CN117723682A - Method for detecting crosslinking degree of crosslinked hyaluronic acid or salt gel thereof - Google Patents
Method for detecting crosslinking degree of crosslinked hyaluronic acid or salt gel thereof Download PDFInfo
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- YWIVKILSMZOHHF-QJZPQSOGSA-N sodium;(2s,3s,4s,5r,6r)-6-[(2s,3r,4r,5s,6r)-3-acetamido-2-[(2s,3s,4r,5r,6r)-6-[(2r,3r,4r,5s,6r)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2- Chemical compound [Na+].CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 YWIVKILSMZOHHF-QJZPQSOGSA-N 0.000 claims description 47
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- BMYNFMYTOJXKLE-UHFFFAOYSA-N 3-azaniumyl-2-hydroxypropanoate Chemical compound NCC(O)C(O)=O BMYNFMYTOJXKLE-UHFFFAOYSA-N 0.000 claims description 13
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Abstract
The invention relates to a method for detecting the crosslinking degree of crosslinked hyaluronic acid or salt gel thereof, belonging to the technical field of chemical analysis. The detection method comprises the steps of acidolysis of crosslinked hyaluronic acid or salt gel thereof at 110-150 ℃ and freeze-drying to obtain a freeze-dried sample; extracting the freeze-dried sample by using ethyl acetate, centrifuging, taking a supernatant, and filtering to obtain a sample solution; the degree of crosslinking of the crosslinked hyaluronic acid or its salt gel is then calculated from the glycerol concentration after acidolysis of the crosslinked hyaluronic acid or its salt gel. According to the detection method provided by the invention, through increasing the acidolysis temperature and freeze-drying the crosslinked hyaluronic acid or the salt gel thereof after acidolysis, and extracting by adopting ethyl acetate, the solubility of inorganic salt and water in a solvent can be greatly reduced, the solution stability and the extraction rate are increased, and the accuracy of a detection result is effectively improved.
Description
Technical Field
The invention relates to a method for detecting the crosslinking degree of crosslinked hyaluronic acid or salt gel thereof, belonging to the technical field of chemical analysis.
Background
Hyaluronic Acid (HA) is a linear polysaccharide composed of disaccharide repeating structural units of D-glucuronic acid and N-acetyl-D-glucosamine linked by β - (1-3) glycosidic bonds, each disaccharide unit being linked to the other disaccharide unit by β - (1-4) glycosidic bonds. HA HAs been widely used clinically because of its excellent moisture retention, lubricity, viscoelasticity, non-immunogenicity, and promotion of drug absorption. However, natural HA HAs the disadvantages of strong water solubility, easy diffusion and enzymatic degradation in tissues, short residence time in vivo, and the modified and crosslinked HA derivatives can compensate for the disadvantages. Some crosslinked HA products have been successfully used for the prevention of post-operative adhesions, the treatment of osteoarthritis, soft tissue augmentation, tissue engineering and controlled drug release.
Hyaluronic acid is generally present in the form of a salt, i.e. most of the time used by sodium hyaluronate. The crosslinking agent is a substance for crosslinking sodium hyaluronate, for example 1, 4-butanediol diglycidyl ether (BDDE). Crosslinking reactions are complex reactions in which macromolecules contain spatial multiple sites. The physicochemical properties of the crosslinked sodium hyaluronate are closely related to the degree of crosslinking. The crosslinked sodium hyaluronate is a gel macromolecule formed by reacting sugar ring active groups on the crosslinked sodium hyaluronate with a crosslinking agent under certain conditions and crosslinking, and is one of crosslinked HA derivatives. The crosslinking degree of the crosslinked sodium hyaluronate refers to the degree of intermolecular bonding through chemical bonds, the higher the crosslinking degree is, the tighter the intermolecular bonding is, the better the stability and durability of the material are, and the slower the in vivo degradation rate is relatively. However, too high a degree of crosslinking can lead to stiffness of the material and loss of some elasticity and softness. Therefore, the degree of crosslinking is one of the indicators controlling the quality of the crosslinked sodium hyaluronate product.
Chinese patent document CN107561179a provides a method for measuring the degree of crosslinking of crosslinked hyaluronic acid or a salt thereof, disclosing that oligosaccharides and disaccharides are produced by enzymatic hydrolysis of crosslinked hyaluronic acid or a salt thereof by hyaluronidase; separating and detecting chromatographic peak areas of the oligosaccharides and the disaccharides by combining liquid chromatography with ultraviolet absorption; and finally, calculating to obtain the crosslinking degree. However, the method needs to determine the enzymolysis product of the gel, and has the defects of more precise instruments and complicated process in the detection process, so that the method is not suitable for rapidly controlling the product quality in the industrial production process.
The Chinese patent document CN114280193A provides a method for detecting the crosslinking degree of gel, and discloses the method for calculating the crosslinking degree of the gel according to the concentration of glycerol after acidolysis of the gel, which comprises the three steps of drawing a glycerol standard working curve, measuring the concentration of glycerol in a sample, and calculating the crosslinking degree of the sample. However, this patent method has several significant drawbacks: firstly, in the measurement process, gel samples are subjected to acidolysis and then are evaporated to dryness at 50-60 ℃ and then are subjected to ultrasonic extraction, but the evaporated samples are hard and are not easy to grind into powder, and partial acidolysis samples can be lost in the ultrasonic extraction process, so that the test result is inaccurate. Secondly, if the ultrasonic time in the extraction process of the patent is too short, the extraction is incomplete; if the ultrasonic time is too long, the extraction solvent is easy to lose and the stability of the solution is affected, so that the test result is inaccurate. Thirdly, the extraction solvent of the patent is absolute ethyl alcohol, a large amount of inorganic salt is generated in the acid-base neutralization process, part of moisture and inorganic salt which are not evaporated can be dissolved in the absolute ethyl alcohol, the instrument response can be lowered or even no response can be caused in the sample injection and collection process, the test result can be inaccurate, and the chromatographic column can be damaged. Fourthly, the acidolysis temperature of the patent is between room temperature and 100 ℃, and the acidolysis effect below 100 ℃ is not obvious during actual detection, so that the test result is inaccurate.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for detecting the crosslinking degree of crosslinked hyaluronic acid or salt gel thereof. Compared with the prior art, the detection method has the advantages of simpler detection process and more accurate result.
