3 - [4- (2-oxiranylmethoxy) butoxy] -1, 2-propanediol

Abstract

The application discloses a detection method of 3-[4-(2-oxiranylmethoxy) butoxy]-1,2-propanediol. The detection method of 3-[4-(2-oxiranylmethoxy) butoxy]-1,2-propanediol comprises the following steps: using a high performance liquid chromatography-mass spectrometry method to qualitatively or quantitatively detect 3-[4-(2-oxiranylmethoxy) butoxy]-1,2-propanediol in a to-be-detected liquid. The application qualitatively and quantitatively detects 3-[4-(2-oxiranylmethoxy) butoxy]-1,2-propanediol in a to-be-detected sample by using a liquid chromatography-mass spectrometry method, and the sensitivity and accuracy are relatively high, and the reproducibility is good. The method can be used for monitoring the quality of cross-linked sodium hyaluronate or a cross-linked product using BDDE as a cross-linking agent, and reducing the safety risk of the above-mentioned materials.

Description

Technical Field

[0001] This invention relates to the field of chemical detection, and in particular to a method for detecting 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol and its application. Background Technology

[0002] Hyaluronic acid (HA) is a naturally occurring, biodegradable polysaccharide composed of alternating D-glucuronic acid and N-acetyl-D-glucosamine units, exhibiting ideal biocompatibility. Hyaluronic acid is currently the most widely used soft tissue filler material, used in cosmetic medicine for correction or to fill defects in therapeutic applications. However, hyaluronic acid is affected by factors such as hyaluronidase and free radicals in vivo, resulting in rapid degradation, short retention time, and poor mechanical strength in aqueous systems. Therefore, BDDE is often used to modify hyaluronic acid, producing cross-linked sodium hyaluronate gel, which shows significant improvements in both physical and chemical properties compared to natural hyaluronic acid.

[0003] BDDE forms stable ether bonds with the hydroxyl groups in HA through the reaction of epoxy groups at both ends of its molecular chain. Although it has lower toxicity compared to crosslinking agents such as divinyl sulfone, the residual BDDE in crosslinked sodium hyaluronate is often considered a potential toxic or carcinogenic substance due to the presence of highly reactive epoxy groups. It is also listed by the State Food and Drug Administration as a group-toxic impurity requiring strict quality control. YY / T0962-2021 stipulates that the BDDE content in crosslinked sodium hyaluronate must not exceed 2 ppm. During the crosslinking process, some BDDE undergoes single-end ring opening, meaning that crosslinked sodium hyaluronate may contain the impurity 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol. This type of impurity also contains epoxy groups and is also classified as a genotoxic impurity. However, this substance has not received widespread attention, and there is currently no effective method for detecting it in crosslinked sodium hyaluronate.

[0004] Therefore, in order to control the quality of cross-linked sodium hyaluronate or other materials using BDDE as a cross-linking agent and reduce the potential risks of the above materials, there is an urgent need to develop a detection and analysis method for 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol. Summary of the Invention

[0005] The technical problem this application aims to solve is to overcome the shortcomings of existing technologies, such as insufficient attention to residual 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol in cross-linked sodium hyaluronate and the lack of suitable detection methods. This application provides a method for detecting 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol. This application uses liquid chromatography-mass spectrometry (LC-MS) for qualitative and quantitative detection of 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol in the sample, achieving high sensitivity, accuracy, and reproducibility. This method can be used to monitor the quality of cross-linked sodium hyaluronate or cross-linked products using BDDE as a cross-linking agent, reducing the potential risks associated with these materials.

[0006] This application solves the above-mentioned technical problems through the following technical solutions.

[0007] This application provides a method for detecting 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol, comprising the following steps: using high performance liquid chromatography-mass spectrometry to qualitatively or quantitatively detect 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol in the test solution.

[0008] In some embodiments, the test solution comprises a test sample containing 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol and a solvent, wherein the solvent comprises an alcohol solvent and / or water.

[0009] In a preferred embodiment, the alcohol solvent is selected from C1-C3 alcohol solvents, preferably methanol.

[0010] In some embodiments, the concentration of 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol in the test solution is 4~85 ng / mL, for example 5 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL or 85 ng / mL.

[0011] In a preferred embodiment, the sample to be tested is a chemically synthesized sample containing 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol.

[0012] In another preferred embodiment, the sample to be tested is a cross-linked product prepared with BDDE as a cross-linking agent, more preferably a cross-linked polysaccharide prepared with BDDE as a cross-linking agent, and even more preferably a cross-linked sodium hyaluronate prepared with BDDE as a cross-linking agent.

[0013] The cross-linked product may further undergo degradation treatment before testing. Preferably, the degradation method includes enzymatic hydrolysis or chemical degradation. The purpose of the degradation is to fully dissolve 3-[4-(2-epoxyethylenemethoxy)butoxy]-1,2-propanediol in the cross-linked product in a solvent to avoid false negatives.

[0014] When the sample to be tested is cross-linked sodium hyaluronate prepared with BDDE as a cross-linking agent, the degradation method includes the following steps: enzymatic hydrolysis of cross-linked sodium hyaluronate in water using hyaluronidase.

[0015] Preferably, the volume ratio of water to solvent in the test solution is (5~20):1, more preferably (8~15):1, for example 9:1.

[0016] Preferably, the amount of hyaluronidase used is 1000~3000 IU / g, for example 2500 IU / g, based on the unit mass of the cross-linked sodium hyaluronate.

[0017] Preferably, the hyaluronidase can be added in the form of an aqueous solution of hyaluronidase in accordance with conventional practices in the art, wherein the concentration of hyaluronidase in the aqueous solution of hyaluronidase is 8000~15000 IU / mL, preferably 10000 IU / mL.

[0018] Preferably, the enzymatic hydrolysis temperature is 30~45°C, for example 37°C.

[0019] Preferably, the enzymatic hydrolysis time is 0.5 to 5 hours, for example, 1 hour.

[0020] Preferably, the enzymatic hydrolysis may further include a filtration operation, wherein the filtration uses a filter membrane with a pore size of 0.2~0.5μm, for example 0.22μm.

[0021] In some embodiments, the detection method is the external standard method.

