A method for detecting trace residues of N-methylmorpholine in gel based on gas chromatography-mass spectrometry

Abstract

This invention provides a method for detecting trace amounts of N-methylmorpholine residues in gels using gas chromatography-mass spectrometry (GC-MS). This method is highly specific, sensitive, linear, precise, accurate, has high solution stability, is durable, and provides accurate and reliable measurement results. It offers strong support for effectively controlling the quality, efficacy, and safety of related medical aesthetic products and ensuring their quality control.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection technology, specifically to a method for detecting N-methylmorpholine residues in gels based on gas chromatography-mass spectrometry (GC-MS). Background Technology

[0002] Aesthetic medicine is the fastest and most effective way to improve skin imperfections and regain a youthful appearance. Hyaluronic acid is a popular ingredient, possessing extremely strong water-locking capabilities. It can bind approximately 1000 times its own weight in water, and when injected into the dermis, it can quickly plump up skin tissue, providing hydration, physical support, and promoting collagen regeneration. Recombinant collagen can promote the proliferation and repair of fibroblasts and has good biocompatibility, biodegradability, and bioactivity. Injecting recombinant collagen into the dermis can replenish collagen in situ, improve skin texture, reduce static wrinkles, improve skin laxity, and enhance skin elasticity.

[0003] Hyaluronic acid and recombinant collagen are both major components of the skin. A recombinant collagen-sodium hyaluronate cross-linked gel could provide immediate repair and contour correction for satisfactory results. Furthermore, the amino acids and peptides produced by the degradation of recombinant collagen in the cross-linked gel could facilitate new collagen synthesis and tissue repair and remodeling. CN118483405A uses sodium hyaluronate and recombinant collagen as raw materials, employing 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM) as a condensing agent. This promotes the condensation reaction between recombinant collagen and sodium hyaluronate, forming amide bonds, resulting in a recombinant collagen-sodium hyaluronate cross-linked gel with high viscoelasticity and shear viscosity, thus better improving skin condition.

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

[0005] Table 1

[0006]

[0007] Currently, gas chromatography (GC) methods have been reported for the detection of N-methylmorpholine residues in gels. For example, patent CN121027356A discloses a GC method using a flame ionization detector (FID). However, the complex gel matrix, especially cross-linked gels containing recombinant collagen and sodium hyaluronate, may suffer from significant matrix interference and insufficient sensitivity to meet lower limit detection requirements when using GC-FID. Gas chromatography-mass spectrometry (GC-MS) combines the high separation capability of GC with the high selectivity and sensitivity of mass spectrometry, effectively eliminating matrix interference. However, there are currently no reports on GC-MS methods for the detection of N-methylmorpholine in complex biological samples such as gels. Therefore, developing a highly specific and sensitive method for the detection of N-methylmorpholine residues that can effectively eliminate gel matrix interference is of great significance for strictly controlling product quality and ensuring the safety of clinical medication. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for detecting trace amounts of N-methylmorpholine in gels based on gas chromatography-mass spectrometry (GC-MS). This method has high specificity, high sensitivity, good linearity, good precision, high accuracy, high solution stability, good durability, and accurate and reliable measurement results.

[0009] To achieve the above-mentioned objective, this invention provides a method for detecting trace residues of N-methylmorpholine in a gel using gas chromatography-mass spectrometry (GC-MS), characterized by the following steps: detection using GC-MS; wherein the chromatographic conditions include: a capillary column with cyanopropylphenyl-polydimethylsiloxane as the stationary phase, an initial temperature of 80–120°C, maintained for 1–3 minutes, and then increased to 230–270°C at a rate of 40–60°C / min, maintained for 3–7 minutes; wherein the mass spectrometry conditions include: a selected ion monitoring (SIM) scan mode, with the quantitative ion of N-methylmorpholine at m / z 101.1 and the qualitative ion at m / z 43.1.

