A method for detecting the metabolic time of sodium hyaluronate gel in animal plasma

Abstract

The present invention discloses a method for detecting the metabolic time of sodium hyaluronate gel in an animal, which comprises the following steps: injecting sodium hyaluronate gel and enzymatically hydrolyzed sodium hyaluronate gel into an experimental animal, then continuously sampling blood from the animal according to a preset time, obtaining a plasma sample, removing the protein in the plasma sample, and obtaining a first plasma sample without protein; using liquid chromatography-quadrupole-time-of-flight mass spectrometry to analyze the mass spectrum characteristic example in the first plasma sample; drawing a curve of the intensity of mass spectrum characteristic ions over time, and analyzing the metabolic time of sodium hyaluronate gel in animal plasma; wherein, the mass spectrum characteristic ions are 356.7168m / z, 367.7046m / z, 402.1026m / z, 133.0859m / z and 177.1121m / z respectively. The mass spectrum characteristic ions of the present invention can be used to track the metabolic time of sodium hyaluronate gel in an animal.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and particularly relates to a method for detecting the metabolism time of sodium hyaluronate gel in animal plasma. Background Art

[0002] Sodium hyaluronate gel is a high molecular polymer formed by the reaction of the hydroxyl groups on the repeating units of hyaluronic acid disaccharide with the hydroxyl groups on both ends of the cross-linking agent after the ring opening, and the removal of a molecule of water for cross-linking. It is a new medical beauty product in recent years. The main routes of administration of sodium hyaluronate gel are percutaneous absorption, subcutaneous injection and local injection. After entering the body, sodium hyaluronate gel can quickly replenish water to the skin to achieve a long-term moisturizing effect. Sodium hyaluronate gel can stimulate the increase of collagen in the skin, thereby tightening the skin. In addition, sodium hyaluronate gel can also be used as a filler for human skin to remove wrinkles and achieve three-dimensional shaping.

[0003] At present, there are few studies on the metabolic pathways of sodium hyaluronate gel in the body (human and animal), and there is a lack of safety data. In addition, sodium hyaluronate gel will be degraded by metabolic enzymes in the body and degraded into fragments with smaller molecular weight. How to track its metabolic process is a difficulty faced by researchers. Summary of the invention

[0004] In order to solve the above-mentioned technical problems, the biological functions and absorption and transport mechanisms of sodium hyaluronate gel are studied in depth to improve its precise targeting effect and targeted application value.

[0005] In a first aspect, the present invention provides a method for detecting the metabolism time of sodium hyaluronate gel in animal plasma, the detection method comprising the following steps:

[0006] Step 1-1, injecting sodium hyaluronate gel and enzymatically hydrolyzed sodium hyaluronate gel into experimental animals, and raising the experimental animals;

[0007] Step 1-2, continuously collecting blood from the animal at preset time intervals to obtain a plasma sample, and then removing protein from the plasma sample to obtain a protein-free first plasma sample;

[0008] Step 1-3, analyzing the mass spectral characteristic examples in the first plasma sample by liquid chromatography-quadrupole-time of flight mass spectrometry;

[0009] Step 1-4, plotting the change curve of the mass spectrometry characteristic ion intensity over time, thereby analyzing the metabolism time of the sodium hyaluronate gel in the animal plasma;

[0010] Among them, the mass spectrum characteristic ions are 356.7168m / z, 367.7046m / z, 402.1026m / z, 133.0859m / z and 177.1121m / z respectively.