The technical scheme of the invention is as follows:
a method for detecting the crosslinking degree of crosslinked hyaluronic acid or salt thereof gel comprises the following steps:
(1) Preparation of test solution
Acidolysis is carried out on crosslinked hyaluronic acid or salt gel thereof for 12-36 hours at 110-150 ℃, cooling is carried out to 20-30 ℃ and then neutralization is carried out to neutrality, and freeze-drying is carried out to obtain a freeze-dried sample; extracting the freeze-dried sample by using ethyl acetate, centrifuging, taking a supernatant, and filtering to obtain a sample solution;
(2) Preparation of glycerol standard solution
Gradually diluting the glycerol standard substance with ethyl acetate to obtain glycerol standard solutions with different concentrations;
(3) Detection of the degree of crosslinking
Carrying out gas chromatography determination on the sample solution and the glycerol standard solution to obtain peak areas of the sample solution and the glycerol standard solution, drawing a glycerol standard working curve according to the peak areas of the glycerol standard solution, and calculating the concentration of glycerol in the sample solution according to the standard curve; and finally, calculating the crosslinking degree of the sample according to the concentration of glycerol in the sample, wherein the calculation formula is as follows:
wherein:
C glycerol -concentration of glycerol in the crosslinked sodium hyaluronate gel, μg/mL;
v-volume of ethyl acetate added, mL;
rho-crosslinked sodium hyaluronate gel density, 1.01 g/mL;
omega-sodium hyaluronate content in crosslinked sodium hyaluronate gel, mg/mL;
m-mass of crosslinked sodium hyaluronate gel, g;
92.094 relative molecular mass of glycerol;
401.3-sodium hyaluronate repeat disaccharide unit relative molecular mass.
According to a preferred embodiment of the present invention, in step (1), the acidolysis is: adding hydrobromic acid or sulfuric acid into the crosslinked hyaluronic acid or the salt gel thereof, and acidolysis is carried out for 18-24 h at 120-130 ℃.
Further preferably, the concentration of hydrobromic acid or sulfuric acid is 0.4-1M; the mass volume ratio of the crosslinked hyaluronic acid or the salt gel thereof to hydrobromic acid or sulfuric acid is (2-3): (5-10), unit: g/mL.
According to a preferred embodiment of the present invention, in step (1), the specific procedure of lyophilization is: pre-freezing for 5-10 h at-30 ℃, heating to-10 ℃ for 2-5 h, heating to-2 ℃ for 2-5 h, continuously heating to 20 ℃ for 6-10 h, heating to 40 ℃ for 20-45 h, and removing water in the sample.
According to the invention, in the step (1), the mass-volume ratio of the crosslinked hyaluronic acid or the salt gel thereof to the ethyl acetate is (2-3): (10-20), unit: g/mL.
According to the invention, in the step (1), the extraction time is 5-10 min; the centrifugation is carried out for 5-10 min at 8000-14000 r/min.
According to a preferred embodiment of the present invention, in the step (2), the concentration of the glycerol standard solution is set to a series of concentrations of 6 to 96. Mu.g/ml.
According to a preferred embodiment of the present invention, in step (2), the gas chromatography conditions are as follows:
chromatographic column: RBX-5, 30 m.times.320 μm.times.0.25 μm; sample inlet temperature: 250-270 ℃; a detector: a hydrogen flame ionization detector; detector temperature: 270-290 ℃; flow rate: 0.5-1.0 ml/min; sample injection amount: 1 μl; column temperature: starting temperature: maintaining at 70-90 deg.c for 5min and maintaining at 10-20 deg.c/min to 260 deg.c for 15min; the split ratio is 1:1.
The beneficial effects are that:
1. according to the detection method provided by the invention, the crosslinked hyaluronic acid or the salt gel thereof is acidolyzed at 110-150 ℃, so that the problem of low solubility of a test sample is solved, and the accuracy of a detection result is improved.
2. According to the detection method provided by the invention, the crosslinked hyaluronic acid or the salt gel thereof after acidolysis is freeze-dried, the obtained freeze-dried sample is loose in state and easy to extract, and the ethyl acetate is used for extraction and extraction in the follow-up process, so that the solubility of inorganic salt and water in a solvent can be greatly reduced, the solution stability and the extraction rate are increased, and the accuracy of a detection result is further improved.
3. The detection method provided by the invention optimizes the gas chromatography conditions, adopts the RBX-5 chromatographic column to carry out gas chromatography, and sets the split ratio to be 1:1, thereby effectively improving the peak type and the separation degree of the gas chromatography and further improving the accuracy of the detection result.
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
A method for detecting the crosslinking degree of crosslinked hyaluronic acid or salt thereof gel comprises the following steps:
(1) Preparation of test solution
Respectively weighing 2.5140g, 2.5066g and 2.5230g of crosslinked sodium hyaluronate gel (the content of sodium hyaluronate in the crosslinked sodium hyaluronate gel is 19.42 mg/mL), respectively adding 5mL of 0.5M sulfuric acid into a headspace bottle (marked as sample 1, sample 2 and sample 3), performing acidolysis for 18 hours at 120 ℃ after capping, adding sodium hydroxide solution to neutralize to neutrality, pre-freezing for 5 hours at-30 ℃, heating to-10 ℃ for 2 hours, heating to-2 ℃ for 2 hours, continuously heating to 20 ℃ for 6 hours, finally heating to 40 ℃ for 22 hours, removing water in the sample, adding 10mL of ethyl acetate into the freeze-dried sample, extracting for 5 minutes, centrifuging for 5 minutes at 14000r/min, collecting supernatant, filtering to obtain sample solutions, and respectively filling into sample injection vials for later use;
(2) Preparation of glycerol standard solution
Precisely weighing 12.21mg of glycerol, placing in a 100ml measuring flask, adding ethyl acetate for dissolution, fixing the volume to a scale, and shaking uniformly to prepare standard substance stock solution with the concentration of 122.1 mug/ml; adding ethyl acetate into the stock solution of the standard substance, respectively diluting the stock solution into glycerol standard solutions with gradient concentrations of 6.10 mug/ml, 12.21 mug/ml, 24.42 mug/ml, 48.84 mug/ml and 97.68 mug/ml step by step, respectively marking the glycerol standard solutions as 1, 2, 3, 4 and 5, and respectively filling the glycerol standard solutions into sample injection vials for standby;
(3) Blank solution preparation
Measuring 5mL of 0.5M sulfuric acid in a headspace bottle, preparing 3 parts in parallel (marked as blank 1-3), performing acidolysis at 120 ℃ for 18 hours after capping, adding sodium hydroxide solution for neutralization to neutrality, pre-freezing at-30 ℃ for 5 hours, heating to-10 ℃ for 2 hours, then heating to-2 ℃ for 2 hours, continuously heating to 20 ℃ for 6 hours, finally heating to 40 ℃ for 22 hours, removing water in a sample, adding 10mL of ethyl acetate for extraction for 5 minutes, centrifuging for 5 minutes at 14000r/min, taking supernatant, filtering to obtain blank solution, and filling in a sample injection vial for standby; the blank solution refers to other components without the test sample;
(4) System suitability determination
Precisely measuring 1 μl of glycerol standard solution 3, injecting into a gas chromatograph, continuously measuring 5 times (each time, one needle is recorded as 3-1, 3-2, 3-3, 3-4 and 3-5), and calculating retention time and RSD of peak area of 5 needles of glycerol standard solution 3; after all samples were collected, 1 μl of glycerol standard solution 3 was precisely measured and injected into a gas chromatograph, and as a return reference, the retention time and RSD of the peak area of the return reference and the previous 5-needle glycerol standard solution 3 were calculated, and the results are shown in table 1.
