CN118348183A - Method for determining molecular weight distribution and content of gelatin in vulcanized colloidal lyophilized product by gel permeation chromatography - Google Patents
Method for determining molecular weight distribution and content of gelatin in vulcanized colloidal lyophilized product by gel permeation chromatography Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 32
- 238000005227 gel permeation chromatography Methods 0.000 title claims abstract description 25
- 238000004458 analytical method Methods 0.000 claims abstract description 16
- AKHNMLFCWUSKQB-UHFFFAOYSA-L sodium thiosulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=S AKHNMLFCWUSKQB-UHFFFAOYSA-L 0.000 claims abstract description 12
- 235000019345 sodium thiosulphate Nutrition 0.000 claims abstract description 12
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 claims abstract description 9
- 239000000523 sample Substances 0.000 claims description 68
- 239000012488 sample solution Substances 0.000 claims description 33
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 claims description 28
- 239000000243 solution Substances 0.000 claims description 28
- 239000013558 reference substance Substances 0.000 claims description 22
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 21
- 239000000084 colloidal system Substances 0.000 claims description 19
- 238000002347 injection Methods 0.000 claims description 19
- 239000007924 injection Substances 0.000 claims description 19
- 235000010344 sodium nitrate Nutrition 0.000 claims description 14
- 239000004317 sodium nitrate Substances 0.000 claims description 14
- 229920003171 Poly (ethylene oxide) Polymers 0.000 claims description 13
- 239000002202 Polyethylene glycol Substances 0.000 claims description 13
- 229920001223 polyethylene glycol Polymers 0.000 claims description 13
- 238000012545 processing Methods 0.000 claims description 10
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- QYYMDNHUJFIDDQ-UHFFFAOYSA-N 5-chloro-2-methyl-1,2-thiazol-3-one;2-methyl-1,2-thiazol-3-one Chemical compound CN1SC=CC1=O.CN1SC(Cl)=CC1=O QYYMDNHUJFIDDQ-UHFFFAOYSA-N 0.000 claims description 7
- 238000010829 isocratic elution Methods 0.000 claims description 7
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims description 7
- 235000019799 monosodium phosphate Nutrition 0.000 claims description 7
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 7
- 238000004364 calculation method Methods 0.000 claims description 6
- 239000012088 reference solution Substances 0.000 claims description 6
- 229920002307 Dextran Polymers 0.000 claims description 2
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- 229920001218 Pullulan Polymers 0.000 claims description 2
- 239000004373 Pullulan Substances 0.000 claims description 2
- UCKMPCXJQFINFW-UHFFFAOYSA-N Sulphide Chemical compound [S-2] UCKMPCXJQFINFW-UHFFFAOYSA-N 0.000 claims description 2
- 239000003242 anti bacterial agent Substances 0.000 claims description 2
- 238000004587 chromatography analysis Methods 0.000 claims description 2
- 230000007717 exclusion Effects 0.000 claims description 2
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- 238000011208 chromatographic data Methods 0.000 claims 1
- 239000000416 hydrocolloid Substances 0.000 claims 1
- 239000004615 ingredient Substances 0.000 claims 1
- 239000003109 Disodium ethylene diamine tetraacetate Substances 0.000 abstract description 8
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- QXNVGIXVLWOKEQ-UHFFFAOYSA-N Disodium Chemical compound [Na][Na] QXNVGIXVLWOKEQ-UHFFFAOYSA-N 0.000 description 3
- GKLVYJBZJHMRIY-OUBTZVSYSA-N Technetium-99 Chemical compound [99Tc] GKLVYJBZJHMRIY-OUBTZVSYSA-N 0.000 description 3
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- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
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- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
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Abstract
The invention relates to the technical field of medicine analysis, and provides a method for determining the molecular weight distribution and the content of gelatin in a vulcanized colloidal lyophilized product by using a gel permeation chromatography. The method can rapidly and accurately detect the molecular weight distribution and the content of the gelatin by using an analysis method, and effectively monitor the quality of the vulcanized colloidal freeze-dried product. The method has better specificity, gelatin molecules are not sheared under specific chromatographic conditions, gelatin in the vulcanized colloidal freeze-dried product can be separated from sodium thiosulfate and disodium ethylenediamine tetraacetate, and the molecular weight distribution and the content of the gelatin can be accurately detected, so that the technical blank in the field is effectively filled.