[0011] Preferably, the screening process of the mass spectrum characteristic ions is as follows:

[0012] Step 2-1, collecting blood from experimental animals that have not been administrated to prepare a second protein-free plasma sample;

[0013] Step 2-2, preparing a mixed sample of a sodium hyaluronate gel sample, a sodium hyaluronate gel sample after enzymatic hydrolysis, the second plasma sample and the sodium hyaluronate gel;

[0014] Step 2-3, respectively analyzing the second plasma sample, the sodium hyaluronate gel sample, the sodium hyaluronate gel sample after enzymatic hydrolysis, and the mixed sample by liquid chromatography-quadrupole-time of flight mass spectrometry;

[0015] Step 2-4, comparing the analysis results of each sample in step 2-3, screening the mass spectrometry marker ions that are not present in the second plasma sample but are present in the other samples, and obtaining the mass spectrometry characteristic ions.

[0016] Preferably, the experimental animal is selected from the plasma of rats, mice, rabbits, horses, and sheep, and more preferably from SD rats.

[0017] Preferably, in the mixture of the sodium hyaluronate gel sample and the second plasma sample, the volume ratio of the blank plasma sample to the sodium hyaluronate gel sample is 1:0.5-2, and more preferably 1:0.5-1.5, for example: 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5.

[0018] Preferably, the mass spectrometry labeled ion results in the sodium hyaluronate gel sample, the sodium hyaluronate gel sample after enzymatic hydrolysis and the mixed sample are: 356.7168m / z, 367.7046m / z, 402.1026m / z, 133.0859m / z, 177.1121m / z, 380.1140m / z, 359.1932m / z, 370.1868m / z, 204.0853m / z and 138.0559m / z.

[0019] Preferably, the mass spectrometry marker ion results in the second plasma sample are: 356.7168 m / z, 367.7046 m / z, 402.1026 m / z, 133.0859 m / z and 177.1121 m / z.

[0020] Preferably, after comparing the analysis results of the sodium hyaluronate gel sample, the second plasma sample, the mixture of the sodium hyaluronate gel sample and the plasma sample, and the sodium hyaluronate gel sample after enzymatic hydrolysis, the mass spectrometry characteristic ions obtained are 356.7168m / z, 367.7046m / z, 402.1026m / z, 133.0859m / z and 177.1121m / z.

[0021] In a specific embodiment of the present invention, the liquid chromatography is size exclusion chromatography, and the chromatographic column is preferably one of SEC-100, SEC-150, SEC-300, SEC-500, SEC-1000, and SEC-2000, more preferably SEC-1000.

[0022] Preferably, the particle size of the chromatographic column is 3-10 μm, for example, 3 μm, 5 μm, 10 μm, and more preferably 5 μm.

[0023] Preferably, the chromatographic column is selected from one of 4.6×150mm, 4.6×250mm, 4.6×300mm, 7.8×150mm, 7.8×250mm, and 7.8×300mm, and more preferably 7.8×300mm.

[0024] Preferably, in the liquid chromatography analysis process, the mobile phase consists of water and acetonitrile, further preferably, the volume ratio of water to acetonitrile is 100-80:0-20, more preferably, the volume ratio of water to acetonitrile is 85:15.

[0025] Preferably, the elution mode of the liquid chromatography is isocratic elution.

[0026] Preferably, the elution time of the liquid chromatography is 0-160 min, for example: 0-140 min, 0-145 min, 0-150 min, 0-155 min, 0-160 min, more preferably 0-150 min.

[0027] Preferably, during the liquid chromatography analysis, the flow rate of the mobile phase is 0.1-1.0 mL / min, more preferably 0.3-0.8 mL / min, for example: 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 mL / min.

[0028] Preferably, the column temperature of the liquid chromatography is 30-50°C, more preferably 30-45°C, for example: 30, 32, 34, 35, 36, 37, 40, 42, 43, 45°C.

[0029] Preferably, the injection volume of the liquid chromatography is 1-20 μL, more preferably 5-15 μL, for example: 5 μL, 7 μL, 9 μL, 10 μL, 12 μL, 13 μL, 15 μL.

[0030] In a specific embodiment of the present invention, the liquid chromatograph is a SCIEX ExionLC liquid phase system.

[0031] Preferably, the ionization mode of the quadrupole-time-of-flight mass spectrometer in step (2) is an ESI source positive ionization mode.

