CN103630536B - A kind of method of measuring polyethylene glycol residual quantity in cross-linking sodium hyaluronate gel - Google Patents
A kind of method of measuring polyethylene glycol residual quantity in cross-linking sodium hyaluronate gel Download PDFInfo
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Abstract
The invention discloses the detection method of polyethylene glycol residual quantity in a kind of cross-linking sodium hyaluronate gel, according to PEG and barium ions and iodide ion form the optical density of compound of 1:1:1 and the content of PEG the linear principle in 535nm place adopt unconventional solvent set up a kind of cross-linked gel of simple to operate, quick, good stability in the detection method of trace P EG residual quantity. Is this detection method at 0-50.0? within the scope of μ g/mL, there is good linear relationship, especially can be used for residual quantity crosslinking agents below 2 μ g/mL and macromolecule and detect. This detection method not only can be used for the trace detection of PEG residual quantity and the quality control of gel products in the crosslinked hyaluronic acid sodium gel of PEG, and in other gel that PEG is cross-linked and other liquid sample that contains PEG, the detection of PEG residual quantity is also significant.
Description
Technical field
The present invention relates to biochemistry, pharmaceutical chemistry and Pharmaceutical Analysis technical field, be specifically related to a kind of mensuration and be cross-linked thoroughlyThe method of polyethylene glycol residual quantity in phaneroplasm acid sodium gel.
Background technology
Hyaluronic acid is a kind of NMF being extensively present in organism, at least water holding of 1g hyaluronic acid600g, hyaluronic acid is large by disaccharide unit's D-Glucose aldehydic acid and the N-acetyl-glucosamine acidity that repeatedly repeated arrangement formsMolecule polysaccharide, is widely used in cosmetics, food and medicine field, and commodity are generally used its sodium salt, i.e. Sodium Hyaluronate (SH).Natural hyaluronic acid sodium gel has good good biocompatibility, but Yin Qiyi is degraded, and RT is short in vivo,Limit its extensive use. Preparing cross-linked gel by chemical modification is the Main Means that improves at present or extend its stability. OrderIt is front that to prepare the method that cross-linked gel is conventional be with BDO diglycidyl ether (l, 4-butanediolDiglycidylether, BDDE), divinylsulfone (Divinylsulphone, DVS), carbodiimide(Biscarbodimide, EDC) etc. makes for crosslinking agent adopts certain technology, certain but these crosslinking agents haveExcitant and toxicity, residual quantity or the content every country of crosslinking agent in gel product has strict regulation. Polyethylene glycolBe a kind of not only edible but also pharmaceutically useful compound of being ratified by countries in the world, at field of medicaments, it is the oral system of Chang Zuowei not onlyThe auxiliary material of agent, or the conventional carrier of some injections, nontoxic, there is good biocompatibility and easily discharged body by bodyOutward. Make crosslinking agent with PEG and prepare cross-linking sodium hyaluronate gel and caused people's extensive concern and great attention, PEG is crosslinkedHyaluronic acid sodium gel there are obvious superiority and wide market prospects.
Although PEG is widely used, no matter be at food or in medicine, PEG is a kind of external concerning bodyCompound, so its content or residual quantity have strict restriction in Related product, must detect. At present both at home and abroadSee the analyzing and testing PEG content of report or the method for residual quantity mainly contains paper chromatography, thin-layer chromatography, gas-chromatography, high-efficient liquidPhase chromatogram, AAS etc., but these methods are mostly as the content of PEG in the liquid such as vaccine, urine for liquid sampleDetect, there is not yet report for the detection of PEG residual quantity in gel. And in the said method of having reported, paper lookThe disengaging time of spectrum is longer, and the same with thin-layer chromatography, is difficult to PEG to carry out accurate quantification. Gas-chromatography and high-efficient liquid phase colorSpectrum is that PEG is carried out to the common method quantitatively detecting, but the PEG molecular weight that gas-chromatography is measured is lower, and 1000Da is forThe high upper limit, and molecular weight is greater than 500 PEG and need carries out detecting after derivatization again, the consuming time and derivative stability of derivative step is notHigh. High performance liquid chromatography can separate with quantitative PEG, and what most of efficient liquid-phase chromatography methods used is differential refraction inspectionSurvey device, and this method is because the low range of application of sensitivity is not general yet, and the equipment investment of liquid chromatogram instrument is large, and instituteNeed reagent consumptive material costliness. Spectrophotometry PEG content mainly contains D reagent method and barium chloride method. Due to D reagent method preparation examinationLoaded down with trivial details and the unstable easy degraded of agent process, bismuth nitrate is expensive, and the LDL of the method higher (being greater than 2 μ g/mL)And make its application fewer and feweri. Childs has set up the barium chloride detection method of PEG first in 1975, its general principle is PEGForm compound (1:1:1) with barium ions and iodide ion, the OD value at 535nm place and the content of PEG linear enterRow detects, by B.Skoog and G.E.C.Sims and T.J.Snape improvement, so far still in application. But B.Skoog reportThe LDL of PEG be 5 μ g/mL, in the result of study of G.E.C.Sims and T.J.Snape, the content of PEG is lower than 2 μWhen g/mL, just can't detect, and the residual quantity of crosslinking agent in the domain-specific cross-linked gel products such as medical cosmetology generally should be littleJust meet the requirements in 2 μ g/mL, also lack and adopt spectrophotometry to go out PEG600-20000 residual quantity in gel at presentBe less than correlation technique and the method for 2 μ g/mL.
