CN109580428A - A kind of method of hyaluronan molecule amount in simplicity Accurate Determining solution - Google Patents
A kind of method of hyaluronan molecule amount in simplicity Accurate Determining solution Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N11/00—Investigating flow properties of materials, e.g. viscosity, plasticity; Analysing materials by determining flow properties
- G01N11/02—Investigating flow properties of materials, e.g. viscosity, plasticity; Analysing materials by determining flow properties by measuring flow of the material
- G01N11/04—Investigating flow properties of materials, e.g. viscosity, plasticity; Analysing materials by determining flow properties by measuring flow of the material through a restricted passage, e.g. tube, aperture
- G01N11/06—Investigating flow properties of materials, e.g. viscosity, plasticity; Analysing materials by determining flow properties by measuring flow of the material through a restricted passage, e.g. tube, aperture by timing the outflow of a known quantity
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/38—Diluting, dispersing or mixing samples
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
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- G—PHYSICS
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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
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
Abstract
The invention discloses a kind of method of hyaluronan molecule amount in easy Accurate Determining fermentation liquid and solution, this method measures the molecular weight of hyaluronic acid wherein included based on the intrinsic viscosity and glucuronic acid content of hyaluronic acid solution or fermentation liquid.This method was not only simple, but also accuracy with higher and reproducibility.It can not only be used for hyaluronic acid fluid molecule amount simplicity accurate detection method, and the hyaluronic acid for the detection of hyaluronic acid manufacturing enterprise fermentation level and production different molecular weight provides operation foundation.It is of great significance to raising product quality and production efficiency, while also providing the method for direct detection molecules amount for hyaluronic acid liquid needed for cosmetics and field of medicaments.
Description
Technical field
The present invention relates to the detection fields of hyaluronic acid, and in particular to it is a kind of simplicity Accurate Determining hyaluronic acid fermentation liquor and
The method of the molecular weight of hyaluronic acid in solution.
Background technique
It is that dissacharide units repeat to hand over that hyaluronic acid (hyaluronic acid, HA), which is by acetylglucosamine and glucuronic acid,
The macromolecule mucopolysaccharide formed for connection has been widely used in making up due to its special biological characteristics and physiological function
Product and medical domain.The relative molecular mass size of HA be characterize its characteristic one of basic parameter, the biological property of HA with
Its relative molecular weight size has close relationship.Different relative molecular weight hyaluronic acids, physiological activity is not also identical, or even tool
There is antipodal effect.For example, HA of the molecular weight greater than 2 000 000 has preferable moisture retention, viscoelasticity, lubrication, suppression
The functions such as inflammatory reaction processed can be used for ophthalmologic operation, Bones and joints ejection preparation；The HA of molecular weight 1 000 000~2 000 000
With good moisture retention, lubricity and medicament slow release effect, it is widely used in cosmetics, eye drops and skin burn concrescence；Point
The HA of son amount 10000~80000 belongs to low molecule, and the deficiency of body HA can be supplemented by intestinal absorption by taking orally, and plays health care and beauty
The effect of beauty treatment；HA oligosaccharide (molecular weight is less than 10000) has antitumor, promotion bone and angiogenesis function, has potential
Medical application prospect.
Currently used hyaluronic acid average molecular quantity measuring method has following three kinds: intrinsic viscosity (intrinsic
Viscosity) method, High Performance Gel Permeation chromatography (high performence gel permeation chromatography,
HPGPC) method, multiple angle laser light scattering instrument attached gel permeation chromatography (multi angle laser light scattering
Combined with gel permeation chromatography, GPC/MALLS) method.
Intrinsic viscosity method is using the rheological property of polymer substance come the molecular weight of substance for calculation, and this method is simple
It is easy, it is widely used so far.For example, in patent document 1, utilization level viscosity number (limiting viscosity) method determines molecular weight and is
100000 low molecular weight hyaluronic acids below.In " European Pharmacopoeia " 7.0 editions and " British Pharmacopoeia " 2013 editions sodium hyaluronate inspections recorded
Cha Xiang controls its molecular weight yet by intrinsic viscosity method.The method does not need reference substance, but by sample purity influenced compared with
Greatly, and the accurate content for learning hyaluronic acid in solution is needed.Intrinsic viscosity method is not due to needing expensive equipment, and detection side
Method is simple, is most common hyaluronan molecule quantity measuring method as a result accurately.Hyaluronic acid is measured by such method at present
When solution molecule amount, solution is subjected to ethanol precipitation first, and collects precipitating, is measured again after dry acquisition solid, the party
The accuracy of method complexity result is poor and takes a long time.