[0032] Preferably, the scanning range of the quadrupole-time-of-flight mass spectrometry is 100-2000 m / z.

[0033] Preferably, the spray voltage of the quadrupole-time-of-flight mass spectrometry is 5500v.

[0034] Preferably, the parameter Time bins to sum of the quadrupole time-of-flight mass spectrometry is 4.

[0035] Preferably, the mass spectrum characteristic ion extraction range of the quadrupole-time-of-flight mass spectrometry analysis is ±0.005Da.

[0036] Preferably, the parameter Accumulation time of the quadrupole-time-of-flight mass spectrometry is 0.15 sec.

[0037] Preferably, the mass spectrometry parameters of the five mass spectrometry characteristic ions of the quadrupole-time-of-flight mass spectrometry analysis are as follows:

[0038] The mass spectrometry parameters of the mass spectrometry characteristic ion 356.7168 m / z are as follows: scanning range 356.2-357.2 m / z, ion source gas 1 pressure 65 psi, ion source gas 2 pressure 65 psi, curtain gas pressure 35 psi, ion source temperature 500° C., spray voltage 5500 v, declustering voltage 60 v, collision energy 10 v, and accumulation time 0.15 s;

[0039] The mass spectrometry parameters of the mass spectrometry characteristic ion 367.7046 m / z are as follows: scanning range 367.2-368.2 m / z, ion source gas 1 pressure 55 psi, ion source gas 2 pressure 55 psi, curtain gas pressure 35 psi, ion source temperature 500° C., spray voltage 5500 v, declustering voltage 60 v, collision energy 5 v, and accumulation time 0.15 s;

[0040] The mass spectrometry parameters of the mass spectrometry characteristic ion 402.1026 m / z are: scanning range 401.5-402.5 m / z, ion source gas 1 pressure 55 psi, ion source gas 2 pressure 55 psi, curtain gas pressure 35 psi, ion source temperature 500° C., spray voltage 5500 v, declustering voltage 50 v, collision energy 110 v, and accumulation time 0.15 s;

[0041] The mass spectrometry parameters of the mass spectrometry characteristic ion 133.0859 m / z are as follows: scanning range 132.5-133.5 m / z, ion source gas 1 pressure 60 psi, ion source gas 2 pressure 60 psi, curtain gas pressure 35 psi, ion source temperature 500° C., spray voltage 5500 v, declustering voltage 180 v, collision energy 40 v, and accumulation time 0.15 s;

[0042] The mass spectrometry parameters of the mass spectrometry characteristic ion 177.1121m / z are as follows: scanning range 176.5-177.5m / z, ion source gas 1 pressure 60psi, ion source gas 2 pressure both 60psi, curtain gas pressure 35psi, ion source temperature 500°C, spray voltage 5500v, declustering voltage 180v, collision energy 40v, and accumulation time 0.15s.

[0043] In a specific embodiment of the present invention, the quadrupole-time of flight mass spectrometer is a Triple TOF 6600 detector.

[0044] In a second aspect, the present invention provides application of the detection method of the first aspect in tracking the metabolism time of sodium hyaluronate gel in animal plasma.

[0045] Beneficial effects of the present invention:

[0046] The mass spectrometry characteristic ions of the present invention can be used to track the metabolic pathway of sodium hyaluronate gel in vivo, providing a technical basis for further research on the efficient utilization of sodium hyaluronate gel.