Summary of the invention
The present invention is directed to the deficiency of prior art, the detection of PEG residual quantity in a kind of cross-linking sodium hyaluronate gel is providedMethod, by a kind of pre-treatment or preprocess method of gel sample are provided, adds a kind of making of unconventional calibration curve,Having set up one can be simply, the trace of PEG residual quantity (being less than 2 μ g/mL) divides in fast detecting cross-linking hyaluronic acid sodium sodium gelAnalysis method. The method is carried out special pretreatment to gel sample, with 95% or absolute ethyl alcohol precipitate this gel, centrifuging and taking supernatantLiquid detects its polyethylene glycol residual quantity. Form optical density and the PEG of the compound of 1:1:1 according to PEG and barium ions and iodide ionContent in the linear principle in 535nm place, detect the residual quantity of PEG in gel sample. The method and additive method phaseRatio, can reduce experimental cost and highly sensitive greatly, and good stability is simple and safe, convenient and swift.
In this mensuration cross-linking sodium hyaluronate gel, the method for PEG residual quantity comprises following processing step:
(1) sample pretreatment
The sample pretreatment of cross-linking sodium hyaluronate gel: by 7-14 times of volume for hyaluronic acid sodium gel crosslinked PEG95% ethanol or absolute ethyl alcohol alcohol precipitation repeatedly, gained precipitation be dried and is pulverized, particle or powder after pulverizing are partly existedAt 108-124 DEG C, redissolve 10-30min, precision takes the gel 1.0g after redissolution, adds 2mL absolute ethyl alcohol, and sealing, through whirlpoolOscillator vibration, makes gel precipitation, then, in the centrifugal 3-30min of 5000-18000r/min, gets supernatant to be measured;
The sample pretreatment of non-crosslinked hyaluronic acid sodium gel: by non-crosslinked hyaluronic acid sodium gel with 7-14 times of volume95% ethanol or absolute ethyl alcohol alcohol precipitation repeatedly, be dried and pulverize gained precipitation, by the particle after pulverizing or powder at 108-124DEG C redissolve 10-30min, precision takes whole gels after redissolution in 1000mL beaker, adds absolute ethyl alcohol, absolute ethyl alcohol withThe volume of gel and mass ratio are 2, and unit is mL/g, stir and make gel precipitation with glass bar, then supernatant are sub-packed inIn 100mL centrifuge tube, at the centrifugal 3-30min of 5000-18000r/min, get supernatant for subsequent use;
(2) preparation of PEG standard liquid
PEG600-20000 standard storage liquid: precision takes the PEG600-20000 of 50mg, adds non-crosslinked hyaluronic acidSodium gel sample dissolves in the prepared supernatant of pretreatment, and constant volume in 100mL volumetric flask is for subsequent use in 4 DEG C of Refrigerator stores;
PEG600-20000 standard operation liquid: draw PEG600-20000 standard storage liquid 5mL in 50mL volumetric flask, useNon-crosslinked hyaluronic acid sodium gel sample is through the prepared supernatant constant volume of pretreatment, and mass concentration is 50 μ g/mL;
(3) preparation of barium chloride solution
Accurately take 5g barium chloride, adding distil water is settled to 100ml, obtains 5% barium chloride solution;
(4) preparation of iodine liquid
Accurately take KI 2.0g and iodine 1.3g, adding distil water is settled to 100ml, obtains the iodine liquid of 0.05mol/L;
(5) making of PEG solution calibration curve
Getting respectively 6-15 group, to have variable concentrations be that the PEG standard of 0,0.5,10.5,20.5,30.5,40.5 μ g/mL is moltenLiquid 1.0ml, concentration range, between 0-50 μ g/ml, adds 5ml distilled water, the each 50-1000 μ of 5% barium chloride solution and iodine liquidL, reaction 8-15min, measures OD value in wavelength 535nm place; Each concentration is done three Duplicate Samples; According to every group of PEG concentrationAnd the relation between the OD value recording, taking PEG concentration as abscissa, OD value is ordinate, drawing standard curve;
(6) calculating of PEG residual quantity in cross-linking sodium hyaluronate gel
Of the present invention as follows for calculating the computing formula of cross-linking sodium hyaluronate gel PEG residual quantity:
Y=0.0108X+0.2047，R2=0.9994
In formula: X---the mass concentration of PEG, unit is μ g/ml;
The mass ratio of Y---iodo-barium chloride-PEG is that the compound of 1:1:1 is at the OD value at 535nm place;
(7) the assay method step of PEG residual quantity in cross-linking sodium hyaluronate gel
By step (5), the supernatant of getting gained in step (1) cross-linked hyaluronic acid gel sample pretreatment adds distillationWater, barium chloride solution and the reaction of iodine liquid, the reaction time is in 15min, measures OD value, according to step in wavelength 535nm placeSuddenly the standard corresponding relation curve between (5) make PEG concentration and corresponding light density value or true by the computing formula of step (6)PEG residual quantity in fixed cross-linking sodium hyaluronate gel to be measured.
Described stepIn pretreatment that cross-linked gel is carried out be the ethanol alcohol precipitation cross-linked gel with 95%, with centrifugalRear obtained supernatant detects the PEG residual quantity in cross-linking sodium hyaluronate gel.
Described stepThe solvent of middle prepared PEG standard liquid is stepMiddle uncrosslinked gel is through coagulating with crosslinkedThe supernatant that 95% ethanol alcohol precipitation of glue same volume umber obtains, uncrosslinked gel is crosslinked except not adding in preparation processOutside agent PEG, other steps are all identical, so, the PEG standard liquid of preparing and the supernatant that will detect residual quantity exceptOutside PEG content is different, other component is all the same.
Described stepIn the making of PEG solution calibration curve zero contrast is stepMiddle non-crosslinked Sodium HyaluronateGel is through the supernatant of 95% ethanol alcohol precipitation centrifugal gained, and stepPlays PEG solution will add 5ml distilled water successively,5% barium chloride solution and iodine liquid, zero contrast is through 95% ethanol alcohol precipitation centrifugal institute to non-crosslinked hyaluronic acid sodium gelThe supernatant obtaining is added distilled water equates gained solution to volume and standard P EG solution gradient series.
The Best Times that described method is measured OD value is 10min.