Description
Technical Field
The invention relates to the technical field of medicine analysis, in particular to a method for determining the molecular weight distribution and the content of gelatin in a vulcanized colloidal lyophilized product by gel permeation chromatography.
Background
The technetium 99m Tc vulcanized colloid injection is mainly used for lymph node positioning in tumor drainage area of breast cancer patient, and is a kind of instant marked diagnostic radioactive medicine, and the technetium 99m Tc vulcanized colloid injection is prepared by heating reaction of pertechnetum 99m Tc sodium acid injection and vulcanized colloid medicine box for injection. The freeze-dried product of vulcanized colloid in the medicine box for injection comprises three components of gelatin, disodium edentate and sodium thiosulfate, wherein the gelatin is used as a carrier for adsorbing and wrapping technetium [ 99m Tc ] and is a key component for preparing technetium [ 99m Tc ] vulcanized colloid injection, and the molecular weight distribution and the content of the gelatin can influence the granularity and distribution of the subsequent technetium [ 99m Tc ] vulcanized colloid injection. Technetium 99m Tc sulfide colloid injection has been marketed in the United states for many years and has not been marketed in China. The quality standard of the vulcanized colloid medicine box for injection is not recorded in the Chinese pharmacopoeia, the United states pharmacopoeia, the Japanese pharmacopoeia and the European pharmacopoeia. The detection and research on the molecular weight distribution or the content of gelatin in the vulcanized colloidal freeze-dried product are not reported at present.
Disclosure of Invention
The invention aims to provide a method for determining the molecular weight distribution and the content of gelatin in a vulcanized colloidal freeze-dried product by gel permeation chromatography.
In order to achieve the object of the present invention, in a first aspect, the present invention provides a method for determining the molecular weight distribution of gelatin and the content thereof in a vulcanized colloidal lyophilized product by gel permeation chromatography, comprising the steps of:
(1) Preparing a molecular weight standard sample solution: respectively weighing at least 5 standard samples with different peak top molecular weights (Mp), wherein the molecular weights of the standard samples are uniformly distributed between 1000 and 1500000, and respectively adding mobile phases to prepare a molecular weight standard sample solution with the concentration of 2-10mg/mL (preferably 5 mg/mL);
(2) Preparing gelatin into gelatin control solution with concentration of 2-20mg/mL (preferably 5 mg/mL) by flow matching;
(3) Dissolving the lyophilized product of the vulcanized colloid in mobile phase to make the gelatin concentration be 2-15mg/mL (preferably 5 mg/mL), and taking the lyophilized product as a sample solution;
(4) Taking the molecular weight standard sample solution, the gelatin reference substance solution and the sample solution, respectively injecting the solutions into a liquid chromatograph, recording the chromatograms, collecting the collected data, and processing the data by GPC software to obtain the relative molecular mass and the distribution information of the relative molecular mass of gelatin in the vulcanized colloidal freeze-dried product; and calculating by an external standard method to obtain the content of gelatin in the vulcanized colloidal freeze-dried product.
In the steps (2) - (3), the mobile phase is a salt solution of sodium nitrate: contains 0.1-0.2mol/L sodium nitrate, 0.01mol/L sodium dihydrogen phosphate, 0.007mol/L sodium hydroxide and 0.05% ProClin 300 antibacterial agent.
In the present invention, the standard sample may be at least one selected from polyethylene glycol, polyethylene oxide, polystyrene, dextran, pullulan, and the like.
Preferably, the standard samples are polyethylene glycol and polyethylene oxide.
More preferably, the number of standard samples is 7.
Further, step (4) comprises the following sub-steps:
1) Using Agilent 1260 high performance liquid chromatograph, the detector is differential refraction detector (RID); adopting AGLIENT GPC exclusion chromatographic columns; the mobile phase is a salt solution of sodium nitrate (containing 0.1-0.2mol/L sodium nitrate, 0.01mol/L sodium dihydrogen phosphate, 0.007mol/L sodium hydroxide and 0.05% ProClin 300 bacteriostatic agent).
The flow rate is 0.8-1.2mL/min, the isocratic elution is carried out, the analysis time is 30-50 minutes, the column temperature is 25-35 ℃, the detector temperature is 40-50 ℃, and the sample injection amount is 5-20 mu L.
Preferably, the flow rate is 1.0mL/min, the isocratic elution is carried out for 35 minutes, the column temperature is 30 ℃, the detector temperature is 40 ℃, and the sample injection amount is 20 mu L.