High Performance Gel Permeation Chromatography (HPGPC method) is a kind of liquid chromatography technology separated according to molecular size,
Substance is linearly negatively correlated by the retention time of chromatographic column and the logarithm of its molecular weight.The method favorable reproducibility, speed is fast, can survey
Obtain average molecular weight and its distribution.However HPGPC method measurement molecular weight needs corresponding reference substance to be corrected, and hyaluronic acid
Reference substance generally hardly results in, therefore the similar sample of structure can only be selected as reference substance, and reference substance structure is different, measures point
Son amount is different, this makes the molecular weight analyte and true value measured have certain deviation.
Multiple angle laser light scattering instrument attached gel permeation chromatography (GPC/MALLS method), to the height of any molecular weight ranges
Molecular sample, regardless of it in mobile phase molecular state, all can get sample elution figure in each grade of branch concentration and
Molecular weight.The method, which does not need reference substance, can be obtained the molecular weight of sample, and measurement result is accurate and reliable.But the instrument price is high
It is expensive, and in super high molecular weight measurement, in tool hole medium shear degradation may occur for sample, and may also block solidifying
Rubber column gel column.Therefore, the application of this method is restricted.
It is mentioned in non-patent literature 1, it is special when measuring hyaluronic acid of the molecular weight ranges in 10KDa~100KDa
Property the molecular weight values that measure of viscosity number method and GPC/MALLS method it is essentially identical, and the value that HPGPC method measures and other two methods are surveyed
It must be worth and differ greatly.
And colorimetry of carbazole is also referred to as alditol acid system, can be used for measuring the content of hyaluronic acid, and in measurement, hyaluronic acid
Uronic acid is generated through hydrolysis, condensation reaction occurs with carbazole reagent in strong acid, generates colored compound, in intensity of colour and sugar
Galacturonic acid content is directly proportional, carrys out the content of colorimetric assay hyaluronic acid.The poor specificity of colorimetry of carbazole, in hyaluronic acid fermentation liquor
Comparison of ingredients it is complicated, directly carries out carbazole colorimetric detection with fermentation liquid, disturbing factor is too many, especially at the beginning of zymotechnique exploitation
Phase, the hyaluronic acid fermentation liquor nature difference for fermenting out using different culture medium is bigger, certainly will will affect the judgement of result.
Therefore need to establish a kind of influence that can have not only reduced impurity, but also can letter that is easy and accurately detecting hyaluronic acid in fermentation liquid
Just feasible method.
Existing technical literature
Patent document
Patent document 1:CN102174122B
Non-patent literature
Non-patent literature 1: " 3 kinds of methods of measurement low-molecular-weight hyaluronic acid relative molecular mass compare ", China is biochemical
Drug magazine, 2007,28 (3), 182-184
Summary of the invention
The present invention is in order to solve the above technical problems, provide in a kind of each molecular weight section hyaluronic acid fermentation liquor and solution thoroughly
The detection method of bright matter acid molecule amount, this method is easy to be accurate, provides operation foundation for the production of different molecular weight hyaluronic acid,
Detection foundation also is provided for the hyaluronic acid solution of different molecular weight simultaneously.To achieve the above object, the present invention is using as follows
Technical solution:
Specifically, the present invention provides a kind of measuring method of hyaluronan molecule amount, which is characterized in that is based on hyaluronic acid
The intrinsic viscosity and glucuronic acid content of solution measure the molecular weight of the hyaluronic acid for including in solution.
Above-mentioned intrinsic viscosity is the viscosity number of the hyaluronic acid solution that hyaluronic acid contents are adjusted to 0.08~12g/L and right
Extrapolated value of the number viscosity numbers in infinite dilution, above-mentioned glucuronic acid content are generated after the hyaluronic acid hydrolysis in hyaluronic acid solution
Uronic acid content.
