CN109613132A - A kind of detection method of heterozygosis chondroitin sulfate - Google Patents
A kind of detection method of heterozygosis chondroitin sulfate Download PDFInfo
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Abstract
The present invention provides a kind of detection method of heterozygosis chondroitin sulfate, include the following steps: that sample to be tested is successively passed through nuclear magnetic resonance spectrometer by (A), infrared spectrometer is detected to carry out the prediction of preliminary structure；(B) sample to be tested is carried out using structure of the high performance liquid chromatography to glucuronic acid, iduronic acid and acetylamino galactosamine qualitative.Detection method of the invention is easy to operate, and accuracy in detection is high, should be widely promoted and is applied.
Description
Technical field
The present invention relates to Sodium Hyaluronate control of product quality fields, in particular to a kind of heterozygosis chondroitin sulfate
Detection method.
Background technique
Sodium Hyaluronate (SODIUM HYALURONATE) is the substance extracted from cockscomb, can also pass through lactic acid horse hammer
Bacterium fermentation is made, for white or off-white color particle or powder, odorless, when dry after, nitrogen content 2.8%-4.0%, glucose
Galacturonic acid content is 37.0%-51.0%.Using more in cosmetic field, there is moisture-keeping function.
Sodium Hyaluronate is very widely used, for the biochemical drug with higher clinical value, is widely used in all kinds of eyes
Section's operation, such as Lens implantation, corneal transplantation and resisting glaucoma operation, it may also be used for treatment of arthritis and accelerating wound healing,
It is used in cosmetics, unique protection skin can be played the role of, skin moisturizing can be kept smooth, it is fine and smooth tender, rich in bullet
Property, have the function of wrinkle resistant, crease-resistant, beauty and health care and restores skin physiology function.
Sodium Hyaluronate can be used in the products such as cream, frost, honey, milk, facial mask, the shampoo of cosmetics, to keep skin, head
The moisture of hair moisturizes the skin, hair, increases gloss, and can prevent the generation of chapped skin and wrinkle.
In the prior art, it can be generated containing the impurity such as albumen, nucleic acid, chondroitin sulfate, source in Sodium Hyaluronate product
The reason of be primarily due to cockscomb and inherently and in extractive technique be brought into Sodium Hyaluronate product, wherein impurity contains
Have and will have a direct impact on the probability that the adverse reaction of product clinically occurs, reduces the safety of Sodium Hyaluronate product,
It is unfavorable for the marketing dynamics of its product.
In view of this, the present invention is specifically proposed.
Summary of the invention
The purpose of the present invention is to provide the detection methods of the heterozygosis chondroitin sulfate in above-mentioned Sodium Hyaluronate product, should
Detection method itself is easy to operate, operating procedure is successive close, and no three wastes generates in operating process, whole environmentally protective,
Operating condition is also relatively milder, good quality control can be carried out to Sodium Hyaluronate Related product, accuracy in detection is high, can
Strong operability improves the safety of Sodium Hyaluronate product itself, therefore should be widely promoted and applied, and is subsequent technique
Research also provides more reference frames, advantageously forms more extensive economic benefit, whole environmentally protective, institute in method
Various reagents, which obtain, to be easy, easy to implement.
In order to realize above-mentioned purpose of the invention, the following technical scheme is adopted:
The present invention provides a kind of detection methods of heterozygosis chondroitin sulfate, mainly include the following steps:
(A) sample to be tested is successively passed through to nuclear magnetic resonance spectrometer, infrared spectrometer is detected to carry out the prediction of preliminary structure；
(B) use high performance liquid chromatography to glucuronic acid, iduronic acid and acetylamino galactosamine sample to be tested
Structure carry out it is qualitative.
In the prior art, Sodium Hyaluronate is very widely used, for the biochemical drug with higher clinical value, answers extensively
For all kinds of ophthalmologic operations, such as Lens implantation, corneal transplantation and resisting glaucoma operation, it may also be used for treatment of arthritis and acceleration
Wound healing is used in cosmetics, can play the role of unique protection skin, skin moisturizing can be kept smooth, fine and smooth soft
Tender, high resilience has the function of wrinkle resistant, crease-resistant, beauty and health care and restores skin physiology function.
Sodium Hyaluronate can be used in the products such as cream, frost, honey, milk, facial mask, the shampoo of cosmetics, to keep skin, head
The moisture of hair moisturizes the skin, hair, increases gloss, and can prevent the generation of chapped skin and wrinkle.