2) Establishing a molecular weight standard sample standard curve: the standard curve is established by taking the peak time (peak top time) of each molecular weight standard sample solution as an abscissa and taking the logarithmic value (lgMp) of the peak top molecular weight as an ordinate.
3) The peak time (peak top time) of the gelatin in the gelatin reference solution is brought into a molecular weight standard sample standard curve to obtain peak top molecular weight (Mp) of the gelatin, and the collected chromatogram data are further processed by using professional GPC (for example, thousands of spectrums HW-2000 chromatographic workstation software) to obtain detailed information of molecular weight and distribution of the gelatin, including peak top molecular weight (Mp), number average molecular weight (Mn), weight average molecular weight (Mw), molecular weight distribution coefficient (Mw/Mn) and detail of slicing.
4) The peak time (peak top time) of the gelatin in the sample solution is carried into a molecular weight standard sample standard curve to obtain peak top molecular weight (Mp) of the gelatin, and the collected chromatogram data are further processed by using professional GPC (GPC) data processing software (such as thousands of spectrums HW-2000 chromatographic workstation software) to obtain detailed information of the molecular weight of the gelatin and the distribution thereof, including peak top molecular weight (Mp), number average molecular weight (Mn), weight average molecular weight (Mw/Mn), molecular weight distribution coefficient (Mw/Mn) and slicing details; calculating the content of gelatin in the sample according to the peak area of the gelatin reference substance; the calculation formula is as follows:
Wherein: cx is the concentration of the sample solution; cr is the concentration of gelatin reference solution; ax is the peak area of the sample; ar is the peak area of the gelatin control.
Preferably, substep 1) uses 1-3 AGLIENT GPC columns of block chromatography in series, with a column size of 8 μm,300mm x 7.5mm.
Further, aglient guard columns (or not) may be used in tandem with the chromatographic column. Preferably Aglient guard columns are 8 μm,50mm by 7.5mm.
Preferably, 7 polyethylene glycol and polyethylene oxide standard samples with different peak top molecular weights (Mp) are weighed in the step (1); wherein, peak top molecular weights of the polyethylene glycol standard samples are 1470, 8500 and 20180 respectively, and peak top molecular weights of the polyethylene oxide standard samples are 73650, 219400, 756500 and 1250000 respectively.
Further, the correlation equation of the standard curve established in sub-step 2) is: y= -0.3272 x+ 13.037, the correlation coefficient R is 0.999.
In the invention, the components of the vulcanized colloid freeze-dried product comprise gelatin, sodium thiosulfate and disodium ethylenediamine tetraacetate.
By means of the technical scheme, the invention has at least the following advantages and beneficial effects:
The invention provides a method for detecting the molecular weight distribution and the content of gelatin in a vulcanized colloidal freeze-dried product by adopting a gel permeation chromatography. The method can rapidly and accurately detect the molecular weight distribution and the content of the gelatin by using an analysis method, and effectively monitor the quality of the vulcanized colloidal freeze-dried product. The method has better specificity, gelatin molecules are not sheared under specific chromatographic conditions, gelatin in the vulcanized colloidal freeze-dried product can be separated from sodium thiosulfate and disodium ethylenediamine tetraacetate, and the molecular weight distribution and the content of the gelatin can be accurately detected; aiming at the detection technology of the molecular weight distribution of gelatin or the content thereof in the vulcanized colloidal freeze-dried product, no related report is yet seen at present, and the invention effectively fills the technical blank in the field; meanwhile, the novel ProClin bacteriostatic agent is used as the mobile phase in the invention, so that the use of sodium azide (extremely toxic and easy to explosion) which is a traditional preservative is avoided.
Drawings
FIG. 1 is a molecular weight standard curve of a preferred embodiment of the present invention.
FIG. 2 is a typical chromatogram of a gelatin control in a preferred embodiment of the invention.
FIG. 3 is a typical chromatogram of a sample in a preferred embodiment of the invention.
FIG. 4 is a molecular weight standard curve (flow rate 0.8 mL/min) of a preferred embodiment of the invention.
FIG. 5 is a typical chromatogram of a gelatin control in a preferred embodiment of the invention.
FIG. 6 is a typical chromatogram of a sample in a preferred embodiment of the invention.
FIG. 7 is a typical chromatogram of a gelatin control in a comparative example of the present invention.
Detailed Description
The invention provides a method for detecting the molecular weight distribution and the content of gelatin in a vulcanized colloidal freeze-dried product by adopting a gel permeation chromatography.
