Method for detecting residual quantity of N-methylmorpholine in gel

Abstract

The method for detecting the residual quantity of N-methylmorpholine in the gel is high in specificity, high in sensitivity, good in linear relation, good in precision, high in accuracy, high in solution stability, good in durability and accurate and reliable in measurement result and aims at effectively controlling the quality, effectiveness and safety of related medical beauty products.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine detection, and particularly relates to a method for detecting residual amount of N-methylmorpholine in gel. BACKGROUND

[0002] Medical beauty is the fastest and most effective method to improve skin defects and return to youth. Hyaluronic acid is a popular ingredient, which has a strong water-locking function and can bind about 1000 times its own weight of water. When injected into the dermis, it can quickly fill the skin tissue, play a role in water retention, physical support and promotion of collagen regeneration. Recombinant collagen can promote the proliferation and repair of fibroblasts, and has good biocompatibility, biodegradability and biological activity. Injection of recombinant collagen into the dermis can supplement collagen in the skin in situ, improve skin quality, reduce static wrinkles, and improve skin laxity and elasticity.

[0003] Hyaluronic acid and recombinant collagen are both main components of the skin. If a recombinant collagen-hyaluronic acid sodium cross-linked gel can be provided, it can not only achieve immediate repair and correction of the profile to achieve satisfactory results, but also enable the amino acids and peptide components produced by the degradation of recombinant collagen in the cross-linked gel to achieve new collagen synthesis and tissue repair and remodeling. CN118483405A uses sodium hyaluronate and recombinant collagen as raw materials, and uses 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (DMTMM) as a condensing agent to promote the condensation reaction between recombinant collagen and sodium hyaluronate, form amide bonds, and obtain a recombinant collagen-hyaluronic acid sodium cross-linked gel with high viscoelasticity and shear viscosity, thereby better improving the skin condition.

[0004] When DMTMM is used as a condensing agent to participate in the cross-linking reaction between recombinant collagen and sodium hyaluronate, two by-products, N-methylmorpholine and 4,6-dimethoxy-1,3,5-triazin-2(1H)-one, are often produced. Both of these by-products are toxic substances, so strict quality control is required. The specific structures of DMTMM, N-methylmorpholine and 4,6-dimethoxy-1,3,5-triazin-2(1H)-one are shown in Table 1.

[0005] Table 1

[0006]

[0007] At present, there is no detection method for the residual amount of N-methylmorpholine in the prior art. Moreover, gas chromatography analysis has specificity and specificity, therefore, it is necessary and urgent to establish a gas chromatography analysis method for detecting the residual amount of N-methylmorpholine in the gel. SUMMARY

[0008] The present application aims to overcome the defects of the prior art, and provide a gas chromatography method for detecting the residual amount of N-methylmorpholine in a gel, which has strong specificity, high sensitivity, good linear relationship, good precision, high accuracy, high solution stability, good durability, and accurate and reliable determination results.

[0009] To achieve the above-mentioned application purposes, the present application provides a method for detecting the residual amount of N-methylmorpholine in a gel, characterized in that the method comprises the following steps:

[0010] The detection is performed by using gas chromatography; the chromatographic conditions include: the chromatographic column is a capillary column with cyano propyl phenyl-dimethyl polysiloxane as the stationary phase, the initial temperature is 35-45℃, the temperature is raised to 80-120℃ at a rate of 7-13℃ / min per minute, and then the temperature is raised to 240-260℃ at a rate of 60-80℃ / min, and maintained for 7-11 minutes.

[0011] In some embodiments, the chromatographic column is a capillary column with 5.5-6.5% cyano propyl phenyl-94.5-93.5% dimethyl polysiloxane as the stationary phase; preferably, the chromatographic column is a capillary column with 6.0% cyano propyl phenyl-94% dimethyl polysiloxane as the stationary phase; more preferably, the chromatographic column is DB-624, 30m x 0.53mm, 5µm.