In the prior art, it can be generated containing the impurity such as albumen, nucleic acid, chondroitin sulfate, source in Sodium Hyaluronate product
The reason of be primarily due to cockscomb and inherently and in extractive technique be brought into Sodium Hyaluronate product, wherein impurity contains
Have and will have a direct impact on the probability that the adverse reaction of product clinically occurs, reduces the safety of Sodium Hyaluronate product,
It is unfavorable for the marketing dynamics of its product.
In order to solve the above technical problems, The present invention provides the heterozygosis chondroitin sulfates in a kind of Sodium Hyaluronate product
Detection method, so that the convenient quality to Sodium Hyaluronate product is managed, the front and back step design of this method rationally, is being carried out
Step-by-step operating method according to the invention is needed to be implemented when specific operation, by being in Sodium Hyaluronate product
It is no be measured containing heterozygosis chondroitin sulfate after, also facilitate subsequent technique research related work to carry on, improve production
The safety of product.
Heterozygosis chondroitin sulfate is a kind of heterozygote, contains glucuronic acid, iduronic acid and acetylamino gala
Sugar is the covalent heterozygosis composition of constitutional repeating unit, then during being specifically measured, by glucuronic acid, Chinese mugwort
The detection of Du uronic acid and acetylamino galactosamine specific structure, so that it may know whether contain heterozygosis chondroitin sulfate.
Preferably as further enforceable scheme, in the step (A), using D2After O dissolves sample to be tested,
It is analyzed in Nuclear Magnetic Resonance；
Preferably, the model DD of the Nuclear Magnetic Resonance2400-MR。
By using Nuclear Magnetic Resonance, there can be a preliminary analysis to its structure, if doubtful in the product contain heterozygosis
Chondroitin sulfate, can show in the result of nuclear magnetic spectrogram the signal, C-2, C-6 that have anomer hydrogen on saccharide residue proton signal,
The signal that acetyl matrix generates.
When specific operation, it takes 10mg impurity sample to be placed in containing 12h dry in phosphorus pentoxide desiccator, uses 0.5mlD2O
Sample dissolution is transferred in nuclear magnetic tube, is analyzed in DD2400-MR Nuclear Magnetic Resonance, heavy water is as internal standard.As the result is shown
The signal for thering is the signal of anomer hydrogen, the proton signal of C-2, C-6, acetyl matrix to generate on saccharide residue.
Preferably as further enforceable scheme, in the step (A), sample to be tested is used into pressing potassium bromide troche
Infrared spectrometer detection is carried out afterwards.
Whether the purpose analyzed using infrared spectrometer is correct also for the result of verifying nuclear-magnetism test, by red
External spectrum instrument analysis, to judge whether contain acetylamino and sulfate in impurity sample.
Preferably as further enforceable scheme, in the step (B), the column temperature of high performance liquid chromatography measurement is
20-40 DEG C, Detection wavelength is between 200-300nm.
Preferably as further enforceable scheme, in the step (B), the mobile phase of high performance liquid chromatography measurement
For the mixed solution of acetonitrile and ammonium acetate solution, flow control is between 0.2-0.4ml/min.
After front carries out preliminary judgement to its structure using the instruments analysis means such as nuclear-magnetism, infrared spectroscopy, behind pass through
It is analyzed using high performance liquid chromatography and determines specific structure, carry out monosaccharide composition detection, inspection using high performance liquid chromatograph
The map visible foreign measured contains iduronic acid, glucuronic acid, amine-galactose.Because iduronic acid is sulfuric acid skin
One of principal monosaccharides composition of element, then impurity contains the structure of similar chondroitin sulfate.
It equally also can use high performance liquid chromatography carries out further iduronic acid and amine-galactose composition two
The detection of sugar.
It, may be only with efficient liquid in the case that comparision contents are low when heterozygosis bulk concentration itself is smaller in sample to be tested
Phase chromatographic determination, accuracy will receive influence, therefore the present invention detects sample to be tested using the method for LC-MS,
The accuracy of detection is improved in this way.
Preferably as further enforceable scheme, in the step (B), using mass spectrum and the high-efficient liquid phase color
Method associated with spectrum carries out qualitative.
Preferably as further enforceable scheme, in the step (B), mass spectrographic gas temperature control is in 300-
Between 400 DEG C, flow rate of carrier gas 10-30L/min.
Preferably as further enforceable scheme, in the step (B), include thes steps that after qualitative quantitative: adopting
With the content of glucuronic acid in UV carbazole method measurement sample to be tested.
Preferably as further enforceable scheme, in the step (B), during UV carbazole method measures, boiling water
After heating sample to be tested 10min or more, carbazole is added after cooling, boiling water heats 15min or more again after shaking up, and observation phenomenon is
It can.