The test principle is as follows: gel Permeation Chromatography (GPC) is a column chromatography separation analysis method in which a chromatographic column is filled with a filler of a porous substance, polymer molecules are separated according to the size of the filler in the chromatographic column when passing through the filler, larger molecules are difficult to enter a small pore size, the elution time in the chromatographic column is shorter, the peak is earlier, and the peak is later for smaller molecules. And (3) establishing a correction curve of the corresponding relation between the retention time and the relative molecular mass by using standard polymers with known relative molecular mass, and calculating the relative molecular mass and the distribution information of the relative molecular mass of the test sample (gelatin in the vulcanized colloidal freeze-dried product) from the GPC spectrogram. And injecting a gelatin standard substance and a vulcanized colloid freeze-dried product respectively, and determining the gelatin content in the vulcanized colloid freeze-dried product by an external standard method according to the peak area of the gelatin standard substance.
The analysis method is as follows:
Chromatographic conditions: the detector is a differential refraction detector by adopting a high performance liquid chromatograph. A AGLIENT GPC-column chromatography (PL aquagel-OH MIXED-H8 μm 300 mm. Times.7.5 mm) was used, and 3 columns were connected in series to increase the degree of separation. A Aglient Guard column (PL aquagel-OH Guard 8 μm 50 mm. Times.7.5 mm) was used in series before the column. The mobile phase was a salt solution (containing 0.2mol/L sodium nitrate, 0.01mol/L sodium dihydrogen phosphate, 0.007mol/L sodium hydroxide, 0.05% ProClin 300 bacteriostatic agent). The flow rate is 0.8-1.2mL/min, preferably 1mL/min, the isocratic elution is carried out, the column temperature is 30 ℃, the detector temperature is 40 ℃, and the sample injection amount is 20 mu l.
Molecular weight standard sample solution: weighing at least 5 polyethylene glycol/polyethylene oxide standard samples with different peak top molecular weights (Mp), wherein the molecular weights are approximately and uniformly distributed between 1000 and 1500000, respectively adding mobile phases to prepare solutions with the concentration of 5mg in each 1mL, and detecting the molecular weights and the distribution thereof, wherein the concentration of the solutions does not influence the molecular weights and the distribution thereof;
Gelatin control solution: weighing a proper amount of gelatin, adding a mobile phase to dissolve so that the gelatin concentration is 5mg/mL, and taking the gelatin as a gelatin reference substance solution;
test solution: taking a1 bottle of vulcanized colloid freeze-dried product (containing 18.1mg of gelatin, 2.0mg of sodium thiosulfate and 2.3mg of disodium ethylenediamine tetraacetate), adding a mobile phase for dissolution, so that the gelatin concentration is 5mg/mL, and taking the mixture as a sample solution;
and respectively injecting a molecular weight standard sample solution, a gelatin reference substance solution and a sample solution into a liquid chromatograph, recording a chromatogram, collecting collected data, and processing the data by GPC software to obtain the relative molecular mass and the distribution information of the relative molecular mass of gelatin in the vulcanized colloidal freeze-dried product. And calculating by an external standard method to obtain the content of gelatin in the vulcanized colloidal freeze-dried product.
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the technical means used in the examples are conventional means well known to those skilled in the art, and all raw materials used are commercially available.
Example 1 method for detecting molecular weight distribution and content of gelatin in a vulcanized colloidal lyophilized product by gel permeation chromatography (normal test conditions)
Chromatographic conditions: using an Agilent 1260 high performance liquid chromatograph, the detector is a differential refractive detector (RID). 3 AGLIENT GPC-column chromatography columns were used in series (PL aquagel-OH MIXED-H8 μm 300 mm. Times.7.5 mm) and Aglient Guard columns (PL aquagel-OH Guard 8 μm 50 mm. Times.7.5 mm) were used in series. The mobile phase is a salt solution of sodium nitrate (containing 0.2mol/L sodium nitrate, 0.01mol/L sodium dihydrogen phosphate, 0.007mol/L sodium hydroxide, 0.05% ProClin 300 bacteriostatic agent). The flow rate is 1.0ml/min, the isocratic elution is carried out, the analysis time is 35 minutes, the column temperature is 30 ℃, the detector temperature is 40 ℃, and the sample injection amount is 20 mu l.
