 (concentration is 20.76 μ g/ml)
Precision measures the 2ml of reference substance storing solution I and put in 5ml measuring bottles, plus 1ml inner mark solutions, and n-hexane is diluted to scale, shaken
It is even, it is used as solution 5 (concentration is 41.52 μ g/ml)
Precision measures 1ml inner mark solutions and put in 5ml measuring bottles, plus reference substance storing solution I shakes up to scale, is used as solution 6
(concentration is 83.04 μ g/ml)；
Detected according to chromatographic condition, record chromatogram, using safrole peak area and internal standard peak area ratio as vertical seat
Mark (Y), safrole concentration (X, μ g/mL) is that abscissa draws standard curve, obtains regression equation Y=0.05571X-0.08767
(R2=0.99981).Safrole is in good line with peak area ratio in the μ g/ml concentration ranges of 2.076 μ g/ml~83.040
Sexual intercourse.
4.7 precision test
4.7.1. the preparation of inner mark solution：Precision weighs n-tetradecane 0.05876g, and 10ml is settled to n-hexane, then
Precision measures 1ml and is settled to 100ml with n-hexane, obtains inner mark solution；
4.7.2. the preparation of reference substance solution：Safrole reference substance sampling amount 0.01038g, with n-hexane and dissolves constant volume
To 10ml, then precision measures 1ml 10ml is settled to n-hexane and obtains safrole reference substance solution；By safrole reference substance solution with
Both inner mark solutions take 1ml in 5ml volumetric flasks respectively, mix, are settled to scale with n-hexane, obtain reference substance solution.
Reference substance solution is taken, according to the pin of chromatographic condition continuous sample introduction 6, internal standard compound, the retention time of safrole and Huang is determined
The value of camphor tree ether peak area/internal standard peak area, the results are shown in Table 9
The precision test measurement result of table 9
Brief summary：Precision test result RSD<2% (RSD=0.678%, n=6), shows that instrument precision is preferable.
4.8 replica test
Precision measures 10ml bupleurum root and asarum herb parenteral solutions, and with petroleum ether, (30-60 °) extracts 3 times, and each 10ml takes upper liquid,
Wave near dry in 20 DEG C of water-baths, n-hexane, which redissolves and adds 1ml inner mark solution, to be settled to 5ml and obtain sample solution 1, repeatedly above-mentioned behaviour
Make, six parts of sample solutions are prepared altogether, is determined according to chromatographic condition, the results are shown in Table 10.
The replica test result of table 10
Brief summary：Replica test result RSD<3% (internal standard method RSD=2.58%, external standard method RSD=2.84%, n=6),
Show that internal mark method determination safrole method repeatability is good.
4.9 sample solution stability tests
Precision measures 10ml bupleurum root and asarum herb parenteral solutions, and with petroleum ether, (30-60 °) extracts 3 times, and each 10ml takes upper liquid,
Wave near dry in 20 DEG C of water-baths, n-hexane, which redissolves and adds 1ml inner mark solution, to be settled to 5ml and obtain sample solution, according to chromatostrip
Part is determined, and the results are shown in Table 11.
The stability test result of table 11
Brief summary：Stability test result RSD<2% (internal standard method RSD=0.273%, external standard method RSD=1.91%, n=
6), illustrate that need testing solution is good in 24 hours internal stabilities.
5.0 average recoveries are tested
Precision measures 10ml bupleurum root and asarum herb parenteral solutions, adds 1ml safrole reference substance solution (equivalent to addition safrole
Amount be about sample 100%), extracted 3 times with (30-60 °) of petroleum ether, each 10ml takes upper liquid, is waved closely in 20 DEG C of water-baths
Dry, n-hexane, which redissolves and adds 1ml inner mark solution, to be settled to 5ml and obtains sample solution, repeats aforesaid operations six parts of samples of preparation
Solution, is determined according to chromatographic condition, the results are shown in Table 12,13.
The average recovery result of the test of table 12 (internal standard method calculating total amount)
The average recovery result of the test of table 13 (external standard method calculating total amount)
Brief summary：Average recovery result of the test, safrole total amount is calculated by internal standard method, and mean sample recovery rate is
98.84%, RSD=2.82% (<3%), illustrate that internal standard method content assaying method is preferable to the safrole rate of recovery.
5th, sample is determined
Precision measures five batches 20160104,20151010,20150826-1,20150826-2,20150826- respectively
3 bupleurum root and asarum herb parenteral solution 10ml, with petroleum ether, (30-60 °) extracts 3 times, and each 10ml takes upper liquid, is waved closely in 20 DEG C of water-baths
Dry, n-hexane, which redissolves and adds 1ml inner mark solution, to be settled to 5ml and obtains sample solution, two parts of sample solutions of each batch preparation,
Determined according to chromatographic condition, the results are shown in Table 14.
The bupleurum root and asarum herb parenteral solution safrole assay result of table 14
In upper table, lot number be 20160104 bupleurum root and asarum herb parenteral solutions in safrole content apparently higher than other batch products,
Through researching and analysing discovery, caused mainly due to the content height of safrole in the Asarum medicinal materials for preparing the batch sample.
Embodiment
Following embodiments are used to illustrate the present invention, but are not limited to the scope of the present invention.