[0047] The detection method of the present invention can be extended to the tracking study of macromolecules similar to sodium hyaluronate gel, and provides a new research method for the study of the biological function and absorption and transport mechanism of macromolecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 Shown is the intensity variation curve of the mass spectrum characteristic ion 356.7168m / z over time in the present invention;

[0049] Figure 2 Shown is the intensity variation curve of the mass spectrum characteristic ion 367.7046m / z over time in the present invention;

[0050] Figure 3 Shown is the intensity variation curve of the mass spectrum characteristic ion 133.0859m / z over time in the present invention;

[0051] Figure 4 Shown is the intensity variation curve of the mass spectrum characteristic ion 177.1121m / z over time in the present invention;

[0052] Figure 5 Shown is the intensity variation curve of the mass spectrum characteristic ion 402.1026m / z over time in the present invention. DETAILED DESCRIPTION

[0053] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0054] It should be noted that the terms "first" and "second" in the specification, claims and abstract of the application document are only used to describe similar objects and do not have to be understood as a specific order or sequence.

[0055] In addition, it should be noted that the chemical reagents in the present invention are all conventional reagents in the art unless otherwise specified.

[0056] Example 1: Analysis of Sodium Hyaluronate Gel, Sodium Hyaluronate Gel Hydrolysate, Plasma Sample, and Mixture of Plasma and Sodium Hyaluronate Gel by Liquid Chromatography-Quadrupole-Time-of-Flight Mass Spectrometry

[0057] 1.1 Sample pretreatment

[0058] 1.1.1 Weigh 50 mg of sodium hyaluronate gel (catalog number HA-YF210913, Tianjin Jiankai Technology Co., Ltd.) sample, add it to 1 mL of water and stir thoroughly to obtain a sodium hyaluronate gel sample solution.

[0059] 1.1.2 The sodium hyaluronate gel samples were treated with 750 U / L active enzyme and 1500 U / L active enzyme (hyaluronidase for injection, batch number: 2202701, Shanghai No. 1 Biochemical Pharmaceutical Co., Ltd.) respectively, and incubated at 37°C for 48 h to degrade the samples, and then fully mixed to obtain sodium hyaluronate gel degradation product samples (1) and (2).

[0060] 1.1.3 Blank plasma sample: collect 100 μL of abdominal plasma from SD rats, add 300 μL of methanol pre-cooled at -4°C, vortex for 3 min to mix, centrifuge for 10 min, discard the precipitate and take the supernatant, blow dry with nitrogen to about 200 μL, and obtain a protein-free blank plasma sample.

[0061] 1.1.4 Take 50 μL of the sodium hyaluronate gel sample solution and 50 μL of the plasma sample respectively, mix them evenly to obtain a mixed sample of plasma and sodium hyaluronate gel.

[0062] 1.2 Sample analysis

[0063] The above samples were tested and analyzed according to the instruments and parameters in Table 1, and the analysis results were recorded, as shown in Table 2.

[0064] Table 1 Instrument and analysis parameters

[0065]

[0066] Table 2 Analysis results of each sample

[0067]

[0068]

[0069] From the results in Table 2, it can be seen that the candidate mass spectrometry marker ions of the plasma sample are: 380.1140m / z, 359.1932m / z, 370.1868m / z, 204.0853m / z, 138.0559m / z. However, it does not contain: 356.7168m / z, 367.7046m / z, 402.1026m / z, 133.0859m / z, 177.1121m / z. The candidate mass spectrometry marker ions in the sodium hyaluronate gel sample and the candidate mass spectrometry marker ions in the mixture of the plasma sample and the sodium hyaluronate gel sample are: 356.7168m / z, 367.7046m / z, 402.1026m / z, 133.0859m / z, 177.1121m / z, 380.1140m / z, 359.1932m / z, 370.1868m / z, 204.0853m / z, and 138.0559m / z. The candidate mass spectrometry marker ion results in the sodium hyaluronate gel enzymatic hydrolysis product (1) are: 356.7168m / z, 367.7046m / z, 402.1026m / z, 133.0859m / z, 177.1121m / z, 380.1140m / z, 359.1932m / z, 370.1868m / z, 204.0853m / z, and 138.0559m / z. The candidate mass spectrometry marker ions in the sodium hyaluronate gel hydrolysate (2) are: 356.7168m / z, 367.7046m / z, 402.1026m / z, 133.0859m / z, 177.1121m / z, 380.1140m / z, 359.1932m / z, 370.1868m / z, 204.0853m / z, 138.0559m / z.

