CN102243215A - Detection method for water-soluble glucomannan - Google Patents
Detection method for water-soluble glucomannan Download PDFInfo
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Abstract
The invention discloses a detection method for water-soluble glucomannan, comprising the following steps of: simultaneously performing a molecular weight qualitative analysis and a purity analysis on glucomannan by using a high-efficiency gel permeation chromatography, and performing a monosaccharide composition and content quantitative analysis on a glucomannan complete acidolysis product by using a high-efficiency liquid chromatography, wherein the detection method disclosed by the invention can perform systematic physicochemical property measurements such as molecular weight analysis and the distribution range analysis of molecular weights, purity analysis, monosaccharide composition and polysaccharide content measurement, acylation degree measurement, intrinsic viscosity measurement and the like, as well as can qualitatively and quantitatively represent the product characteristics of glucomannan comprehensively; the detection method disclosed by the invention has the advantages of exact result, strong operability and the like, which is adaptive to the product quality control of natural-source water-soluble glucomannan (for example, konjac glucomannan, rhizoma bletillae polysaccharide, etc.).
Description
Technical field
The present invention relates to medical detection range, a kind of detection method of water-soluble glucomannan is provided especially.
Background technology
Polysaccharide is that the special boiomacromolecule of enriching structure diversity is formed, had to a class by monosaccharide unit, and in vivo, polysaccharide exists with the energy storage macromolecule, or passes through the supermolecule effect of different modes, the big molecule of formative tissue structure; In addition, polysaccharide more and more in depth is familiar with by people as the key substance in some bio-transformation identification (as activity and selectivity) process.Numerous organisms such as plant, animal, fungi, marine alga and microorganism can be by the synthetic in a large number polysaccharide of biological approach.The polysaccharide of natural origin has better biocompatibility and degradability, pass through chemical modification, can obtain function polysaccharide derivates more widely, be widely used at aspects such as modern food, cosmetics, medical supplies, industrial materials, representational as starch, cellulose, glucosan, shitosan, konjaku glucomannan, Arabic gum and tragacanth etc. and chemical modification spin-off thereof.
Glucomannan is a kind of common natural stickiness polysaccharide, and its main chain is that D-mannose and D-glucose are formed, and generally by β 1-4 position glycosidic bond, and often has part branched structure and acetyl group to modify.Such polysaccharide distributes comparatively extensive, mainly finds as SKGM (konjac gel), bletilla polysaccharide (from bletilla Bletilla striata) etc., discovery is arranged also in microorganism in plant.The good wholefood thickening agent of such polysaccharide Chang Zuowei uses, and the application in medical material, pharmaceutical carrier and biomedical material simultaneously also has report.
The polysaccharide composition is because molecular weight is big, complex structure, even homogeneous polysaccharide also has the microcosmic heterogencity, there is certain difficulty in the qualitative and quantitative detection of polysaccharide.The sign and the assay method of polysaccharide molecular weight distribution range are a lot, mainly contain osmometry, viscosity method, light scattering method and gel filtration etc., and the whole bag of tricks all needs to have relevant device, and the molecular weight data that records is not quite similar.In recent years it is very ripe to adopt high performance liquid chromatogram one gel permeation chromatography (HPGPC) to measure the method and the technology of polysaccharide molecular weight distribution range, and is recorded and be one of standards of pharmacopoeia method.In the prior art, the also normal monose that adopts complete acid-hydrolysis method to analyze in the polysaccharide is formed, and generally adopt vapor-phase chromatography to detect, but sample needs through derivatization treatment.