Solution in the present invention can be any solution comprising hyaluronic acid, such as can be raw using microbial fermentation
The obtained fermentation liquid comprising hyaluronic acid is produced, any drug solution, cosmetics stoste etc. comprising well-illuminated matter acid is also possible to
Deng.
Specifically, the measuring method of hyaluronan molecule amount of the invention, comprising the following steps:
Step 1: pre-treatment is diluted the solution comprising hyaluronic acid, filters, and obtaining extension rate is n1For examination
Liquid 1；
Step 2: Intrinsic Viscosity Measurements measure test liquid 1 with viscosimeter, record blank delivery time t0When with sample outflow
Between t1；
Step 3: glucuronic acid content measurement takes test liquid 1 to dilute, and obtaining extension rate is n2Test liquid 2, with alditol acid system
Measure the glucuronic acid content C of test liquid 21；With
Step 4: it calculates, the molecular weight for the hyaluronic acid for including in above-mentioned solution is calculated according to following formula,
The molecular weight of hyaluronic acid sample
Wherein, the intrinsic viscosity of hyaluronic acid sample
Hyaluronic acid solution content C (g/L)=C of test liquid 11×2.0675×n2。
Specifically, in step 1, it is diluted with the NaCl of 0.15~0.20mol/L, and crosses 0.22 μm~0.45 μm
It is n that filter membrane, which obtains extension rate,1Test liquid 1.
Further, when above-mentioned solution is the fermentation liquid comprising hyaluronic acid produced using microbial fermentation,
In step 1, above-mentioned pre-treatment further includes alcohol precipitation and filtering.Specifically, above-mentioned alcohol precipitation is the precipitating for using the ethyl alcohol greater than 95%,
It is above-mentioned to be filtered into using 0.22 μm~0.45 μm membrane filtration.
More specifically, in step 1, the fermentation liquid NaCl solution of 0.15~0.20mol/L being diluted 9~11 times, is obtained
To dilution；It takes the above-mentioned dilution in part in a reservoir, dehydrated alcohol is added, precipitates it sufficiently；By aforesaid liquid with 0.22 μ
M~0.45 μm membrane filtration, and with dehydrated alcohol repeated flushing container inner wall, it is transferred to precipitating on filter membrane, collects precipitating；It will
The precipitating of above-mentioned collection is fully transferred in other containers, is sufficiently dissolved with the NaCl solution of 0.15~0.20mol/L；It uses again
0.22 μm~0.45 μm membrane filtration, obtaining extension rate is n1Test liquid 1.
Specifically, solution extension rate n1It is following to determine: at 25 ± 0.03 DEG C, above-mentioned sample delivery time t1It is above-mentioned
Blank delivery time t01.3~1.5 times.
Further, blank delivery time t0At least twice, measured value answers identical, and t to replication0Greater than 100s, sample flow
Time t out1For the delivery time of test liquid 1, replication at least twice, is averaged as t1。
Further, hyaluronic acid solution extension rate to be measured is the dense of test liquid 2 after making the dilution when alditol acidity test
Degree is 20~100 μ g/ml, preferably 60 μ g/ml.
The beneficial effects of the present invention are:
Hyaluronic acid fermentation liquor molecular weight detection method provided by the present invention, for the hyaluronic acid for producing different molecular weight
Operation foundation is provided, while also providing detection foundation for the hyaluronic acid solution of different molecular weight, this method was not only easy but also quasi-
Really.
The specific embodiment of invention
The present invention will be described in detail below.
The measuring method of hyaluronan molecule amount provided by the invention, intrinsic viscosity and alditol based on hyaluronic acid solution
Acid content measures the molecular weight of the hyaluronic acid for including in solution, and intrinsic viscosity method and alditol acid system are combined, thus, it is possible to
It enough overcomes and needs that dry time-consuming and accuracy difference by hyaluronic acid precipitating in solution when intrinsic viscosity method is used alone
Defect, and easy accurately measurement hyaluronan molecule amount.
Above-mentioned intrinsic viscosity, also referred to as inherent viscosity are the hyalomitomes that hyaluronic acid contents are adjusted to 0.08~12g/L
Extrapolated value of the viscosity number and logarithm viscosity number of acid solution in infinite dilution, intrinsic viscosity independent of hyaluronic acid solution concentration,
Wherein, logarithm viscosity number is also known as the ratio between logarithmic viscosity number, the natural logrithm and concentration of the viscosity ratio (also referred to as relative viscosity) of solution.