[0022] In some embodiments of high performance liquid chromatography, the mobile phase includes mobile phase A and mobile phase B; mobile phase A is an aqueous solution of a volatile acid, wherein the mass percentage of the volatile acid in the aqueous solution is 0.05% to 0.2%; and mobile phase B is methanol.

[0023] Preferably, the aqueous solution of the volatile acid is selected from aqueous formic acid and / or aqueous acetic acid.

[0024] Preferably, the volatile acid in the aqueous solution is 0.08% to 0.12% by mass, for example, 0.1%.

[0025] In some embodiments of high performance liquid chromatography, the flow rate of the mobile phase is 0.15~0.5 mL / min, preferably 0.3 mL / min.

[0026] In some embodiments of high performance liquid chromatography, the elution method is isocratic elution or gradient elution.

[0027] When isocratic elution is used, the mobile phase A accounts for more than 85% of the volume percentage of the mobile phase, preferably 85% to 95%, for example 85%. When the volume percentage of mobile phase A in the mobile phase is less than 85%, for example 80%, the separation effect is poor and the detection accuracy is low.

[0028] When using the gradient elution, from 0 to 8.5 min, the volume percentage of mobile phase A is 85% to 92% and the volume percentage of mobile phase B is 8% to 25%; from 8.6 to 11 min, the volume percentage of mobile phase A is 10% to 30% and the volume percentage of mobile phase B is 70% to 90%; from 11.1 to 15 min, the volume percentage of mobile phase A is 75% to 92% and the volume percentage of mobile phase B is 8% to 25%.

[0029] In some embodiments of high performance liquid chromatography, the injector temperature is 5-30°C, preferably 8-15°C, for example 10°C.

[0030] In some embodiments of high performance liquid chromatography, the injection volume of the test solution is 0.5~5μL, for example 1μL.

[0031] In some embodiments of high-performance liquid chromatography, the chromatographic column is an octadecylsilane-bonded silica column, preferably an Agilent Infinitylab Poroshell 120 EC-18 100mm column. 3mm, 1.9µm.

[0032] In a preferred embodiment of high performance liquid chromatography, the octadecylsilane-bonded silica column has a packing particle size of 1.5~5μm, for example 1.9μm.

[0033] In a preferred embodiment of high performance liquid chromatography, the octadecylsilane-bonded silica column has a column length of 50-250 mm, for example, 100 mm.

[0034] In a preferred embodiment of high performance liquid chromatography, the inner diameter of the octadecylsilane-bonded silica column is 1.8 to 4.6 mm, for example, 3 mm.

[0035] In some embodiments of high performance liquid chromatography, the column temperature is 30~50°C, for example 40°C.

[0036] In some embodiments of mass spectrometry detection, the eluent with a high performance liquid chromatography retention time of 5 to 8.5 min is detected.

[0037] In some embodiments of the mass spectrometry, the ion mode is positive ion mode.

[0038] In some embodiments of the mass spectrometry, the scanning mode is multiple reaction monitoring mode.

[0039] In some embodiments of the mass spectrometry, the ion source is an electrospray ion source.

[0040] In some embodiments of the mass spectrometer, the drying gas is nitrogen.

[0041] In some embodiments of the mass spectrometer, the temperature of the drying gas is 100~350°C, preferably 300~340°C, for example 325°C.

[0042] In some embodiments of the mass spectrometer, the flow rate of the drying gas is 1 to 13 L / min, for example 10 L / min.

[0043] In some embodiments of the mass spectrometer, the nebulizer gas pressure is 10 to 40 psi, for example, 40 psi.

[0044] In some embodiments of the mass spectrometry, the sheath gas is nitrogen.

[0045] In some embodiments of the mass spectrometry, the temperature of the sheath gas is 250-400°C, preferably 380-400°C, for example 400°C.

[0046] In some embodiments of the mass spectrometer, the flow rate of the sheath gas is 10-14 L / min, for example 11 L / min.

[0047] In some embodiments of the mass spectrometer, the capillary voltage is 3500~6000V, preferably 3500~4500V, for example 4000V.

[0048] In some embodiments of the mass spectrometer, the fragmentation voltage is 0~200V, preferably 60~150V, more preferably 70~100V, for example 60V, 70V, 80V, 90V, 100V, 110V, 120V, 130V, 140V, 150V, 160V, 170V, 180V, 190V or 200V.

[0049] In some embodiments of the mass spectrometer, the collision energy is 5 to 35 V, preferably 10 to 20 V, such as 5 V, 8 V, 10 V, 12 V, 14 V, 16 V, 18 V, 20 V, 22 V, 24 V, 26 V, 28 V, 30 V, 32 V, 34 V or 35 V.

[0050] In the mass spectrometry of some embodiments, the detection ion pair for 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol is at least one of 221 / 57.1, 221 / 73.2 and 221 / 129.

[0051] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of this application.

[0052] All reagents and raw materials used in this application are commercially available.

[0053] The positive advancements of this application are as follows: The liquid chromatography-mass spectrometry (LC-MS) method provided here can perform qualitative and quantitative detection of 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol in the sample to be tested, with high sensitivity and accuracy, and good reproducibility. This method can also be used to monitor the quality of cross-linked sodium hyaluronate or cross-linked products using BDDE as a cross-linking agent, reducing the potential risks associated with these materials. Attached Figure Description

[0054] Figure 1 The gas chromatogram of 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol under the detection conditions of Comparative Example 1;

[0055] Figure 2 The gas chromatogram of BDDE under the detection conditions of Comparative Example 1 is shown.

[0056] Figure 3 The gas chromatogram of 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol under the detection conditions of Comparative Example 2;

[0057] Figure 4 The gas chromatogram of BDDE under the detection conditions of Comparative Example 2 is shown.

[0058] Figure 5 The gas chromatogram of the mixture of 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol and BDDE under the detection conditions of Comparative Example 2;

[0059] Figure 6 The detection graph of the reference solution at a collision energy of 15V is shown in Example 3.

[0060] Figure 7 The detection graph of the reference solution at a collision energy of 5V is shown in Example 3.

[0061] Figure 8 The detection graph of the reference solution at a collision energy of 10V is shown in Example 3.

[0062] Figure 9The detection graph of the reference solution at a collision energy of 20V is shown in Example 3.

[0063] Figure 10 The characteristic spectrum of the blank solution in Example 4 is shown.