The specific operating procedure of UV carbazole method can be carried out according to following operation: being pipetted 1.0ml sample solution and passed through
0.025mol/L borax sulfuric acid solution 5.0ml, boiling water bath heat 10 minutes, cooling rapidly, add the dehydrated alcohol of 0.125% carbazole
Solution 0.2ml, shakes up, and heats 15 minutes (centre shaking is primary), is cooled to room temperature in boiling water bath.(explanation: sample passes through strong acid
UV absorption can just be had by being degraded into glucuronic acid and carbazole colour developing, and color and concentration are directly proportional).
It further include pair in the quantitative step in the step (B) preferably as further enforceable scheme
The step of sulfate radical content in sample to be tested is measured；
Preferably, the sulfate radical content is measured using ion chromatograph.
Pass through the specific measuring method of above-mentioned each step, it may be determined that whether impurity contains glucuronic acid, idose aldehyde
Acid and acetylamino galactosamine are the chondroitin sulfates of the covalent heterozygosis composition of constitutional repeating unit.
Compared with prior art, the invention has the benefit that
(1) detection method of the invention itself is easy to operate, operating procedure is successive close, no three wastes in operating process
It generates, whole environmentally protective, operating condition is also relatively milder, can carry out good quality pipe to Sodium Hyaluronate Related product
Control, accuracy in detection is high, and strong operability improves the safety of Sodium Hyaluronate product itself；
(2) detection method of the invention is that subsequent technique research also provides more reference frames, is advantageously formed more
Whole environmentally protective for wide economic benefit, various reagents used in method, which obtain, to be easy, and is implemented more convenient.
Detailed description of the invention
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
It puts, is also possible to obtain other drawings based on these drawings.
Fig. 1 is the hydrogen nuclear magnetic resonance spectrogram of the sample to be tested of the embodiment of the present invention 1；
Fig. 2 is the infrared spectrogram of the sample to be tested of the embodiment of the present invention 1；
Fig. 3 is the standard High Performance liquid chromatogram of monosaccharide PMP derivative products；
Fig. 4 is the standard High Performance liquid chromatogram after dermatan sulfate hydrolysate PMP is checked and accepted；
Fig. 5 is the standard High Performance liquid chromatogram after amine-galactose is derivative；
Fig. 6 is that the sample to be tested of the embodiment of the present invention 1 hydrolyzes the high-efficient liquid phase chromatogram after PMP derivatization；
Fig. 7 is the mass spectrogram hydrolyzed after PMP derivatization using the sample to be tested of the embodiment of the present invention 1.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
The conventional products that can be obtained by commercially available purchase.
Embodiment 1
The detection method of heterozygosis chondroitin sulfate carries out in accordance with the following steps:
1, it takes 10mg Sodium Hyaluronate sample to be tested to be placed in containing 12h dry in phosphorus pentoxide desiccator, uses
0.5mlD2O sample dissolution, is transferred in nuclear magnetic tube, is analyzed in DD2400-MR Nuclear Magnetic Resonance, heavy water as internal standard,
Hydrogen spectrogram is specifically shown in proton signal, the acetyl group of the signal, C-2, C-6 that have anomer hydrogen shown in attached drawing 1 on saccharide residue as the result is shown
The signal that matter generates.
2,2mg impurity sample is taken, after pressing potassium bromide troche, by infrared spectrum analysis, specific spectrogram the results are shown in attached figure 2 institute
Show, it is known that contain acetylamino and sulfate in impurity sample.
3, after the hydrolysis of impurity sample, monosaccharide composition detection, the result detected such as Fig. 6 are carried out using high performance liquid chromatograph
It is shown, after being compared by the standard diagram with Fig. 3-5, it is seen that impurity contains iduronic acid, glucuronic acid, amino half
Lactose.Because iduronic acid is one of the principal monosaccharides composition of dermatan sulfate, then impurity contains the knot of similar chondroitin sulfate
Structure.
4, go out the disaccharides of iduronic acid and amine-galactose composition, the condition of liquid chromatogram by LC-MS combination analysis
Are as follows: 30 DEG C of column temperature, sample volume 1ul, mobile phase is acetonitrile: 0.01mol/L ammonium acetate solution=15:85 (volume ratio), flow velocity are
0.3ml/min；Detector DAD, Detection wavelength 250nm；Mass spectrographic condition are as follows: range 50-1500m/s, nitrogen 10L/min, gas
350 DEG C of temperature, nebulizer pressure 30psig, second order ms collision voltage 40V, the result of specific mass spectrogram is as shown in Figure 7.
In Fig. 6-7, a- disaccharides, b- glucuronic acid, c- iduronic acid, d- amine-galactose.