[0010] In some embodiments, the chromatographic conditions include: a capillary column with 5.5–6.5% cyanopropylphenyl-94.5–93.5% polydimethylsiloxane as the stationary phase; preferably, a capillary column with 6.0% cyanopropylphenyl-94% polydimethylsiloxane as the stationary phase; and more preferably, a VF-624ms column with dimensions of 30m × 0.25mm and a diameter of 1.4µm.

[0011] In some embodiments, the chromatographic conditions are as follows: column temperature: initial temperature 90-110°C, maintained for 1.5-2.5 minutes, then increased to 240-260°C at a rate of 45-55°C / min, maintained for 4-6 minutes; preferably, column temperature: initial temperature 100°C, maintained for 2 minutes, then increased to 250°C at a rate of 50°C / min, maintained for 5 minutes.

[0012] In some embodiments, the chromatographic conditions include an injection port temperature of 245–255°C, preferably 250°C.

[0013] In some embodiments, the carrier gas is helium under chromatographic conditions.

[0014] In some embodiments, the chromatographic conditions include a flow rate of 0.5 to 2.5 mL / min, preferably 1.5 mL / min.

[0015] In some embodiments, the split ratio in the chromatographic conditions is 5:1 to 15:1; preferably 10:1.

[0016] In some embodiments, the injection volume is 1~5µL; preferably 1µL.

[0017] In some embodiments, the mass spectrometry conditions use an EI source as the ion source.

[0018] In some embodiments, the mass spectrometry conditions include an ion source temperature of 220-240°C; preferably 230°C.

[0019] In some embodiments, the mass spectrometry conditions include a quadrupole temperature of 140-160°C; preferably 150°C.

[0020] In some examples of mass spectrometry conditions, the solvent delay is 1.5 to 2.5 minutes; preferably 1.8 minutes.

[0021] In some embodiments, the specific steps of the detection method are as follows: prepare blank solution, reference solution and test solution, inject blank solution, reference solution and test solution into gas chromatography-mass spectrometry (GC-MS) instrument in sequence, record the chromatogram and calculate the content of N-methylmorpholine by peak area according to the external standard method.

[0022] In some embodiments, the preparation of the test solution includes: mixing, dispersing, centrifuging, collecting the supernatant, and filtering to obtain the test solution.

[0023] In some embodiments, the concentration of the test solution is 0.05 g / mL to 0.5 g / mL, preferably 0.08 g / mL to 0.2 g / mL, for example 0.1 g / mL.

[0024] In some embodiments, the concentration of the reference solution is 0.1 μg / mL to 10 μg / mL, preferably 0.1 μg / mL to 3 μg / mL, for example 1 μg / mL.

[0025] The present invention also provides an application of the detection method described above for analyzing the residual amount of N-methylmorpholine in a gel. Preferably, the gel is a cross-linked gel of recombinant collagen and sodium hyaluronate.

[0026] In some embodiments, the gel is made from sodium hyaluronate and recombinant collagen, using 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM) as a condensing agent to induce a condensation reaction between the recombinant collagen and sodium hyaluronate.

[0027] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0028] Compared with the prior art, the present invention has the following beneficial technical effects:

[0029] This invention establishes a gas chromatography-mass spectrometry (GC-MS) method for detecting trace N-methylmorpholine residues in gels. This method is highly specific, sensitive, linear, precise, accurate, stable, and robust, providing accurate and reliable results. It offers strong support for effectively controlling the quality, efficacy, and safety of related medical aesthetic products, and lays a solid foundation for establishing quality control standards for related products. It also has broad prospects for industrialization and promotion. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0031] Figure 1 The spectrum of the blank solution in Experimental Example 1 of this invention;

[0032] Figure 2 This is the spectrum of the reference solution in Experimental Example 1 of the present invention;

[0033] Figure 3 This is the spectrum of the test solution in Experimental Example 1 of the present invention;

[0034] Figure 4 This is the spectrum of the spiked solution of the test sample in Experimental Example 1 of the present invention;

[0035] Figure 5This is a detection diagram of the reference standard positioning solution in Experimental Example 1 of the present invention. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0037] The reagents and raw materials used in the following examples are all commercially available. Information on reagents and reference standards is shown in Table 2.