Embodiment 1
Radix bupleuri 2500g asarums 250g
1000ml is made in above-mentioned raw materials as follows：
Method 1：The taste medicinal material of the above two, is added water and is distilled, and collects distillate about 5500ml, and weight is collected in distillate redistillation
Distillate about 950ml, plus 30ml propane diols, shaking are completely dissolved oil, then add 8g sodium chloride, are adjusted with 10% sodium hydroxide solution
PH value is saved to 6.8, ormal weight is injected water to, after the activated carbon for adding 0.5%, is sufficiently stirred for, is filtered, embedding, sterilizing is produced
Sample I.
Method 2：The taste medicinal material of the above two, adds water and is distilled, first circulate 30min, regathers distillate about 5500ml, distillate
Redistillation, collects re-distilled liquid about 950ml, plus 30ml propane diols, and shaking is completely dissolved oil, then adds 8g sodium chloride, with 10%
Sodium hydroxide solution adjusts pH value to 6.8, injects water to ormal weight, after the activated carbon for adding 0.5%, is sufficiently stirred for, and filters,
Embedding, sterilizing, produces sample II.
Method 3：The taste medicinal material of the above two, adds water and is distilled, first circulate 60min, regathers distillate about 5500ml, distillate
Redistillation, collects re-distilled liquid about 950ml, plus 30ml propane diols, and shaking is completely dissolved oil, then adds 8g sodium chloride, with 10%
Sodium hydroxide solution adjusts pH value to 6.8, injects water to ormal weight, after the activated carbon for adding 0.5%, is sufficiently stirred for, and filters,
Embedding, sterilizing, produces sample III.
【Differentiate】I 20ml is sampled, is extracted 2 times with ether shaking, each 15ml divides and takes ether solution, waves near and does, residue is stood
I.e. plus petroleum ether 0.5ml makes dissolving, it is used as need testing solution；Asarum control medicinal material 1g separately is taken, is put in round-bottomed flask, connection volatilization
Oily analyzer, adds water from analyzer upper end, untill overflow enters in flask, adds petroleum ether 1ml, connects reflux condenser,
Boiling is heated to, and keeps micro-boiling 2 hours, lets cool, takes petroleum ether liquid as control medicinal material solution；According to《Chinese Pharmacopoeia》Version in 2015
The one B thin-layered chromatography of annex VI experiment, draws the μ l of need testing solution 10, the μ l of control medicinal material solution 1, puts respectively in same silica gel
On G lamellaes, n-hexane-ethyl acetate using volume ratio as 15: 1 deploys for solvent, takes out, dries, and sprays with 5% vanilla
Aldehyde sulfuric acid solution, it is clear that hot blast is blown to spot development；As a result：Safrole spot colors are than asarum control medicinal material in test sample chromatogram
Safrole spot colors are shallow in chromatogram.
【Limit detection】Determined according to high performance liquid chromatography (one D of annex VI of Chinese Pharmacopoeia version in 2015).
Chromatographic condition is tested using octadecylsilane chemically bonded silica as filler with system availability；With acetonitrile-water (42:
58) it is mobile phase；Detection wavelength is 235nm；Column temperature：30℃；Theoretical cam curve is calculated by safrole peak should be not less than 2000.
The preparation of reference substance solution takes safrole reference substance appropriate, accurately weighed, plus every 1mL μ containing safrole 2 are made in methanol
G reference substance solution, is produced.
The preparation of need testing solution takes sample I-III, respectively with (0.22 μm) filtration of miillpore filter, takes subsequent filtrate, produces.
Determination method is accurate respectively to draw reference substance solution and each 10 μ L of need testing solution, injects liquid chromatograph, determines, i.e.,
.
Results sample I is per 1ml parenteral solutions μ g containing safrole 6.93；Sample II is per 1ml parenteral solutions μ g containing safrole 2.76；Sample
Product III are per 1ml parenteral solutions μ g containing safrole 1.07.
Claims (1)
1. a kind of detection method of bupleurum root and asarum herb parenteral solution, it is characterised in that described including the thin-layer chromatography inspection to safrole
Thin-layer chromatography inspection method is as follows：Bupleurum root and asarum herb parenteral solution 20ml is taken, is extracted 2 times with ether shaking, each 15ml divides and takes ether
Liquid, waves near and does, residue adds petroleum ether 0.5ml to make dissolving immediately, is used as need testing solution；Asarum control medicinal material 1g separately is taken, is put
In round-bottomed flask, volatile oil determination apparatus is connected, is added water from analyzer upper end, untill overflow enters in flask, adds petroleum ether
1ml, connects reflux condenser, is heated to boiling, and keeps micro-boiling 2 hours, lets cool, takes petroleum ether liquid as control medicinal material solution；
According to《Chinese Pharmacopoeia》The B thin-layered chromatography of one annex of version in 2015 VI is tested, and draws the μ l of need testing solution 10, control medicinal material molten
The μ l of μ l of liquid 1~10, put on same silica gel g thin-layer plate, being expansion by 15: 1 n-hexane-ethyl acetate of volume ratio respectively
Agent, deploys, and takes out, dries, and sprays with 5% vanillin-sulfuric acid solution, it is clear that hot blast is blown to spot development；As aobvious in test sample chromatogram
Safrole spot, is compared with the safrole spot shown in asarum control medicinal material chromatogram, must not be deeper.
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