[0012] In some embodiments, the column temperature is: the initial temperature is 38-42℃, the temperature is raised to 85-115℃ at a rate of 8-12℃ / min per minute, and then the temperature is raised to 245-255℃ at a rate of 65-75℃ / min, and maintained for 8-10 minutes; preferably, the column temperature is: the initial temperature is 40℃, the temperature is raised to 100℃ at a rate of 10℃ / min per minute, and then the temperature is raised to 250℃ at a rate of 70℃ / min, and maintained for 9 minutes.

[0013] In some embodiments, the injection port temperature is 245-255℃; preferably 250℃.

[0014] In some embodiments, the detector is a hydrogen flame ionization detector (FID).

[0015] In some embodiments, the detector temperature is 245-255℃; preferably 250℃.

[0016] In some embodiments, the carrier gas is nitrogen.

[0017] In some embodiments, the flow rate is 0.5-5.0mL / min; preferably, the flow rate is 0.8-3.0mL / min; more preferably, the flow rate is 1.0mL / min.

[0018] In some embodiments, the split ratio is 1:1-5:1, preferably 5:1.

[0019] In some embodiments, the sample volume is 1-5 µL, preferably 2 µL.

[0020] In some embodiments, the specific steps of the detection method are as follows: preparing blank solution, control solution and test solution, respectively taking the blank solution, control solution and test solution into the sample, detecting under the gas chromatography conditions, recording the chromatogram and calculating the content of N-methylmorpholine by peak area according to the external standard method.

[0021] In some embodiments, the preparation of the test solution includes mixing, dispersing and centrifuging the gel to be tested with N,N-dimethylformamide, collecting the supernatant, filtering and obtaining the test solution.

[0022] Preferably, the preparation of the test solution includes weighing the gel to be tested, adding an appropriate amount of N,N-dimethylformamide, vortexing and dispersing, adding N,N-dimethylformamide to constant volume, shaking well, centrifuging, taking the supernatant, filtering and collecting the filtrate.

[0023] In some embodiments, the concentration of the test solution is 0.05 g / mL-0.5 g / mL, and the concentration of the control solution is 0.005 mg / mL-0.1 mg / mL; preferably, the concentration of the test solution is 0.08 g / mL-0.2 g / mL, and the concentration of the control solution is 0.008 mg / mL-0.05 mg / mL; more preferably, the concentration of the test solution is 0.1 g / mL, and the concentration of the control solution is 0.01 mg / mL.

[0024] The application also provides a use of the detection method as described above for analyzing the residual amount of N-methylmorpholine in the gel. Preferably, the gel is a cross-linked gel of recombinant collagen and sodium hyaluronate.

[0025] On the basis of common sense in the art, the above-mentioned preferred conditions can be combined arbitrarily, thereby obtaining each preferred example of the application.

[0026] Compared with the prior art, the application has the following beneficial technical effects:

[0027] The application establishes a gas chromatography detection method for the residual amount of N-methylmorpholine in the gel, which has strong specificity, high sensitivity, good linear relationship, good precision, high accuracy, high solution stability, good durability and accurate and reliable determination results, thereby effectively controlling the quality, effectiveness and safety of related medical and beauty products, providing a strong guarantee for realizing controllable quality, laying a solid foundation for establishing related product quality control standards and having a broad industrialization and popularization prospect. BRIEF DESCRIPTION OF DRAWINGS

[0028] In order to more clearly illustrate the technical solutions of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings described below are some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0029] Figure 1 The chromatogram of the blank solution in the experimental example 1 of the present application;

[0030] Figure 2 The chromatogram of the control solution in the experimental example 1 of the present application;

[0031] Figure 3 The chromatogram of the by-product 2 positioning solution in the experimental example 1 of the present application;

[0032] Figure 4 The chromatogram of the test solution in the experimental example 1 of the present application;

[0033] Figure 5 The chromatogram of the test solution in the experimental example 1 of the present application;

[0034] Figure 6 The linear curve of N-methylmorpholine in the experimental example 3 of the present application.