Although it is polysaccharide is more to the detection method of polysaccharide both at home and abroad, less to the method for comprehensive detection report of glucomannan.Existing document only limits to the evaluation work of glucomannan more, can not directly apply in the product qualitative and quantitative analysis.There is report to adopt the molecular weight of light scattering method and viscosity method qualitative analysis SKGM, adopts ultraviolet method to measure the total content of bletilla polysaccharide.At document " high effective liquid chromatography for measuring konjak glucomannan " (East China University of Science's journal, 2002,28 (4): reported 406-409) with high performance liquid chromatography and differential refraction detector, adopt aminopropyl modified silica-gel chromatographic column that konjak glucomannan is carried out the method for assay, but this method is not separated mannose and glucose.Document " high substituted degree water soluble polymer konjak glucomannan acetate preparation technology's research " (food research and development, 2006, (5): 49-51) once reported the acetyl degree of substitution that adopts oxammonium hydrochloride method mensuration konjaku glucomannan acetic acid esters.But the purity of measuring glucomannan is not appeared in the newspapers, and adopts the monose composition of HPLC quantitative analysis glucomannan and the method for content not to appear in the newspapers.
Summary of the invention
The detection method that the purpose of this invention is to provide a kind of water-soluble glucomannan.
The present invention be directed to existing glucomannan product lack thoroughly evaluating method of quality control present situation and launch to study.The glucomannan detection method of having reported purity and monose comparatively simple, that be difficult to definite this polysaccharide are formed.By the systematic study to glucomannan architectural feature and detection method, we have set up practicable glucomannan mass analysis method, are applicable to detect the glucomannan product performance.
The invention provides a kind of combined detection method of water-soluble glucomannan, the detection of described glucomannan is made up of following method step:
(1) adopt efficient gel permeation chromatography method that glucomannan is carried out molecular weight qualitative analysis and purity analysis simultaneously
Chromatographic condition and system suitability test: adopt the high performance liquid chromatogram system, gel chromatographic columns, aqueous mobile phase, flow velocity 0.4-1.0mL/min, column temperature 20-40 ℃, differential or evaporative light-scattering detector;
The preparation of reference substance solution: precision takes by weighing polysaccharide series reference substance, and water is mixed with the serial reference substance solution of 0.5-5.0mg/mL;
The preparation of sample solution: precision takes by weighing testing sample, and water is mixed with the testing sample solution of 0.5-5.0mg/mL;
Determination method: accurate respectively reference substance solution and each 5-20 μ l of need testing solution of drawing, inject liquid chromatograph, measure, promptly.
(2) adopting high performance liquid chromatography that the complete acid hydrolysis products of glucomannan is carried out monose forms and quantitative test
Chromatographic condition and system suitability test: adopt the high performance liquid chromatogram system, modified silica-gel matrix chromatogram post, moving phase is acetonitrile-water, flow velocity 0.8-1.0mL/min, column temperature 20-40 ℃ ℃, differential or evaporative light-scattering detector;
The preparation of reference substance solution: precision takes by weighing mannose and glucose reference substance, and water is mixed with the serial reference substance solution of 0.05-2.0mg/mL;
The preparation of need testing solution: precision takes by weighing glucomannan sample 0.2g in the 25ml measuring bottle, separates and constant volume with water-soluble.Precision is measured above-mentioned solution 0.50ml in sample bottle, adds trifluoroacetic acid solution to final concentration 2mol/L, at 120 ℃ of following hydrolysis reaction 2h.Reaction finishes the back except that desolvating, and the residue water is mixed with the sample solution of 0.1-1.0mg/mL;
Determination method: accurate respectively reference substance solution and each 5-20 μ l of need testing solution of drawing, inject liquid chromatograph, measure, promptly.
Used detecting device preferably evaporates photodetector in the detection method of bletilla polysaccharide of the present invention (1); Used moving phase preferred low concentration volatile salt solusion or pure water solution, more preferably pure water solution; Used reference substance preferably glucomannan class reference substance.
The silica matrix chromatogram post of the preferred aminopropyl modification of used chromatographic column filler in the combined detection method of bletilla polysaccharide of the present invention (2), the preferred ethane nitrile content of moving phase is 75-85%; Used detecting device preferably evaporates photodetector.