(5) Detection of the degree of crosslinking
Carrying out gas chromatography determination on the sample solution, the blank solution and the glycerol standard solution to obtain peak areas of the sample solution, the blank solution and the glycerol standard solution;
the gas chromatography conditions were as follows: chromatographic column: RBX-5, 30 m.times.320 μm.times.0.25 μm; sample inlet temperature: 250 ℃; a detector: FID; detector temperature: 270 ℃; flow rate: 1ml/min; sample injection amount: 1 μl; column temperature: starting temperature: maintaining at 80deg.C for 5min, and heating to 260deg.C at 20deg.C/min for 15min; the split ratio is 1:1;
then drawing a glycerol standard working curve according to the peak area of the glycerol standard solution, wherein the peak area of the glycerol standard solution is specifically shown in table 2;
table 1, system applicability results
As is clear from Table 1, the retention times of glycerol standard solution 3 for the continuous 5-needle and the return control were 0.013% and 0.012%, respectively, and the peak areas were 0.84% and 1.10%, respectively, indicating that the system applicability was good.
TABLE 2 retention time and peak area of glycerol standard solutions
| Standard glycerol solution | Retention time/min | Peak area |
| 1 | 6.758 | 62.05 |
| 2 | 6.758 | 114.98 |
| 3 | 6.759 | 242.61 |
| 4 | 6.757 | 507.06 |
| 5 | 6.759 | 960.75 |
The glycerol standard working curve was plotted according to the data in table 2, with the peak area of glycerol as Y-axis, the concentration of the glycerol standard solution as X-axis, and the curve was y=9.8949x+2.9614, r 2 ＝0.9989；
Then calculating the concentration of glycerol in the sample according to a linear equation of a standard working curve; and finally, calculating the crosslinking degree of the crosslinked sodium hyaluronate gel according to the concentration of glycerol in the sample, wherein the calculation formula is as follows:
wherein:
C glycerol -concentration of glycerol in the crosslinked sodium hyaluronate gel, μg/mL;
v-volume of ethyl acetate added, mL;
rho-crosslinked sodium hyaluronate gel density, 1.01 g/mL;
omega-sodium hyaluronate content in crosslinked sodium hyaluronate gel, 19.42mg/mL;
m-mass of crosslinked sodium hyaluronate gel, g;
92.094 relative molecular mass of glycerol;
401.3-sodium hyaluronate repeat disaccharide unit relative molecular mass.
The calculation results of the blank solution (refer to other components not containing the sample), the peak area of the sample solution, and the degree of crosslinking are shown in Table 3.
TABLE 3 calculation results of peak area and crosslinking degree of sample solution
| Test article | Retention time | Sample weight/g | Peak area | Degree of crosslinking% |
| Blank 1 | / | / | / | / |
| Blank 2 | / | / | / | / |
| Blank 3 | / | / | / | / |
| Sample 1 | 6.757 | 2.5140 | 47.19 | 0.20 |
| Sample 2 | 6.759 | 2.5066 | 45.77 | 0.19 |
| Sample 3 | 6.757 | 2.5230 | 48.11 | 0.20 |
Example 2
A method for detecting the crosslinking degree of crosslinked hyaluronic acid or salt thereof gel comprises the following steps:
(1) Preparation of test solution
Weighing 2.5114g, 2.5127g and 2.5168g of crosslinked sodium hyaluronate gel (the content of sodium hyaluronate in the crosslinked sodium hyaluronate gel is 19.42 mg/mL) in a headspace bottle (marked as a sample 1, a sample 2 and a sample 3), respectively adding 5mL of 1M hydrobromic acid, performing acidolysis at 130 ℃ for 18 hours after capping, cooling to 25 ℃ after taking out, adding sodium hydroxide solution to neutralize to neutrality, pre-freezing at-30 ℃ for 5 hours, heating to-10 ℃ for 2 hours, heating to-2 ℃ for 2 hours, continuously heating to 20 ℃ for 6 hours, finally heating to 40 ℃ for 22 hours, removing water in the sample, adding 10mL of ethyl acetate into a freeze-dried sample for extraction for 5 minutes, centrifuging at 10000r/min for 5 minutes, taking supernatant, filtering to obtain sample solutions, and respectively filling into sample injection vials for later use;
(2) Preparation of glycerol standard solution
Precisely weighing 12.17mg of glycerol, placing in a 100ml measuring flask, adding ethyl acetate for dissolution, fixing the volume to a scale, and shaking uniformly to prepare a standard stock solution with the concentration of 121.7 mug/ml; adding ethyl acetate into the stock solution of the standard substance, respectively diluting the stock solution into glycerol standard solutions with gradient concentrations of 6.08 mug/ml, 12.17 mug/ml, 24.34 mug/ml, 48.68 mug/ml and 97.36 mug/ml step by step, respectively marking the glycerol standard solutions as 1, 2, 3, 4 and 5, and respectively filling the glycerol standard solutions into sample injection vials for standby;
(3) System suitability determination
Precisely measuring 1 μl of glycerol standard solution 3, injecting into a gas chromatograph, continuously measuring 5 times (each time, one needle is recorded as 3-1, 3-2, 3-3, 3-4 and 3-5), and calculating retention time and RSD of peak area of 5 needles of glycerol standard solution 3; after all samples were collected, 1 μl of glycerol standard solution 3 was precisely measured and injected into a gas chromatograph, and as a return reference, the retention time and RSD of the peak area of the return reference and the previous 5-needle glycerol standard solution 3 were calculated, and the results are shown in table 4.
(4) Detection of the degree of crosslinking
Carrying out gas chromatography determination on the sample solution and the glycerol standard solution to obtain peak areas of the sample solution and the glycerol standard solution;
the gas chromatography conditions were as follows: chromatographic column: RBX-5, 30 m.times.320 μm.times.0.25 μm; sample inlet temperature: 250 ℃; a detector: FID; detector temperature: 270 ℃; flow rate: 1ml/min; sample injection amount: 1 μl; column temperature: starting temperature: maintaining at 80deg.C for 5min, and heating to 260deg.C at 20deg.C/min for 15min; the split ratio is 1:1;
then drawing a glycerol standard working curve according to the peak area of the glycerol standard solution, wherein the peak area of the glycerol standard solution is specifically shown in table 5;
table 4, system applicability results
As is clear from Table 4, the retention times of glycerol standard solution 3 for the continuous 5-needle and the return-needle controls were 0.014% and 0.013% respectively, and the peak areas were 0.21% and 0.34% respectively, indicating that the system applicability was good.