Molecular weight standard sample solution: 7 polyethylene glycol/polyethylene oxide standard samples (Table 1) with different peak top molecular weights (Mp) are weighed, the molecular weights are uniformly distributed between 1000 and 1500000, and mobile phases are respectively added to prepare a solution with the concentration of 5mg in each 1 mL.
TABLE 1 molecular weight series Standard samples
Gelatin control solution: weighing 50mg of gelatin, adding 10mL of mobile phase for dissolution, so that the gelatin concentration is 5mg/mL, and taking the gelatin as gelatin reference substance solution; two portions were prepared in parallel.
Test solution: taking a 1 bottle of vulcanized colloid freeze-dried product (containing 18.1mg of gelatin, 2.0mg of sodium thiosulfate and 2.3mg of disodium ethylenediamine tetraacetate), adding 3.62mL of mobile phase for dissolution, so that the gelatin concentration is 5mg/mL, and taking the mixture as a test sample solution.
Taking 1 needle of each sample of the molecular weight standard sample solution; two parts of gelatin reference substance solution, each part is injected with two needles; and (3) injecting two samples of the sample solution respectively, recording a chromatogram, collecting collected data, and processing the data by GPC software to obtain the relative molecular mass and the distribution information of the relative molecular mass of gelatin in the vulcanized colloidal freeze-dried product. And calculating by an external standard method to obtain the content of gelatin in the vulcanized colloidal freeze-dried product.
Results:
Establishment of a standard curve: establishing a standard curve (figure 1) by taking the peak time (peak top time) of a molecular weight standard sample as an abscissa and taking a logarithmic value (lgMp) of the peak top molecular weight as an ordinate, wherein a correlation equation of the standard curve is y= -0.3272 x+ 13.037, and a correlation coefficient R of the standard curve is 0.999.
The peak time (peak top time) of the gelatin in the gelatin control solution can be obtained after the gelatin control is injected, the peak top molecular weight Mp of the gelatin can be obtained by introducing the peak time (peak top time) into a molecular weight standard sample standard curve, and further, the molecular weight of the gelatin and detailed information of the distribution thereof can be obtained by processing the acquired chromatogram data by using a thousand-spectrum HW-2000 chromatographic workstation software, including peak top molecular weight (Mp), number average molecular weight (Mn), weight average molecular weight (Mw/Mn), molecular weight distribution coefficient (Mw/Mn), slicing details and the like. The peak area of the obtained gelatin can be used for calculating the content of gelatin in the test sample. A typical chromatogram of the gelatin control is shown in FIG. 2.
The peak time (peak top time) of the gelatin in the sample solution can be obtained after sample injection of the sample, the peak top molecular weight Mp of the gelatin can be obtained by introducing the peak time (peak top time) into a molecular weight standard sample standard curve, and further, the molecular weight of the gelatin and detailed information of the distribution thereof can be obtained by processing the acquired chromatogram data by using a thousand-spectrum HW-2000 chromatographic workstation software, including peak top molecular weight (Mp), number average molecular weight (Mn), weight average molecular weight (Mw/Mn), molecular weight distribution coefficient (Mw/Mn), slicing details and the like. And obtaining the peak area information of the gelatin, and calculating the content of the gelatin in the sample according to the peak area information of the gelatin reference substance.
The calculation formula is as follows: content of
Wherein: cx is the concentration of the sample; cr is the concentration of gelatin reference substance; ax is the peak area of the sample; ar is the peak area of the gelatin control.
A typical chromatogram of the test sample is shown in FIG. 3. The experimental result shows that the retention time of the gelatin peak in the test sample is 25.44min, the separation degree of the gelatin peak from the disodium edentate and the sodium thiosulfate peak is more than 1, the two peaks are basically separated, the analysis requirement can be met, and the method has better specificity.
The test results are shown in Table 2, and the average value of the retention time of the gelatin control and the average value of the retention time of the test sample are taken into a standard curve of a molecular weight standard sample, so that the molecular weights of the gelatin control and the test sample relative to polyethylene glycol/polyethylene oxide are 51258 and 51645 respectively. The detailed information of the molecular weight and the distribution of the gelatin can be obtained by processing the collected chromatogram data by using the thousand-spectrum HW-2000 chromatographic workstation software, and the representative sample information is shown in Table 3. The gelatin content in the test sample was calculated by external standard method to be 5.09mg/ml.