[0035] Figure 7 The chromatogram of the control solution in the experimental example 2 of the present application;

[0036] Figure 8 The chromatogram of the blank solution in the comparative example 1 of the present application;

[0037] Figure 9 The chromatogram of the positioning solution in the comparative example 1 of the present application. DETAILED DESCRIPTION

[0038] In order to make the objects, technical solutions and advantages of the present application more clear, the technical solutions in the present application will be described clearly and completely below. Obviously, the described embodiments are some embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

[0039] The reagents and raw materials used in the following embodiments are commercially available. The reagent and control information is shown in Table 2.

[0040] Table 2

[0041]

[0042] The following examples were detected using a gas chromatograph of Agilent Company, model 7890B. The test sample solution was vortexed and dispersed using a Vortex 3000 Elite vortex of WIGGENS Company, and centrifuged using a Hunan Xiangyi centrifuge.

[0043] The test sample mentioned in the embodiments of the present application is a self-researched gel product of Meiyuan Space (Hebei) Biotechnology Co., Ltd., batch number: S20241201, which is prepared as follows: taking sodium hyaluronate and recombinant collagen as raw materials, taking DMTMM as a condensing agent, performing cross-linking reaction in a phosphate buffer, then dialyzing to remove DMTMM added in the cross-linking reaction and by-products N-methylmorpholine (by-product 1) and 4,6-dimethoxy-1,3,5-triazin-2(1H)-one (by-product 2) produced, and then screening and sterilizing to obtain the gel product.

[0044] In order to better monitor the quality of the prepared gel product, the residual amount of N-methylmorpholine is monitored, and therefore the content of N-methylmorpholine in the product is determined to better monitor the quality of the product.

[0045] Example 1

[0046] 1. Chromatographic conditions

[0047] ① Chromatographic column: Agilent DB-624 (30 m x 0.53 mm, 5 µm or equivalent performance) capillary column with 6% cyanopropyl phenyl-94% dimethyl polysiloxane as stationary phase as chromatographic column;

[0048] ② Column temperature: initial temperature 40℃, increased to 100℃ at a rate of 10℃ / min, and then increased to 250℃ at a rate of 70℃ / min, and maintained for 9 min;

[0049] ③ Injection port temperature: 250℃;

[0050] ④ Detector: hydrogen flame ionization detector (FID);

[0051] ⑤ Detector temperature: 250℃;

[0052] ⑥ Carrier gas: nitrogen;

[0053] ⑦ Flow rate: 1.0 mL / min;

[0054] ⑧ Split ratio: 5:1;

[0055] ⑨ Injection volume: 2 µL.

[0056] 2. Solution preparation

[0057] Reference solution

[0058]

[0059] ②Reference solution: accurately measure 0.1 mL of the reference stock solution and place it in a 10 mL volumetric flask, dilute to the mark with DMF, shake well, and you get it.

[0060] Test solution

[0061] Take about 1 g of the product (self-researched gel product), accurately weigh it, place it in a 10 mL volumetric flask, add DMF, vortex to disperse, then add DMF to the mark, shake well; centrifuge at 10000 r / min for 10 min, take the supernatant, filter, and collect the filtrate, and you get it.

[0062] Blank solution: N,N-dimethylformamide (DMF).

[0063] 3. Determination method

[0064] After the baseline of the gas chromatograph is balanced, the test solution and the reference solution are injected according to the specified chromatographic conditions, the chromatogram is recorded, and the peak area is calculated by external standard method.

[0065] Limit: N-methylmorpholine in gel is 0.01%.

[0066] Effect verification

[0067] According to the "Guiding Principles for Analysis Method Verification" in Chinese Pharmacopoeia 2020 edition, the specificity, detection limit and quantitative limit, linearity and range, precision, accuracy, solution stability and durability of the method described in Example 1 were verified, as follows:

[0068] The test verification process and results of the method of the application are shown in Experimental Examples 1-7.