When described method reaction, adding the optimised quantity of barium chloride solution and iodine liquid is 250 μ l.
The features and advantages of the invention be the PEG600-20000 that uses of production standard Curves standard liquid preparation withConventional method difference, conventional method is to be mixed with certain density solution with distilled water as dissolution with solvents PEG600-20000,Add again the distilled water diluting of different proportion to become needed concentration gradient, and the preparation PEG600-20000 that this research institute usesThe method of standard liquid is as dissolution with solvents PEG600-20000 with the supernatant after non-crosslinked hyaluronic acid derivatives alcohol precipitationBe mixed with certain density solution, then add the supernatant of different proportion to be diluted to needed concentration gradient. Except non-crosslinkedAfter hyaluronic acid sodium gel alcohol precipitation, in supernatant, do not contain PEG600-20000, the supernatant of non-crosslinked hyaluronic acid sodium gel usedThe constituent of liquid cross-linking sodium hyaluronate gel supernatant during with sample detection is consistent, has as much as possiblely got rid of impact and detectsThe interference factor of result and is different by the method that distilled water carrys out preparing standard solution in conventional method.
Blank of the present invention is also different from conventional method in addition, and the contrast of conventional method empty is distilled water, lastStep will add developer barium chloride and iodine liquid, and the blank of method therefor of the present invention is to be the acid cure of non-crosslinked hyalomitomeSupernatant after glue alcohol precipitation, in the end step does not add developer, with the alternative developer of distilled water. The concrete operations of this method are to useNon-crosslinked hyaluronic acid sodium gel alcohol precipitation supernatant adds solution after 5.5mL distilled water as blank, with conventional methodIn blank be that in distilled water, to add the solution of developer be different, these class methods are higher for measuring PEG contentSolution is suitable, is difficult to detect for the lower sample of PEG content. Because the blank of this method does not add colour developingAgent, so can obtain desirable OD value in the situation that of lower in PEG concentration (≤2 μ g/mL). Therefore, of the present inventionMethod can be used for the detection of lower PEG residual quantity (≤2 μ g/mL), can be directly used in the crosslinked gel products production process of PEGThe trace detection of middle PEG residual quantity and the quality control of gel products.
The beneficial effect of content of the present invention
After the present invention explores through long term test, finally find a kind of pre-treatment or preprocess method of gel sample,By a kind of making of unconventional calibration curve, having set up one can be simply, in fast detecting cross-linking hyaluronic acid sodium sodium gelThe trace analysis method of PEG residual quantity (being less than 2 μ g/mL). This detection method can be in the crosslinked hyaluronic acid sodium gel of PEGThe trace detection of PEG residual quantity and the quality control of gel products lay the foundation, to other crosslinked gel of PEG and contain PEGOther liquid sample in the detection of PEG residual quantity have certain reference value, in gel product production process, other is crosslinkedThe trace detection of agent and control of product quality also have reference function.
Meanwhile, the present invention by alcohol precipitation technology the supernatant after with alcohol precipitation to detect crosslinking agent in gel sample residualAmount, both to raw sample without any pollution or destruction, can further refine raw sample again, by detect after gel sampleProduct alcohol hypostasis also can be made gel again by certain glue technology, has the effect of achieving many things at one stroke.
The present invention uses barium chloride and iodine liquid as developer, and PEG and barium ions and iodide ion (1:1:1) generate compoundThing is linear in relative broad range at the OD value of 535nm and the concentration of PEG. This method does not need expensive instrument, largeSave greatly experimental cost, simple and convenient, be convenient to large-scale promotion application.
Detailed description of the invention
Below introduce in detail embodiments of the invention.
Embodiment 1:
Key instrument: FA1204B type electronic balance, Shanghai Precision Scientific Apparatus Co., Ltd; The clean work of SW-CJ-1FDPlatform, Su Jing Aetna Inc.; 722N type visible spectrophotometer, Shanghai Precision Scientific Apparatus Co., Ltd; TGL-16C typeLuxuriant and rich with fragrance just that centrifuge, Shanghai Fei Qiaer Analytical Instrument Co., Ltd; Vortex shaker, the north is with positive Co., Ltd; Pipettor, moralEppendorf company of state;
Sample pretreatment
The sample pretreatment of cross-linking sodium hyaluronate gel: by the cross-linking sodium hyaluronate gel of preparation with 14 times of volumes95% ethanol alcohol precipitation repeatedly, is dried and pulverizes gained precipitation, and the particle after part is pulverized or powder are 121 DEG C of redissolution20min, precision takes the gel 1.0g after redissolution, adds 2mL absolute ethyl alcohol, and sealing, through the vibration of vortex oscillation device, makes gelPrecipitation, then, in the centrifugal 15min of 10000r/min, gets supernatant to be measured;
The sample pretreatment of non-crosslinked hyaluronic acid sodium gel: by 14 times of bodies for non-crosslinked hyaluronic acid sodium gel of preparation95% long-pending ethanol alcohol precipitation repeatedly, be dried and pulverizes gained precipitation, by the particle after pulverizing or powder 121 DEG C of redissolution20min, precision takes whole gels after redissolution in 1000mL beaker, adds absolute ethyl alcohol, the body of absolute ethyl alcohol and gelLong-pending and mass ratio is 2, and unit is mL/g, stirs and makes gel precipitation with glass bar, then supernatant is sub-packed in to 100mL centrifugalIn pipe, at the centrifugal 15min of 10000r/min, get supernatant for subsequent use;
The preparation of PEG standard liquid
PEG20000 standard storage liquid: precision takes the PEG20000 of 50mg, adds non-crosslinked hyaluronic acid sodium gelSample dissolves in the prepared supernatant of pretreatment, and constant volume in 100mL volumetric flask is for subsequent use in 4 DEG C of Refrigerator stores;