[0038] Table 2

[0039] name Grade / Purity (%) Dimethyl sulfoxide (DMSO) Chromatographic grade N-Methylmorpholine (byproduct 1) 99.189

[0040] The following examples use an Agilent gas chromatography-mass spectrometry (GC-MS) system, model 7697A / 7890B / 5977BMSD.

[0041] The test sample mentioned in this embodiment of the invention is a self-developed gel product of Meiyan Space (Hebei) Biotechnology Co., Ltd., batch number: S20250801. Its preparation process is as follows: Sodium hyaluronate and recombinant collagen are used as raw materials, DMTMM is used as a condensing agent, and a cross-linking reaction is carried out in phosphate buffer. Then, the DMTMM added in the cross-linking reaction and the byproducts N-methylmorpholine (byproduct 1) and 4,6-dimethoxy-1,3,5-triazine-2(1H)-one (byproduct 2) are removed. The product is then sieved and sterilized to obtain the gel product.

[0042] In order to better monitor the quality of the synthesized gel product and to monitor the residue of N-methylmorpholine, the content of N-methylmorpholine in this product is determined to better monitor the quality of this product.

[0043] Example 1

[0044] 1. Chromatographic conditions

[0045] ①Chromatographic column: Agilent VF-624ms capillary column (30m×0.25mm, 1.4µm) with 6% cyanopropylphenyl-94% polydimethylsiloxane as the stationary phase was used as the chromatographic column;

[0046] ② Column temperature: Initial temperature 100℃, maintain for 2 minutes, then increase to 250℃ at a rate of 50℃ / min, maintain for 5 minutes;

[0047] ③ Inlet temperature: 250℃;

[0048] ④ Carrier gas: Helium; Carrier gas flow rate: 1.5 mL / min;

[0049] ⑤ Flow split ratio: 10:1;

[0050] ⑥ Injection volume: 1µL.

[0051] 2. Mass spectrometry conditions

[0052] The ion source was an EI source with a temperature of 230℃ and a quadrupole temperature of 150℃. The acquisition mode was Selected Ion Monitoring (SIM), with a quantitative ion density of 101.1 and a qualitative ion density of 43.1. The solvent delay was 1.8 minutes and the detector shutdown time was 3.8 minutes.

[0053] 3. Solution preparation

[0054] Reference solution

[0055] ① Reference stock solution: Weigh approximately 20 mg of N-methylmorpholine accurately, place it in a 20 mL volumetric flask, dissolve and dilute it to the mark with dimethyl sulfoxide, shake well, then take 1 mL of this solution into a 100 mL volumetric flask, dilute it to the mark with dimethyl sulfoxide, shake well, and the solution is ready;

[0056] ② Reference solution: Accurately measure 1 mL of the reference stock solution, place it in a 10 mL volumetric flask, dilute to the mark with dimethyl sulfoxide, and shake well to obtain the solution.

[0057] Test solution

[0058] Weigh approximately 1g of this product (self-developed gel product), accurately weigh it, place it in a 10mL volumetric flask, add an appropriate amount of dimethyl sulfoxide, vortex to disperse, then add dimethyl sulfoxide to make up to volume and shake well; then centrifuge at a rate of 10000r / min for 10 minutes, take the supernatant and filter it, collect the filtrate, and you have the product.

[0059] Blank solution: dimethyl sulfoxide (DMSO).

[0060] 4. Measurement Method

[0061] The blank solution, reference solution, and test solution were injected sequentially into the gas chromatography-mass spectrometry (GC-MS) instrument. The chromatograms were recorded according to the specified chromatographic and mass spectrometric conditions, and the results were calculated by peak area using the external standard method.