Above-mentioned glucuronic acid content is the content of the uronic acid generated after the hyaluronic acid hydrolysis in hyaluronic acid solution.
In a specific embodiment, the measuring method of hyaluronan molecule amount of the invention, comprising the following steps:
Step 1: pre-treatment is diluted the solution comprising hyaluronic acid, filters, and obtaining extension rate is n1For examination
Liquid 1；
Step 2: Intrinsic Viscosity Measurements measure test liquid 1 with viscosimeter, record blank delivery time t0When with sample outflow
Between t1；
Step 3: glucuronic acid content measurement takes test liquid 1 to dilute, and obtaining extension rate is n2Test liquid 2, with alditol acid system
Measure the glucuronic acid content C of test liquid 21；With
Step 4: it calculates, the molecular weight for the hyaluronic acid for including in above-mentioned solution is calculated according to following formula,
The molecular weight of hyaluronic acid sample
Wherein, the intrinsic viscosity of hyaluronic acid sample
Hyaluronic acid solution content C (g/L)=C of test liquid 11×2.0675×n2。
Above-mentioned formula is to People's Republic of China (PRC) light industry professional standard cosmetics raw material Sodium Hyaluronate QB/
T4416-2012 standard improves.It needs first to do the fermentation liquid of alcohol precipitation when measuring in QB/T4416-2012 standard
After dry, obtain loss on drying and obtain accurate hyaluronic acid contents again, and the present processes do not need alcohol precipitation fermentation liquid then
It is dry, hyaluronic acid contents are directly measured by alditol acid system.Due to method of the invention do not need by hyaluronic acid into
Row drying reduces drying steps, therefore can easily be measured.
By utilizing above-mentioned formula, hyaluronan molecule amount can be more accurately measured.
In a specific embodiment, in step 1, it is diluted with the NaCl of 0.15~0.20mol/L, and mistake
It is n that 0.22 μm~0.45 μm of filter membrane, which obtains extension rate,1Test liquid 1.Thus, it is possible to reduce influence of the impurity to measurement.
In a specific embodiment, when above-mentioned solution is to be produced using microbial fermentation comprising hyalomitome
When the fermentation liquid of acid, in step 1, above-mentioned pre-treatment further includes alcohol precipitation and filtering.As long as wherein microorganism used in fermentation
It is able to produce hyaluronic acid to be just not particularly limited, can be bacterium, fungi etc..Specifically, above-mentioned alcohol precipitation is that use is greater than
The precipitating of 95% ethyl alcohol, it is above-mentioned to be filtered into using 0.22 μm~0.45 μm membrane filtration.According to step 1, fermentation liquid pre-treatment
In the process, alcohol precipitation and the step of membrane filtration, can eliminate the shadow that impurity measures intrinsic viscosity and glucuronic acid content in fermentation liquid
It rings.When collecting precipitating, hyaluronic acid is collected completely by filtering precipitating and repeated flushing dissolution, to guarantee measurement result
Accuracy.
More specifically, in step 1, the fermentation liquid NaCl solution of 0.15~0.20mol/L being diluted 9~11 times, is obtained
To dilution；It takes the above-mentioned dilution in part in a reservoir, dehydrated alcohol is added, precipitates it sufficiently；By aforesaid liquid with 0.22 μ
M~0.45 μm membrane filtration, and with dehydrated alcohol repeated flushing container inner wall, it is transferred to precipitating on filter membrane, collects precipitating；It will
The precipitating of above-mentioned collection is fully transferred in other containers, is sufficiently dissolved with the NaCl solution of 0.15~0.20mol/L；It uses again
0.22 μm~0.45 μm membrane filtration, obtaining extension rate is n1Test liquid 1.