TABLE 2 gelatin control and test results for test samples
TABLE 3 molecular weight and distribution details of gelatin control and sample typical samples
Example 2 method System suitability investigation
The same analysis conditions as in example 1 were used. Preparing gelatin reference substance solution and test substance solution in one portion. Gelatin control solution: weighing 50mg of gelatin, adding 10mL of mobile phase for dissolution, so that the gelatin concentration is 5mg/mL, and taking the gelatin as a gelatin reference solution. Test solution: taking a1 bottle of vulcanized colloid freeze-dried product (containing 18.1mg of gelatin, 2.0mg of sodium thiosulfate and 2.3mg of disodium ethylenediamine tetraacetate), adding 3.62mL of mobile phase for dissolution, so that the gelatin concentration is 5mg/mL, and taking the mixture as a test sample solution. Each sample is sampled for 6 times in parallel, the peak retention time and the peak area of gelatin are recorded, and the RSD of the peak retention time and the peak area is analyzed by statistics, wherein the RSD is less than or equal to 5 percent.
The experimental results are shown in tables 4 and 5, and the results show that the peak areas and the RSD of the peak retention time of the gelatin reference substance and the tested substance are less than 1%, and the method has better system applicability and repeatability precision.
Table 4 results of the suitability test of the gelatin control system
TABLE 5 test results of test line System suitability test
Example 3 method accuracy investigation
The present example mainly examined the accuracy of the method in the calculation of the gelatin content.
Preparing a solution: gelatin control solution: weighing 50mg of gelatin, adding 10mL of mobile phase for dissolution, so that the gelatin concentration is 5mg/mL, and taking the gelatin as a gelatin reference solution. Adding a standard solution to a test sample: taking a1 bottle of vulcanized colloid freeze-dried product (containing 18.1mg of gelatin, 2.0mg of sodium thiosulfate and 2.3mg of disodium ethylenediamine tetraacetate), adding 3.62mL of gelatin reference substance solution for dissolution, and taking the mixture as a standard test substance solution.
The accuracy of the test sample labeling inspection method is adopted. The control is tested for 2 times in parallel, the standard adding test sample is tested for 6 times in parallel, and the standard adding recovery rate of the test sample is calculated. The recovery rate of gelatin in 6 parts of the standard sample solution is 90.0-110.0%, and RSD is less than or equal to 5.0%.
The recovery rate calculation formula is: recovery rate
Wherein: p is the sample labeling recovery rate; c 1 is the concentration of the sample to be tested and the standard sample; c 2 is the concentration of the test sample; c 3 is the addition concentration.
Table 6 accuracy verification of gelatin content measurement method
The experimental results are shown in Table 6, and the results show that the average recovery rate of gelatin in 6 parts of the labeled sample solution is 95.3%, the RSD is less than or equal to 5.0%, the requirements are met, and the method has good accuracy.
Example 4 method durability investigation (flow 0.8 ml/min)
Chromatographic conditions: using an Agilent 1260 high performance liquid chromatograph, the detector is a differential refractive detector (RID). 3 AGLIENT GPC-column chromatography columns were used in series (PL aquagel-OH MIXED-H8 μm 300 mm. Times.7.5 mm) and Aglient Guard columns (PL aquagel-OH Guard 8 μm 50 mm. Times.7.5 mm) were used in series. The mobile phase is a salt solution of sodium nitrate (containing 0.2mol/L sodium nitrate, 0.01mol/L sodium dihydrogen phosphate, 0.007mol/L sodium hydroxide, 0.05% ProClin 300 bacteriostatic agent). The flow rate is 0.8ml/min, the isocratic elution is carried out, the analysis time is 45 minutes, the column temperature is 30 ℃, the detector temperature is 40 ℃, and the sample injection amount is 20 mu l.
Molecular weight standard sample solution: 7 polyethylene glycol/polyethylene oxide standard samples (Table 1) with different peak top molecular weights (Mp) are weighed, the molecular weights are uniformly distributed between 1000 and 1500000, and mobile phases are respectively added to prepare a solution with the concentration of 5mg in each 1 mL.
Gelatin control solution: weighing 50mg of gelatin, adding 10mL of mobile phase for dissolution, so that the gelatin concentration is 5mg/mL, and taking the gelatin as a gelatin reference solution.
Test solution: taking a 1 bottle of vulcanized colloid freeze-dried product (containing 18.1mg of gelatin, 2.0mg of sodium thiosulfate and 2.3mg of disodium ethylenediamine tetraacetate), adding 3.62mL of mobile phase for dissolution, so that the gelatin concentration is 5mg/mL, and taking the mixture as a test sample solution.