[0064] Figure 11 The characteristic spectrum of the reference solution in Example 4 is shown.

[0065] Figure 12 The characteristic spectrum of the test solution in Example 4 is shown.

[0066] Figure 13 The characteristic spectrum of the spiked test solution in Example 4 is shown.

[0067] Figure 14 The standard curve graph is plotted in Example 9 to illustrate the effect. Detailed Implementation

[0068] The present application is further illustrated below by way of embodiments, but these embodiments are not intended to limit the scope of the present application. Experimental methods not specifically described in the following embodiments are performed according to conventional methods and conditions, or as selected in accordance with the product instructions.

[0069] In the following examples and comparative examples, hyaluronidase was purchased from Anhui Zesheng Technology Co., Ltd., product model 005XR5FH;

[0070] In the following examples and comparative examples, the formic acid and methanol were of chromatographic purity.

[0071] In the following examples and comparative examples, the water used is ultrapure water;

[0072] In the examples and comparative examples below, the purity of the 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol standard was 96.46%.

[0073] The following method is used to prepare cross-linked sodium hyaluronate: 100 μL of 1,4-butanediol diglycidyl ether (BDDE) is added to 8.5 mL of 1 wt% sodium hydroxide aqueous solution and mixed evenly to obtain material A. 1 g of sodium hyaluronate with a weight-average molecular weight of 130 WDa is weighed and added to material A. After mixing evenly, a first cross-linking reaction is carried out at 40°C and allowed to stand for 4 h, followed by a second cross-linking reaction at 25°C and allowed to stand for 12 h. The gel obtained after the second cross-linking reaction is cut into suitable small pieces. The cut gel is then dialyzed and washed using a phosphate buffer solution with an osmotic pressure of 250 mOsm / L and a pH of 7. The dialysate is changed every 8 h for a total of 72 h. The washed gel is diluted to prepare a gel with a sodium hyaluronate concentration of 20 mg / g. The diluted gel is granulated twice using a 200-mesh sieve to obtain cross-linked sodium hyaluronate.

[0074] Example 1

[0075] (1) Solution preparation

[0076] Preparation of the test solution: Weigh 200 mg of cross-linked sodium hyaluronate and place it in a 10 mL volumetric flask. Add 1 mL of water and 50 µL of hyaluronidase aqueous solution. The concentration of hyaluronidase in the hyaluronidase aqueous solution is 10000 IU / mL. Incubate in a water bath at 37 °C for 1 hour to obtain the enzymatically hydrolyzed test sample. Make up to 10 mL with methanol, shake well, and filter through a 0.22 µm filter membrane.

[0077] Preparation of reference solution: Dissolve 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol in water to prepare a solution containing 400 ng of 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol per mL. Accurately measure 1 mL of the solution and place it in a 10 mL volumetric flask. Dilute to the mark with methanol and shake well.

[0078] (2) High performance liquid chromatography-mass spectrometry conditions

[0079] Equipment: Agilent liquid chromatography-mass spectrometry system, model 1290HPLC / G6470BMSD;

[0080] Chromatographic column: Octadecylsilane bonded silica column, model Agilent Infinitylab poroshell120 EC-18, column length 100mm, inner diameter 3mm, packing particle size 1.9µm.

[0081] Chromatographic conditions: injector temperature 10℃; column temperature 40℃; injection volume 1µL; mobile phase A was 0.1% formic acid aqueous solution, mobile phase B was methanol, and the flow rate of the mobile phase was 0.3mL / min; the elution gradient of the mobile phase is shown in Table 1 below.

[0082] Table 1

[0083]

[0084] Mass spectrometry conditions:

[0085] The ion source was an ESI source (electrospray ionization source); the drying gas was nitrogen, with a temperature of 325℃ and a flow rate of 10 L / min; the nebulizing gas pressure was 40 psi; the sheath gas was nitrogen, with a temperature of 400℃ and a flow rate of 11 L / min; the capillary voltage was 4000 V; the fragmentation voltage was 70 V; the collision energy (CE) was 15 V; the ionization mode was positive ion mode; the scanning mode was multiple reaction monitoring (MRM); the quantitative ion pair was 221 / 73.2, and the qualitative ion pair was 221 / 57.1.

[0086] The valve switching mode is as follows: 0 min to waste liquid, 5 min to mass spectrometer, and 8.5 min to waste liquid.

[0087] (3) Detection and calculation methods: The test solution and reference solution were detected using the high performance liquid chromatography-mass spectrometry conditions in step (2). The concentration of 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol in the test solution was calculated using the external standard method. The concentration of 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol in cross-linked sodium hyaluronate was further calculated.

[0088] Comparative Example 1: Gas chromatography was used.

[0089] Gas chromatography column: DB-624 column (30m) 0.53mm (3.0 μm), the stationary phase is 6% cyanopropyl-94% dimethylpolysiloxane;

[0090] Gas chromatography conditions: initial temperature 150℃, increased to 240℃ at a rate of 30℃ / min, held for 9 min, then increased to 250℃ at a rate of 10℃ / min, held for 12 min;

[0091] Detector: FID;

[0092] Carrier gas: Nitrogen;

[0093] Carrier gas flow rate: 2 mL / min;

[0094] Inlet temperature: 250℃;

[0095] Detector temperature: 280℃;

[0096] Flow split ratio: 1:1;

[0097] Injection volume: 2 μL;

[0098] Detector: FID;

[0099] 3-[4-(2-epoxyethylenemethoxy)butoxy]-1,2-propanediol and BDDE were tested under the above gas chromatography conditions, and the results are shown in the figures below. Figure 1 and Figure 2 .

[0100] It is evident that the peak positions of 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol and BDDE are both within the retention time range of 8–10 min, making separation impossible. Furthermore, the peak area of ​​BDDE is 1,511,836, while the peak area of ​​3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol is only 106,229, significantly lower than that of BDDE. When both substances are present in the system, the detection sensitivity of the analyte 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol is low, rendering the method unsuitable.