[0062] Limit: The amount of N-methylmorpholine in this product shall not exceed 10 µg / g, calculated by peak area using the external standard method.

[0063] Effect verification

[0064] In accordance with the "Guidelines for Validation of Analytical Methods 9101" in the 2025 edition of the Chinese Pharmacopoeia, the method described in Example 1 underwent methodological validation for specificity, limit of detection and limit of quantitation, linearity and range, accuracy, precision, solution stability, and robustness, as detailed below:

[0065] The experimental verification process and results of the method of the present invention are shown in Experiment Examples 1 to 7.

[0066] Specificity in Experiment Example 1

[0067] 1. Solution preparation

[0068] ① Blank solution: Dimethyl sulfoxide (DMSO);

[0069] ② Reference stock solution: Weigh approximately 20 mg of N-methylmorpholine accurately, place it in a 20 mL volumetric flask, dissolve and dilute to the mark with dimethyl sulfoxide, shake well, then take 1 mL of this solution into a 100 mL volumetric flask, dilute to the mark with dimethyl sulfoxide, shake well, and the solution is ready;

[0070] ③ Reference solution: Accurately measure 1 mL of the reference stock solution and place it in a 10 mL volumetric flask. Dilute to the mark with dimethyl sulfoxide and shake well. The solution is labeled as STD.

[0071] ④ Reference standard positioning solution: Weigh approximately 20 mg of N-methylmorpholine accurately, place it in a 10 mL volumetric flask, dissolve and dilute it to the mark with dimethyl sulfoxide, shake well, then take 1 mL of this solution into a 10 mL volumetric flask, dilute it to the mark with dimethyl sulfoxide, shake well, and the solution is ready.

[0072] ⑤ Test solution: Weigh about 1g of this product (self-developed gel product), accurately weigh it, place it in a 10mL volumetric flask, add an appropriate amount of dimethyl sulfoxide, vortex to disperse, then add dimethyl sulfoxide to make up to volume and shake well; then centrifuge at 10000r / min for 10 minutes, take the supernatant and filter, collect the filtrate, and it is the test solution, labeled as SPL.

[0073] ⑥ Spiked solution for test sample: Weigh about 1g of this product (self-developed gel product), accurately weigh it, place it in a 10mL volumetric flask, add an appropriate amount of dimethyl sulfoxide, vortex to disperse, then add 1mL of the reference stock solution, dilute to volume with dimethyl sulfoxide and shake well; then centrifuge at 10000r / min for 10 minutes, take the supernatant and filter, collect the filtrate, and it is the test sample, labeled as SPL-STD.

[0074] 2. Experimental Results

[0075] The spectrum of the blank solution is shown below. Figure 1 The chromatogram of the reference solution is shown below. Figure 2 The spectrum of the test solution is shown in [reference needed]. Figure 3 The spectrum of the spiked solution of the test sample is shown in [reference needed]. Figure 4The results are shown in Table 3.

[0076] Table 3. Results of Specificity Experiment

[0077] name Retention time (min) Does it interfere? blank solution N / A no Reference solution 3.116 N / A Test solution 3.116 no Test sample spiking solution 3.119 no

[0078] Conclusion: Neither the blank solution nor the test solution interfered with the determination of N-methylmorpholine, and the specificity met the requirements, indicating that the gas chromatography-mass spectrometry method for detecting N-methylmorpholine residues in gels provided by this invention has good specificity.

[0079] The detection chromatogram of the reference standard positioning solution is shown below. Figure 5 .

[0080] Figure 5 The detection chromatogram for the reference standard localization solution is shown below. A full-scan analysis of the N-methylmorpholine reference standard localization solution yielded its mass spectrum. Based on the abundance and characteristics of fragment ions in the mass spectrum, fragment ions with high response values ​​and low interference were selected as monitoring ions. Ultimately, the quantitative ion was determined to be m / z 101.1 and the qualitative ion to be m / z 43.1 in the selected ion monitoring (SIM) mode of mass spectrometry, for subsequent verification experiments on specificity, limit of quantitation, and linearity.