PEG20000 standard operation liquid: draw PEG20000 standard storage liquid 5mL in 50mL volumetric flask, saturating with non-crosslinkedThe sample of phaneroplasm acid sodium gel is through the prepared supernatant constant volume of pretreatment, and mass concentration is 50 μ g/mL;
The preparation of barium chloride solution
Accurately take 5g barium chloride, adding distil water is settled to 100ml, obtains 5% barium chloride solution;
The preparation of iodine liquid
Accurately take KI 2.0g and iodine 1.3g, adding distil water is settled to 100ml, obtains the iodine liquid of 0.05mol/L;
The making of PEG solution calibration curve
Get respectively 6 groups and there is the PEG standard liquid that variable concentrations is 0,0.5,10.5,20.5,30.5,40.5 μ g/mL1.0ml, concentration range, between 0-50 μ g/ml, adds 5ml distilled water, the each 250 μ l of 5% barium chloride solution and iodine liquid, reaction15min, measures OD value in wavelength 535nm place; Each concentration is done three Duplicate Samples; According to every group of PEG concentration with recordRelation between OD value, taking PEG concentration as abscissa, OD value is ordinate, drawing standard curve;
(6) calculating of PEG residual quantity in cross-linking sodium hyaluronate gel
Of the present invention as follows for calculating the computing formula of cross-linking sodium hyaluronate gel PEG residual quantity:
Y=0.0108X+0.2047，R2=0.9994
In formula: X---the mass concentration of PEG, unit is μ g/ml;
The mass ratio of Y---iodo-barium chloride-PEG is that the compound of 1:1:1 is at the OD value at 535nm place;
(7) the assay method step of PEG residual quantity in cross-linking sodium hyaluronate gel
By step (5), the supernatant of getting gained in step (1) cross-linked hyaluronic acid gel sample pretreatment adds steaming in right amountHeat up in a steamer water, barium chloride solution and the reaction of iodine liquid, the reaction time is in 15min, measures OD value in wavelength 535nm place, according toStandard corresponding relation curve between PEG concentration and corresponding light density value that step (5) is made or press the computing formula of step (6)Determine PEG residual quantity in cross-linking sodium hyaluronate gel to be measured.
Embodiment 2:
Key instrument: FA1204B type electronic balance, Shanghai Precision Scientific Apparatus Co., Ltd; The clean work of SW-CJ-1FDPlatform, Su Jing Aetna Inc.; 722N type visible spectrophotometer, Shanghai Precision Scientific Apparatus Co., Ltd; TGL-16C typeLuxuriant and rich with fragrance just that centrifuge, Shanghai Fei Qiaer Analytical Instrument Co., Ltd; Vortex shaker, the north is with positive Co., Ltd; Pipettor, moralEppendorf company of state;
Sample pretreatment
The sample pretreatment of cross-linking sodium hyaluronate gel: by the cross-linking sodium hyaluronate gel of preparation with 7 times of volumesAbsolute ethyl alcohol alcohol precipitation repeatedly, is dried and pulverizes gained precipitation, and the particle after part is pulverized or powder are 108 DEG C of redissolution30min, precision takes the gel 1.0g after redissolution, adds 2mL absolute ethyl alcohol, and sealing, through the vibration of vortex oscillation device, makes gelPrecipitation, then, in the centrifugal 30min of 5000r/min, gets supernatant to be measured;
The sample pretreatment of non-crosslinked hyaluronic acid sodium gel: by 7 times of bodies for non-crosslinked hyaluronic acid sodium gel of preparationLong-pending absolute ethyl alcohol alcohol precipitation repeatedly, be dried and pulverizes gained precipitation, by the particle after pulverizing or powder 108 DEG C of redissolution30min, precision takes whole gels after redissolution in 1000mL beaker, adds absolute ethyl alcohol, the body of absolute ethyl alcohol and gelLong-pending and mass ratio is 2, and unit is mL/g, stirs and makes gel precipitation with glass bar, then supernatant is sub-packed in to 100mL centrifugalIn pipe, at the centrifugal 30min of 5000r/min, get supernatant for subsequent use;
The preparation of PEG standard liquid
PEG600 standard storage liquid: precision takes the PEG600 of 50mg, adds the sample of non-crosslinked hyaluronic acid sodium gelIn the prepared supernatant of pretreatment, dissolve, constant volume in 100mL volumetric flask, for subsequent use in 4 DEG C of Refrigerator stores;
PEG600 standard operation liquid: draw PEG600 standard storage liquid 5mL in 50mL volumetric flask, use non-crosslinked hyalomitomeThe sample of acid sodium gel is through the prepared supernatant constant volume of pretreatment, and mass concentration is 50 μ g/mL;
The preparation of barium chloride solution
Accurately take 5g barium chloride, adding distil water is settled to 100ml, obtains 5% barium chloride solution;
The preparation of iodine liquid
Accurately take KI 2.0g and iodine 1.3g, adding distil water is settled to 100ml, obtains the iodine liquid of 0.05mol/L;
The making of PEG solution calibration curve
Get respectively 15 groups and there is the PEG standard liquid that variable concentrations is 0,0.5,10.5,20.5,30.5,40.5 μ g/mL1.0ml, concentration range, between 0-50 μ g/ml, adds 5ml distilled water, the each 50 μ l of 5% barium chloride solution and iodine liquid, reaction8min, measures OD value in wavelength 535nm place; Each concentration is done three Duplicate Samples; According to every group of PEG concentration and the light recordingRelation between density value, taking PEG concentration as abscissa, OD value is ordinate, drawing standard curve;
(6) calculating of PEG residual quantity in cross-linking sodium hyaluronate gel
Of the present invention as follows for calculating the computing formula of cross-linking sodium hyaluronate gel PEG residual quantity:
Y=0.0108X+0.2047，R2=0.9994
In formula: X---the mass concentration of PEG, unit is μ g/ml;
The mass ratio of Y---iodo-barium chloride-PEG is that the compound of 1:1:1 is at the OD value at 535nm place;
(7) the assay method step of PEG residual quantity in cross-linking sodium hyaluronate gel
By step (5), the supernatant of getting gained in step (1) cross-linked hyaluronic acid gel sample pretreatment adds steaming in right amountHeat up in a steamer water, barium chloride solution and the reaction of iodine liquid, the reaction time is in 15min, measures OD value in wavelength 535nm place, according toStandard corresponding relation curve between PEG concentration and corresponding light density value that step (5) is made or press the computing formula of step (6)Determine PEG residual quantity in cross-linking sodium hyaluronate gel to be measured.