The detection of glucomannan of the present invention also comprises adopts oxammonium hydrochloride colour developing ultraviolet method to measure degree of acetylation in the glucomannan; The preferred monose acetate esters of used reference substance reference substance, more preferably penta-acetyl glucose reference substance.
The detection of glucomannan of the present invention also comprises measures the glucomannan intrinsic viscosity.
Outstanding feature of the present invention is that this detection method can be carried out physicochemical properties such as molecular weight and distribution range, purity analysis, monose composition and determination of polysaccharide, degree of acetylation mensuration, intrinsic viscosity mensuration to glucomannan and carried out system measurement; can be qualitative and the product performance of comprehensive characterization glucomannan quantitatively; have advantages such as the result is accurate, workable; be applicable to that the water-soluble glucomannan of natural origin is (as SKGM; bletilla polysaccharide, microbe-derived glucomannan etc.) production quality control.
The key content that the present invention is claimed is to carry out finishing on the systematic study basis in architectural feature and detection method to glucomannan.Major technique of the present invention is characterized as following research contents.
(1), the molecular weight of glucomannan and the research of purity analysis method thereof
The molecular weight of polysaccharide and the sign of distribution range thereof are the important physical and chemical parameters of polysaccharide.Adopting efficient gel permeation chromatography (HPGPC) to measure has been maturation method of the prior art.But because polysaccharide only has terminal the absorption, differential detector sensitivity commonly used is also lower.The evaporative light-scattering detector of developed recently maturation (ELSD) is the mass type all-purpose detector, is applicable to the polysaccharide component analysis.The polysaccharide of different structure classification, the elution volume on the HPGPC spectrogram has difference, and the present invention adopts the glucomannan reference substance of same structure type by discovering, and typical curve of being measured and specimen molecular weight match.In addition, all adopting electrolytic salt in the general analysis method is moving phase, finds in the present invention experiment that the glucomannan sample is chromatogram retention behavior and ammonium acetate buffer salt solution consistent of moving phase with the pure water.On this basis, the present invention has set up the HPGPC-ELSD method first and has measured the molecular weight of glucomannan and the method for distribution range thereof.
(2), the monose of glucomannan is formed and content assaying method research
The content Determination of Polysaccharide method mainly contains chemical method (colourimetry, titrimetry etc.), chromatography (vapor-phase chromatography, high performance liquid chromatography) etc.General chemical method can only be measured the total amount of polysaccharide, can not measure the composition of polysaccharide.Though vapor-phase chromatography can be measured the composition of polysaccharide, must carry out derivatization to sugar and just can detect.Use high performance liquid chromatography to survey sugar, because this does not have absorption in the ultraviolet region sugar, can adopt pre-column derivatization ultraviolet or fluorescence detection to carry out, also can select for use the versatility detecting device directly to analyze, detect, evaporate light detection, the detection of electric mist formula, pulse Amperometric Detection Coupled, Mass Spectrometer Method etc. as differential refraction.Wherein, evaporative light-scattering detector is a kind of universal mass detector, have the compound that to measure no uv absorption, can carry out gradient elution, good stability, highly sensitive, characteristics such as solvent-free peak interference have remedied the deficiency of UV-detector and differential refraction detector, are widely used at the carbohydrate analysis field in recent years.
Saccharide compound is because polarity is strong, isomeride is complicated and changeable, physicochemical property is similar, and its component separating is difficulty comparatively.(hydrophilic interaction chromatography HILIC), is a kind of employing polar stationary phase (as silica gel or a bonded silica gel), to contain the chromatogram mode that high concentration polar organic solvent and low concentration aqueous solution are moving phase to hydrophilic Interaction Chromatography.HILIC is very suitable for the separation to polar compound, and development is very fast in carbohydrate content is analyzed in recent years.The present invention adopts the silica matrix chromatogram post of aminopropyl modification, has set up hydrophilic Interaction Chromatography first---and evaporative light-scattering detects the contents of monosaccharides of (HILIC-ELSD) quantitative test glucomannan and the method for composition thereof.