[0069] Experimental Example 1 Specificity

[0070] 1. Solution preparation

[0071] ①Blank solution: N,N-dimethylformamide (DMF);

[0072] ②By-product 1 stock solution: take about 50 mg of by-product 1 reference substance, accurately weigh it, place it in a 50 mL volumetric flask, add DMF, dissolve and dilute to the mark, shake well;

[0073] ③Reference solution: accurately measure 1 mL of the reference stock solution and place it in a 100 mL volumetric flask, dilute to the mark with DMF, shake well;

[0074] ​(4) Sub-product 2 positioning solution: about 10 mg of the sub-product 2 reference substance was precisely weighed, placed in a 10 mL volumetric flask, and dissolved and diluted to the mark with DMF; 0.1 mL of the above solution was precisely measured and placed in a 10 mL volumetric flask, and diluted to the mark with DMF, and shaken well;

[0075] (5) Test sample solution: about 1 g of the product was precisely weighed, placed in a 10 mL volumetric flask, and vortexed and dispersed with an appropriate amount of DMF, and then diluted to the mark with DMF, and shaken well; and then centrifuged at a speed of 10,000 r / min for 10 min, and the supernatant was taken, filtered, and the filtrate was collected, and the test sample solution was obtained;

[0076] (6) Spiked test sample solution: about 1 g of the product was precisely weighed, placed in a 10 mL volumetric flask, and vortexed and dispersed with an appropriate amount of the reference substance solution, and then diluted to the mark with the reference substance solution, and shaken well; and then centrifuged at a speed of 10,000 r / min for 10 min, and the supernatant was taken, filtered, and the filtrate was collected, and the spiked test sample solution was obtained.

[0077] 2. Experimental results

[0078] The chromatogram of the blank solution is shown in Figure 1 The chromatogram of the reference substance solution is shown in Figure 2 The chromatogram of the sub-product 2 positioning solution is shown in Figure 3 The chromatogram of the test sample solution is shown in Figure 4 The chromatogram of the spiked test sample solution is shown in Figure 5 The results are shown in Table 3.

[0079] Table 3. Results of specificity experiment

[0080]

[0081] Conclusion: The blank solution does not interfere with the detection; there is no interference near the main peak, DMTMM is not dissolved, and sub-product 2 does not peak, which does not interfere with the detection; the main peak retention time of the test sample solution and the spiked test sample solution is consistent with that of the reference substance solution, and the residual amount of N-methylmorpholine in the gel is calculated by the external standard method, and it is calculated that the residual amount of N-methylmorpholine is 0.0044%, which is within the specified limit of 0.01%. The specificity of the method meets the detection requirements, and it is shown that the detection method for determining the residual amount of N-methylmorpholine in the gel by the gas chromatography method provided by the application has good specificity.

[0082] Detection limit and quantification limit of experimental example 2

[0083] When S / N is about 3, it is the detection limit, and when S / N is about 10, it is the quantification limit.

[0084] 1. Solution preparation

[0085] (1) Blank solution: N,N-dimethylformamide (DMF);

[0086] 2. The control solution: take the control solution under the specificity item of experimental example 1;

[0087] 3. The limit of quantification solution: accurately take 2 mL of the control solution into a 10 mL volumetric flask, dilute to the mark with DMF, shake well, and prepare 6 parallel samples;

[0088] 4. The limit of detection solution: accurately take 3 mL of the limit of quantification solution into a 10 mL volumetric flask, dilute to the mark with DMF, and shake well.

[0089] 2. Experimental results

[0090] The experimental results of the limit of detection and the limit of quantification are shown in Table 4.

[0091] Table 4 Experimental results of the limit of detection and the limit of quantification

[0092]

[0093] Conclusion: the RSD value of the peak area of the limit of quantification solution is 8.43%, which is less than 10%; S / N is 13.09, 11.89, 12.05, 18.42, 14.79, and 16.66, respectively, all of which are greater than 10; the limit of quantification concentration is 1.97 μg / mL; which is 19.65% of the limit concentration, less than 30% of the limit concentration; the signal-to-noise ratio of the limit of detection solution is 3.33, which is about 3; the limit of detection concentration is 0.59 μg / mL; which is 5.90% of the limit concentration. The limit of quantification and the limit of detection of the method meet the detection requirements, indicating that the gas chromatography method provided by the application for determining the residual amount of N-methylmorpholine in the gel has high sensitivity.