[0101] Comparative Example 2

[0102] Gas chromatography column: RTX-50 column (30m) 0.32mm 0.25 μm), the stationary phase is 50% phenyl + dimethyl polysiloxane;

[0103] Gas chromatography conditions: initial temperature 150℃, increased to 260℃ at a rate of 30℃ / min, and held for 10 min;

[0104] Carrier gas: Nitrogen;

[0105] Carrier gas flow rate: 1.5 mL / min;

[0106] Inlet temperature: 250℃;

[0107] Detector temperature: 280℃;

[0108] Flow split ratio: 1:1;

[0109] Injection volume: 2 μL;

[0110] Detector: FID;

[0111] 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol test solution: Mix 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol with methanol to prepare a test solution with a concentration of 1 mg / mL;

[0112] BDDE test solution: Mix BDDE with methanol to prepare a test solution with a concentration of 1 mg / mL;

[0113] Mixed test solution: Mix 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol, BDDE and methanol to prepare a test solution with a concentration of 100 μg / mL for each analyte;

[0114] The three test solutions prepared above were tested under the above gas chromatography conditions, and the results are shown in the figure below. Figures 3-5 .

[0115] It can be seen that, according to Figure 3 and Figure 4 The results showed that both 3-[4-(2-epoxyethylenemethoxy)butoxy]-1,2-propanediol and BDDE produced impurities under gas-phase detection conditions, suggesting that the two analytes may have undergone an epoxy group ring-opening reaction during the detection process. Additionally, according to... Figure 5 The data results show that when the concentrations of BDDE and 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol in the test solution are 1 mg / mL, the signal-to-noise ratios are 16.6 and 4.4, respectively. It is evident that the signal-to-noise ratios are low at a concentration of 1 mg / mL for both substances, and this concentration is significantly higher than the upper limit for BDDE content control in the industry standard YY / T0962-2021 (2 mg / mL). 10 -3 (mg / mL). When this method is used to detect the content of both in cross-linked sodium hyaluronate, the signal-to-noise ratio will be even lower, the detection sensitivity will be low, and the method will not be applicable.

[0116] Example 2

[0117] Compared with Example 1, the only difference is that the solvent in the test solution prepared in step (1) is different. Specifically, methanol is replaced with an equal amount of water, and the temperature of the injector in step (2) is adjusted to 25°C. Other conditions and parameters are the same as in Example 1. The concentration of 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol in cross-linked sodium hyaluronate is tested and calculated.

[0118] Examples 3-12

[0119] Compared with Example 1, the only difference is the rupture voltage in step (2). The rupture voltage is adjusted to 80V, 90V, 100V, 110V, 120V, 130V, 140V, 150V, 180V or 200V respectively. Other conditions and parameters are the same as in Example 1. The concentration of 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol in cross-linked sodium hyaluronate is tested and calculated.

[0120] Examples 13-15

[0121] Compared with Example 1, the only difference is the collision energy in step (2). The collision energy is adjusted to 5V, 10V or 20V respectively, and other conditions and parameters are the same as in Example 1. The concentration of 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol in cross-linked sodium hyaluronate is tested and calculated.

[0122] Example 1: Recovery Rate

[0123] The recovery rates of the detection methods in Examples 1 and 2 above were verified. The specific experimental steps are as follows:

[0124] (1) Solution preparation

[0125] Test solution: Prepared in the same way as the test solution in Examples 1 and 2 above;

[0126] Example 1 Reference solution: Dissolve 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol in water to prepare a solution containing 400 ng of 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol per 1 mL as a stock solution; then take 1 mL of the stock solution into a 10 mL volumetric flask, dilute to volume with methanol and shake well;

[0127] Example 1: Spiked solution for test sample: Weigh 200 mg of cross-linked sodium hyaluronate and place it in a 10 mL volumetric flask. Add 1 mL of stock solution and 50 µL of hyaluronidase aqueous solution. The concentration of hyaluronidase in the aqueous solution is 10,000 IU / mL. Incubate in a water bath at 37 °C for 1 hour. Make up to 10 mL with methanol, shake well, and filter through a 0.22 µm filter membrane.

[0128] Example 2 Reference Solution: Dissolve 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol in water to prepare a solution containing 400 ng of 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol per 1 mL as a stock solution; then take 1 mL of the stock solution into a 10 mL volumetric flask, dilute to volume with water and shake well.

[0129] Example 2: Spiked solution for test sample: Weigh 200 mg of cross-linked sodium hyaluronate and place it in a 10 mL volumetric flask. Add 1 mL of stock solution and 50 µL of hyaluronidase aqueous solution. The concentration of hyaluronidase in the aqueous solution is 10,000 IU / mL. Incubate in a water bath at 37 °C for 1 hour. Make up to 10 mL with water, shake well, and filter through a 0.22 µm filter membrane.

[0130] (2) The reference solution, test solution and spiked solution of the test sample were detected using the high performance liquid chromatography-mass spectrometry conditions in Example 1 or Example 2 above. The concentration of (3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol) in the test solution and spiked solution of the test sample was calculated using the external standard method. The mass of 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol in 200 mg of cross-linked sodium hyaluronate was calculated as the background amount. The mass of 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol in the spiked solution of the test sample prepared with 200 mg of cross-linked sodium hyaluronate was calculated and labeled as the test amount. The recovery rate was calculated. The test was performed in parallel for three times and the average value was taken. The results are shown in Table 2.

[0131] Recovery rate = (Test volume - Background volume) / Added volume 100%, wherein the amount added is the mass of 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol in 1 mL of stock solution.

[0132] Table 2

[0133]

[0134] Example 2: Effect of Rupture Voltage on Response Value

[0135] The test solutions in Examples 1 and 3-12 were replaced with reference solutions, and the reference solutions were detected using the detection methods corresponding to the above examples. The ion abundance at a mass-to-charge ratio of 221 was recorded, and the results are shown in Table 3.

[0136] Preparation of reference solution: Dissolve 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol in water to prepare a solution containing 50 μg of 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol per mL.

[0137] Table 3

[0138]

[0139] According to the results in Table 3, the smaller the rupture voltage, the greater the ion abundance at mass-to-charge ratio 221, and the higher the detection sensitivity.

[0140] Example 3: Effect of Collision Energy on Response Value

[0141] In Examples 1 and 13-15 above, the test solution was replaced with a reference solution, and the reference solution was tested using the detection methods corresponding to the above examples. The detection results are shown in the figures below. Figures 6-9 The ion abundances at mass-to-charge ratios of 57.2 and 73.1 were recorded, and the results are shown in Table 4.