(3), the degree of substitution with acetyl group study on determination method of glucomannan
Often contain a small amount of acetyl group in the glucomannan and replace, degree of substitution with acetyl group is one of characteristic parameter that characterizes glucomannan.The common method of measuring the polysaccharide degree of substitution with acetyl group is saponification method and azanol colourimetry.Saponification method is a classical way, but big, the consuming time length of required sample size is bigger to the measured value relative error of low degree of substitution polysaccharide.The azanol colourimetry is comparatively simple and efficient, highly sensitive, but is subjected to coloration method and interference such as time, sample purity and other esterified group easily.The present invention selects the similar monose acetate esters reference substance of easy acquisition for use by research, i.e. penta-acetyl glucose has been set up the method for azanol colorimetric method for determining glucomannan degree of substitution with acetyl group.
Acetyl content is at 15.4～92.4 μ gmL in mensuration β-D-five acetyl glucose
-1Scope in have good linear relationship (r=0.9996), detect and to be limited to 11.1 μ gmL-1, average recovery rate is 101.6% (n=9), method precision experiment RSD=0.08%.Record that degree of substitution with acetyl group is 0.0358 in the SKGM sample, the result shows this law quick and precisely, and favorable reproducibility is simple and easy to do, is applicable to the assay determination of acetyl degree of substitution in the polysaccharide.
(4), the intrinsic viscosity study on determination method of glucomannan
To the macromolecular material of a certain definite type, its molecular weight size viscosity (as intrinsic viscosity) specific with it has quantitative relationship.According to this principle,, can prepare the product of required viscosity specification, corresponding to the glucomannan of corresponding molecular weight specification by the viscosity of product in the Monitoring and Controlling course of reaction.The present invention studies the relative viscosity of having set up the bletilla polysaccharide sample and the corresponding relation of intrinsic viscosity and its molecular weight, is applicable to the intrinsic viscosity that characterizes this sample.
Description of drawings
The present invention is further detailed explanation below in conjunction with drawings and the embodiments:
Fig. 1 is the HPGPC-ELSD chromatogram of bletilla polysaccharide sample;
Fig. 2 is that (1 for mannose for the HPLC-ELSD chromatogram of mannose and glucose mixing reference substance; 2 is glucose);
Fig. 3 is that (1 for mannose for the HPLC-ELSD chromatogram of the complete acid hydrolysis sample of bletilla polysaccharide; 2 is glucose).
Embodiment
Glucomannan combined detection method of the present invention is an example with bletilla polysaccharide and SKGM, is that involved method is the technological means that those skilled in the art can grasp and use by represented method manufacturing of following embodiment or discovery.
Embodiment 1
The described HPGPC-ELSD method of present embodiment is measured the molecular weight and the distribution range thereof of bletilla polysaccharide:
Chromatographic condition: get the about 10mg of each specification bletilla polysaccharide sample, dissolve the concentration that is mixed with 1.0mg/mL, adopt TSK PW4000 type chromatographic column with suitable quantity of water, GPC-ELSD analyzes (Alltech company) system, with water is moving phase, and flow velocity 0.6mL/min, chromatographic column column temperature are that room temperature is analyzed; With the series specification bletilla polysaccharide is that reference substance is formulated typical curve, testing sample GPC computed in software result.
The absolute molecular weight of reference substance BT01-07 adopts the MALLS method to measure.Get the solution of BT01-07, after 0.22 μ m millipore filter filters, adopt HPGPC-ELSD efficient gel permeation chromatography system and selected chromatographic condition, according to molecular exclusion chromatography (2010 editions or 2005 editions appendix V H of Pharmacopoeia of People's Republic of China) sample introduction analysis.Write down the retention time t of each standard items chromatographic peak
R(min), the result is referring to table 2.With lgM separately
wTo t
RDo linear regression, obtaining regression equation is y=-0.2244x+4.5749, R
2=0.9855, illustrate that bletilla polysaccharide is lgM in the molecular weight ranges of 24～178kDa in employed efficient gel permeation chromatography system
wTo t
RBe better linearity.