[0094] Experimental example 3 linearity and range

[0095] 1. Solution preparation

[0096] 1 (20%): take the limit of quantification solution under the limit of detection and the limit of quantification item of experimental example 2;

[0097] 2 (50%): accurately take 25 mL of the control solution under the specificity item of experimental example 1 into a 50 mL volumetric flask, dilute to the mark with DMF, and shake well;

[0098] 3 (80%): accurately take 8 mL of the control solution under the specificity item of experimental example 1 into a 10 mL volumetric flask, dilute to the mark with DMF, and shake well;

[0099] 4 (100%): accurately take the control solution under the specificity item of experimental example 1.

[0100] 5. Linear solution 5 (120%): precisely pipette 1.2 mL of the by-product 1 stock solution under the specificity item of experimental example 1 into a 10 mL volumetric flask, dilute to the mark with DMF, shake well;

[0101] 6. Linear solution 6 (150%): precisely pipette 1.5 mL of the by-product 1 stock solution under the specificity item of experimental example 1 into a 10 mL volumetric flask, dilute to the mark with DMF, shake well;

[0102] 7. Linear solution 7 (200%): precisely pipette 10 mL of the by-product 1 stock solution under the specificity item of experimental example 1 into a 50 mL volumetric flask, dilute to the mark with DMF, shake well;

[0103] 8. Linear solution 8 (500%): precisely pipette 5 mL of the by-product 1 stock solution under the specificity item of experimental example 1 into a 10 mL volumetric flask, dilute to the mark with DMF, shake well;

[0104] 9. Linear solution 9 (800%): precisely pipette 8 mL of the by-product 1 stock solution under the specificity item of experimental example 1 into a 10 mL volumetric flask, dilute to the mark with DMF, shake well;

[0105] 10. Linear solution 10 (1000%): precisely pipette the by-product 1 stock solution under the specificity item of experimental example 1.

[0106] 2. Experimental results

[0107] The linear experimental results are shown in Table 5.

[0108] Table 5 Linear and range experimental results

[0109]

[0110] Conclusion: N-methylmorpholine is linear in the range of the quantitative limit to the limit concentration of 1000%; the linear equation is y=2.2143x+3.9301; the R value is 0.9997, which is greater than 0.995. The linearity of the method meets the detection requirements, the linear range is good, and the linear relationship is good.

[0111] Experimental example 4 precision

[0112] Repeatability

[0113] 1. Solution preparation

[0114] 1. Solution preparation

[0115] 2. Control solution: take the control solution under the specificity item of experimental example 1;

[0116] Test solution: about 1 g of the product was accurately weighed into a 10 mL volumetric flask, and dissolved with DMF. The solution was diluted to the mark with DMF, and vortexed. The solution was centrifuged at 10000 r / min for 10 min. The supernatant was collected, and filtered. The filtrate was collected. The procedure was repeated 6 times.

[0117] 2. Experimental results

[0118] The results of the repeatability experiment are shown in Table 6.

[0119] Table 6. Results of the repeatability experiment

[0120]

[0121] Conclusion: The content of N-methylmorpholine in the 6 test solutions was 0.0040%, 0.0038%, 0.0041%, 0.0042%, 0.0041%, and 0.0043%, respectively, as calculated by the external standard method and peak area. The average content was 0.004%, which was less than 0.01%. The RSD value of the 6 test solutions was 3.78%, which was less than 5%. The repeatability of the method met the detection requirements and was good.

[0122] Intermediate precision

[0123] Different times and different personnel were used to operate according to the same method under repeatability.

[0124] 1. Solution preparation

[0125] Blank solution: N,N-dimethylformamide (DMF);

[0126] Test solution: about 1 g of the product was accurately weighed into a 10 mL volumetric flask, and dissolved with DMF. The solution was diluted to the mark with DMF, and vortexed. The solution was centrifuged at 10000 r / min for 10 min. The supernatant was collected, and filtered. The filtrate was collected. The procedure was repeated 6 times.