[0142] Preparation of reference solution: Dissolve 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol in water to prepare a solution containing 50 μg of 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol per mL.

[0143] Table 4

[0144]

[0145] According to the results in Table 4, when using the detection method of the example, both the qualitative ion pair (221 / 57.1) and the quantitative ion pair (221 / 73.2) have ideal response values, and the detection sensitivity is the highest when the collision energy is 15V.

[0146] Example 4: Specificity

[0147] The specificity of the detection method was verified with reference to the "Guiding Principles for Registration Review of Qualitative and Quantitative Studies and In Vitro Release of Drugs in Drug-Device Combination Products with Medical Device Function as the Main Function".

[0148] (1) Solution preparation

[0149] Blank solution: Methanol and water are mixed in a volume ratio of 90:10 and labeled as Blank.

[0150] Reference solution: Accurately weigh approximately 20 mg of 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol standard, place it in a 10 mL volumetric flask, dissolve and dilute to the mark with water, and shake well. Accurately measure 2 mL of this solution, place it in a 100 mL volumetric flask, dilute to the mark with water, and shake well. Accurately measure 1 mL of this solution, place it in a 100 mL volumetric flask, dilute to the mark with water, and shake well to obtain the stock solution. Accurately measure 2 mL of the stock solution, place it in a 20 mL volumetric flask, dilute to the mark with methanol, and shake well to obtain the final product, labeled as STD.

[0151] Test solution: Accurately weigh approximately 200 mg of cross-linked sodium hyaluronate and place it in a 10 mL volumetric flask. Add 1 mL of water and 50 µL of hyaluronidase aqueous solution (10000 IU / mL). Seal the flask and incubate it in a 37°C water bath for approximately 1 hour. Dilute to the mark with methanol, shake well, and filter through a 0.22 µm filter membrane. Collect the filtrate and label it as SPL.

[0152] Spiked test solution: Accurately weigh approximately 200 mg of cross-linked sodium hyaluronate and place it in a 10 mL volumetric flask. Accurately add 1 mL of stock solution, 1 mL of water, and 50 µL of hyaluronidase aqueous solution (10000 IU / mL). Seal and incubate at 37°C for approximately 1 hour. Dilute to the mark with methanol, shake well, and filter through a 0.22 µm filter membrane. Collect the filtrate. Label as SPL-STD.

[0153] (2) The blank solution, reference solution, test solution, and spiked test solution were tested using the high performance liquid chromatography-mass spectrometry conditions in step (2) of Example 1. The retention times of the peaks were observed, and the results are shown in Table 5. Figures 10-13 .

[0154] Table 5

[0155]

[0156] (3) Verification conclusion

[0157] According to the results in Table 5, neither the blank solution nor cross-linked sodium hyaluronate interfered with the determination of 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol, indicating that the detection method in Example 1 is specific.

[0158] Example 5: Limit of Quantitation and Limit of Detection

[0159] The limits of quantitation and limits of detection of the detection method were verified with reference to the "Guiding Principles for Registration Review of Qualitative and Quantitative Studies and In Vitro Release of Drugs in Drug-Device Combination Products with Medical Device Function as the Main Function".

[0160] (1) Solution preparation

[0161] Limit of Quantitation (LOQ) Solution: Accurately weigh approximately 20 mg of 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol standard, place it in a 10 mL volumetric flask, dissolve and dilute to the mark with water, and mix well. Accurately measure 2 mL of this solution and place it in a 100 mL volumetric flask, dilute to the mark with water, and mix well. Accurately measure 1 mL of this solution and place it in a 100 mL volumetric flask, dilute to the mark with water, and mix well to obtain the stock solution. Accurately measure 2 mL of the stock solution and place it in a 20 mL volumetric flask, dilute to the mark with methanol, and mix well. Label this solution as STD. Take 1 mL of the STD solution, place it in a 10 mL volumetric flask, dilute to the mark with methanol, and mix well to obtain the final solution. Prepare six parallel aliquots, labeled LOQ-1 to LOQ-6.

[0162] Limit of Detection Solution: Take 3 mL of the above-prepared limit of quantitation solution, place it in a 10 mL volumetric flask, dilute to the mark with methanol, shake well, and label it as LOD.

[0163] (2) The limit of quantitation solution and the limit of detection solution were tested using the high performance liquid chromatography-mass spectrometry conditions in step (2) of Example 1, and the signal-to-noise ratio was calculated. The results of the limit of quantitation are shown in Table 6, and the results of the limit of detection are shown in Table 7.

[0164] Table 6

[0165]

[0166] Table 7

[0167]

[0168] (3) Verification conclusion

[0169] The LOQ solution concentration was 4.01 ng / mL, which is equivalent to 10.0% of the limit concentration and not greater than 20% of the limit concentration. The signal-to-noise ratio of 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol in the 6 LOQ solutions was between 10 and 14, and not less than 10. The limit of quantitation verification results met the standard.

[0170] The LOD solution concentration is 1.20 ng / mL, which is equivalent to 3.0% of the limit concentration and not greater than 10% of the limit concentration. The signal-to-noise ratio of 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol in the LOD solution is 5, which is not less than 3. The detection limit verification results meet the standard.

[0171] Effect Example 6: Solution Stability

[0172] The stability of the detection method was verified with reference to the "Guiding Principles for Registration Review of Qualitative and Quantitative Studies and In Vitro Release of Drugs in Drug-Device Combination Products with Medical Device Function as the Main Function".

[0173] (1) Solution preparation

[0174] Reference solution: Accurately weigh approximately 20 mg of 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol standard, place it in a 10 mL volumetric flask, dissolve and dilute to the mark with water, and shake well. Accurately measure 2 mL of this solution, place it in a 100 mL volumetric flask, dilute to the mark with water, and shake well. Accurately measure 1 mL of this solution, place it in a 100 mL volumetric flask, dilute to the mark with water, and shake well to obtain the stock solution. Accurately measure 2 mL of the stock solution, place it in a 20 mL volumetric flask, dilute to the mark with methanol, and shake well to obtain the final product, labeled as STD.