The HPGPC-ELSD analysis result of table 1 reference substance BT01-07
The described HPGPC-ELSD method of present embodiment is measured the purity of bletilla polysaccharide:
Chromatographic condition: with embodiment 1;
The range of linearity: get the series standard solution of bletilla polysaccharide reference substance BT40, after 0.22 μ m millipore filter filters, sample introduction analysis, the peak area A of chromatographic peak in the recording solution GPC spectrogram.With lnA lnm is carried out linear regression, the regression equation that obtains bletilla polysaccharide reference substance BT40 is: lnA=1.4924lnm+1.7227, R
2=0.9984, show that bletilla polysaccharide lnA in the concentration range of 0.5～5mg/mL is favorable linearity to lnm.Calculate by signal to noise ratio (S/N ratio) S/N=3 and 10, the detection that the HPGPC-ELSD method is analyzed bletilla polysaccharide is limited to 2.33 μ g, quantitatively is limited to 5.48 μ g.
Sample determination: get BT40-1 sample to be measured and measure, recording its content is 96.63%.
The methodology confirmatory experiment is as follows:
(1) range of linearity
Get assay with bletilla polysaccharide reference substance BT40S series standard solution, after 0.22 μ m millipore filter filtered, the peak area A of chromatographic peak in each solution GPC spectrogram was write down in the sample introduction analysis.With lgA lgm is carried out linear regression, regression equation is: y=1.4924x+0.7482, R
2Be 0.9984.Show that bletilla polysaccharide lgA in the concentration range of 0.5～5mg/mL is favorable linearity to lgm.
(2) accuracy
Get 9 parts of the same a BT40-1 need testing solutions of 2mg/mL, each 2mL places the 5mL volumetric flask respectively.Per 3 parts one group, accurate BT40S titer 0.2,1.2, the 2.2mL that adds 5mg/mL in 1,2,3 group adds the pure water constant volume respectively, shakes up, make 1,2, the mixed solution of 3mg/mL, concentration is respectively 50%, 100%, 150% of analytical concentration (2mg/mL).After 0.22 μ m millipore filter filtered, the chromatographic peak peak area was write down in the sample introduction analysis, calculates the recovery, RSD (%) and the average recovery rate of basic, normal, high 3 kinds of additions, and data see Table 2.The result meets the methodology requirement.
Table 2 recovery experimental result
* the recovery=(recording total amount-test sample content)/reference substance addition * 100%
(3) precision
Get the peak area A of precision test gained chromatogram under the molecular weight determination item, calculating the average A value is that 1,714 ten thousand, RSD% are 1.67 (tables 3), and the result shows that this method meets the methodology requirement.
The experiment of table 3 sample introduction precision
(4) reappearance
Get the peak area A of reappearance test gained chromatogram under the molecular weight determination item, calculating the average A value is 1392, and RSD% is 1.41 (tables 4), and the result shows that this method meets the methodology requirement.
The experiment of table 4 method reappearance
(5) stability
Get the peak area A of stability test gained chromatogram under the molecular weight determination item, record in a few days and in the daytime RSD% be respectively 1.69 and 3.41 (table 5-6), meet the methodology requirement.
Table 5 day internal stability
Stability between table 6 day
(6) detectability LOD, quantitative limit LOQ
Get the BT40S titer of 0.50mg/mL, adopt that dilution method makes 0.10,0.15,0.20,0.25,0.30,0.35, the serial low concentration solution of 0.40mg/mL, after 0.22 μ m millipore filter filters, sample introduction analysis, record chromatographic peak peak height (table 7).Calculate by signal to noise ratio (S/N ratio) S/N=3 and 10, the detection that the HPGPC-ELSD method is analyzed bletilla polysaccharide is limited to 2.33 μ g, quantitatively is limited to 5.48 μ g.