TABLE 5 retention time and peak area of glycerol standard solutions
| Standard glycerol solution | Retention time/min | Peak area |
| 1 | 6.765 | 57.61 |
| 2 | 6.764 | 121.07 |
| 3 | 6.765 | 238.50 |
| 4 | 6.765 | 510.39 |
| 5 | 6.764 | 992.17 |
The standard working curve of glycerol was plotted according to the data in table 5, with the peak area of glycerol as Y-axis, the concentration of glycerol standard solution as X-axis, and the curve was y= 10.2813X-3.9354, r 2 ＝0.9995；
Then calculating the concentration of glycerol in the test sample according to a linear equation; and finally, calculating the crosslinking degree of the crosslinked sodium hyaluronate gel according to the concentration of glycerol in the sample, wherein the calculation formula is as follows:
wherein:
C glycerol -concentration of glycerol in the crosslinked sodium hyaluronate gel, μg/mL;
v-volume of ethyl acetate added, mL;
rho-crosslinked sodium hyaluronate gel density, 1.01 g/mL;
omega-sodium hyaluronate content in crosslinked sodium hyaluronate gel, 19.42mg/mL;
m-mass of crosslinked sodium hyaluronate gel, g;
92.094 relative molecular mass of glycerol;
401.3-sodium hyaluronate repeat disaccharide unit relative molecular mass.
The peak areas and the results of the calculation of the degree of crosslinking of the sample solutions are shown in Table 6.
TABLE 6 calculation results of peak area and crosslinking degree of sample solution
| Test article | Retention time | Sample weight/g | Peak area | Degree of crosslinking% |
| Sample 1 | 6.765 | 2.5114 | 121.92 | 0.55 |
| Sample 2 | 6.765 | 2.5127 | 124.75 | 0.56 |
| Sample 3 | 6.766 | 2.5168 | 128.87 | 0.58 |
Comparative example 1
The degree of crosslinking of the crosslinked sodium hyaluronate gel was determined by reference to the method for detecting the degree of crosslinking of gel provided in chinese patent document CN114280193a, specifically as follows:
(1) Preparation of test solution
Weighing 2.5480g, 2.5405g and 2.5490g of crosslinked sodium hyaluronate gel (the content of sodium hyaluronate in the crosslinked sodium hyaluronate gel is 19.42 mg/mL), respectively adding 5mL of 0.5M sulfuric acid into a headspace bottle (marked as sample 1, sample 2 and sample 3), performing acidolysis for 15 hours at normal temperature after capping, adding sodium hydroxide solution to neutralize to neutrality, evaporating to dryness in a constant-temperature magnetic stirrer (55 ℃), cooling to 25 ℃, adding 10mL of absolute ethyl alcohol for extraction, centrifuging for 5 minutes at 14000r/min, taking supernatant, filtering to obtain a sample solution, and filling the sample solution into a sample injection vial for standby;
(2) Preparation of glycerol standard solution
Precisely weighing 11.91mg of glycerol, placing into a 100ml measuring flask, adding absolute ethanol for dissolution, fixing the volume to a scale, and shaking uniformly to prepare a standard stock solution with the concentration of 119.1 mug/ml; adding absolute ethyl alcohol into stock solution of standard substance, respectively diluting stepwise into glycerol standard solution with gradient concentration of 5.96 μg/ml, 11.91 μg/ml, 23.82 μg/ml, 47.64 μg/ml, 95.28 μg/ml, respectively marking as 1, 2, 3, 4, 5, respectively, and respectively filling into sample injection vials for use;
(3) Blank solution preparation
Weighing 5ml of 0.5M sulfuric acid in a headspace bottle, preparing 3 parts (marked as blank 1-3) in parallel, performing acidolysis at normal temperature for 15 hours after capping, adding sodium hydroxide solution for neutralization to neutrality, evaporating to dryness in a constant temperature magnetic stirrer (55 ℃), cooling to 25 ℃, adding 10ml of absolute ethyl alcohol for extraction, centrifuging for 5min at 14000r/min, taking supernatant, filtering to obtain blank solution, and filling in a sample injection vial for later use; the blank solution refers to other components without the test sample;
(4) System suitability determination
Precisely measuring 1 μl of glycerol standard solution 3, injecting into a gas chromatograph, continuously measuring 5 times (each time, one needle is recorded as 3-1, 3-2, 3-3, 3-4 and 3-5), and calculating retention time and RSD of peak area of 5 needles of glycerol standard solution 3; after all samples were collected, 1 μl of glycerol standard solution 3 was precisely measured and injected into a gas chromatograph, and as a return reference, the retention time and RSD of the peak area of the return reference and the previous 5-needle glycerol standard solution 3 were calculated, and the results are shown in table 7.
(5) Detection of the degree of crosslinking
Carrying out gas chromatography determination on the sample solution, the blank solution and the glycerol standard solution to obtain peak areas of the sample solution, the blank solution and the glycerol standard solution;
the gas chromatography conditions were as follows: chromatographic column: DB-FFAP,30m x 320 m x 0.5 m; sample inlet temperature: 250 ℃; a detector: FID; detector temperature: 270 ℃; flow rate: 2ml/min; sample injection amount: 2 μl; column temperature: starting temperature: raising the temperature to 220 ℃ at the speed of 30 ℃/min and keeping the temperature for 5min; split ratio: not split;
then drawing a glycerol standard working curve according to the peak area of the glycerol standard solution, wherein the peak area of the glycerol standard solution is specifically shown in table 8;
TABLE 7 System applicability results
As can be seen from Table 7, the retention times of glycerol standard solution 3 for the continuous 5-needle and the return-needle controls were 0.021% and 0.018% respectively, and the peak areas were 1.13% and 41.39% respectively. The method of comparative example 1 showed poor system applicability and reduced instrument response.
TABLE 8 retention time and peak area of glycerol standard solutions
| Standard glycerol solution | Retention time/min | Peak area |
| 1 | 8.216 | 8953.89 |
| 2 | 8.218 | 18210.56 |
| 3 | 8.216 | 37695.86 |
| 4 | 8.215 | 79161.31 |
| 5 | 8.217 | 150406.48 |
The peak area of glycerol was taken as the Y axis, and the concentration of the glycerol standard solution was taken as the X axis, and the data shown in Table 8 were plottedGlycerol standard working curve, y=1591.7978x+114.8514, r 2 ＝0.9990；
Then calculating the concentration of glycerol in the test sample according to a linear equation; and finally, calculating the crosslinking degree of the crosslinked sodium hyaluronate gel according to the concentration of glycerol in the sample and a formula provided by Chinese patent document CN 114280193A.