[0175] Spiked test solution: Weigh approximately 200 mg of cross-linked sodium hyaluronate accurately and place it in a 10 mL volumetric flask. Accurately add 1 mL of stock solution, 1 mL of water, and 50 µL of hyaluronidase aqueous solution (10000 IU / mL). Seal and incubate at 37°C for approximately 1 hour. Dilute to the mark with methanol, shake well, and filter through a 0.22 µm filter membrane. Collect the filtrate and label it as SPL-STD.

[0176] (2) The peak areas of the reference solution were tested at room temperature for 0h, 4h and 8.5h using the high performance liquid chromatography-mass spectrometry conditions in step (2) of Example 1, and the percentage change in peak area at 4h and 8.5h relative to 0h was calculated. The results are shown in Table 8.

[0177] The peak areas of the spiked test solution were tested at room temperature for 0h, 4h and 8.5h using the high performance liquid chromatography-mass spectrometry conditions in step (2) of Example 1, and the percentage change in peak area at 4h and 8.5h relative to 0h was calculated. The results are shown in Table 9.

[0178] Table 8

[0179]

[0180] Table 9

[0181]

[0182] (4) Verification conclusion

[0183] The reference solution was placed at room temperature for 8.5 hours. The peak area at different time points was between 95.0% and 97.3% of the peak area at 0 hours, and all were between 80% and 120%. The verification results met the acceptance criteria.

[0184] After the spiked test solution was placed at room temperature for 8.5 hours, the peak area at different time points was between 92.1% and 95.3% of the peak area at 0 hours, and all were between 80% and 120%. The verification results met the acceptance criteria.

[0185] Example 7: Accuracy and Precision

[0186] The accuracy and precision of the testing methods were verified in accordance with the "Guiding Principles for Registration Review of Qualitative and Quantitative Studies and In Vitro Release of Drugs in Drug-Device Combination Products with Medical Device Function as the Main Function".

[0187] (1) Solution preparation

[0188] Blank solution: Methanol and water are mixed in a volume ratio of 90:10 and labeled as Blank.

[0189] Reference solution: Accurately weigh approximately 20 mg of 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol standard, place it in a 10 mL volumetric flask, dissolve and dilute to the mark with water, and shake well. Accurately measure 2 mL of this solution, place it in a 100 mL volumetric flask, dilute to the mark with water, and shake well. Accurately measure 1 mL of this solution, place it in a 100 mL volumetric flask, dilute to the mark with water, and shake well to obtain the stock solution. Accurately measure 1 mL of the stock solution, place it in a 10 mL volumetric flask, dilute to the mark with methanol, and shake well to obtain the final product, labeled as STD.

[0190] Test solution: Accurately weigh approximately 200 mg of cross-linked sodium hyaluronate and place it in a 10 mL volumetric flask. Add 1 mL of water and 50 µL of hyaluronidase aqueous solution (10000 IU / mL). Seal the flask and incubate it in a 37°C water bath for approximately 1 hour. Dilute to the mark with methanol, shake well, and filter through a 0.22 µm filter membrane. Collect the filtrate and label it as SPL.

[0191] LOQ concentration spiking solution: Accurately weigh approximately 200 mg of cross-linked sodium hyaluronate and place it in a 10 mL volumetric flask. Accurately add 1 mL of the reference solution, 1 mL of water, and 50 µL of hyaluronidase aqueous solution (10000 IU / mL). Seal and incubate at 37°C for approximately 1 hour. Dilute to the mark with methanol, shake well, and filter through a 0.22 µm filter membrane. Collect the filtrate. Prepare three parallel portions, labeled as SPL-LOQ-1 to SPL-LOQ-3, respectively.

[0192] 100% concentration spiked solution: Accurately weigh approximately 200 mg of cross-linked sodium hyaluronate and place it in a 10 mL volumetric flask. Accurately add 1 mL of stock solution, 1 mL of water, and 50 µL of hyaluronidase aqueous solution (10000 IU / mL). Seal and incubate at 37°C for approximately 1 hour. Dilute to the mark with methanol, shake well, and filter through a 0.22 µm filter membrane. Collect the filtrate. Prepare 6 parallel portions, labeled as SPL-100STD-1 to SPL-100STD-6.

[0193] 150% concentration spiked solution: Accurately weigh approximately 200 mg of cross-linked sodium hyaluronate and place it in a 10 mL volumetric flask. Accurately add 1.5 mL of stock solution, 1 mL of water, and 50 µL of hyaluronidase aqueous solution (10000 IU / mL). Seal and incubate at 37°C for approximately 1 hour. Dilute to the mark with methanol, shake well, and filter through a 0.22 µm filter membrane. Collect the filtrate. Prepare three parallel aliquots, labeled as SPL-150STD-1 to SPL-150STD-3, respectively.

[0194] (2) The solutions prepared above were detected using the high performance liquid chromatography-mass spectrometry conditions in step (2) of Example 1. The concentration of 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol in each solution was calculated using the external standard method. The mass of 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol in 200 mg of cross-linked sodium hyaluronate was then calculated as the background amount. The mass of 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol in each spiked solution was calculated and labeled as the test amount. The recovery rate was calculated. The test was performed in parallel three times, and the average value was taken. The results are shown in Table 10.

[0195] Recovery rate = (Test volume - Background volume) / Added volume 100%, wherein the amount added is the mass of 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol in 1 mL of stock solution.

[0196] Table 10

[0197]

[0198] (3) Verification conclusion

[0199] The recoveries of 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol at LOQ%, 100%, and 150% concentration levels ranged from 84.9% to 105.0%, with an average recovery of 96.1%, all within the range of 75% to 120%. The RSD was 7.6%, not exceeding 8%, and the accuracy test results met the standards.

[0200] The RSD of 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol content in SPL-100STD-1 to SPL-100STD-6 is 7.2%, which is no more than 8%. Therefore, the precision test results meet the standard.

[0201] Example 8: Durability

[0202] The robustness of the testing method was verified with reference to the "Guiding Principles for Registration and Review of Qualitative and Quantitative Studies of Drugs and In Vitro Release in Drug-Device Combination Products with Medical Device Function as the Main Function".