[0127] Test solution: about 1 g of the product was accurately weighed into a 10 mL volumetric flask, and dissolved with DMF. The solution was diluted to the mark with DMF, and vortexed. The solution was centrifuged at 10000 r / min for 10 min. The supernatant was collected, and filtered. The filtrate was collected. The procedure was repeated 6 times.

[0128] 2. Experimental results

[0129] The results of the intermediate precision experiment are shown in Table 7.

[0130] Table 7. Results of the intermediate precision experiment

[0131]

[0132] Conclusion: Different time, different personnel, according to the external standard method with peak area calculation content, the content of 6 test solution is 0.0040%, 0.0043%, 0.0040%, 0.0042%, 0.0044%, 0.0044%, the average content is 0.004%, RSD value is 3.75%, less than 5%; the average content of 12 test solution is 0.004%, RSD value is 4.42%, less than 5%. The intermediate precision meets the detection requirements.

[0133] Experimental example 5 accuracy

[0134] 1, solution configuration

[0135] ① Blank solution: N, N-dimethylformamide (DMF);

[0136] ② Control solution: take the control solution under the specificity item of experimental example 1;

[0137] ③ Background solution: take the test solution under the repeatability item of experimental example 4 precision;

[0138] ④ 50% recovery solution: take about 1g of the product, accurately weigh, put it in a 10mL volumetric flask, add linear solution 2, vortex dispersion, add linear solution 2 to the mark, shake well; again at the speed of 10000r / min centrifugation for 10min, take the supernatant, filter, collect the filtrate, that is, parallel preparation of 3;

[0139] ⑤ 100% recovery solution: take about 1g of the product, accurately weigh, put it in a 10mL volumetric flask, add control solution, vortex dispersion, add control solution to the mark, shake well; again at the speed of 10000r / min centrifugation for 10min, take the supernatant, filter, collect the filtrate, that is, parallel preparation of 3;

[0140] ⑥ 200% recovery solution: take about 1g of the product, accurately weigh, put it in a 10mL volumetric flask, add linear solution 7, vortex dispersion, add linear solution 7 to the mark, shake well; again at the speed of 10000r / min centrifugation for 10min, take the supernatant, filter, collect the filtrate, that is, parallel preparation of 3.

[0141] 2, experimental results

[0142] The accuracy test results are shown in table 8.

[0143] Table 8 accuracy test results

[0144]

[0145] Conclusion: At the limit concentration of 50%, 100%, 200%, the recovery rates of N-methyl morpholine were 94.50%, 94.87%, 96.04%, 93.79%, 95.13%, 92.70%, 94.49%, 95.64%, 94.45% respectively, the average recovery rates were 95.1%, 93.9%, 94.9% respectively, the average recovery rate was 94.6%, all within the range of 85%-110%, and the RSD value was 1.04%, less than 5%. The recovery rate of the method met the detection requirements, and the accuracy was high.

[0146] Solution stability of experimental example 6

[0147] 1. Solution preparation

[0148] ① Control solution: the control solution under the specificity item of experimental example 1 was taken;

[0149] ② Standard sample solution: the standard sample solution under the specificity item of experimental example 1 was taken.

[0150] 2. Experimental results

[0151] The solution stability experimental results are shown in Table 9.

[0152] Table 9 Solution stability experimental results

[0153]

[0154] Conclusion: The RSD value of the peak area of the control solution was 3.32% within 30h at room temperature, less than 5%; the RSD value of the peak area of the standard sample solution was 3.08% within 26h at room temperature, less than 5%; the control solution was stable within 30h, and the standard sample solution was stable within 26h.

[0155] Durability of experimental example 7

[0156] 1. Solution preparation

[0157] ① Blank solution: N,N-dimethylformamide (DMF);

[0158] ② Control solution: the control solution under the specificity item of experimental example 1 was taken;

[0159] ③ Standard sample solution: the 100% recovery rate solution under the accuracy item of experimental example 5 was taken.