The calculation results of the blank solution (refer to other components not containing the sample), the peak area of the sample solution, and the degree of crosslinking are shown in Table 9.
TABLE 9 calculation results of peak area and crosslinking degree of blank and sample solution
| Test article | Retention time | Sample weight/g | Peak area | Degree of crosslinking% |
| Blank 1 | 8.266 | / | 23.11 | / |
| Blank 2 | 8.264 | / | 28.89 | / |
| Blank 3 | 8.268 | / | 21.07 | / |
| Sample 1 | 8.214 | 2.5480 | 1300.08 | 0.033 |
| Sample 2 | 8.216 | 2.5405 | 970.63 | 0.024 |
| Sample 3 | 8.214 | 2.5490 | 265.08 | 0.004 |
As can be seen from the comparison of the data of the comparative example 1 and the data of the example 1, the white solution in the comparative example 1 has interference near the peak of the test sample, and the peak area of the glycerol in 3 test sample solutions in the comparative example 1 gradually and obviously decreases, which means that as the sample collection times increase, the response of the instrument gradually decreases, and the gap between the system applicability result and the test sample solution before collection is larger. The blank solution obtained by the method of the embodiment 1 is basically free of interference, the peak area of glycerol in 3 parts of sample solution is normal, the response of the instrument is normal, and the applicability of the system before and after the sample solution is collected is qualified.
Test example 1, repeatability verification
1. And (3) repeatability verification of the reference substance:
(1) Preparation of test solutions
Weighing 12.20mg, 11.63mg, 12.50mg, 11.51mg, 12.30mg and 12.02mg of glycerol respectively in a headspace bottle (marked as samples 1-6), adding 5mL of 1M hydrobromic acid respectively, acidolyzing at 120 ℃ for 18 hours after capping, cooling to 25 ℃, adding sodium hydroxide solution for neutralization, pre-freezing at-30 ℃ for 5 hours, heating to-10 ℃ for 2 hours, heating to-2 ℃ for 2 hours, continuously heating to 20 ℃ for 6 hours, heating to 40 ℃ for 22 hours, removing water in the samples, adding 100mL of ethyl acetate into the freeze-dried samples for extraction for 5 minutes, centrifuging at 8000r/min for 10 minutes, taking supernatant, filtering to obtain test solutions, and respectively filling the test solutions into sample injection vials for later use;
(2) Preparation of glycerol standard solution
Precisely weighing 25.07mg of glycerol, placing in a 100ml measuring flask, adding ethyl acetate for dissolution, fixing the volume to a scale, and shaking uniformly to prepare a standard substance stock solution with the concentration of 250.7 mug/ml; adding ethyl acetate into the stock solution of the standard substance, respectively diluting the stock solution into glycerol standard solutions with gradient concentrations of 50.14 mug/ml, 100.28 mug/ml, 120.34 mug/ml, 150.42 mug/ml and 250.70 mug/ml in a stepwise manner, respectively marking the glycerol standard solutions as 1, 2, 3, 4 and 5, and respectively filling the glycerol standard solutions into sample injection vials for later use;
(3) System suitability determination
Precisely measuring 1 μl of glycerol standard solution 3, injecting into a gas chromatograph, continuously measuring 5 times (each time, one needle is recorded as 3-1, 3-2, 3-3, 3-4 and 3-5), and calculating retention time and RSD of peak area of 5 needles of glycerol standard solution 3; after all samples were collected, 1 μl of glycerol standard solution 3 was precisely measured and injected into a gas chromatograph, and as a return reference, the retention time and RSD of the peak area of the return reference and the previous 5-needle glycerol standard solution 3 were calculated, and the results are shown in table 10.
(4) Detection of the degree of crosslinking
Carrying out gas chromatography determination on the test solution and the glycerol standard solution to obtain peak areas of the test solution and the glycerol standard solution;
the gas chromatography conditions were as follows: chromatographic column: RBX-5, 30 m.times.320 μm.times.0.25 μm; sample inlet temperature: 250 ℃; a detector: FID; detector temperature: 270 ℃; flow rate: 1ml/min; sample injection amount: 1 μl; column temperature: starting temperature: maintaining at 80deg.C for 5min, and heating to 260deg.C at 20deg.C/min for 15min; the split ratio is 1:1;
then drawing a glycerol standard working curve according to the peak area of the glycerol standard solution, wherein the peak area of the glycerol standard solution is specifically shown in table 11;
table 10, system applicability results
As is clear from Table 10, the retention times of glycerol standard solution 3 for continuous 5-needle and return-needle controls were 0.043% and 0.040% for RSD, and 0.47% and 0.53% for peak area, respectively, and the system applicability results were good.
TABLE 11 retention time and peak area of glycerol standard solutions
| Standard glycerol solution | Retention time/min | Peak area |
| 1 | 6.764 | 502.49 |
| 2 | 6.761 | 1021.32 |
| 3 | 6.763 | 1256.23 |
| 4 | 6.765 | 1545.16 |
| 5 | 6.766 | 2512.45 |
The glycerol standard working curve was plotted according to the data in table 11, with the peak area of glycerol as Y-axis, the concentration of the glycerol standard solution as X-axis, and the curve was y=9.9922x+24.8299, r 2 ＝0.9991；
Then calculating the concentration of glycerol in the test solution according to a linear equation; and finally, calculating the recovery rate according to the concentration of glycerol in the test solution, wherein the calculation formula is as follows:
wherein:
C glycerol -concentration of glycerol in the test sample, μg/mL;
m-mass of glycerol, mg.
The peak area and recovery rate calculation results of the test solutions are specifically shown in table 12.
TABLE 12 Peak area and recovery calculation results for test solutions
As shown in Table 12, the average recovery rate was 100.4% and RSD was 0.80% as measured by continuous sample injection of 6 parts of the test solution, indicating that the detection method provided by the invention has good repeatability.