[0081] Experimental Example 2: Limit of Detection and Limit of Quantification

[0082] 1. Solution preparation

[0083] ① Limit of Quantitation Solution: Take 1 mL of the reference solution under the specificity item of Experiment Example 1 into a 10 mL volumetric flask, dilute to the mark with dimethyl sulfoxide, shake well, and use it as the limit of quantitation solution. Prepare 6 parallel aliquots and label them as QL-1 to QL-6.

[0084] ② Detection limit solution: Accurately measure 3 mL of QL-1 solution into a 10 mL volumetric flask, dilute to the mark with dimethyl sulfoxide, shake well, and use this as the detection limit solution, labeled as DL.

[0085] 2. Experimental Results

[0086] The experimental results for the limits of detection and limits of quantitation are shown in Tables 4 and 5.

[0087] Table 4 Detection Limit Results

[0088] name DL Concentration (µg / mL) 0.03 Equivalent to the limit concentration (%) 3 S / N 11

[0089] Table 5 Results of Limit of Quantification

[0090]

[0091] Conclusion: The detection limit (LOD) is 3% of the limit concentration, which is less than 10% of the limit concentration. The signal-to-noise ratio (SNR) of N-methylmorpholine in the LOD solution is 11, which is greater than 3, and the LOD meets the requirements. The quantitation limit (LOQ) is 10% of the limit concentration, which is less than 20% of the limit concentration. The SNR of N-methylmorpholine in all six quantitation limit solutions is greater than 10. The RSD of the peak area in the six quantitation limit solutions is 2.1%, which is less than 10%, and the quantitation limit meets the requirements. The quantitation limit and detection limit of this method meet the detection requirements, indicating that the gas chromatography-mass spectrometry (GC-MS) method for detecting N-methylmorpholine residues in gels provided by this invention has high sensitivity.

[0092] Experiment Example 3: Linearity and Range

[0093] 1. Solution preparation

[0094] Stock solution ①: The stock solution ① under the specificity item of Experiment Example 1 is used.

[0095] L-200%: Accurately measure 2 mL of the stock solution ① into a 10 mL volumetric flask, dilute to the mark with dimethyl sulfoxide, shake well, and label as L-200.

[0096] L-150%: Accurately measure 1.5 mL of the stock solution ① into a 10 mL volumetric flask, dilute to the mark with dimethyl sulfoxide, shake well, and label as L-150.

[0097] L-100%: Accurately measure 1.0 mL of the stock solution ① into a 10 mL volumetric flask, dilute to the mark with dimethyl sulfoxide, shake well, and label as L-100.

[0098] L-50%: Accurately measure 0.5 mL of the stock solution ① into a 10 mL volumetric flask, dilute to the mark with dimethyl sulfoxide, shake well, and label as L-50.

[0099] L-Limit of Quantification: Accurately measure 1 mL of L-100% solution into a 10 mL volumetric flask, dilute to the mark with dimethyl sulfoxide, shake well, and label as L-QL.

[0100] Within the range of the quantitation limit to 200% limit solution, the linear equation and correlation coefficient R of each analyte were obtained by plotting the concentration of N-methylmorpholine as the abscissa and the peak area as the ordinate.

[0101] 2. Experimental Results

[0102] The results of the linear experiment are shown in Table 6.

[0103] Table 6. Experimental Results on Linearity and Range

[0104]

[0105] Conclusion: N-methylmorpholine exhibits linearity in the concentration range from the limit of quantitation to 200% of the limit; the linear equation is y = 31799x + 1250; the R-value is 0.9997, greater than 0.995, and the intercept at the limit concentration is 3.5%, less than 10%, indicating that the linearity meets the requirements. This method demonstrates good linearity, meets the detection requirements, has a good linear range, and exhibits a favorable linear relationship.