Embodiment 3:
Key instrument: FA1204B type electronic balance, Shanghai Precision Scientific Apparatus Co., Ltd; The clean work of SW-CJ-1FDPlatform, Su Jing Aetna Inc.; 722N type visible spectrophotometer, Shanghai Precision Scientific Apparatus Co., Ltd; TGL-16C typeLuxuriant and rich with fragrance just that centrifuge, Shanghai Fei Qiaer Analytical Instrument Co., Ltd; Vortex shaker, the north is with positive Co., Ltd; Pipettor, moralEppendorf company of state;
Sample pretreatment
The sample pretreatment of cross-linking sodium hyaluronate gel: by the cross-linking sodium hyaluronate gel of preparation with 10 times of volumes95% ethanol alcohol precipitation repeatedly, is dried and pulverizes gained precipitation, and the particle after part is pulverized or powder are 110 DEG C of redissolution18min, precision takes the gel 1.0g after redissolution, adds 2mL absolute ethyl alcohol, and sealing, through the vibration of vortex oscillation device, makes gelPrecipitation, then, in the centrifugal 5min of 15000r/min, gets supernatant to be measured;
The sample pretreatment of non-crosslinked hyaluronic acid sodium gel: by 10 times of bodies for non-crosslinked hyaluronic acid sodium gel of preparation95% long-pending ethanol alcohol precipitation repeatedly, be dried and pulverizes gained precipitation, by the particle after pulverizing or powder 110 DEG C of redissolution18min, precision takes whole gels after redissolution in 1000mL beaker, adds absolute ethyl alcohol, the body of absolute ethyl alcohol and gelLong-pending and mass ratio is 2, and unit is mL/g, stirs and makes gel precipitation with glass bar, then supernatant is sub-packed in to 100mL centrifugalIn pipe, at the centrifugal 5min of 15000r/min, get supernatant for subsequent use;
The preparation of PEG standard liquid
PEG2000 standard storage liquid: precision takes the PEG2000 of 50mg, adds the sample of non-crosslinked hyaluronic acid sodium gelProduct dissolve in the prepared supernatant of pretreatment, and constant volume in 100mL volumetric flask is for subsequent use in 4 DEG C of Refrigerator stores;
PEG2000 standard operation liquid: draw PEG2000 standard storage liquid 5mL in 50mL volumetric flask, transparent with non-crosslinkedThe sample of matter acid sodium gel is through the prepared supernatant constant volume of pretreatment, and mass concentration is 50 μ g/mL;
The preparation of barium chloride solution
Accurately take 5g barium chloride, adding distil water is settled to 100ml, obtains 5% barium chloride solution;
The preparation of iodine liquid
Accurately take KI 2.0g and iodine 1.3g, adding distil water is settled to 100ml, obtains the iodine liquid of 0.05mol/L;
The making of PEG solution calibration curve
Get respectively 10 groups and there is the PEG standard liquid that variable concentrations is 0,0.5,10.5,20.5,30.5,40.5 μ g/mL1.0ml, concentration range, between 0-50 μ g/ml, adds 5ml distilled water, the each 1000 μ l of 5% barium chloride solution and iodine liquid, reaction9min, measures OD value in wavelength 535nm place; Each concentration is done three Duplicate Samples; According to every group of PEG concentration and the light recordingRelation between density value, taking PEG concentration as abscissa, OD value is ordinate, drawing standard curve;
(6) calculating of PEG residual quantity in cross-linking sodium hyaluronate gel
Of the present invention as follows for calculating the computing formula of cross-linking sodium hyaluronate gel PEG residual quantity:
Y=0.0108X+0.2047，R2=0.9994
In formula: X---the mass concentration of PEG, unit is μ g/ml;
The mass ratio of Y---iodo-barium chloride-PEG is that the compound of 1:1:1 is at the OD value at 535nm place;
(7) the assay method step of PEG residual quantity in cross-linking sodium hyaluronate gel
By step (5), the supernatant of getting gained in step (1) cross-linked hyaluronic acid gel sample pretreatment adds steaming in right amountHeat up in a steamer water, barium chloride solution and the reaction of iodine liquid, the reaction time is in 15min, measures OD value in wavelength 535nm place, according toStandard corresponding relation curve between PEG concentration and corresponding light density value that step (5) is made or press the computing formula of step (6)Determine PEG residual quantity in cross-linking sodium hyaluronate gel to be measured.