2. Test article repeatability verification:
(1) Preparation of glycerol standard solution
Precisely weighing 12.59mg of glycerol, placing in a 100ml measuring flask, adding ethyl acetate for dissolution, fixing the volume to a scale, and shaking uniformly to prepare a standard stock solution with the concentration of 125.9 mug/ml; adding ethyl acetate into the stock solution of the standard substance, respectively diluting the stock solution into glycerol standard solutions with gradient concentrations of 6.30 mug/ml, 12.59 mug/ml, 25.18 mug/ml, 50.36 mug/ml and 100.72 mug/ml step by step, respectively marking the glycerol standard solutions as 1, 2, 3, 4 and 5, and respectively filling the glycerol standard solutions into sample injection vials for standby;
(2) Preparation of test solutions
Weighing 2.5106g, 2.5119g, 2.5006g, 2.5214g, 2.5149g and 2.5318g of crosslinked sodium hyaluronate gel respectively, putting the gel into a headspace bottle (marked as a sample 1-6), adding 1mL of glycerol standard solution 2 respectively, adding 5mL of 1M hydrobromic acid respectively, acidolyzing the gel at 130 ℃ for 18h after capping, taking out the gel, cooling the gel to 25 ℃, adding sodium hydroxide solution for neutralizing the gel, pre-freezing the gel at-30 ℃ for 5h, heating the gel to-10 ℃ for 2h, heating the gel to-2 ℃ for 2h, continuing to heat the gel to 20 ℃ for 6h, finally heating the gel to 40 ℃ for 22h, removing water in the sample, adding 10mL of ethyl acetate into the freeze-dried sample for extraction for 5min, centrifuging the gel at 10000r/min, filtering the supernatant to obtain a test solution, and respectively putting the test solution into a sample injection vial for standby;
(3) System suitability determination
Precisely measuring 1 μl of glycerol standard solution 3, injecting into a gas chromatograph, continuously measuring 5 times (each time, one needle is recorded as 3-1, 3-2, 3-3, 3-4 and 3-5), and calculating retention time and RSD of peak area of 5 needles of glycerol standard solution 3; after all samples were collected, 1 μl of glycerol standard solution 3 was precisely measured and injected into a gas chromatograph, and as a return reference, the retention time and RSD of the peak area of the return reference and the previous 5-needle glycerol standard solution 3 were calculated, and the results are shown in table 13.
(4) Detection of the degree of crosslinking
Carrying out gas chromatography determination on the test solution and the glycerol standard solution to obtain peak areas of the test solution and the glycerol standard solution;
the gas chromatography conditions were as follows: chromatographic column: RBX-5, 30 m.times.320 μm.times.0.25 μm; sample inlet temperature: 250 ℃; a detector: FID; detector temperature: 270 ℃; flow rate: 1ml/min; sample injection amount: 1 μl; column temperature: starting temperature: maintaining at 80deg.C for 5min, and heating to 260deg.C at 20deg.C/min for 15min; the split ratio is 1:1;
then drawing a glycerol standard working curve according to the peak area of the glycerol standard solution, wherein the peak area of the glycerol standard solution is specifically shown in table 14;
TABLE 13 System applicability results
As is clear from Table 13, the retention times of glycerol standard solution 3 for the continuous 5-needle and the return-needle controls were 0.013% and 0.018% respectively, and the peak areas were 0.86% and 1.16% respectively, and the system applicability results were good.
TABLE 14 retention time and peak area of glycerol standard solutions
| Standard glycerol solution | Retention time/min | Peak area |
| 1 | 6.761 | 62.18 |
| 2 | 6.761 | 124.36 |
| 3 | 6.760 | 261.16 |
| 4 | 6.763 | 496.21 |
| 5 | 6.759 | 1044.65 |
The glycerol standard working curve is drawn according to the data in table 14, with the peak area of glycerol as Y axis, the concentration of the glycerol standard solution as X axis, and the curve is y= 10.3668X-6.8918, r 2 ＝0.9992；
Then calculating the concentration of glycerol in the test solution according to a linear equation; and finally, calculating the recovery rate of the test solution according to the concentration of glycerol in the test sample, wherein the calculation formula is as follows:
wherein:
C measured quantity -the concentration of glycerol in the test solution, μg/mL;
C amount of detection -concentration of glycerol in the test sample, 12.5583 μg/mL;
C addition amount of -glycerol concentration added, μg/mL;
the peak area and recovery rate calculation results of the test solutions are specifically shown in table 15.
TABLE 15 Peak area and recovery calculation results for test solutions
As shown in Table 15, the average recovery rate was 98.9% and RSD was 0.87% as measured by continuous sample injection of 6 parts of the test solution, indicating that the detection method provided by the invention has good repeatability.
Test example 2, verification of accuracy
The invention adopts commercial crosslinked sodium hyaluronate gel with the crosslinking degree of 1 percent (the content of sodium hyaluronate in the crosslinked sodium hyaluronate gel is 20.13 mg/mL) for accuracy verification, and adopts the method of the invention and the method of Chinese patent document CN114280193A for verification respectively, and the specific process is as follows:
1. the method of the invention comprises the following steps:
(1) Preparation of test solution
Weighing 2.5084g, 2.5153g, 2.5065g, 2.5206g, 2.5332g and 2.5184g of crosslinked sodium hyaluronate gel in a headspace bottle (marked as sample 1, sample 2, sample 3, sample 4, sample 5 and sample 6), respectively adding 5mL of 1M hydrobromic acid, carrying out acidolysis for 24h (sample 1, sample 2 and sample 3) and 36h (sample 4, sample 5 and sample 6) at 120 ℃ after capping, cooling to 25 ℃ after taking out, adding sodium hydroxide solution for neutralization, pre-freezing for 5h at-30 ℃, heating to-10 ℃ for 2h, heating to-2 ℃ for 2h, continuing to heat to 20 ℃ for 6h, finally heating to 40 ℃ for 22h, removing water in the samples, then adding 10mL of ethyl acetate into the freeze-dried samples for extraction for 5min, centrifuging for 5min at 14000r/min, taking supernatant, filtering to obtain sample solutions, and respectively loading the sample solutions into sample injection vials for later use;
(2) Preparation of glycerol standard solution
Precisely weighing 12.36mg of glycerol, placing in a 100ml measuring flask, adding ethyl acetate for dissolution, fixing the volume to a scale, and shaking uniformly to prepare a standard stock solution with the concentration of 123.6 mug/ml; adding ethyl acetate into standard stock solution, respectively diluting stepwise into glycerol standard solutions with gradient concentration of 6.18 μg/ml, 12.36 μg/ml, 24.72 μg/ml, 49.44 μg/ml and 98.88 μg/ml, respectively marking as 1, 2, 3, 4 and 5, and filling into sample injection vials for standby;
(3) Blank solution preparation
Measuring 5mL of 1M hydrobromic acid in a headspace bottle, preparing 3 parts in parallel (marked as blank 1-3), performing acidolysis at 120 ℃ for 36 hours after capping, adding sodium hydroxide solution for neutralization to neutrality, performing prefreezing at-30 ℃ for 5 hours, heating to-10 ℃ for 2 hours, then heating to-2 ℃ for 2 hours, continuously heating to 20 ℃ for 6 hours, finally heating to 40 ℃ for 22 hours, removing water in a sample, adding 10mL of ethyl acetate for extraction for 5 minutes, performing centrifugation at 14000r/min for 5 minutes, taking supernatant, filtering to obtain blank solution, and filling the blank solution in a sample injection vial for later use; the blank solution refers to other components without the test sample;
(4) System suitability determination
Precisely measuring 1 μl of glycerol standard solution 3, injecting into a gas chromatograph, continuously measuring 5 times (each time, one needle is recorded as 3-1, 3-2, 3-3, 3-4 and 3-5), and calculating retention time and RSD of peak area of 5 needles of glycerol standard solution 3; after all samples were collected, 1 μl of glycerol standard solution 3 was precisely measured and injected into a gas chromatograph, and as a return reference, the retention time and RSD of the peak area of the return reference and the previous 5-needle glycerol standard solution 3 were calculated, and the results are shown in table 16.