[0203] (1) Solution preparation

[0204] Reference solution: Accurately weigh approximately 20 mg of 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol standard, place it in a 10 mL volumetric flask, dissolve and dilute to the mark with water, and shake well. Accurately measure 2 mL of this solution, place it in a 100 mL volumetric flask, dilute to the mark with water, and shake well. Accurately measure 1 mL of this solution, place it in a 100 mL volumetric flask, dilute to the mark with water, and shake well to obtain the stock solution. Accurately measure 1 mL of the stock solution, place it in a 10 mL volumetric flask, dilute to the mark with methanol, and shake well.

[0205] Spiked test solution: Weigh approximately 200 mg of cross-linked sodium hyaluronate accurately and place it in a 10 mL volumetric flask. Accurately add 1 mL of stock solution, 1 mL of water, and 50 µL of hyaluronidase aqueous solution (10000 IU / mL). Seal and incubate at 37°C for approximately 1 hour. Dilute to the mark with methanol, shake well, and filter through a 0.22 µm filter membrane. Collect the filtrate and label it as SPL-STD.

[0206] (2) The above reference solution and spiked test solution were detected using the high performance liquid chromatography-mass spectrometry conditions in step (2) of Example 1. The concentration of 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol in the spiked test solution was calculated according to the external standard method. The results are shown in Table 11.

[0207] The robustness of the method was observed by changing the concentration of formic acid in mobile phase A by 0.1±0.01%, the column temperature by 40±2℃, and the flow rate of mobile phase by 0.3±0.01mL / min. The results are shown in Table 11.

[0208] Table 11

[0209]

[0210] (3) Verification conclusion

[0211] By changing the concentration of the mobile phase A formic acid aqueous solution, the column temperature, and the mobile phase flow rate, compared with Example 1, the difference in the content of 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol in the spiked test solution was between 0.00 ppm and 0.04 ppm, none of which was greater than 0.2 ppm. Therefore, the durability test results met the standard.

[0212] Example 9: Linear

[0213] The linearity of the detection method was verified with reference to the "Guiding Principles for Registration Review of Qualitative and Quantitative Studies and In Vitro Release of Drugs in Drug-Device Combination Products with Medical Device Function as the Main Function".

[0214] (1) Solution preparation

[0215] Weigh approximately 20 mg of 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol standard accurately, place it in a 10 mL volumetric flask, dissolve and dilute to the mark with water, and shake well. Accurately measure 2 mL of this solution, place it in a 100 mL volumetric flask, dilute to the mark with water, and shake well. Accurately measure 1 mL of this solution, place it in a 100 mL volumetric flask, dilute to the mark with water, and shake well to prepare a stock solution.

[0216] L-200%: Accurately measure 2 mL of the stock solution, place it in a 10 mL volumetric flask, dilute to the mark with methanol, and shake well;

[0217] L-150%: Accurately measure 1.5 mL of the stock solution, place it in a 10 mL volumetric flask, dilute to the mark with methanol, and shake well;

[0218] L-100%: Accurately measure 1.0 mL of the stock solution, place it in a 10 mL volumetric flask, dilute to the mark with methanol, and shake well;

[0219] L-50%: Accurately measure 0.5 mL of the stock solution, place it in a 10 mL volumetric flask, dilute to the mark with methanol, and shake well;

[0220] L-LOQ: Accurately measure 2 mL of the stock solution and place it in a 20 mL volumetric flask. Dilute to the mark with methanol and shake well. Take 1 mL of this solution and place it in a 10 mL volumetric flask. Dilute to the mark with methanol and shake well.

[0221] The HPLC-MS conditions of step (2) in Example 1 were used to detect the above-mentioned test solutions containing different concentrations of standard (i.e., analytes). Within the LOQ~200% limit solution range, the linear equation and correlation coefficient R of the analyte were obtained by plotting the concentration of the analyte as the abscissa and the peak area (ion abundance ratio) as the ordinate. The results are shown in Table 12 and 13. Figure 14 The results show that the peak area is positively correlated with the concentration of 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol, with a correlation coefficient R=0.9994 and an intercept / Y100%=2.1%; where R is not less than 0.990 and the intercept / Y100% is not greater than 25%, the linearity results meet the acceptance criteria.

[0222] Table 12

[0223]

[0224] Application Example 1

[0225] Preparation of cross-linked sodium hyaluronate: 100 μL of 1,4-butanediol diglycidyl ether (BDDE) was added to 8.5 mL of a 1 wt% sodium hydroxide aqueous solution and mixed thoroughly to obtain material A. 1 g of sodium hyaluronate with a weight-average molecular weight of 130 WDa was weighed and added to material A. After mixing thoroughly, a first cross-linking reaction was carried out at 40°C under static conditions for 4 h, followed by a second cross-linking reaction at 25°C under static conditions for 12 h. The gel obtained after the second cross-linking reaction was cut into suitable small pieces; the gel was then subjected to dialysis. The method involved dialysis and washing the sheared gel with a phosphate buffer solution of 250 mOsm / L and pH 7. The phosphate buffer solution was changed every 8 hours. Gels were collected after dialysis for 32 hours, 48 ​​hours, and 64 hours, respectively. The washed gels were diluted to prepare a gel with a sodium hyaluronate concentration of 20 mg / g. The diluted gels were then granulated twice through a 200-mesh sieve to obtain cross-linked sodium hyaluronate, which were named cross-linked sodium hyaluronate A, cross-linked sodium hyaluronate B, and cross-linked sodium hyaluronate C, respectively.

[0226] The content of 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol in the cross-linked sodium hyaluronate A~C prepared above was tested using the method of Example 1. The test results of the reference standard are shown in Table 13, and the test results of the sample to be tested are shown in Table 14.

[0227] Table 13

[0228]

[0229] Table 14

[0230]

[0231] The results show that the cross-linked sodium hyaluronate system contains a large amount of the impurity 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol after the cross-linking reaction. The impurity content in the gel gradually decreases with increasing dialysis cycles. Therefore, this method can be used to detect the content of this impurity in the sample, assess product quality, monitor dialysis progress, determine the end time of dialysis, and avoid increased production costs and risk of bacterial contamination due to over-dialysis.

[0232] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof in this application 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 process, method, article, or apparatus.

[0233] Although this application has been disclosed above through the description of specific embodiments, it should be understood that those skilled in the art can devise various modifications, improvements, or equivalents to this application within the spirit and scope of the appended solutions. Such modifications, improvements, or equivalents should also be considered to be included within the scope of protection claimed in this application.