[0070] Due to the influence of candidate mass spectrometry marker ions 380.1140m / z, 359.1932m / z, 370.1868m / z, 204.0853m / z, and 138.0559m / z of plasma samples, 356.7168m / z, 367.7046m / z, 402.1026m / z, 133.0859m / z, and 177.1121m / z were selected as mass spectrometry characteristic ions for tracking and analyzing the metabolic time of sodium hyaluronate gel in animals.

[0071] Example 2: Detection of sodium hyaluronate gel or its metabolites in SD rats

[0072] 2.1 Sample pretreatment

[0073] 2.1.1 Two groups of SD rats were selected, with 7 male 200-250g SD rats in each group. The intact sodium hyaluronate and the enzymatically hydrolyzed sodium hyaluronate were respectively administered with subcutaneous multiple injections at 20mL / kg at 0h, and blood samples were collected at the time points shown in Table 3 below (this part of the experiment was commissioned by Tianjin Youji Pharmaceutical Technology Development Co., Ltd.).

[0074] Table 3 Blood sample collection of SD rats

[0075] Complete Sodium Hyaluronate Group 1h 2h 4h 8h 24h 48h 72h 96h 120h 144h 168h Enzymatic hydrolysis of sodium hyaluronate group 1h 2h 4h 8h 24h 48h 72h 96h 120h 144h 168h

[0076] 2.1.1 Take 100 μL of abdominal plasma from the above SD rats, add 300 μL of methanol pre-cooled at -4°C, vortex for 3 min to mix, centrifuge for 10 min, discard the precipitate, take the supernatant, blow dry with nitrogen to about 200 μL, and obtain protein-free plasma samples for Q-TOF detection.

[0077] 2.2 Sample analysis

[0078] The above samples were tested and analyzed according to the instruments and parameters in Table 1 and Table 4, and the analysis results were recorded. The results of each mass spectrometry characteristic ion are shown in Figure 1-Figure 5 .

[0079] Table 4 Q-TOF parameters

[0080]

[0081] Depend on Figure 1 The results show that within 0-1h after administration to SD rats, the intensity value of the mass spectrometry characteristic ion 356.7168m / z in the enzymatically hydrolyzed hyaluronic acid plasma sample increased from 0 to a peak value of 16000, and then gradually decreased, and the intensity value dropped to 0 at 4h. This indicates that the use of the mass spectrometry characteristic ion 356.7168m / z can track the metabolic process of the enzymatic hydrolysis products of sodium hyaluronate gel in SD rats. Within 0-1.5h after administration to SD rats, the intensity value of the mass spectrometry characteristic ion 356.7168m / z in the intact sodium hyaluronate gel sample increased from 0 to a peak value of 20000, and then gradually decreased, and the intensity value dropped to 0 at 8h. This indicates that the use of the mass spectrometry characteristic ion 356.7168m / z can track the metabolic process of sodium hyaluronate gel in SD rats.

[0082] Depend on Figure 2The results show that within 0-1h after administration to SD rats, the intensity value of the mass spectrometry characteristic ion 367.7046m / z in the enzymatically hydrolyzed hyaluronic acid plasma sample increased from 0 to a peak value of 13000, and then gradually decreased, and the intensity value dropped to 0 at 4h. This indicates that the use of the mass spectrometry characteristic ion 367.7046m / z can track the metabolic process of the enzymatic hydrolysis products of sodium hyaluronate gel in SD rats. Within 0-2h after administration to SD rats, the intensity value of the mass spectrometry characteristic ion 367.7046m / z in the intact sodium hyaluronate gel sample increased from 0 to a peak value of 35000, and then gradually decreased, and the intensity value dropped to 0 at 24h. This indicates that the use of the mass spectrometry characteristic ion 367.7046m / z can track the metabolic process of sodium hyaluronate gel in SD rats.