[0106] Accuracy of Experiment Example 4

[0107] 1. Solution preparation

[0108] ① Blank solution: Dimethyl sulfoxide (DMSO);

[0109] ② Reference solution: Take the reference solution from the specificity section of Experiment Example 1;

[0110] ③ Test solution: Accurately measure 1 mL of the reference stock solution under the specificity item of Experiment Example 1, place it in a 10 mL volumetric flask, dilute to the mark with dimethyl sulfoxide, and shake well to obtain the solution;

[0111] ④ 10% recovery solution: Weigh about 1g of this product accurately, place it in a 10mL volumetric flask, add an appropriate amount of dimethyl sulfoxide, vortex to disperse, then add 1mL of reference solution, dilute to volume with dimethyl sulfoxide and shake well; then centrifuge at 10000r / min for 10min, take the supernatant and filter, collect the filtrate, and prepare 3 parallel portions.

[0112] ⑤ 100% recovery solution: Weigh about 1g of this product accurately, place it in a 10mL volumetric flask, add an appropriate amount of dimethyl sulfoxide, vortex to disperse, then add 1mL of the reference stock solution under the specificity item of Experiment Example 1, and dilute to volume with dimethyl sulfoxide and shake well; then centrifuge at 10000r / min for 10min, take the supernatant, filter, collect the filtrate, and prepare 6 parallel portions;

[0113] ⑥ 150% recovery solution: Weigh approximately 1 g of this product accurately and place it in a 10 mL volumetric flask. Add an appropriate amount of dimethyl sulfoxide, vortex to disperse, then add 1.5 mL of the reference stock solution under the specificity item of Experiment Example 1, and dilute to volume with dimethyl sulfoxide and shake well. Centrifuge at 10000 r / min for 10 min, take the supernatant, filter, and collect the filtrate. Prepare 3 parallel portions.

[0114] 2. Experimental Results

[0115] The results of the accuracy experiment are shown in Table 7.

[0116] Table 7. Accuracy Experiment Results

[0117]

[0118] Conclusion: The recovery rate of N-methylmorpholine at the limit of quantitation, 100%, and 150% concentration levels was 89.8%, with a 95% confidence interval of 86.5%–93.1%, all within the range of 80%–115%. The RSD was 5.8%, less than 10%, and the accuracy met the requirements. This method has a recovery rate that meets the detection requirements and high accuracy.

[0119] Experimental Example 5 Precision

[0120] Repeatability

[0121] 1. Solution preparation

[0122] Weigh approximately 1 g of this product accurately and place it in a 10 mL volumetric flask. Add an appropriate amount of dimethyl sulfoxide, vortex to disperse, and then add 1 mL of the reference stock solution under the specificity item of Experiment Example 1. Make up to volume with dimethyl sulfoxide and shake well. Centrifuge at 10000 r / min for 10 min, take the supernatant, filter, and collect the filtrate. Prepare 6 parallel portions and label them as SPL-100STD-1 to SPL-100STD-6, respectively.

[0123] 2. Experimental Results

[0124] The results of the repeatability experiment are shown in Table 8.

[0125] intermediate precision

[0126] For different times and different people, follow the same procedure for repetitive items.

[0127] 1. Solution preparation

[0128] Weigh approximately 1 g of this product accurately and place it in a 10 mL volumetric flask. Add an appropriate amount of dimethyl sulfoxide, vortex to disperse, and then add 1 mL of the reference stock solution under the specificity item of Experiment Example 1. Make up to volume with dimethyl sulfoxide and shake well. Centrifuge at 10000 r / min for 10 min, take the supernatant, filter, and collect the filtrate. Prepare 6 parallel portions and label them as SPL-100STD-inter-prec-1 to SPL-100STD-inter-prec-6, respectively.