In a specific embodiment, solution extension rate n1It is following to determine: at 25 ± 0.03 DEG C, sample outflow
Time t1It is blank delivery time t01.3~1.5 times.According to step 2, the extension rate of fermentation liquid pre-treatment be should be, 25 ±
At 0.03 DEG C, delivery time t of the sample in dark type viscometer (internal diameter 0.53mm)1It is NaCl solution delivery time t01.3~
It 1.5 times, is surveyed again after otherwise should adjusting 1 concentration of test liquid.Thus it enables that result is more accurate.Intrinsic viscosity method measures molecular weight
When, the concentration of solution was adjusted according to the sample delivery time, and the delivery time herein is related to solution viscosity, and exists with sample
Content in solution is not directly relevant to, and under normal circumstances, the molecular weight of sample to be tested is higher, and solution viscosity is got over when same concentrations
Greatly, then required solution concentration is lower.Therefore, fermentation liquid higher for molecular weight, when dilution is preferably 100 times or so, solution
Viscosity is more appropriate.Wherein, dilution post-fermentation liquid concentration is preferably the dense of 0.9~1.1% or the test liquid 1 of original fermentation liquor
Degree is preferably adjusted to the content of hyaluronic acid preferably between 0.08~12g/L.
Further, blank delivery time t0At least twice, measured value answers identical, and t to replication0Greater than 100s, sample flow
Time t out1For the delivery time of test liquid 1, replication at least twice, is averaged as t1.Thus it enables that result is more quasi-
Really.
Further, hyaluronic acid solution extension rate to be measured is the about 60 μ g/ of concentration for making test liquid 2 when alditol acidity test
Ml, this is because the concentration of the uronic acid of assay process standard curve is 10 μ g/ml, 20 μ g/ml, 30 μ g/ml, 40 μ g/
Ml, 50 μ g/ml, correspond to hyaluronic acid concentration be about 20.07 μ g/ml, 41.35 μ g/ml, 62.03 μ g/ml, 82.70 μ g/
Ml, 103.38 μ g/ml, under normal circumstances, result is most accurate when measured value falls in intermediate point, therefore hyaluronic acid concentration should be 20
~100 μ g/ml, preferably 60 μ g/ml.
As described below is the preferred embodiment of the present invention, and what the present invention was protected is not limited to following preferred implementation side
Formula.It should be pointed out that for those skilled in the art on the basis of the inventive concept, several deformations for making and
It improves, belongs to protection scope of the present invention.
The present invention will be further described combined with specific embodiments below, and unspecified operating procedure is asked in embodiment
Referring to " Chinese Pharmacopoeia " (2015 editions) or the specification refering to instrument, wherein used reagent sulfuric acid is excellent pure grade
Outside, remaining is that analysis is pure.
Embodiment
The measurement of hyaluronan molecule amount in 1 hyaluronic acid fermentation liquor of embodiment
(1) fermentation liquid pre-treatment
Taking lot number is that 180903 fermentation liquid 25.00g is set in the volumetric flask of 250ml, weighs after peeling, 0.2mol/L is added
NaCl solution to 250.00g, i.e. fermentation liquid dilutes 10 times for this.When dilution, magneton is added in volumetric flask, in magnetic stirring apparatus
On stir evenly.
It takes above-mentioned dilution 25.01g to set in 250ml volumetric flask, magneton is added in volumetric flask, be added while stirring anhydrous
Ethyl alcohol stirs 5 minutes to 250ml groove.
Above-mentioned mixed liquor is poured into filter bowl, crosses 0.22 μm~0.45 μm of filter membrane, and held with dehydrated alcohol repeated flushing
Measuring bottle and filter bowl are transferred to precipitating on filter membrane completely.
100ml 0.2mol/L is added in magneton when above-mentioned precipitating and filter membrane being put into 250ml beaker, and being put into precipitating
NaCl solution in magnetic stirring apparatus, (model: 84-1 magnetic stirring apparatus, producer: Shanghai Mei Yingpu instrument and meter manufactures limited public affairs
Department) on stir and be completely dissolved to precipitating, solution is then poured into 250ml volumetric flask, the NaCl solution for adding 0.2mol/L is anti-
Filter membrane and beaker inner wall are rinsed in multiple stirring, are settled to groove.
By 0.45 μm of membrane filtration of above-mentioned solution, test liquid 1 is obtained.
(2) Intrinsic Viscosity Measurements
Use Ubbelohde viscometer (capillary inner diameter 0.53mm, viscometer constant 0.008389mm2/S2, producer: Shanghai Shen is vertical
Glass apparatus Co., Ltd), under the conditions of 25 ± 0.03 DEG C, measure blank delivery time t0With sample delivery time t1, make t1/
t0Between 1.3~1.5.