Table 7 detectability, quantitative limit experimental result
Embodiment 3
The described HPLC-ELSD method of present embodiment is measured the monose of bletilla polysaccharide and SKGM and is formed and content:
The preparation of sample solution: precision takes by weighing bletilla polysaccharide sample 0.1806g in the 25ml measuring bottle, separates and constant volume with water-soluble.Precision is measured above-mentioned solution 0.50ml in sample bottle, adds 10mol/L trifluoroacetic acid solution 125 μ L, seals, at 120 ℃ of following hydrolysis reaction 2h.Reaction finishes after the room temperature blowing air dries up, and residue is dissolved in water, and is transferred in the 5mL measuring bottle, and constant volume, solution detect for HPLC behind 0.22 μ m filtering with microporous membrane.
The preparation of reference substance solution: precision takes by weighing reference substance mannose 0.01553g, anhydrous dextrose 0.01662g and places the 10mL measuring bottle, separates and constant volume, product storing solution in contrast with water-soluble.Precision is measured above-mentioned storing solution 0.25,0.50,0.75,1.00,1.50,2.00, and 2.50mL places the 5mL volumetric flask, the water constant volume.Obtain the serial reference substance solution of mannose and glucose, put 4 ℃ of refrigerators and preserve standby.
Chromatographic condition: chromatographic column is a Sugar-D Waters post (4.6 * 250mm); Moving phase: acetonitrile-water (volume ratio is 85: 25); Flow velocity 1.0mL/min; Column temperature: 30 ℃; Sample size 20 μ L.ELSD parameter: 40 ℃ of drift tube temperatures; Atomization gas (air) pressure 350kPa.
Measurement result: record that mannose and glucose content are respectively 0.1333 in the bletilla polysaccharide sample, 0.0273mg, the constitutive molar ratio of two kinds of glycosyls are 83: 17.Record with method that the constitutive molar ratio of mannose and glucose is 63: 37 in the SKGM sample.
The methodology confirmatory experiment is as follows
(1) linear relationship and detectability are investigated
Get serial mannose and glucose reference substance solution sample introduction 20 μ L respectively, logarithm (Y) with peak area A is an ordinate, the logarithm (X) of sample introduction quality m (μ g) is the horizontal ordinate mapping, and the equation of linear regression that gets mannose, glucose is followed successively by Y=1.1882X+5.7316 (r
2=0.9983), Y=1.3122X+5.5745 (r
2=0.9988), its range of linearity is 0.07765-1.553mg/mL, 0.08112-1.622mg/mL.Calculate with 3 times of signal to noise ratio (S/N ratio)s (S/N), the detection limit of mannose and glucose is respectively 0.4659 μ g, 0.4730 μ g.
(2) precision test
Get mannose and glucose series reference substance solution and repeat sample introduction 6 times, the record chromatogram is measured mannose and glucose peaks area, and its relative standard deviation (RSD) is respectively 0.44%, 0.11%.The precision that shows instrument is better.
(3) replica test
Get same sample according to 6 parts of the parallel preparations of sample solution preparation method, carry out efficient liquid phase chromatographic analysis, the peak area RSD that records mannose and glucose is respectively 1.60%, 1.33%, and the RSD of retention time is respectively 0.09%, 0.15%.
(4) stability test
Get same duplicate samples solution, respectively at placing 0,2,4,6, sample introduction carries out study on the stability behind the 8h.The result shows that the peak area of mannose and glucose does not have significant change in 8h, and RSD is respectively 1.40%, 1.98%, and sample solution is good at 8h internal stability analysis time.
(5) average recovery test
Get 9 parts in the bletilla mannan sample that oneself knows content, be divided into 3 groups.Every group of sample adds a certain amount of mannose and glucose reference substance solution respectively, handles and measures by the method for being set up, and calculates average recovery, sees Table 8.The result shows that the recovery satisfies the methodology requirement.