[0129] 2. Experimental Results

[0130] The results of the intermediate precision experiment are shown in Table 8.

[0131] Table 8. Repeatability and Intermediate Precision Results of N-Methylmorpholine

[0132]

[0133] Conclusion: The RSD of N-methylmorpholine content in the six 100% spiked solutions was 1.1%, less than 10%, and the repeatability met the requirements. The method exhibits good repeatability and meets the detection requirements.

[0134] The RSD of N-methylmorpholine content in 12 100% spiked solutions measured by different experimenters at different times was 3.7%, which is less than 10%, and the intermediate precision meets the requirements.

[0135] Experimental Example 6 Solution Stability

[0136] 1. Solution preparation

[0137] ① Reference solution: Take the reference solution from the specificity section of Experiment Example 1;

[0138] ②Spiked solution of test sample: Take the spiked solution of test sample under the specificity item of Experiment Example 1.

[0139] The stability of the reference solution and the spiked solution of the test sample were investigated at room temperature. The peak area of ​​the reference solution chromatogram at each time interval was calculated as a percentage (S) of the peak area at 0°C. The time intervals and corresponding results were entered into a table. The investigation was expected to last 20 hours and cover 4 points.

[0140] 2. Experimental Results

[0141] The results of the solution stability experiments are shown in Tables 9 and 10.

[0142] Table 9. Stability Results of Reference Solution

[0143] Time interval (h) Peak area S% 0.0 32085 100.0 4.0 34064 106.2 14.0 34820 108.5 24.0 32020 99.8

[0144] Table 10 Stability Results of Spiked Solutions of Test Samples

[0145] Time interval (h) Peak area S% 0.0 34935 100.0 2.0 34459 98.6 13.0 33321 95.4 22.0 34744 99.5

[0146] Conclusion: When comparing the peak area of ​​the reference solution obtained within 24 hours at room temperature with the peak area at 0 h, the percentage S was between 90% and 110%, indicating that the reference solution was stable within 24 hours.

[0147] The peak area of ​​the spiked solution of the test sample obtained within 22 hours at room temperature was compared with the peak area at 0 h. The percentage S was between 90% and 110%, indicating that the spiked solution of the test sample was stable within 22 hours.

[0148] Experimental Example 7 Durability

[0149] 1. Solution preparation

[0150] ① Blank solution: Dimethyl sulfoxide (DMSO);

[0151] ② Reference solution: Weigh approximately 20 mg of N-methylmorpholine accurately and place it in a 20 mL volumetric flask. Dissolve and dilute to the mark with dimethyl sulfoxide and shake well. Then take 1 mL of this solution into a 100 mL volumetric flask, dilute to the mark with dimethyl sulfoxide, and shake well to obtain stock solution ①. Then take 1 mL of stock solution ① into a 10 mL volumetric flask, dilute to the mark with dimethyl sulfoxide, and shake well to obtain the reference solution.

[0152] ③Spiked solution for test sample: Weigh about 1g of this product (self-developed gel product), accurately weigh it, place it in a 10mL volumetric flask, add an appropriate amount of dimethyl sulfoxide, vortex to disperse, then add 1mL of stock solution, centrifuge at 10000r / min for 10 minutes, take the supernatant and filter it, collect the filtrate, and prepare 2 parallel portions, labeled as SPL-STD-robu-1~SPL-STD-robu-2 respectively.

[0153] Keeping other method conditions unchanged, the injection port temperature was changed to 250±5℃, the initial column temperature to 100±5℃, and the flow rate to 1.5±0.1mL / min. The robustness of the method was then investigated.

[0154] 2. Experimental Results

[0155] The results of the durability test are shown in Table 11.