Embodiment 4:
Key instrument: FA1204B type electronic balance, Shanghai Precision Scientific Apparatus Co., Ltd; The clean work of SW-CJ-1FDPlatform, Su Jing Aetna Inc.; 722N type visible spectrophotometer, Shanghai Precision Scientific Apparatus Co., Ltd; TGL-16C typeLuxuriant and rich with fragrance just that centrifuge, Shanghai Fei Qiaer Analytical Instrument Co., Ltd; Vortex shaker, the north is with positive Co., Ltd; Pipettor, moralEppendorf company of state;
Sample pretreatment
The sample pretreatment of cross-linking sodium hyaluronate gel: by the cross-linking sodium hyaluronate gel of preparation with 12 times of volumesAbsolute ethyl alcohol alcohol precipitation repeatedly, is dried and pulverizes gained precipitation, and the particle after part is pulverized or powder are 115 DEG C of redissolution25min, precision takes the gel 1.0g after redissolution, adds 2mL absolute ethyl alcohol, and sealing, through the vibration of vortex oscillation device, makes gelPrecipitation, then, in the centrifugal 25min of 8000r/min, gets supernatant to be measured;
The sample pretreatment of non-crosslinked hyaluronic acid sodium gel: by 10 times of bodies for non-crosslinked hyaluronic acid sodium gel of preparationLong-pending absolute ethyl alcohol alcohol precipitation repeatedly, be dried and pulverizes gained precipitation, by the particle after pulverizing or powder 115 DEG C of redissolution25min, precision takes whole gels after redissolution in 1000mL beaker, adds absolute ethyl alcohol, the body of absolute ethyl alcohol and gelLong-pending and mass ratio is 2, and unit is mL/g, stirs and makes gel precipitation with glass bar, then supernatant is sub-packed in to 100mL centrifugalIn pipe, at the centrifugal 25min of 8000r/min, get supernatant for subsequent use;
The preparation of PEG standard liquid
PEG8000 standard storage liquid: precision takes the PEG8000 of 50mg, adds the sample of non-crosslinked hyaluronic acid sodium gelProduct dissolve in the prepared supernatant of pretreatment, and constant volume in 100mL volumetric flask is for subsequent use in 4 DEG C of Refrigerator stores;
PEG8000 standard operation liquid: draw PEG8000 standard storage liquid 5mL in 50mL volumetric flask, transparent with non-crosslinkedThe sample of matter acid sodium gel is through the prepared supernatant constant volume of pretreatment, and mass concentration is 50 μ g/mL;
The preparation of barium chloride solution
Accurately take 5g barium chloride, adding distil water is settled to 100ml, obtains 5% barium chloride solution;
The preparation of iodine liquid
Accurately take KI 2.0g and iodine 1.3g, adding distil water is settled to 100ml, obtains the iodine liquid of 0.05mol/L;
The making of PEG solution calibration curve
Get respectively 11 groups of PEG standard liquid 1.0ml with variable concentrations, concentration range, between 0-50 μ g/ml, adds5ml distilled water, the each 500 μ l of 5% barium chloride solution and iodine liquid, reaction 11min, measures OD value in wavelength 535nm place; OftenIndividual concentration is done three Duplicate Samples; According to the relation between every group of PEG concentration and the OD value that records, taking PEG concentration as horizontal seatMark, OD value is ordinate, drawing standard curve;
(6) calculating of PEG residual quantity in cross-linking sodium hyaluronate gel
Of the present invention as follows for calculating the computing formula of cross-linking sodium hyaluronate gel PEG residual quantity:
Y=0.0108X+0.2047，R2=0.9994
In formula: X---the mass concentration of PEG, unit is μ g/ml;
The mass ratio of Y---iodo-barium chloride-PEG is that the compound of 1:1:1 is at the OD value at 535nm place;
(7) the assay method step of PEG residual quantity in cross-linking sodium hyaluronate gel
By step (5), the supernatant of getting gained in step (1) cross-linked hyaluronic acid gel sample pretreatment adds steaming in right amountHeat up in a steamer water, barium chloride solution and the reaction of iodine liquid, the reaction time is in 15min, measures OD value in wavelength 535nm place, according toStandard corresponding relation curve between PEG concentration and corresponding light density value that step (5) is made or press the computing formula of step (6)Determine PEG residual quantity in cross-linking sodium hyaluronate gel to be measured.
Embodiment 5:
Key instrument: FA1204B type electronic balance, Shanghai Precision Scientific Apparatus Co., Ltd; The clean work of SW-CJ-1FDPlatform, Su Jing Aetna Inc.; 722N type visible spectrophotometer, Shanghai Precision Scientific Apparatus Co., Ltd; TGL-16C typeLuxuriant and rich with fragrance just that centrifuge, Shanghai Fei Qiaer Analytical Instrument Co., Ltd; Vortex shaker, the north is with positive Co., Ltd; Pipettor, moralEppendorf company of state;
Sample pretreatment
The sample pretreatment of cross-linking sodium hyaluronate gel: by the cross-linking sodium hyaluronate gel of preparation with 9 times of volumes95% ethanol alcohol precipitation repeatedly, is dried and pulverizes gained precipitation, and the particle after part is pulverized or powder are 120 DEG C of redissolution19min, precision takes the gel 1.0g after redissolution, adds 2mL absolute ethyl alcohol, and sealing, through the vibration of vortex oscillation device, makes gelPrecipitation, then, in the centrifugal 10min of 12000r/min, gets supernatant to be measured;
The sample pretreatment of non-crosslinked hyaluronic acid sodium gel: by 9 times of bodies for non-crosslinked hyaluronic acid sodium gel of preparation95% long-pending ethanol alcohol precipitation repeatedly, be dried and pulverizes gained precipitation, by the particle after pulverizing or powder 120 DEG C of redissolution19min, precision takes whole gels after redissolution in 1000mL beaker, adds absolute ethyl alcohol, the body of absolute ethyl alcohol and gelLong-pending and mass ratio is 2, and unit is mL/g, stirs and makes gel precipitation with glass bar, then supernatant is sub-packed in to 100mL centrifugalIn pipe, at the centrifugal 10min of 12000r/min, get supernatant for subsequent use;
The preparation of PEG standard liquid
PEG10000 standard storage liquid: precision takes the PEG10000 of 50mg, adds non-crosslinked hyaluronic acid sodium gelSample dissolves in the prepared supernatant of pretreatment, and constant volume in 100mL volumetric flask is for subsequent use in 4 DEG C of Refrigerator stores;