[0083] Depend on Figure 3 The results show that within 0-1h after administration to SD rats, the intensity value of the mass spectrometry characteristic ion 133.0859m / z in the enzymatically hydrolyzed hyaluronic acid plasma sample increased from 900 to a peak value of 1400, and then gradually decreased, and the intensity value dropped to 0 at 48h. This indicates that the use of the mass spectrometry characteristic ion 133.0859m / z can track the metabolic process of the enzymatic hydrolysis products of sodium hyaluronate gel in SD rats. Within 0-1.5h after administration to SD rats, the intensity value of the mass spectrometry characteristic ion 133.0859m / z in the intact sodium hyaluronate gel sample increased from 400 to a peak value of 5800, and then gradually decreased, and the intensity value dropped to 0 at 24h, but there was a slight fluctuation within 24-48h, and the intensity value dropped to 0 at 72h. This indicates that the use of the mass spectrometry characteristic ion 133.0859m / z can track the metabolic process of sodium hyaluronate gel in SD rats.

[0084] Depend on Figure 4 The results show that within 0-1h after administration to SD rats, the intensity value of the mass spectrometry characteristic ion 177.1121m / z in the enzymatically hydrolyzed hyaluronic acid plasma sample increased from 400 to a peak value of 1000, and then gradually decreased, and the intensity value dropped to 0 at 48h. This indicates that the use of the mass spectrometry characteristic ion 177.1121m / z can track the metabolic process of the enzymatic hydrolysis products of sodium hyaluronate gel in SD rats. Within 0-1h after administration to SD rats, the intensity value of the mass spectrometry characteristic ion 177.1121m / z in the intact sodium hyaluronate gel sample increased from 500 to a peak value of 4500, and then gradually decreased, and the intensity value dropped to 0 at 24h. This indicates that the use of the mass spectrometry characteristic ion 177.1121m / z can track the metabolic process of sodium hyaluronate gel in SD rats.

[0085] Depend on Figure 5The results show that within 0-1h after administration to SD rats, the intensity value of the mass spectrometry characteristic ion 402.1026m / z in the enzymatically hydrolyzed hyaluronic acid plasma sample increased from 1500 to a peak value of 8000, and then gradually decreased, and the intensity value dropped to 0 at 48h. This indicates that the use of the mass spectrometry characteristic ion 402.1026m / z can track the metabolic process of the enzymatic hydrolysis products of sodium hyaluronate gel in SD rats. Within 0-1h after administration to SD rats, the intensity value of the mass spectrometry characteristic ion 402.1026m / z in the intact sodium hyaluronate gel sample increased from 1500 to a peak value of 2300, and then gradually decreased, and the intensity value dropped to 0 at 24h. This indicates that the use of the mass spectrometry characteristic ion 402.1026m / z can track the metabolic process of sodium hyaluronate gel in SD rats.

[0086] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for detecting the metabolic time of sodium hyaluronate gel in animal plasma, characterized in that: The detection method comprises the following steps: Step 1-1, injecting sodium hyaluronate gel and enzymatically hydrolyzed sodium hyaluronate gel into experimental animals, and raising the experimental animals; Step 1-2, continuously collecting blood from the animal at preset time intervals to obtain a plasma sample, and then removing protein from the plasma sample to obtain a protein-free first plasma sample; Step 1-3, analyzing mass spectrometry characteristic ions in the first plasma sample using liquid chromatography-quadrupole-time of flight mass spectrometry; Step 1-4, plotting the change curve of the mass spectrometry characteristic ion intensity over time, thereby analyzing the metabolism time of the sodium hyaluronate gel in the animal plasma; The chromatographic column in the liquid chromatography is SEC-1000; The mobile phase flow rate in the liquid chromatography was 0.5 mL / min, the column temperature was 35°C, and the injection volume was 10 μL; The mobile phase of the liquid chromatography consists of water and acetonitrile in a volume ratio of 85:15; The elution mode of the liquid chromatography is isocratic elution, and the elution time is 0-150min; The ionization mode of the quadrupole-time-of-flight mass spectrometry analysis is an ESI source positive ionization mode; The scanning range of the quadrupole-time of flight mass spectrometry analysis is 100-2000 m / z; Among them, the mass spectrum characteristic ions are 356.7168m / z, 367.7046m / z, 402.1026m / z, 133.0859m / z and 177.1121m / z respectively.