[0156] Table 11 Durability Test Results

[0157]

[0158] Conclusion: The RSD of N-methylmorpholine content in the six solutions before and after changes in different robustness conditions (injector temperature 250±5℃, initial column temperature 100±5℃, flow rate 1.5±0.1mL / min) was less than 10%. The robustness meets the requirements. The robustness of this method meets the detection requirements.

[0159] Finally, it should be noted that in this invention, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus.

[0160] Although the invention has been disclosed above through the description of specific embodiments, it should be understood that those skilled in the art can design various modifications, improvements, or equivalents to the invention within the spirit and scope of the appended embodiments. These modifications, improvements, or equivalents should also be considered to be included within the scope of protection claimed by the invention.

Claims

1. A method for detecting trace amounts of N-methylmorpholine residues in gels using gas chromatography-mass spectrometry (GC-MS), characterized in that: The analysis was performed using gas chromatography-mass spectrometry (GC-MS). The chromatographic conditions included: a capillary column with cyanopropylphenyl-polydimethylsiloxane as the stationary phase; an initial temperature of 80–120 °C for 1–3 minutes; followed by a temperature increase to 230–270 °C at a rate of 40–60 °C / min for 3–7 minutes; and mass spectrometry conditions including: selected ion monitoring (SIM) mode; a quantitative ion of N-methylmorpholine at m / z 101.1 and a qualitative ion at m / z 43.

1.

2. The method according to claim 1, 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 5.5-6.5% cyanopropylphenyl-94.5-93.5% polydimethylsiloxane as the stationary phase was used as the chromatographic column; 2) Column temperature: The initial temperature is 90-110℃, maintained for 1.5-2.5 minutes, and then the temperature is increased to 240-260℃ at a rate of 45-55℃ / min, and maintained for 4-6 minutes; 3) Inlet temperature: 245~255℃; 4) Carrier gas: Helium; 5) Flow rate: 0.5~2.5 mL / min; 6) Flow split ratio: 5:1~15:1; 7) Injection volume: 1~5µL.

3. The method according to claim 2, characterized in that, The chromatographic conditions must satisfy at least one of the following conditions 1) to 6): 1) Chromatographic column: The chromatographic column is a capillary column with 6.0% cyanopropylphenyl-94% polydimethylsiloxane as the stationary phase, preferably VF-624ms, 30m×0.25mm, 1.4µm; 2) Column temperature: The initial temperature is 100℃, maintained for 2 minutes, and then increased to 250℃ at a rate of 50℃ / min, maintained for 5 minutes; 3) Inlet temperature: 250℃; 4) Flow rate: 1.5 mL / min; 5) Flow split ratio: 10:1; 6) Injection volume: 1µL.

4. The method according to claim 1, characterized in that, The mass spectrometry conditions must satisfy at least one of the following conditions 1) to 4): 1) The ion source is an EI source; 2) The ion source temperature is 220~240℃; 3) The quadrupole temperature is 140~160℃; 4) Solvent delay is 1.5~2.5 minutes.

5. The method according to claim 4, characterized in that, The mass spectrometry conditions must satisfy at least one of the following conditions 1) to 3): 1) The ion source temperature is 230℃; 2) The quadrupole temperature is 150℃; 3) Solvent delay is 1.8 minutes.

6. The method according to claim 1, 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 gas chromatography-mass spectrometry (GC-MS) instrument in sequence, record the chromatograms and calculate the content of N-methylmorpholine by peak area according to the external standard method.

7. The method according to claim 6, characterized in that, The preparation of the test solution includes: mixing, dispersing, and centrifuging the gel to be tested with dimethyl sulfoxide, collecting the supernatant, filtering, and obtaining the test solution.

8. The method according to claim 7, 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 solution is 0.1 μg / mL to 10 μg / mL.

9. The method according to any one of claims 1 to 8 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.

10. The application according to claim 9, characterized in that, The gel is made from sodium hyaluronate and recombinant collagen, using 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM) as a condensing agent to induce a condensation reaction between the recombinant collagen and sodium hyaluronate.

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