PEG10000 standard operation liquid: draw PEG10000 standard storage liquid 5mL in 50mL volumetric flask, saturating with non-crosslinkedThe sample of phaneroplasm acid sodium gel is through the prepared supernatant constant volume of pretreatment, and mass concentration is 50 μ g/mL;
The preparation of barium chloride solution
Accurately take 5g barium chloride, adding distil water is settled to 100ml, obtains 5% barium chloride solution;
The preparation of iodine liquid
Accurately take KI 2.0g and iodine 1.3g, adding distil water is settled to 100ml, obtains the iodine liquid of 0.05mol/L;
The making of PEG solution calibration curve
Get respectively 13 groups of PEG standard liquid 1.0ml with variable concentrations, concentration range, between 0-50 μ g/ml, adds5ml distilled water, the each 750 μ l of 5% barium chloride solution and iodine liquid, reaction 13min, measures OD value in wavelength 535nm place; OftenIndividual concentration is done three Duplicate Samples; According to the relation between every group of PEG concentration and the OD value that records, taking PEG concentration as horizontal seatMark, OD value is ordinate, drawing standard curve;
(6) calculating of PEG residual quantity in cross-linking sodium hyaluronate gel
Of the present invention as follows for calculating the computing formula of cross-linking sodium hyaluronate gel PEG residual quantity:
Y=0.0108X+0.2047，R2=0.9994
In formula: X---the mass concentration of PEG, unit is μ g/ml;
The mass ratio of Y---iodo-barium chloride-PEG is that the compound of 1:1:1 is at the OD value at 535nm place;
(7) the assay method step of PEG residual quantity in cross-linking sodium hyaluronate gel
By step (5), the supernatant of getting gained in step (1) cross-linked hyaluronic acid gel sample pretreatment adds steaming in right amountHeat up in a steamer water, barium chloride solution and the reaction of iodine liquid, the reaction time is in 15min, measures OD value in wavelength 535nm place, according toStandard corresponding relation curve between PEG concentration and corresponding light density value that step (5) is made or press the computing formula of step (6)Determine PEG residual quantity in cross-linking sodium hyaluronate gel to be measured.
Embodiment 6:
Key instrument: FA1204B type electronic balance, Shanghai Precision Scientific Apparatus Co., Ltd; The clean work of SW-CJ-1FDPlatform, Su Jing Aetna Inc.; 722N type visible spectrophotometer, Shanghai Precision Scientific Apparatus Co., Ltd; TGL-16C typeLuxuriant and rich with fragrance just that centrifuge, Shanghai Fei Qiaer Analytical Instrument Co., Ltd; Vortex shaker, the north is with positive Co., Ltd; Pipettor, moralEppendorf company of state;
Sample pretreatment
The sample pretreatment of cross-linking sodium hyaluronate gel: by the cross-linking sodium hyaluronate gel of preparation with 13 times of volumesAbsolute ethyl alcohol alcohol precipitation repeatedly, is dried and pulverizes gained precipitation, and the particle after part is pulverized or powder are 124 DEG C of redissolution10min, precision takes the gel 1.0g after redissolution, adds 2mL absolute ethyl alcohol, and sealing, through the vibration of vortex oscillation device, makes gelPrecipitation, then, in the centrifugal 3min of 18000r/min, gets supernatant to be measured;
The sample pretreatment of non-crosslinked hyaluronic acid sodium gel: by 13 times of bodies for non-crosslinked hyaluronic acid sodium gel of preparation95% long-pending ethanol alcohol precipitation repeatedly, be dried and pulverizes gained precipitation, by the particle after pulverizing or powder 124 DEG C of redissolution10min, precision takes whole gels after redissolution in 1000mL beaker, adds absolute ethyl alcohol, the body of absolute ethyl alcohol and gelLong-pending and mass ratio is 2, and unit is mL/g, stirs and makes gel precipitation with glass bar, then supernatant is sub-packed in to 100mL centrifugalIn pipe, at the centrifugal 3min of 18000r/min, get supernatant for subsequent use;
The preparation of PEG standard liquid
PEG16000 standard storage liquid: precision takes the PEG16000 of 50mg, adds non-crosslinked hyaluronic acid sodium gelSample dissolves in the prepared supernatant of pretreatment, and constant volume in 100mL volumetric flask is for subsequent use in 4 DEG C of Refrigerator stores;
PEG16000 standard operation liquid: draw PEG16000 standard storage liquid 5mL in 50mL volumetric flask, saturating with non-crosslinkedThe sample of phaneroplasm acid sodium gel is through the prepared supernatant constant volume of pretreatment, and mass concentration is 50 μ g/mL;
The preparation of barium chloride solution
Accurately take 5g barium chloride, adding distil water is settled to 100ml, obtains 5% barium chloride solution;
The preparation of iodine liquid
Accurately take KI 2.0g and iodine 1.3g, adding distil water is settled to 100ml, obtains the iodine liquid of 0.05mol/L;
The making of PEG solution calibration curve
Get respectively 9 groups of PEG standard liquid 1.0ml with variable concentrations, concentration range, between 0-50 μ g/ml, adds5ml distilled water, the each 200 μ l of 5% barium chloride solution and iodine liquid, reaction 10min, measures OD value in wavelength 535nm place; OftenIndividual concentration is done three Duplicate Samples; According to the relation between every group of PEG concentration and the OD value that records, taking PEG concentration as horizontal seatMark, OD value is ordinate, drawing standard curve;
(6) calculating of PEG residual quantity in cross-linking sodium hyaluronate gel
Of the present invention as follows for calculating the computing formula of cross-linking sodium hyaluronate gel PEG residual quantity:
Y=0.0108X+0.2047，R2=0.9994
In formula: X---the mass concentration of PEG, unit is μ g/ml;
The mass ratio of Y---iodo-barium chloride-PEG is that the compound of 1:1:1 is at the OD value at 535nm place;
(7) the assay method step of PEG residual quantity in cross-linking sodium hyaluronate gel
By step (5), the supernatant of getting gained in step (1) cross-linked hyaluronic acid gel sample pretreatment adds steaming in right amountHeat up in a steamer water, barium chloride solution and the reaction of iodine liquid, the reaction time is in 15min, measures OD value in wavelength 535nm place, according toStandard corresponding relation curve between PEG concentration and corresponding light density value that step (5) is made or press the computing formula of step (6)Determine PEG residual quantity in cross-linking sodium hyaluronate gel to be measured.