(3) glucuronic acid content measures
A. required reagent
0.025mol/L borax sulfuric acid solution: weighing 4.77g borax, is dissolved in the concentrated sulfuric acid (excellent pure grade) 500ml, shakes up i.e.
Obtain borax sulfuric acid solution.
Carbazole test solution: it weighs carbazole 0.125g and adds dehydrated alcohol 100ml, shaking makes it dissolve to get carbazole test solution.This liquid
It should set in Brown Glass Brown glass bottles and jars only, close plug saves.
B. reference substance
Glucuronic acid
C. prepared by reference substance solution
Precision weighs the glucuronic acid reference substance about 100mg that constant weight is dried under vacuum to through 105 DEG C, sets in 100ml measuring bottle,
It is dissolved in water and is diluted to scale, shake up, as stock solution；With preceding accurate measurement stock solution 5.0ml, sets in 100ml measuring bottle, add water
The solution for containing 50 μ g in every 1ml is made, shakes up.
D. prepared by test liquid
It uses purified water to dilute about 1.5 times test liquid 1 to shake up as test liquid 2.
E. the preparation of standard curve
Precision measures 0.0,0.2,0.4,0.6,0.8 and 1.0ml of reference substance solution, sets in tool plug test tube respectively, respectively plus water
It to 1.0ml, mixes, is cooled to 4 DEG C hereinafter, 0.025mol/L borax sulfuric acid solution 5.0ml is slowly added dropwise under shaking, mixes,
It sets in boiling water bath and heats 10 minutes, set in ice water and let cool.Carbazole test solution 0.20ml is added in precision, shakes up, sets in boiling water bath and heat
It 15 minutes, sets in ice water and lets cool.According to ultraviolet-uisible spectrophotometer, (model: UV-1800, producer: Shimadzu instrument (Suzhou) is limited
Company) operating instruction, measuring absorbance at 530nm wavelength must be returned with the micrograms per millilitre and absorbance of glucuronic acid
EQUATION x=60.7484y+0.243697 and correlation coefficient r2=0.99990.
F. sample measures
It is accurate to measure test liquid 1.0ml (parallel 2 pipe of preparation), it sets in tool plug test tube, " is cooled to certainly under sighting target directrix curve item
4 DEG C or less " it rises, it is operated with method, concentration (taking two pipe average values) (C of glucuronic acid is obtained by regression equation1) (unit: μ g/
Ml), sodium hyaluronate content C in test liquid 2 is calculated according to extension rate.
(4) molecular weight calculates
Hyaluronic acid fermentation liquor molecular weight is measured according to following formula:
1 hyaluronic acid contents of test liquid (μ g/L) C=C1×n2×2.0675
The intrinsic viscosity of hyaluronic acid
The molecular weight of hyaluronic acid solution
(5) GPC/MALLS method:
Chromatographic condition: TSK-GEL G3000PWXLWith TSK-GEL G4000PWXLCoupled columns；Flow velocity: 1.0ml/
min；Mobile phase: 0.2mol/L sodium chloride solution；Sample volume: 100 μ l
Test sample preparation: taking above-mentioned fermentation liquid appropriate, be configured to the test solution that concentration is about 5mg/mL with mobile phase,
With 0.22 μm of membrane filtration, sample introduction.
Molecular weight determination: the test sample concentration and test sample that differential refraction detector and laser detector measure are recorded respectively
In the light scatter intensity of different angle, dn/dc value is 0.165, and molecular weight is calculated by data processing software.
Measurement lot number is 180903 fermentation liquid 3 times respectively according to the method described above, as a result as shown in the table:
Table 1
| Measure number | (1) | (2) | (3) |
| t0(s) | 108.09 | 108.09 | 108.09 |
| t1(s) | 161.970 | 161.175 | 162.010 |
| n2 | 50.3694/33.0897 | 50.2926/33.1525 | 50.2249/33.2424 |
| C1(μg/L) | 30.0485 | 30.1945 | 30.3265 |
| C(μg/L) | 94.5676 | 94.7024 | 94.7315 |
| Mr(KDa) | 3.50×103 | 3.44×103 | 3.50×103 |
Same batch of sample is measured into 3 molecular weight respectively it can be seen from above-mentioned table 1, result is roughly the same, illustrates this
Method measurement hyaluronic acid fermentation liquor has good stability.With the fermentation liquid of GPC/MALLS measurement lot number 180903, molecular weight is
3.48×103KDa illustrates that this method measurement hyaluronic acid fermentation liquor accuracy is good.