Taking 1 needle of each sample of the molecular weight standard sample solution; injecting gelatin reference substance solution into two needles; and (3) injecting the sample solution into two needles, recording a chromatogram, collecting collected data, and processing the data by GPC software to obtain the relative molecular mass and the distribution information of the relative molecular mass of gelatin in the vulcanized colloidal freeze-dried product. And calculating by an external standard method to obtain the content of gelatin in the vulcanized colloidal freeze-dried product.
Results: establishment of a standard curve: the molecular weight standard sample peak time (peak top time) is taken as an abscissa, the logarithmic value (lgMp) of the peak top molecular weight is taken as an ordinate to establish a standard curve (figure 4), the correlation equation of the standard curve is y= -0.2594 x+ 12.953, and the correlation coefficient R of the standard curve is 0.999.
The peak time (peak top time) of the gelatin in the gelatin reference substance solution can be obtained after the gelatin reference substance is injected, and the peak top molecular weight Mp of the gelatin can be obtained after the gelatin reference substance is injected into a molecular weight standard sample standard curve. The peak area of the obtained gelatin can be used for calculating the content of gelatin in the test sample. A typical chromatogram of the gelatin control is shown in FIG. 5.
The peak time (peak top time) of gelatin in the sample solution can be obtained after sample injection of the sample, and the peak top molecular weight Mp of gelatin can be obtained by putting the peak time into a standard curve of a molecular weight standard sample. And obtaining the peak area information of the gelatin, and calculating the content of the gelatin in the sample according to the peak area information of the gelatin reference substance.
The calculation formula is as follows: content of
Wherein: cx is the concentration of the sample; cr is the concentration of gelatin reference substance; ax is the peak area of the sample; ar is the peak area of the gelatin control.
A typical chromatogram of the test sample is shown in FIG. 6. The experimental result shows that the retention time of the gelatin peak in the test sample is 31.04min, the separation degree of the gelatin peak from the disodium edentate and the sodium thiosulfate peak is more than 1, the two peaks are basically separated, the analysis requirement can be met, and the method has better specificity.
The test results are shown in Table 7, and the average value of the retention time of the gelatin control and the average value of the retention time of the test sample are taken into a standard curve of a molecular weight standard sample, so that the molecular weights of the gelatin control and the test sample relative to polyethylene glycol/polyethylene oxide are 81583 and 79433 respectively. The gelatin content in the test sample was calculated to be 5.17mg/mL by the external standard method. Representative sample results are shown in Table 8.
TABLE 7 gelatin control and test results for test samples
TABLE 8 molecular weight and distribution details of gelatin control and sample typical samples
Comparative example:
Chromatographic conditions: using an Agilent 1260 high performance liquid chromatograph, the detector is a differential refractive detector (RID). The use of a （PL aquagel-OH 30,8μm 300mm×7.5mm;PL aquagel-OH 40,8μm 300mm×7.5mm;PL aquagel-OH 50,8μm 300mm×7.5mm）, column in tandem with a AGLIENT GPC-column block with 3 different single pore sizes was preceded by a Aglient Guard column (PL aquagel-OH Guard 8 μm 50 mm. Times.7.5 mm). The mobile phase is a salt solution of sodium nitrate (containing 0.2mol/L sodium nitrate, 0.01mol/L sodium dihydrogen phosphate, 0.007mol/L sodium hydroxide, 0.05% ProClin 300 bacteriostatic agent). The flow rate is 1.0ml/min, the isocratic elution is carried out, the analysis time is 35 minutes, the column temperature is 30 ℃, the detector temperature is 40 ℃, and the sample injection amount is 20 mu l.