[0160] 2. Experimental results

[0161] The durability experimental results are shown in Table 10.

[0162] Table 10 Durability experimental results

[0163]

[0164] Conclusion: Under the conditions, the absolute difference of N-methyl morpholine content between the conditions of slight changes of sample inlet temperature (250±5℃), detector temperature (250±5℃), initial column temperature (40±2℃), and flow rate (1±0.2ml / min) and the content under the proposed conditions was 0.0006%, 0.0003%, 0.0004%, 0.0010%, 0.0001%, 0.0002%, 0.0017%, and 0.0005%, respectively, all less than 0.002%. The durability of the method met the detection requirements.

[0165] Example 2

[0166] 1. Chromatographic conditions

[0167] Other chromatographic conditions were the same as in Example 1, except for the following conditions:

[0168] 1. Flow rate: 3.0 mL / min;

[0169] 2. Split ratio: 1:1.

[0170] 2. Solution preparation

[0171] Reference solution

[0172] 1. Reference stock solution: About 10 mg of N-methyl morpholine reference substance was accurately weighed and placed in a 10 mL volumetric flask. Appropriate amount of N,N-dimethylformamide (DMF) was added to dissolve and dilute to the mark, and then shaken to obtain the reference stock solution.

[0173] 2. Reference solution: 0.2 mL of the reference stock solution was accurately measured and placed in a 1000 mL volumetric flask. DMF was added to dilute to the mark, and then shaken to obtain the reference solution.

[0174] 3. Experimental results

[0175] The chromatogram of the reference solution is shown in Figure 7 .

[0176] Conclusion: Compared with Example 1, in this example, the flow rate in the chromatographic conditions was adjusted from 1.0 mL / min to 3.0 mL / min, and the split ratio was adjusted from 5:1 to 3:1. The main peak retention time of the reference solution was adjusted to 7.760 min, the peak shape was good, and the resolution met the detection requirements.

[0177] Comparative Example 1

[0178] 1. Chromatographic conditions

[0179] Other chromatographic conditions were the same as in Example 1, except for the following conditions:

[0180] Column temperature: initial temperature 150℃, maintain for 5 min, increase to 250℃ at a rate of 20℃ / min, maintain for 5 min.

[0181] 2. Solution preparation

[0182] 1. Blank solution: N,N-dimethylformamide (DMF);

[0183] 2. By-product 1 positioning solution (0.2 mg / mL): about 10 mg of by-product 1 reference substance was accurately weighed, placed in a 10 mL volumetric flask, and dissolved and diluted to the mark with DMF. 2 mL of the above solution was accurately measured and placed in a 10 mL volumetric flask, and diluted to the mark with DMF, and shaken well.

[0184] 3. Experimental results

[0185] The chromatogram of the blank solution is shown in Figure 7 , and the chromatogram of the by-product 1 positioning solution is shown in Figure 8 . The main peak in the chromatogram of the by-product 1 positioning solution cannot be completely separated from the solvent peak.

[0186] Comparative Example 2

[0187] 1. Chromatographic conditions

[0188] Chromatographic column: capillary column Agilent HP-5 (30 m x 0.53 mm, 5 µm or equivalent performance) with 5% phenyl-95% methylpolysiloxane as stationary phase;

[0189] 2. Solution preparation

[0190] 1. Blank solution: N,N-dimethylformamide (DMF);

[0191] 2. By-product 1 positioning solution (0.2 mg / mL): about 10 mg of by-product 1 reference substance was accurately weighed, placed in a 10 mL volumetric flask, and dissolved and diluted to the mark with DMF. 2 mL of the above solution was accurately measured and placed in a 10 mL volumetric flask, and diluted to the mark with DMF, and shaken well.

[0192] 3. Experimental results

[0193] The main peak in the chromatogram of the by-product 1 positioning solution is severely tailing.

[0194] Finally, it should be noted that in the present application, the terms "comprise", "include" or any other variant thereof are intended to cover non-exclusive inclusion, so that processes, methods, articles or equipment including a series of elements not only include those elements, but also include other elements not explicitly listed or inherent to such processes, methods, articles or equipment.