The measurement of hyaluronan molecule amount in 2 hyaluronic acid fermentation liquor of embodiment
The hyaluronic acid fermentation liquor that lot number is 180903 carries out molecular weight determination according to the method for embodiment 1, is as a result shown in
Table 2.
Table 2
| Measure number | (1) | (2) | (3) |
| t0(s) | 107.03 | 107.03 | 107.03 |
| t1(s) | 159.040 | 158.895 | 159.125 |
| n2 | 50.2694/30.0792 | 50.1482/30.4839 | 50.1447/30.3936 |
| C1(μg/L) | 21.8235 | 21.8942 | 21.9322 |
| C(μg/L) | 75.4062 | 74.4662 | 74.8119 |
| Mr(KDa) | 4.55×103 | 4.61×103 | 4.61×103 |
With the fermentation liquid of GPC/MALLS measurement lot number 180903, molecular weight is 4.65 × 103KDa。
The measurement of hyaluronan molecule amount in 3 hyaluronic acid solution of embodiment
In process of production, it is also necessary to molecular weight determination be carried out to fermentation treatment fluid, fermentation treatment fluid herein is transparent
Intermediate product in matter acid production process, be by re-dissolving acquisition after fermentation liquid alcohol precipitation, actually its processing mode with it is above-mentioned
Fermentation liquor treatment mode is identical, because also needing to measure the molecular weight of this step in the actual production process.Hyalomitome at this time
Acid solution purity is higher, can directly carry out intrinsic viscosity and glucuronic acid content measurement, as a result be shown in table 3.
Taking lot number is that 180904 hyaluronic acid solution 18.04g is set in 250ml volumetric flask, adds the NaCl of 0.2mol/L molten
Liquid is settled to 250ml, and weight is 250.87g after peeling, shook up 0.22 μm~0.45 μm filter membrane and obtains test liquid 1.25 ±
At 0.03 DEG C, blank delivery time t is measured0For 107.25s, the sample delivery time is respectively 168.28s, 168.29s, i.e. t1For
168.285s。
Test liquid 1 is diluted, 28.6409g is taken to set 50ml volumetric flask, after adding the NaCl solution of 0.2mol/L to be settled to 50ml
Going bottle weight is 50.2035g, shakes up to obtain test liquid 2, with the C of alditol acid system measurement test liquid hyaluronic acid1Content is
28.8720μg/ml。
1 hyaluronic acid solution content of test liquid (μ g/L) C=C1×n2× 2.0675=28.8720 × (50.2035/
28.6409) × 2.0675=104.6333
The intrinsic viscosity of hyaluronic acid
The molecular weight of hyaluronic acid solution
With the hyaluronic acid solution of GPC/MALLS measurement lot number 180904, molecular weight is 3.00 × 103KDa, further
Illustrate that the accuracy of this method measurement hyaluronic acid solution is good.
The measurement of hyaluronan molecule amount in 4 hyaluronic acid solution of embodiment
The hyaluronic acid solution that lot number is 180905 carries out molecular weight determination, extension rate according to the method for embodiment 3
As a result difference is shown in table 3.
The measurement of hyaluronan molecule amount in 5 hyaluronic acid solution of embodiment
The hyaluronic acid solution that lot number is 180906 carries out molecular weight determination, extension rate according to the method for embodiment 3
As a result difference is shown in table 3.
The measurement of hyaluronan molecule amount in 6 hyaluronic acid solution of embodiment
The hyaluronic acid solution that lot number is 180907 carries out molecular weight determination, extension rate according to the method for embodiment 3
As a result difference is shown in table 3.
The measurement of hyaluronan molecule amount in 7 hyaluronic acid solution of embodiment
The hyaluronic acid solution that lot number is 180910 carries out molecular weight determination, extension rate according to the method for embodiment 3
As a result difference is shown in table 3.
Table 3
The measuring method of hyaluronan molecule amount of the invention known to above-mentioned table 3, for the saturating of nearly all molecular weight
Bright matter acid can be easy and accurately measures its molecular weight.