Embodiment 2
The detection method of heterozygosis chondroitin sulfate carries out in accordance with the following steps:
1, it takes 10mg Sodium Hyaluronate sample to be tested to be placed in containing 12h dry in phosphorus pentoxide desiccator, uses
0.5mlD2O sample dissolution, is transferred in nuclear magnetic tube, is analyzed in DD2400-MR Nuclear Magnetic Resonance, heavy water as internal standard,
The signal for thering is the signal of anomer hydrogen, the proton signal of C-2, C-6, acetyl matrix to generate on saccharide residue as the result is shown.
2,2mg Sodium Hyaluronate sample to be tested is taken to pass through infrared spectrum analysis after pressing potassium bromide troche, it is known that impurity sample
Contain acetylamino and sulfate in product.
3, after the hydrolysis of Sodium Hyaluronate sample to be tested, monosaccharide composition detection, liquid phase color are carried out using high performance liquid chromatograph
The condition of spectrum are as follows: 20 DEG C of column temperature, sample volume 1ul, mobile phase is acetonitrile: 0.01mol/L ammonium acetate solution=15:85 (volume
Than), flow velocity 0.2ml/min；Detector DAD, Detection wavelength 200nm；Visible foreign contains iduronic acid, grape alditol
Acid, amine-galactose.Because iduronic acid is one of the principal monosaccharides composition of dermatan sulfate, then it is soft to contain similar sulfuric acid for impurity
The structure of ossein.
4, go out the disaccharides of iduronic acid and amine-galactose composition, the condition of liquid chromatogram by LC-MS combination analysis
Are as follows: 20 DEG C of column temperature, sample volume 1ul, mobile phase is acetonitrile: 0.01mol/L ammonium acetate solution=15:85 (volume ratio), flow velocity are
0.2ml/min；Detector DAD, Detection wavelength 200nm；Mass spectrographic condition are as follows: range 50-1500m/s, nitrogen 30L/min, gas
300 DEG C of temperature, nebulizer pressure 30psig, second order ms collision voltage 40V.
5,20% is detected as using glucuronic acid content in UV carbazole method measurement Sodium Hyaluronate sample to be tested.
1.0ml Sodium Hyaluronate testing sample solution is pipetted by 0.025mol/L borax sulfuric acid solution 5.0ml, boiling water bath
Heat 10min, it is cooling rapidly, add the ethanol solution 0.2ml of 0.125% carbazole, shake up, heated in boiling water bath 15min (in
Between shake it is primary), be cooled to room temperature.(explanation: sample by strong acid be degraded into glucuronic acid and carbazole develop the color can just have it is ultraviolet
It absorbs, color and concentration are directly proportional)
6, Sodium Hyaluronate sample to be tested carries out the measurement of ion chromatography sulfate radical content, obtains content after sour water solution
It is 19.5%.
It is measured by above-mentioned project, it may be determined that contain glucuronic acid, iduronic acid in Sodium Hyaluronate sample to be tested
And acetylamino galactosamine, it is the chondroitin sulfate of the covalent heterozygosis composition of constitutional repeating unit.
Embodiment 3
The detection method of heterozygosis chondroitin sulfate carries out in accordance with the following steps:
1, it takes 10mg Sodium Hyaluronate sample to be tested to be placed in containing 12h dry in phosphorus pentoxide desiccator, uses
0.5mlD2O sample dissolution, is transferred in nuclear magnetic tube, is analyzed in DD2400-MR Nuclear Magnetic Resonance, heavy water as internal standard,
The signal for thering is the signal of anomer hydrogen, the proton signal of C-2, C-6, acetyl matrix to generate on saccharide residue as the result is shown.
2,2mg Sodium Hyaluronate sample to be tested is taken to pass through infrared spectrum analysis after pressing potassium bromide troche, it is known that impurity sample
Contain acetylamino and sulfate in product.
3, after the hydrolysis of Sodium Hyaluronate sample to be tested, monosaccharide composition detection, liquid phase color are carried out using high performance liquid chromatograph
The condition of spectrum are as follows: 40 DEG C of column temperature, sample volume 1ul, mobile phase is acetonitrile: 0.01mol/L ammonium acetate solution=15:85 (volume
Than), flow velocity 0.4ml/min；Detector DAD, Detection wavelength 400nm；Visible foreign contains iduronic acid, grape alditol
Acid, amine-galactose.Because iduronic acid is one of the principal monosaccharides composition of dermatan sulfate, then it is soft to contain similar sulfuric acid for impurity
The structure of ossein.