[0195] While the application has been disclosed by reference to specific embodiments thereof, it will be understood that various other modifications, applications, or equivalents thereof will become apparent to those skilled in the art after considering the foregoing description. It will be appreciated that such modifications, applications, or equivalents fall within the scope of the application as defined by the appended claims.

Claims

1. A method for detecting N-methylmorpholine residues in a gel, characterized in that: The detection was performed using gas chromatography; the chromatographic conditions included: a capillary column with cyanopropylphenyl-dimethylpolysiloxane as the stationary phase, an initial temperature of 35–45°C, a temperature increase to 80–120°C at a rate of 7–13°C / min, and a further increase to 240–260°C at a rate of 60–80°C / min, which was maintained for 7–11 min.

2. The detection method according to claim 1, characterized in that, The chromatographic conditions shall satisfy at least one of the following conditions 1) to 9): 1) Chromatographic column: A capillary column with 5.5-6.5% cyanopropylphenyl-94.5-93.5% dimethylpolysiloxane as the stationary phase was used as the chromatographic column; 2) Column temperature: The initial temperature is 38-42℃, and the temperature is increased to 85-115℃ at a rate of 8-12℃ / min, and then increased to 245-255℃ at a rate of 65-75℃ / min, and maintained for 8-10min. 3) Inlet temperature: 245~255℃; 4) Detector: Flame Ionization Detector (FID); 5) Detector temperature: 245~255℃; 6) Carrier gas: Nitrogen; 7) Flow rate: 0.5~5.0 mL / min; 8) Flow split ratio: 1:1~5:1; 9) Injection volume: 1~5µL.

3. The detection method according to claim 2, characterized in that, The chromatographic conditions shall satisfy at least one of the following conditions 1) to 7): 1) Chromatographic column: A capillary column with 6.0% cyanopropylphenyl-94% dimethylpolysiloxane as the stationary phase was used as the chromatographic column; 2) Column temperature: The initial temperature is 40℃, and the temperature is increased to 100℃ at a rate of 10℃ / min, and then increased to 250℃ at a rate of 70℃ / min, and maintained for 9min. 3) Inlet temperature: 250℃; 4) Detector temperature: 250℃; 5) Flow rate: 0.8~3.0 mL / min; 6) Flow split ratio: 5:1; 7) Injection volume: 2µL.

4. The detection method according to claim 3, characterized in that, The chromatographic conditions must satisfy at least one of the following conditions 1) to 2): 1) Column: DB-624, 30m × 0.53mm, 5µm; 2) Flow rate: 1.0 mL / min.

5. The detection method according to any one of claims 1-4, characterized in that, The specific steps are as follows: Prepare blank solution, reference solution and test solution, inject blank solution, reference solution and test solution into the gas chromatography, detect according to the gas chromatography conditions, record the chromatogram and calculate the content of N-methylmorpholine by peak area according to the external standard method.

6. The detection method according to claim 5, characterized in that, The preparation of the test solution includes: mixing, dispersing, and centrifuging the gel to be tested with N,N-dimethylformamide, collecting the supernatant, filtering, and obtaining the test solution.

7. The detection method according to claim 6, characterized in that, The concentration of the test solution is 0.05 g / mL to 0.5 g / mL, and the concentration of the reference solvent is 0.005 mg / mL to 0.1 mg / mL.

8. The detection method according to claim 7, characterized in that, The concentration of the test solution is 0.08 g / mL to 0.2 g / mL, and the concentration of the reference solvent is 0.008 mg / mL to 0.05 mg / mL.

9. The detection method according to claim 8, characterized in that, The concentration of the test solution is 0.1 g / mL, and the concentration of the reference solvent is 0.01 mg / mL.

10. The detection method according to any one of claims 1 to 9 is used to analyze the residual amount of N-methylmorpholine in a gel; preferably, the gel is a cross-linked gel of recombinant collagen and sodium hyaluronate.

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