The average recovery of monose in the table 8 bletilla polysaccharide sample
Embodiment 4
The described oxammonium hydrochloride method of present embodiment is measured the degree of substitution with acetyl group of bletilla polysaccharide and SKGM:
Need testing solution: precision takes by weighing SKGM 0.02901g, puts in the 10mL volumetric flask, adds an amount of distilled water, places heating for dissolving in 60 ℃ of water-baths, is cooled to room temperature after treating to dissolve fully, adding distil water constant volume again, promptly.
Accurately draw a certain amount of polysaccharide solution in the brown volumetric flask of 50mL, accurately add 0.1molL
-1New complex salt acid hydroxylamine solution 5mL adds 1.5molL
-1NaOH solution 5mL, mixing, leave standstill 20min after, add 2molL
-1Hydrochloric acid solution 3.5mL leaves standstill 20min with the excessive alkali that neutralizes behind the mixing, drip 0.37molL
-1FeCl
3Solution 10mL mixing is used the deionized water constant volume, leave standstill 10min after, replace polysaccharide to operate with the deionized water of same amount with method, measure absorbance in 500nm wavelength place.
The result: record that the quality of acetyl group is respectively 202.7,204.7 in the SKGM, 199.0 μ g, acetyl content is respectively 0.944%, 0.953%, 0.930%, and average DS value is 0.0358.
Embodiment 5
Described bletilla polysaccharide relative viscosity of present embodiment and molecular weight correlativity are investigated:
The mensuration of bletilla polysaccharide relative viscosity:, adopt black formula kapillary viscosity apparatus to carry out with reference to Chinese Pharmacopoeia appendix regulation.Get the about 1.0g of each bletilla polysaccharide test product, add suitable quantity of water dissolving and be mixed with 0.50～2.0% initial concentration, solution filters through the G4 sintered filter funnel, filtrate is the reference solvent with water, measure its viscosity in 25 ℃ of water-baths, adopt progressively that the thin up method obtains each sample series concentration solution, measure its viscosity, calculate the relative viscosity η of each concentration solution
r
Determining of bletilla polysaccharide intrinsic viscosity: according to η
Sp=η
r-1 obtains the specific viscosity η of each concentration solution of bletilla polysaccharide
SpAccording to Huggins's equation η
Sp/ C=[η]+k ' [η]
2C is with the η of each sample/reference substance
Sp/ C maps to C, is extrapolated to infinite dilution solution gained intercept and is its intrinsic viscosity, sees Table 1.
Claims (10)
1. the detection method of a water-soluble glucomannan, it is characterized in that: the detection method of described water-soluble glucomannan is:
(1) adopt efficient gel permeation chromatography method that glucomannan is carried out molecular weight qualitative analysis and purity analysis simultaneously; Chromatographic condition and system suitability test: adopt the high performance liquid chromatogram system, gel chromatographic columns, aqueous mobile phase, flow velocity 0.4-1.0mL/min, column temperature 20-40 ℃, differential or evaporative light-scattering detector; The preparation of reference substance solution: precision takes by weighing polysaccharide series reference substance, and water is mixed with the serial reference substance solution of 0.5-5.0mg/mL; The preparation of sample solution: precision takes by weighing testing sample, and water is mixed with the testing sample solution of 0.5-5.0mg/mL; Determination method: accurate respectively reference substance solution and each 5-20 μ l of need testing solution of drawing, inject liquid chromatograph, measure, promptly;
(2) adopting high performance liquid chromatography that the complete acid hydrolysis products of glucomannan is carried out monose forms and quantitative test; Chromatographic condition and system suitability test: adopt the high performance liquid chromatogram system, modified silica-gel matrix chromatogram post, moving phase is acetonitrile-water, flow velocity 0.8-1.0mL/min, column temperature 20-40 ℃ ℃, differential or evaporative light-scattering detector; The preparation of reference substance solution: precision takes by weighing mannose and glucose reference substance, and water is mixed with the serial reference substance solution of 0.05-2.0mg/mL;
(3) preparation of need testing solution: precision takes by weighing glucomannan sample 0.2g in the 25ml measuring bottle, separates and constant volume with water-soluble.Precision is measured above-mentioned solution 0.50ml in sample bottle, adds trifluoroacetic acid solution to final concentration 2mol/L, at 120 ℃ of following hydrolysis reaction 2h.Reaction finishes the back except that desolvating, and the residue water is mixed with the sample solution of 0.1-1.0mg/mL;
(4) determination method: accurate respectively reference substance solution and each 5-20 μ l of need testing solution of drawing, inject liquid chromatograph, measure, promptly.