(5) Detection of the degree of crosslinking
Carrying out gas chromatography determination on the sample solution, the blank solution and the glycerol standard solution to obtain peak areas of the sample solution, the blank solution and the glycerol standard solution;
the gas chromatography conditions were as follows: chromatographic column: RBX-5, 30 m.times.320 μm.times.0.25 μm; sample inlet temperature: 250 ℃; a detector: FID; detector temperature: 270 ℃; flow rate: 1ml/min; sample injection amount: 1 μl; column temperature: starting temperature: maintaining at 80deg.C for 5min, and heating to 260deg.C at 20deg.C/min for 15min; the split ratio is 1:1;
then drawing a glycerol standard working curve according to the peak area of the glycerol standard solution, wherein the peak area of the glycerol standard solution is specifically shown in table 17;
table 16, system applicability results
As is clear from Table 16, the retention times of glycerol standard solution 3 for the continuous 5-needle and the return-needle controls were 0.014% and 0.013% respectively, and the peak areas were 0.77% and 0.69% respectively, indicating that the system applicability results were good.
TABLE 17 retention time and peak area of glycerol standard solutions
| Standard glycerol solution | Retention time/min | Peak area |
| 1 | 6.757 | 62.31 |
| 2 | 6.757 | 119.31 |
| 3 | 6.756 | 251.74 |
| 4 | 6.757 | 498.45 |
| 5 | 6.758 | 979.27 |
The glycerol standard working curve was plotted according to the data in table 17, with the peak area of glycerol as Y-axis, the concentration of the glycerol standard solution as X-axis, and the curve was y=9.9122x+2.4193, r 2 ＝0.9998；
Then calculating the concentration of glycerol in the test sample according to a linear equation; and finally, calculating the crosslinking degree of the crosslinked sodium hyaluronate gel according to the concentration of glycerol in the sample, wherein the calculation formula is as follows:
wherein:
C glycerol -concentration of glycerol in the crosslinked sodium hyaluronate gel, μg/mL;
v-volume of ethyl acetate added, mL;
rho-crosslinked sodium hyaluronate gel density, 1.01 g/mL;
omega-sodium hyaluronate content in crosslinked sodium hyaluronate gel, 20.13mg/mL;
m-mass of crosslinked sodium hyaluronate gel, g;
92.094 relative molecular mass of glycerol;
401.3-sodium hyaluronate repeat disaccharide unit relative molecular mass.
The calculation results of the blank solution (refer to the other components not containing the sample), the peak area of the sample solution, and the degree of crosslinking are shown in table 18.
TABLE 18 calculation results of peak area and crosslinking degree of blank and sample solution
2. The method of the Chinese patent document CN114280193A is specifically as follows:
(1) Preparation of test solution
Weighing 2.5067g, 2.5055g, 2.5149g, 2.5142g, 2.5231g and 2.5160g of crosslinked sodium hyaluronate gel, respectively adding 5ml of 1M hydrobromic acid into a headspace bottle (marked as sample 1, sample 2, sample 3, sample 4, sample 5 and sample 6), performing acidolysis for 24 hours (sample 1, sample 2, sample 3) and 36 hours (sample 4, sample 5 and sample 6) at 120 ℃ after capping, adding sodium hydroxide solution to neutralize to neutrality, evaporating to dryness at a constant temperature magnetic stirrer (55 ℃), cooling to 25 ℃, adding 10ml of absolute ethyl alcohol for extraction, centrifuging for 5 minutes at 14000r/min, collecting supernatant, filtering to obtain sample solution, and putting into a sample injection vial for standby;
(2) Preparation of glycerol standard solution
Precisely weighing 12.24mg of glycerol, placing in a 100ml measuring flask, adding absolute ethanol for dissolution, fixing the volume to a scale, and shaking uniformly to prepare standard stock solution with the concentration of 122.4 mug/ml; adding absolute ethyl alcohol into stock solution of standard substance, respectively diluting into glycerol standard solution with gradient concentration of 6.12 μg/ml, 12.24 μg/ml, 24.48 μg/ml, 48.96 μg/ml and 97.92 μg/ml, respectively marking as 1, 2, 3, 4 and 5, respectively filling into sample injection vials for use;
(3) Blank solution preparation
Weighing 5ml of 1M hydrobromic acid in a headspace bottle, preparing 3 parts (marked as blank 1-3) in parallel, performing acidolysis for 36 hours at 120 ℃ after capping, adding sodium hydroxide solution for neutralization to neutrality, evaporating to dryness at a constant temperature magnetic stirrer (55 ℃), cooling to 25 ℃, adding 10ml of absolute ethyl alcohol for extraction, centrifuging for 5min at 14000r/min, taking supernatant, filtering to obtain blank solution, and filling in a sample injection vial for later use; the blank solution refers to other components without the test sample;
(4) System suitability determination
Precisely measuring 1 μl of glycerol standard solution 3, injecting into a gas chromatograph, continuously measuring 5 times (each time, one needle is recorded as 3-1, 3-2, 3-3, 3-4 and 3-5), and calculating retention time and RSD of peak area of 5 needles of glycerol standard solution 3; after all samples were collected, 1 μl of glycerol standard solution 3 was precisely measured and injected into a gas chromatograph, and as a return reference, the retention time and RSD of the peak area of the return reference and the previous 5-needle glycerol standard solution 3 were calculated, and the results are shown in table 19.
(5) Detection of the degree of crosslinking
Carrying out gas chromatography determination on the sample solution, the blank solution and the glycerol standard solution to obtain peak areas of the sample solution, the blank solution and the glycerol standard solution;
the gas chromatography conditions were as follows: chromatographic column: DB-FFAP,30m x 320 m x 0.5 m; sample inlet temperature: 250 ℃; a detector: FID; detector temperature: 270 ℃; flow rate: 2ml/min; sample injection amount: 2 μl; column temperature: starting temperature: raising the temperature to 220 ℃ at the speed of 30 ℃/min and keeping the temperature for 5min; split ratio: not split;
then drawing a glycerol standard working curve according to the peak area of the glycerol standard solution, wherein the peak area of the glycerol standard solution is specifically shown in table 20;
table 19, system applicability results
As can be seen from Table 20, the retention times of glycerol standard solution 3 for the continuous 5-needle and the return-needle controls were 0.024% and 0.023%, respectively, and the peak areas were 0.80% and 47.50% respectively. The method described in the Chinese patent document CN114280193A has poor system applicability and reduced instrument response.