4, go out the disaccharides of iduronic acid and amine-galactose composition, the condition of liquid chromatogram by LC-MS combination analysis
Are as follows: 40 DEG C of column temperature, sample volume 1ul, mobile phase is acetonitrile: 0.01mol/L ammonium acetate solution=15:85 (volume ratio), flow velocity are
0.4ml/min；Detector DAD, Detection wavelength 400nm；Mass spectrographic condition are as follows: range 50-1500m/s, nitrogen 20L/min, gas
400 DEG C of temperature, nebulizer pressure 30psig, second order ms collision voltage 40V.
5,20% is detected as using glucuronic acid content in UV carbazole method measurement Sodium Hyaluronate sample to be tested.
1.0ml Sodium Hyaluronate testing sample solution is pipetted by 0.025mol/L borax sulfuric acid solution 5.0ml, boiling water bath
Heat 15min, it is cooling rapidly, add the ethanol solution 0.2ml of 0.125% carbazole, shake up, heated in boiling water bath 10min (in
Between shake it is primary), be cooled to room temperature.(explanation: sample by strong acid be degraded into glucuronic acid and carbazole develop the color can just have it is ultraviolet
It absorbs, color and concentration are directly proportional)
6, Sodium Hyaluronate sample to be tested carries out the measurement of ion chromatography sulfate radical content, obtains content after sour water solution
It is 19.5%.
It is measured by above-mentioned project, it may be determined that contain glucuronic acid, iduronic acid in Sodium Hyaluronate sample to be tested
And acetylamino galactosamine, it is the chondroitin sulfate of the covalent heterozygosis composition of constitutional repeating unit.
Compared with prior art, the invention has the benefit that
(1) detection method of the invention itself is easy to operate, operating procedure is successive close, no three wastes in operating process
It generates, whole environmentally protective, operating condition is also relatively milder, can carry out good quality pipe to Sodium Hyaluronate Related product
Control, accuracy in detection is high, and strong operability improves the safety of Sodium Hyaluronate product itself；
(2) detection method of the invention is that subsequent technique research also provides more reference frames, is advantageously formed more
Whole environmentally protective for wide economic benefit, various reagents used in method, which obtain, to be easy, and is implemented more convenient.
Although illustrate and describing the present invention with specific embodiment, it will be appreciated that without departing substantially from of the invention
Many other change and modification can be made in the case where spirit and scope.It is, therefore, intended that in the following claims
Including belonging to all such changes and modifications in the scope of the invention.
Claims (10)
1. a kind of detection method of heterozygosis chondroitin sulfate, which is characterized in that mainly include the following steps:
(A) sample to be tested is successively passed through to nuclear magnetic resonance spectrometer, infrared spectroscopy is detected to carry out the prediction of preliminary structure；
(B) use high performance liquid chromatography to the knot of glucuronic acid, iduronic acid and acetylamino galactosamine sample to be tested
Structure carries out qualitative.
2. detection method according to claim 1, which is characterized in that in the step (A), using D2O dissolves sample to be tested
Afterwards, it is analyzed on nuclear magnetic resonance spectrometer；
Preferably, the model DD of the nuclear magnetic resonance spectrometer2400-MR。
3. detection method according to claim 1, which is characterized in that in the step (A), sample to be tested is used bromination
Infrared spectroscopy detection is carried out after potassium tabletting.
4. detection method according to claim 1, which is characterized in that in the step (B), high performance liquid chromatography measurement
Column temperature is 20-40 DEG C, and Detection wavelength is between 200-300nm.
5. detection method according to claim 4, which is characterized in that in the step (B), high performance liquid chromatography measurement
Mobile phase is the mixed solution of acetonitrile and ammonium acetate solution, and flow control is between 0.2-0.4ml/min.
6. detection method according to claim 1, which is characterized in that in the step (B), using mass spectrum and it is described efficiently
Method associated with liquid chromatogram carries out qualitative.
7. detection method according to claim 6, which is characterized in that in the step (B), mass spectrographic gas temperature control
Between 300-400 DEG C, flow rate of carrier gas 10-30L/min.
8. detection method according to claim 1, which is characterized in that further include quantitative after qualitative in the step (B)
Step: using the content of glucuronic acid in UV carbazole method measurement sample to be tested.
9. detection method according to claim 9, which is characterized in that in the step (B), the process of UV carbazole method measurement
In, after boiling water heats sample to be tested 10min or more, carbazole is added after cooling, boiling water heats 15min again after shaking up, and observes phenomenon
?.
10. detection method according to claim 8, which is characterized in that in the step (B), in the quantitative step
Further include the steps that being measured the sulfate radical content in sample to be tested；
Preferably, the sulfate radical content is measured using ion chromatograph.
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