2. The detection method according to claim 1, characterized in that: The screening process of the mass spectrometry characteristic ions is as follows: Step 2-1, collecting blood from experimental animals that have not been administrated to prepare a second protein-free plasma sample; Step 2-2, preparing a mixed sample of a sodium hyaluronate gel sample, a sodium hyaluronate gel sample after enzymatic hydrolysis, the second plasma sample and the sodium hyaluronate gel; Step 2-3, respectively analyzing the second plasma sample, the sodium hyaluronate gel sample, the sodium hyaluronate gel sample after enzymatic hydrolysis, and the mixed sample by liquid chromatography-quadrupole-time of flight mass spectrometry; Step 2-4, comparing the analysis results of each sample in step 2-3, screening the mass spectrometry marker ions that are not present in the second plasma sample but are present in the other samples, and obtaining the mass spectrometry characteristic ions.

3. The detection method according to claim 2, characterized in that: The concentration of the sodium hyaluronate gel sample is 5-70 mg / mL, and the volume ratio of the second plasma sample to the sodium hyaluronate gel sample is 1:0.5-2.

4. The detection method according to claim 3, characterized in that: The mass spectrometry parameters of the mass spectrometry characteristic ion 356.7168 m / z are as follows: scanning range 356.2-357.2 m / z, ion source gas 1 pressure 65 psi, ion source gas 2 pressure 65 psi, curtain gas pressure 35 psi, ion source temperature 500° C., spray voltage 5500 v, declustering voltage 60 v, collision energy 10 v, and accumulation time 0.15 s; The mass spectrometry parameters of the mass spectrometry characteristic ion 367.7046 m / z are as follows: scanning range 367.2-368.2 m / z, ion source gas 1 pressure 55 psi, ion source gas 2 pressure 55 psi, curtain gas pressure 35 psi, ion source temperature 500° C., spray voltage 5500 v, declustering voltage 60 v, collision energy 5 v, and accumulation time 0.15 s; The mass spectrometry parameters of the mass spectrometry characteristic ion 402.1026 m / z are: scanning range 401.5-402.5 m / z, ion source gas 1 pressure 55 psi, ion source gas 2 pressure 55 psi, curtain gas pressure 35 psi, ion source temperature 500° C., spray voltage 5500 v, declustering voltage 50 v, collision energy 110 v, and accumulation time 0.15 s; The mass spectrometry parameters of the mass spectrometry characteristic ion 133.0859 m / z are as follows: scanning range 132.5-133.5 m / z, ion source gas 1 pressure 60 psi, ion source gas 2 pressure 60 psi, curtain gas pressure 35 psi, ion source temperature 500° C., spray voltage 5500 v, declustering voltage 180 v, collision energy 40 v, and accumulation time 0.15 s; The mass spectrometry parameters of the mass spectrometry characteristic ion 177.1121m / z are as follows: scanning range 176.5-177.5m / z, ion source gas 1 pressure 60psi, ion source gas 2 pressure both 60psi, curtain gas pressure 35psi, ion source temperature 500°C, spray voltage 5500v, declustering voltage 180v, collision energy 40v, and accumulation time 0.15s.

5. Use of the detection method according to any one of claims 1 to 4 in tracking the metabolism time of sodium hyaluronate gel in animal plasma.

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