The same gelatin control sample injection analysis as in example 1 was used and the test results are shown in fig. 7. The results indicate that the single pore size column does not give the desired results. This is probably because the use of a single pore size column, the sample is sheared during the analysis, resulting in an excessively wide peak profile, and the gelatin control sample has a very poor peak profile, making it difficult to obtain information on their molecular weight, their distribution, their content, etc.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (10)
1. The method for determining the molecular weight distribution and the content of gelatin in the vulcanized colloidal lyophilized product by using the gel permeation chromatography is characterized by comprising the following steps:
(1) Preparing a molecular weight standard sample solution: respectively weighing at least 5 standard samples with different peak top molecular weights, wherein the molecular weights of the standard samples are uniformly distributed between 1000 and 1500000, and respectively adding mobile phases to prepare a molecular weight standard sample solution with the concentration of 2-10 mg/mL;
(2) Preparing gelatin into gelatin reference substance solution with concentration of 2-20mg/mL by flow matching;
(3) Dissolving the lyophilized product of the vulcanized colloid in mobile phase to make the gelatin concentration be 2-15mg/mL, and taking the lyophilized product of the vulcanized colloid as a sample solution;
(4) Taking the molecular weight standard sample solution, the gelatin reference substance solution and the sample solution, respectively injecting the solutions into a liquid chromatograph, recording the chromatograms, collecting the collected data, and processing the data by GPC software to obtain the relative molecular mass and the distribution information of the relative molecular mass of gelatin in the vulcanized colloidal freeze-dried product; calculating to obtain the content of gelatin in the vulcanized colloidal lyophilized product by an external standard method;
in the steps (2) - (3), the mobile phase is a salt solution of sodium nitrate: contains 0.1-0.2mol/L sodium nitrate, 0.01mol/L sodium dihydrogen phosphate, 0.007mol/L sodium hydroxide and 0.05% ProClin 300 antibacterial agent.
2. The method of claim 1, wherein the standard sample is selected from at least one of polyethylene glycol, polyethylene oxide, polystyrene, dextran, and pullulan.
3. The method of claim 2, wherein the standard samples are polyethylene glycol and polyethylene oxide.
4. A method according to claim 3, wherein step (4) comprises the sub-steps of:
1) Adopting an Agilent 1260 high performance liquid chromatograph, wherein the detector is a differential refraction detector; adopting AGLIENT GPC exclusion chromatographic columns; the mobile phase is a salt solution of sodium nitrate; the flow rate is 0.8-1.2mL/min, the isocratic elution is carried out, the analysis time is 30-50 minutes, the column temperature is 25-35 ℃, the detector temperature is 40-50 ℃, and the sample injection amount is 5-20 mu L;
2) Establishing a molecular weight standard sample standard curve: establishing a standard curve by taking the peak outlet time of each molecular weight standard sample solution as an abscissa and the logarithmic value of the peak top molecular weight as an ordinate;
3) Bringing the peak-out time of gelatin in the gelatin reference substance solution into a molecular weight standard sample standard curve to obtain peak top molecular weight of gelatin, and further processing the acquired chromatogram data by using professional GPC (GPC) data processing software to obtain detailed information of molecular weight and distribution of the gelatin, wherein the detailed information comprises peak top molecular weight, number average molecular weight, weight average molecular weight, molecular weight distribution coefficient and slicing detail;
4) Bringing the peak-out time of gelatin in the sample solution into a standard curve of a molecular weight standard sample to obtain the peak top molecular weight of gelatin, and further processing the acquired chromatogram data by using professional GPC (GPC) data processing software to obtain detailed information of the molecular weight and the distribution of the gelatin, wherein the detailed information comprises the peak top molecular weight, the number average molecular weight, the weight average molecular weight, the molecular weight distribution coefficient and the slicing detail; calculating the content of gelatin in the sample according to the peak area of the gelatin reference substance; the calculation formula is as follows:
；
wherein: cx is the concentration of the sample solution; cr is the concentration of gelatin reference solution; ax is the peak area of the sample; ar is the peak area of the gelatin reference substance;
The mobile phase is as claimed in claim 1.
5. The method according to claim 4, wherein the substep 1) uses 1 to 3 AGLIENT GPC columns of column size of 8 μm,300mm x 7.5mm in series.
6. The method of claim 4, wherein a Aglient guard column is used in series before the substep 1) chromatography column.
7. The method of claim 4, wherein the specialized GPC chromatographic data processing software in sub-steps 3) and 4) is kilospectral HW-2000 chromatographic workstation software.
8. The method according to claim 4, wherein 7 polyethylene glycol and polyethylene oxide standard samples with different peak top molecular weights are weighed in the step (1); wherein, peak top molecular weights of the polyethylene glycol standard samples are 1470, 8500 and 20180 respectively, and peak top molecular weights of the polyethylene oxide standard samples are 73650, 219400, 756500 and 1250000 respectively.
9. The method of claim 8, wherein the correlation equation for the standard curve established in sub-step 2) is:
y= -0.3272 x+ 13.037, the correlation coefficient R is 0.999.
10. The method of any one of claims 1-9, wherein the ingredients of the hydrocolloid sulfide freeze-dried product comprise gelatin, sodium thiosulfate, and disodium edetate.
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