2. according to the detection method of the described water-soluble glucomannan of claim 1, it is characterized in that: used detecting device preferably evaporates photodetector in the described method (1).
3. according to the detection method of the described water-soluble glucomannan of claim 1, it is characterized in that: used moving phase preferred low concentration volatile salt solusion or pure water solution, more preferably pure water solution in the described method (1).
4. according to the detection method of the described water-soluble glucomannan of claim 1, it is characterized in that: used reference substance preferably glucomannan class reference substance in the described method (1).
5. according to the detection method of the described water-soluble glucomannan of claim 1, it is characterized in that: the silica matrix chromatogram post of the preferred aminopropyl modification of used chromatographic column filler in the described method (2), the preferred ethane nitrile content of moving phase is 75-85%.
6. according to the detection method of the described water-soluble glucomannan of claim 1, it is characterized in that: used detecting device preferably evaporates photodetector in the described method (2).
7. according to the detection method of the described water-soluble glucomannan of claim 1, it is characterized in that: the detection of described glucomannan also comprises adopts oxammonium hydrochloride colour developing ultraviolet method to measure degree of acetylation in the glucomannan; The preferred monose acetate esters of used reference substance reference substance, more preferably penta-acetyl glucose reference substance.
8. according to the detection method of the described water-soluble glucomannan of claim 1, it is characterized in that: the detection of described glucomannan also comprises measures the glucomannan intrinsic viscosity.
9. according to the detection method of the described glucomannan of claim 1, it is characterized in that: the detection method of described glucomannan can be used in the glucomannan production quality control.
10. according to the detection method of the described water-soluble glucomannan of claim 1, it is characterized in that: the detection method of described glucomannan can be used in SKGM and the bletilla polysaccharide production quality control.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104215703A (en) * | 2013-06-04 | 2014-12-17 | 天津天士力之骄药业有限公司 | Detection method of glycoside macro-molecule substances in qi-tonifying pulse-restoring injection preparation |
| CN109459505A (en) * | 2017-09-06 | 2019-03-12 | 上海绿谷制药有限公司 | A method of measurement mannuronic acid substance weight average molecular weight and content |
| CN112485344A (en) * | 2020-11-04 | 2021-03-12 | 华测检测认证集团湖北有限责任公司 | Ultra-high performance liquid chromatography detection method for mannose in honey |
-
2010
- 2010-05-10 CN CN2010101666878A patent/CN102243215A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104215703A (en) * | 2013-06-04 | 2014-12-17 | 天津天士力之骄药业有限公司 | Detection method of glycoside macro-molecule substances in qi-tonifying pulse-restoring injection preparation |
| CN104215703B (en) * | 2013-06-04 | 2017-06-23 | 天津天士力之骄药业有限公司 | The detection method of glycoside macromolecular substances in a kind of injection Yiqi and vein recovery |
| CN109459505A (en) * | 2017-09-06 | 2019-03-12 | 上海绿谷制药有限公司 | A method of measurement mannuronic acid substance weight average molecular weight and content |
| CN112485344A (en) * | 2020-11-04 | 2021-03-12 | 华测检测认证集团湖北有限责任公司 | Ultra-high performance liquid chromatography detection method for mannose in honey |
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