Although embodiment of the present invention and specific embodiment are described, the invention is not limited to above-mentioned
Specific embodiments and applications field, above-mentioned specific embodiment are only schematical, directiveness, rather than restricted
's.Those skilled in the art are under the enlightenment of this specification and in the range for not departing from the claims in the present invention and being protected
In the case where, a variety of forms can also be made, these belong to the column of protection of the invention.
Claims (10)
1. a kind of measuring method of hyaluronan molecule amount, which is characterized in that intrinsic viscosity and sugar based on hyaluronic acid solution
Galacturonic acid content measures the molecular weight of the hyaluronic acid for including in solution.
2. the measuring method of hyaluronan molecule amount according to claim 1, which is characterized in that
The intrinsic viscosity is that the viscosity number for the hyaluronic acid solution that hyaluronic acid contents are adjusted to 0.08~12g/L and logarithm glue
Extrapolated values of the number in infinite dilution, the glucuronic acid content are the sugar generated after the hyaluronic acid hydrolysis in hyaluronic acid solution
The content of aldehydic acid.
3. the measuring method of hyaluronan molecule amount according to claim 1 or claim 2, which comprises the following steps:
Step 1: pre-treatment is diluted the solution comprising hyaluronic acid, filters, and obtaining extension rate is n1Test liquid 1；
Step 2: Intrinsic Viscosity Measurements measure test liquid 1 with viscosimeter, record blank delivery time t0With sample delivery time t1；
Step 3: glucuronic acid content measurement takes test liquid 1 to dilute, and obtaining extension rate is n2Test liquid 2, with alditol acid system measure
The glucuronic acid content C of test liquid 21；
Step 4: it calculates, the molecular weight for the hyaluronic acid for including in the solution is calculated according to following formula,
The molecular weight of hyaluronic acid sample
Wherein, the intrinsic viscosity of hyaluronic acid sample
Hyaluronic acid solution content C (g/L)=C of test liquid 11×2.0675×n2。
4. measuring method according to claim 3, which is characterized in that
It is in step 1, described when the solution is the fermentation liquid comprising hyaluronic acid produced using microbial fermentation
Pre-treatment further includes alcohol precipitation and filtering.
5. measuring method according to claim 3 or 4, which is characterized in that solution extension rate n1It is following to determine: 25 ±
At 0.03 DEG C, the sample delivery time t1It is the blank delivery time t01.3~1.5 times.
6. the measuring method according to any one of claim 3~5, which is characterized in that blank delivery time t0Replication
At least twice, measured value is answered identical, and t0Greater than 100s, sample delivery time t1For the delivery time of test liquid 1, replication
At least twice, it is averaged as t1。
7. the measuring method according to any one of claim 3~6, which is characterized in that be measured transparent when alditol acidity test
Matter acid solution extension rate is 20~100 μ g/ml of concentration, preferably 60 μ g/ml for making the test liquid 2 after dilution.
8. measuring method according to claim 4, which is characterized in that
The alcohol precipitation is the precipitating for using the ethyl alcohol greater than 95%, described to be filtered into using 0.22 μm~0.45 μm membrane filtration.
9. the measuring method according to any one of claim 4~8, which is characterized in that
In step 1, it is diluted with the NaCl of 0.15~0.20mol/L, and the filter membrane for crossing 0.22 μm~0.45 μm must dilute
Multiple is n1Test liquid 1.
10. the measuring method according to claim 4 or 8, which is characterized in that
In step 1, it when the solution is the fermentation liquid comprising hyaluronic acid produced using microbial fermentation, will send out
Zymotic fluid dilutes 9~11 times with the NaCl solution of 0.15~0.20mol/L, obtains dilution；Take the above-mentioned dilution in part in container
In, dehydrated alcohol is added, precipitates it sufficiently；By 0.22 μm~0.45 μm membrane filtration of aforesaid liquid, and use dehydrated alcohol
Repeated flushing container inner wall is transferred to precipitating on filter membrane, collects precipitating；The precipitating of above-mentioned collection is fully transferred to other appearances
In device, sufficiently dissolved with the NaCl solution of 0.15~0.20mol/L；Again with 0.22 μm~0.45 μm membrane filtration, must dilute
Multiple is n1Test liquid 1.
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