The invention is not restricted to aforesaid operations, all variations of making, remodeling, interpolation or replace in essential scope of the present inventionChange, all should belong to protection scope of the present invention.
Claims (4)
1. a method of measuring polyethylene glycol (PEG) residual quantity in cross-linking sodium hyaluronate gel, comprises following processing step:
(1) sample pretreatment
The sample pretreatment of cross-linking sodium hyaluronate gel: by 95% of 7-14 times of volume for hyaluronic acid sodium gel crosslinked PEGEthanol or absolute ethyl alcohol alcohol precipitation repeatedly, are dried and pulverize gained precipitation, and the particle after part is pulverized or powder are at 108-124At DEG C, redissolve 10-30min, precision takes the gel 1.0g after redissolution, adds 2mL absolute ethyl alcohol, and sealing, through vortex oscillation deviceVibration, makes gel precipitation, then, in the centrifugal 3-30min of 5000-18000r/min, gets supernatant to be measured;
The sample pretreatment of non-crosslinked hyaluronic acid sodium gel: by 95% of 7-14 times of volume for non-crosslinked hyaluronic acid sodium gelEthanol or absolute ethyl alcohol alcohol precipitation repeatedly, are dried and pulverize gained precipitation, by multiple at 108-124 DEG C to the particle after pulverizing or powderMolten 10-30min, precision takes whole gels after redissolution in 1000mL beaker, adds absolute ethyl alcohol, absolute ethyl alcohol and gelVolume and mass ratio be 2, unit is mL/g, with glass bar stir make gel precipitation, then supernatant is sub-packed in to 100mLIn centrifuge tube, at the centrifugal 3-30min of 5000-18000r/min, get supernatant for subsequent use;
(2) preparation of PEG standard liquid
PEG600-20000 standard storage liquid: precision takes the PEG600-20000 of 50mg, adds non-crosslinked Sodium Hyaluronate solidifyingGlue sample dissolves in the prepared supernatant of pretreatment, and constant volume in 100mL volumetric flask is for subsequent use in 4 DEG C of Refrigerator stores;
PEG600-20000 standard operation liquid: draw PEG600-20000 standard storage liquid 5mL in 50mL volumetric flask, use non-friendshipConnection hyaluronic acid sodium gel sample is through the prepared supernatant constant volume of pretreatment, and mass concentration is 50 μ g/mL;
(3) preparation of barium chloride solution
Accurately take 5g barium chloride, adding distil water is settled to 100ml, obtains 5% barium chloride solution;
(4) preparation of iodine liquid
Accurately take KI 2.0g and iodine 1.3g, adding distil water is settled to 100ml, obtains the iodine liquid of 0.05mol/L;
(5) making of PEG solution calibration curve
Get respectively 6-15 group and there is the PEG standard liquid that variable concentrations is 0,0.5,10.5,20.5,30.5,40.5 μ g/mL1.0ml, concentration range, between 0-50 μ g/ml, adds 5ml distilled water, 5% barium chloride solution and the each 50-1000 μ of iodine liquid l,Reaction 8-15min, measures OD value in wavelength 535nm place; Each concentration is done three Duplicate Samples; According to every group of PEG concentration withRelation between the OD value recording, taking PEG concentration as abscissa, OD value is ordinate, drawing standard curve;
(6) calculating of PEG residual quantity in cross-linking sodium hyaluronate gel
Of the present invention as follows for calculating the computing formula of cross-linking sodium hyaluronate gel PEG residual quantity:
Y=0.0108X+0.2047，R2=0.9994
In formula: X---the mass concentration of PEG, unit is μ g/ml;
The mass ratio of Y---iodo-barium chloride-PEG is that the compound of 1:1:1 is at the OD value at 535nm place;
(7) the assay method step of PEG residual quantity in cross-linking sodium hyaluronate gel
By step (5), the supernatant of getting gained in step (1) cross-linked hyaluronic acid gel sample pretreatment adds distilled water, chlorineChange barium solution and the reaction of iodine liquid, the reaction time is in 15min, measures OD value, according to step (5) in wavelength 535nm placeStandard corresponding relation curve between PEG concentration and the corresponding light density value of making or treat by the computing formula of step (6) is definiteSurvey PEG residual quantity in cross-linking sodium hyaluronate gel.
2. the method for polyethylene glycol (PEG) residual quantity in mensuration cross-linking sodium hyaluronate gel according to claim 1, itsFeature is: the solvent of the PEG standard liquid of preparing in described step (2) is that in step (1), uncrosslinked gel warp coagulates with crosslinkedThe supernatant that 95% ethanol of glue same volume umber or absolute ethyl alcohol alcohol precipitation obtain, uncrosslinked gel is except in preparation processDo not add outside crosslinking agent PEG, other steps are all identical, so, the PEG standard liquid of preparing with to detect the upper of residual quantityClear liquid is except PEG content is different, and other component is all the same.
3. the method for polyethylene glycol (PEG) residual quantity in mensuration cross-linking sodium hyaluronate gel according to claim 1, itsFeature is: the blank in the making of described step (5) PEG solution calibration curve is to non-crosslinked hyalomitome in step (1)It is molten to volume and standard P EG that acid sodium gel is added distilled water through the supernatant of 95% ethanol or absolute ethyl alcohol alcohol precipitation centrifugal gainedLiquid gradient series equates the solution of gained.
4. the method for polyethylene glycol (PEG) residual quantity in mensuration cross-linking sodium hyaluronate gel according to claim 1, itsFeature is: the molecular weight ranges of measurable PEG is 600-20000Da.
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