TABLE 20 retention time and peak area of glycerol standard solutions
| Standard glycerol solution | Retention time/min | Peak area |
| 1 | 8.211 | 9158.58 |
| 2 | 8.213 | 18915.46 |
| 3 | 8.211 | 37641.77 |
| 4 | 8.211 | 79047.71 |
| 5 | 8.214 | 150190.65 |
The glycerol standard working curve was plotted according to the data in table 20, with the peak area of glycerol as Y-axis, the concentration of the glycerol standard solution as X-axis, and the curve was y=1542.0453x+479.4669, r 2 ＝0.9991；
Then calculating the concentration of glycerol in the test sample according to a linear equation; finally, the crosslinking degree of the sample is calculated according to the concentration of glycerol in the sample and a formula provided in Chinese patent document CN 114280193A.
The calculation results of the blank solution (refer to the other components not containing the sample), the peak area of the sample solution, and the degree of crosslinking are shown in table 21.
Table 21, blank, results of calculation of peak area and crosslinking degree of sample solution
| Test article | Retention time | Sample weight/g | Peak area | Degree of crosslinking% |
| Blank 1 | 8.221 | / | 1986.24 | / |
| Blank 2 | 8.220 | / | 1793.76 | / |
| Blank 3 | 8.219 | / | 1680.51 | / |
| Sample 1 | 8.210 | 2.5067 | 80835.83 | 1.14 |
| Sample 2 | 8.213 | 2.5055 | 60963.52 | 0.86 |
| Sample 3 | 8.212 | 2.5149 | 45632.28 | 0.64 |
| Sample 4 | 8.212 | 2.5142 | 40667.35 | 0.57 |
| Sample 5 | 8.214 | 2.5231 | 28943.07 | 0.40 |
| Sample 6 | 8.216 | 2.5160 | 9365.66 | 0.13 |
As can be seen from the comparison of the data in Table 18 and Table 21 of the experimental example, the blank solution in the detection method provided by the invention has no interference at the peak of glycerol, and the crosslinking degree of the test sample after acidolysis for 24h and 36h at 120 ℃ has no obvious difference, which indicates that the test sample is completely acidolyzed at 24h, and the crosslinking degree is 1.09-1.10% and the error is not more than 10%. In the method described in the Chinese patent document CN114280193A, the blank solution is obviously interfered at the position of the glycerol peak, the crosslinking degree is finally measured to be 0.13-1.14%, the error is obviously more than 10%, and the error is larger and larger along with the increase of the acquisition times. According to the detection method provided by the invention, through increasing the acidolysis temperature and freeze-drying the crosslinked hyaluronic acid or the salt gel thereof after acidolysis, the sample state is loose and easy to extract, and the ethyl acetate is adopted for extraction and extraction in the follow-up process, the solubility of inorganic salt and water in a solvent can be greatly reduced, the solution stability and the extraction rate are increased, and the accuracy of a detection result is effectively improved.
Claims (8)
1. The method for detecting the crosslinking degree of the crosslinked hyaluronic acid or the salt gel thereof is characterized by comprising the following steps:
(1) Preparation of test solution
Acidolysis is carried out on crosslinked hyaluronic acid or salt gel thereof for 12-36 hours at 110-150 ℃, cooling is carried out to 20-30 ℃ and then neutralization is carried out to neutrality, and freeze-drying is carried out to obtain a freeze-dried sample; extracting the freeze-dried sample by using ethyl acetate, centrifuging, taking a supernatant, and filtering to obtain a sample solution;
(2) Preparation of glycerol standard solution
Gradually diluting the glycerol standard substance with ethyl acetate to obtain glycerol standard solutions with different concentrations;
(3) Detection of the degree of crosslinking
Carrying out gas chromatography determination on the sample solution and the glycerol standard solution to obtain peak areas of the sample solution and the glycerol standard solution, drawing a glycerol standard working curve according to the peak areas of the glycerol standard solution, and calculating the concentration of glycerol in the sample solution according to the standard curve; and finally, calculating the crosslinking degree of the sample according to the concentration of glycerol in the sample, wherein the calculation formula is as follows:
wherein:
C glycerol -concentration of glycerol in the crosslinked sodium hyaluronate gel, μg/mL;
v-volume of ethyl acetate added, mL;
rho-crosslinked sodium hyaluronate gel density, 1.01 g/mL;
omega-sodium hyaluronate content in crosslinked sodium hyaluronate gel, mg/mL;
m-mass of crosslinked sodium hyaluronate gel, g;
92.094 relative molecular mass of glycerol;
401.3-sodium hyaluronate repeat disaccharide unit relative molecular mass.
2. The method of claim 1, wherein in step (1), the acidolysis is: adding hydrobromic acid or sulfuric acid into the crosslinked hyaluronic acid or the salt gel thereof, and acidolysis is carried out for 18-24 h at 120-130 ℃.
3. The method according to claim 2, wherein the concentration of hydrobromic or sulfuric acid is 0.4 to 1M; the mass volume ratio of the crosslinked hyaluronic acid or the salt gel thereof to hydrobromic acid or sulfuric acid is (2-3): (5-10), unit: g/mL.
4. The method of claim 1, wherein in step (1), the lyophilization process is: pre-freezing for 5-10 h at-30 ℃, heating to-10 ℃ for 2-5 h, heating to-2 ℃ for 2-5 h, continuously heating to 20 ℃ for 6-10 h, heating to 40 ℃ for 20-45 h, and removing water in the sample.
5. The method according to claim 1, wherein in the step (1), the mass/volume ratio of the crosslinked hyaluronic acid or its salt gel to ethyl acetate is (2 to 3): (10-20), unit: g/mL.
6. The method according to claim 1, wherein in the step (1), the extraction time is 5 to 10 minutes; the centrifugation is carried out for 5-10 min at 8000-14000 r/min.
7. The method according to claim 1, wherein in the step (2), the concentration of the glycerol standard solution is set to a series of concentrations of 6 to 96 μg/ml.
8. The method of claim 1, wherein in step (2), the gas chromatography conditions are as follows:
chromatographic column: RBX-5, 30 m.times.320 μm.times.0.25 μm; sample inlet temperature: 250-270 ℃; a detector: a hydrogen flame ionization detector; detector temperature: 270-290 ℃; flow rate: 0.5-1.0 ml/min; sample injection amount: 1 μl; column temperature: starting temperature: maintaining at 70-90 deg.c for 5min and maintaining at 10-20 deg.c/min to 260 deg.c for 15min; the split ratio is 1:1.
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