Claims

1. A method for detecting 3-[4-(2-epoxyethylenemethoxy)butoxy]-1,2-propanediol, characterized in that, The steps include: using high performance liquid chromatography-mass spectrometry to qualitatively or quantitatively detect 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol in the test solution.

2. The detection method as described in claim 1, characterized in that, The test solution meets the following conditions (1) and / or (2): (1) The test solution includes a test sample containing 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol and a solvent, wherein the solvent includes alcohol solvents and / or water; (2) The concentration of 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol in the test solution is 4~85 ng / mL.

3. The detection method as described in claim 2, characterized in that, The test solution meets the following conditions (1) and / or (2): (1) The alcohol solvent is selected from C1~C3 alcohol solvents, preferably methanol; (2) The sample to be tested is a chemically synthesized sample containing 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol or a cross-linked product prepared by using BDDE as a cross-linking agent.

4. The detection method as described in claim 3, characterized in that, The sample to be tested is a cross-linked polysaccharide prepared with BDDE as a cross-linking agent, preferably a cross-linked sodium hyaluronate prepared with BDDE as a cross-linking agent.

5. The detection method as described in claim 3 or 4, characterized in that, The cross-linked product further includes a degradation treatment before testing; Preferably, the degradation method includes enzymatic hydrolysis or chemical degradation.

6. The detection method as described in claim 5, characterized in that, The sample to be tested is cross-linked sodium hyaluronate prepared with BDDE as a cross-linking agent. The degradation method includes the following steps: enzymatic hydrolysis of cross-linked sodium hyaluronate in water using hyaluronidase. Preferably, the volume ratio of water to solvent in the test solution is (5~20):1; Preferably, the amount of hyaluronidase used is 1000~3000 IU / g, based on the unit mass of the cross-linked sodium hyaluronate. Preferably, the hyaluronidase is added in the form of an aqueous solution of hyaluronidase, wherein the concentration of hyaluronidase in the aqueous solution of hyaluronidase is 8000~15000 IU / mL; Preferably, the enzymatic hydrolysis temperature is 30~45℃; Preferably, the enzymatic hydrolysis time is 0.5~5 hours; Preferably, the enzymatic hydrolysis is further followed by a filtration operation, wherein the filtration uses a filter membrane with a pore size of 0.2~0.5μm.

7. The detection method according to any one of claims 1 to 6, characterized in that, The detection method satisfies at least one of the following conditions (1) to (7): (1) In high performance liquid chromatography, the mobile phase includes mobile phase A and mobile phase B; mobile phase A is an aqueous solution of a volatile acid, and the mass percentage of the volatile acid in the aqueous solution is 0.05%~0.2%; mobile phase B is methanol; (2) In high performance liquid chromatography, the flow rate of the mobile phase is 0.15~0.5 mL / min; (3) In high performance liquid chromatography, the elution method is isocratic elution or gradient elution; (4) In high performance liquid chromatography, the temperature of the injector is 5~30℃; (5) In high performance liquid chromatography, the injection volume of the test solution is 0.5~5μL; (6) In high performance liquid chromatography, the chromatographic column is an octadecylsilane-bonded silica gel column; (7) In high performance liquid chromatography, the column temperature is 30~50℃.

8. The detection method as described in claim 7, characterized in that, The detection method satisfies at least one of the following conditions (1) to (6): (1) The aqueous solution of the volatile acid is selected from formic acid aqueous solution and / or acetic acid aqueous solution; (2) The mass percentage of the volatile acid in the aqueous solution of the volatile acid is 0.08%~0.12%; (3) The isocratic elution is performed, wherein the mobile phase A accounts for more than 85% of the volume percentage of the mobile phase, preferably 85% to 95%; (4) Using the gradient elution, from 0 to 8.5 min, the volume percentage of mobile phase A is 85% to 92% and the volume percentage of mobile phase B is 8% to 25%; from 8.6 to 11 min, the volume percentage of mobile phase A is 10% to 30% and the volume percentage of mobile phase B is 70% to 90%; from 11.1 to 15 min, the volume percentage of mobile phase A is 75% to 92% and the volume percentage of mobile phase B is 8% to 25%. (5) In high performance liquid chromatography, the temperature of the injector is 8~15℃; (6) In high performance liquid chromatography, the chromatographic column was an Agilent Infinitylab poroshell 120 EC-18 100mm. 3mm, 1.9µm.

9. The detection method according to any one of claims 1 to 8, characterized in that, The detection method satisfies at least one of the following conditions (1) to (15): (1) In mass spectrometry detection, the eluent with a retention time of 5 to 8.5 min in the high performance liquid chromatography is detected; (2) In the mass spectrometer, the ion mode is positive ion mode; (3) In mass spectrometry, the scanning mode is multiple reaction monitoring mode; (4) In the mass spectrometry, the ion source is an electrospray ion source; (5) In the mass spectrometer, the drying gas is nitrogen; (6) In mass spectrometry, the temperature of the drying gas is 100~350℃; (7) In the mass spectrometer, the flow rate of the drying gas is 1~13 L / min; (8) In the mass spectrometer, the pressure of the nebulizing gas is 10~40 psi; (9) In the mass spectrometer, the sheath gas is nitrogen; (10) In mass spectrometry, the temperature of the sheath gas is 250~400℃; (11) In the mass spectrometer, the flow rate of the sheath gas is 10~14 L / min; (12) In mass spectrometry, the capillary voltage is 3500~6000V; (13) In mass spectrometry, the fragmentation voltage is 0~200V; (14) In mass spectrometry, the collision energy is 5~35V; (15) In the mass spectrometry, the detection ion pair of 3-[4-(2-epoxyethylene methoxy)butoxy]-1,2-propanediol is at least one of 221 / 57.1, 221 / 73.2 and 221 / 129.

10. The detection method as described in claim 9, characterized in that, The detection method satisfies at least one of the following conditions (1) to (5): (1) In mass spectrometry, the temperature of the drying gas is 300~340℃; (2) In mass spectrometry, the temperature of the sheath gas is 380~400℃; (3) In mass spectrometry, the capillary voltage is 3500~4500V; (4) In mass spectrometry, the fragmentation voltage is 60~150V; (5) In mass spectrometry, the collision energy is 10~20V.

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